phenylacetylcarbinol (PAC) Synthesis in Aqueous/Organic ...

Bioprocess Development for (R)-phenylacetylcarbinol

(PAC) Synthesis in Aqueous/Organic Two-Phase System

Cindy Gunawan, B.E.

A thesis submitted in fulfillment of the requirements for the Degree of

Doctor of Philosophy

School of Biotechnology and Biomolecular Sciences

University of New South Wales

Sydney, Australia

March 2006

Declaration I hereby declare that this submission is my own work and to the best of my knowledge it

contains no material previously published or written by another person, nor material

which to a substantial extent has been accepted for the award of any degree or diploma at

UNSW or any other educational institution, except where due acknowledgement is made

in this thesis. Any contribution made to the research by others, with whom I have worked

at UNSW or elsewhere, is explicitly acknowledged in the thesis.

I also declare that the intellectual content of this thesis is the product of my own work,

except to the extent that assistance from others in the project’s design and conception or in

style, presentation and linguistic expression is acknowledged.

__________________________

Cindy Gunawan

for papi and mami

Acknowledgements

i

ACKNOWLEDGEMENTS

I would like to express my deepest gratitude to my supervisor Professor Peter L. Rogers

and co-supervisor Dr Bettina Rosche for their endless guidance, support, and patience

throughout my project and in the preparation of this thesis.

I am very thankful to the Australian Government for awarding me the Endeavour

International Postgraduate Research Scholarship, which has given me priceless

opportunities and invaluable experience in research. I would also like to thank BASF

Ludwigshafen for their sponsorhip of the project.

I am very grateful to Dr. Martin Zarka, Dr. Russel Cail, and Malcolm Noble for their

technical support and to Gerry Ferhard, Peter, and John of the Faculty of Science

Workshop for constructing the Lewis cell.

A warm thank you to all my colleagues Lia, Allen, Noppol, André, and Richard for their

opinions, support, and those happy times. I would also like to thank all my friends Eny,

Ronachai, Onn, Yin, Nico, Adrian and all the staff in the School of Biotechnology and

Biomolecular Sciences (BABS) for their friendship.

My greatest gratitude to my parents, sisters and brothers; Lingling, Mona, Cangcang, and

Penpen for their kindness and warm love. Finally, I wish to thank my husband Frans for

his beautiful heart and endless encouragement.

Abstract

ii

ABSTRACT

(R)-phenylacetylcarbinol or R-PAC is a chiral precursor for the synthesis of

pharmaceuticals ephedrine and pseudoephedrine. PAC is produced through

biotransformation of pyruvate and benzaldehyde catalyzed by pyruvate decarboxylase

(PDC) enzyme. The present research project aims at characterizing a two-phase

aqueous/organic process for enzymatic PAC production.

In a comparative study of several selected yeast PDCs, the highest PAC formation was

achieved in systems with relatively high benzaldehyde concentrations when using C.

utilis PDC. C. tropicalis PDC was associated with the lowest by-product acetoin

formation although it also produced lower PAC concentrations. C. utilis PDC was

therefore selected as the biocatalyst for the development of the two-phase PAC

production.

From an enzyme stability study it was established that PDC deactivation rates in the two-

phase aqueous/octanol-benzaldehyde system were affected by: (1) soluble octanol and

benzaldehyde in the aqueous phase, (2) agitation rate, (3) aqueous/organic interfacial

area, and (4) initial enzyme concentration. PDC deactivation was less severe in the

slowly stirred phase-separated system (low interfacial area) compared to the rapidly

stirred emulsion system (high interfacial area), however the latter system was presumably

associated with a faster rate of organic-aqueous benzaldehyde transfer.

To find a balance between maintaining enzyme stability while enhancing PAC

productivity, a two-phase system was designed to reduce the interfacial contact by

decreasing the organic to aqueous phase volume ratio. Lowering the ratio from 1:1 to

0.43:1 resulted in increased overall PAC production at 4°C and 20°C (2.5 M MOPS,

partially purified PDC) with a higher concentration at the higher temperature. The PAC

was highly concentrated in the organic phase with 212 g/L at 0.43:1 in comparison to 111

g/L at 1:1 ratio at 20°C.

The potential of further two-phase process simplification was evaluated by reducing the

expensive MOPS concentration to 20 mM (pH controlled at 7.0) and employment of

Abstract

iii

whole cell PDC. It was found that 20°C was the optimum temperature for PAC

production in such a system, however under these conditions lowering the phase ratio

resulted in decreased overall PAC production. Two-phase PAC production was relatively

low in 20 mM MOPS compared to biotransformations in 2.5 M MOPS. Addition of 2.5

M dipropylene glycol (DPG) into the aqueous phase with 20 mM MOPS at 0.25:1 ratio

and 20°C improved the production with organic phase containing 95 g/L PAC. Although

the productivity was lower, the system may have the benefit of a reduction in production

cost.

Publications

iv

PUBLICATIONS

Published Paper

C. Gunawan, G. Satianegara, A.K. Chen, M. Breuer, B..Hauer, P.L. Rogers, B. Rosche.

(2006). Yeast Pyruvate Decarboxylases: Variation in Biocatalytic Characteristics for (R)-

phenylacetylcarbinol Production. FEMS Yeast Research: doi:10.1111/j.1567-

1364.2006.00138.x (with pending volume, issue and page numbers).

Paper in preparation

C. Gunawan, M. Breuer, B..Hauer, P.L. Rogers, B. Rosche. (2006). Key Factors

Influencing Enzyme Stability and Biotransformation in Two-Phase Aqueous/Organic

System for (R)-phenylacetylcarbinol Production. In preparation for submission to

Biotechnology and Bioengineering Journal.

Poster and oral presentations

C. Gunawan, M. Breuer, B. Hauer, P.L. Rogers, B. Rosche. (2003). Impact of Process

Parameters on R-phenylacetylcarbinol (PAC) Production in Aqueous/Organic Two-Phase

Biotransformation, Fermentation and Bioprocessing Conference. The Garvan Institute for

Medical Research, Sydney, Australia, 14 – 15 April, poster presentation p. 52, ISBN 0

7334 2023 0.

C. Gunawan, M. Breuer, B. Hauer, P.L. Rogers, B. Rosche. (2003). Process

Development for R-phenylacetylcarbinol (PAC) Production in Aqueous/Organic Two-

Phase Biotransformation. 6th International Symposium on Biocatalysis and

Biotransformations. BIOTRANS 2003, Palacky University, Olomouc, Czech Republic,

28 June – 3 July, poster presentation number 245, p. 507, ISSN 0009-2770.

Publications

v

C. Gunawan, M. Breuer, B. Hauer, P.L. Rogers, B. Rosche. (2003). Investigation on R-

phenylacetylcarbinol (PAC) Production in Aqueous/Organic Two-Phase

Biotransformation, School of Biotechnology and Biomolecular Sciences Third Annual

Symposium, Sydney, Australia, 7 November 2003, poster presentation P-12, ISBN 0

7334 1581 4.

C. Gunawan, M. Breuer, B. Hauer, P.L. Rogers, B. Rosche. (2004). Effect of Organic to

Aqueous Phase Volume Ratio in Two-Phase System for R-phenylacetylcarbinol

Biosynthesis, Fermentation and Bioprocessing Conference. UQ Centre University of

Queensland, Brisbane, Australia, 5 – 6 July 2004, poster presentation number 21, p.55,

ISBN 0 646 43707 0.

C. Gunawan, M. Breuer, B. Hauer, P.L. Rogers, B. Rosche. (2004). Bioprocess

Development for R-phenylacetylcarbinol (PAC) Production in Aqueous/Organic Two-

Phase System, School of Biotechnology and Biomolecular Sciences Third Annual

Symposium, Sydney, Australia, 5 November 2004, oral presentation 2-1, ISBN 0 7334

2162 8.

C. Gunawan, M. Breuer, B. Hauer, P.L. Rogers, B. Rosche. (2005). Optimization of

Aqueous/Organic Two-Phase System for (R)-phenylacetylcarbinol (PAC) Biosynthesis,

7th International Symposium on Biocatalysis and Biotransformations. BIOTRANS 2005,

TU Delft, Delft, The Netherlands, 3 – 8 July, poster presentation number 147, p. 147,

ISBN 90 809691 17.

G. Satianegara, C. Gunawan, A.K. Chen, M. Breuer, B. Hauer, P.L. Rogers, B. Rosche.

(2003). R-phenylacetylcarbinol (R-PAC) Production and Stability Study with Pyruvate

Decarboxylase from Four Yeast Strains, Fermentation and Bioprocessing Conference.

The Garvan Institute for Medical Research, Sydney, Australia, 14 – 15 April, poster

presentation p. 63, ISBN 0 7334 2023 0.

Publications

vi

G. Satianegara, C. Gunawan, A.K. Chen, M. Breuer, B. Hauer, P.L. Rogers, B. Rosche.

Comparison of Four Yeast Pyruvate Decarboxylase for R-phenylacetylcarbinol

Production, 6th International Symposium on Biocatalysis and Biotransformations.

BIOTRANS 2003, Palacky University, Olomouc, Czech Republic, 28 June – 3 July,

poster presentation number 119, p. 430, ISSN 0009-2770.

B. Rosche, V. Sandford, N. Leksawasdi, A. Chen, G. Satianegara, C. Gunawan, M.

Breuer, B. Hauer, P.L. Rogers. (2003). Bioprocess development for ephedrine production,

6th International Symposium on Biocatalysis and Biotransformations. BIOTRANS 2003,

Palacky University, Olomouc, Czech Republic, 28 June – 3 July, poster presentation

P224, ISSN 0009-2770.

Table of Contents

vii

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ............................................................................................i

ABSTRACT ...................................................................................................................ii

PUBLICATIONS ..........................................................................................................iv

TABLE OF CONTENTS ...........................................................................................viii

LIST OF TABLES ....................................................................................................xivv

LIST OF FIGURES ...................................................................................................xvii

PROJECT SCOPE AND OBJECTIVES...............................................................xxixx

1. LITERATURE REVIEW .........................................................................................1

1.1 Introduction ...............................................................................................................2

1.2 Development of Biotransformation Processes ............................................................2

1.3 Ephedrine and Pseudoephedrine Synthesis.................................................................6

1.3.1 Pharmacological Values ..............................................................................6

1.3.2 Traditional Production.................................................................................6

1.3.3 (R)-phenylacetylcarbinol (PAC) as a Precursor............................................7

1.4 Biotransformation of Pyruvate and Benzaldehyde to PAC..........................................9

1.4.1 Reaction Mechanisms..................................................................................9

1.4.1.1 Early Findings ....................................................................................9

1.4.1.2 PAC Formation...................................................................................9

1.4.2 Pyruvate Decarboxylase Enzyme (PDC)....................................................12

1.4.2 1 Natural Role of PDC.........................................................................12

1.4.2.2 Structure of PDC ..............................................................................12

1.4.2.3 Role of Thiamine Pyrophosphate (TPP) ............................................14

1.4.2.4 PDC Isozymes ..................................................................................15

1.4.2.5 Factors Influencing PDC Stability…………………………………...15

1.4.3 Formation of By-Products .......................................................................166

1.4.4 Microorganisms for PAC Production.........................................................16

1.5 Factors Influencing Biocatalysis for PAC Production...............................................18

1.5.1 Enzyme Activity........................................................................................18

1.5.2 Toxicity Effect of Benzaldehyde ...............................................................19

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viii

1.5.3 Effect of Dissolved Oxygen Concentration ................................................19

1.5.4 Effect of pH...............................................................................................20

1.5.5 Biomass Condition ....................................................................................20

1.5.5.1 Effect of Cell Age.............................................................................20

1.5.5.2 Effect of Respiratory Quotient (RQ) .................................................21

1.6 Two-Phase Aqueous/Organic Extractive Bioconversion with Organic Solvent.........22

1.6.1 Definition ..................................................................................................22

1.6.2 Advantages and Disadvantages of the Two-Phase Aqueous/Organic

Biotransformation ..............................................................................................22

1.6.3 Organic Solvent Selection .........................................................................23

1.6.3.1 Solvent Biocompatibility ..................................................................23

1.6.3.2 Solvent Toxicity ...............................................................................25

1.6.3.3 Extraction Efficiency ........................................................................28

1.6.3.4 Ease of Solvent Recovery .................................................................28

1.7 Current Status of Two-Phase Aqueous/Organic Biotransformation for PAC

Production .....................................................................................................................29

1.8 Strategy for Two-Phase Model Development………………………………………..33

2. MATERIALS AND METHODS ...........................................................................355

2.1 Microorganisms.....................................................................................................366

2.2 Chemicals, Enzymes and Sources ..........................................................................366

2.3 Buffer Compositions..............................................................................................399

2.4 PDC Enzyme Production .........................................................................................40

2.4.1 General Steps in the Fermentation Processes .............................................40

2.4.1.1 Media Preparation.............................................................................40

2.4.1.2 Growth on Agar Media .....................................................................42

2.4.1.3 Preseed and Seed ..............................................................................42

2.4.1.4 Final Fermentation............................................................................43

2.4.2 Fermentation Processes .............................................................................44

2.4.2.1 Shake Flask Fermentation.................................................................44

2.4.2.2 Aerobic-Partially Anaerobic Two–Stage Fermentation .....................45

2.4.2.3 pH Shift Fermentation ......................................................................46

2.4.2.4 Sampling Procedure..........................................................................47

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ix

2.4.3 PDC Enzyme Preparations.........................................................................48

2.4.3.1 Whole Cell PDC ...............................................................................48

2.4.3.2 Crude Extract PDC ...........................................................................48

2.4.3.3 Partially Purified PDC ......................................................................48

2.5 Biotransformation Systems for PAC Production ......................................................49

2.5.1 Yeast Pyruvate Decarboxylases: Variation in Biocatalytic Characteristics .49

2.5.2 Effect of Organic to Aqueous Phase Volume Ratio on Two-Phase

Aqueous/Organic PAC Synthesis (2.5 M MOPS)...............................................51

2.5.3 Biotransformation Systems........................................................................51

2.5.3.1 MOPS Buffer System .......................................................................51

2.5.3.2 Aqueous (Soluble Benzaldehyde) and Aqueous/Benzaldehyde

Emulsion Systems ........................................................................................53

2.5.3.3 Two-Phase Aqueous/Octanol-Benzaldehyde System.........................54

2.5.3.4 PDC Enzyme Stock Solution Preparation..........................................55

2.5.4 Biotransformation Experiments .................................................................55

2.5.4.1 Set Up of Biotransformation Systems ...............................................55

2.5.4.2 Controls............................................................................................56

2.5.4.3 Sampling ..........................................................................................56

2.5.5 Determination of Residual PDC Enzyme Activities in Biotransformation

Systems..............................................................................................................57

2.6 PDC Enzyme Deactivation and Organic-Aqueous Benzaldehyde Transfer Studies in

the Two-Phase Aqueous/Octanol-Benzaldehyde System................................................60

2.6.1 Construction of the Aqueous/Organic Phase-Separated System – A

Temperature Controlled Lewis Cell System .......................................................60

2.6.1.1 Lewis Cell Construction ...................................................................60

2.6.1.2 Temperature Control System ............................................................61

2.6.2 PDC Enzyme Deactivation ........................................................................62

2.6.2.1 Experimental Details.........................................................................62

2.6.2.2 Effects of Soluble Octanol and Benzaldehyde in the Aqueous Phase

and Agitation Rate on PDC Deactivation......................................................66

2.6.2.3 Effect of Ratio of Organic Phase Interfacial Area to Aqueous Phase

Volume on PDC Deactivation ......................................................................66

2.6.2.4 Effect of Initial Enzyme Concentration on PDC Deactivation ...........67

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2.6.3 Estimation of Organic-Aqueous Benzaldehyde Transfer in Two-Phase

System ...............................................................................................................68

2.6.3.1 Experimental Details.........................................................................68

2.6.3.2 Organic-Aqueous Benzaldehyde Transfer Experiments.....................68

2.7 Two-Phase Aqueous/Organic PAC Synthesis at Lower Buffer Concentration (20 mM

MOPS, Larger Scale).....................................................................................................69

2.7.1 Experimental Details .................................................................................69

2.7.2 Biotransformation Experiments .................................................................71

2.8 Analytical Methods..................................................................................................72

2.8.1 Determination of Cell Culture Optical Density (OD660) ...........................72

2.8.2 Determination of Glucose concentration....................................................72

2.8.3 Determination of Dissolved Oxygen Concentration ...................................72

2.8.4 Determination of Respiratory Quotient ......................................................73

2.8.5 Determination of Dry Biomass ..................................................................74

2.8.6 Determination of Pyruvate Concentration ..................................................74

2.8.7 Determination of Acetaldehyde Concentration...........................................75

2.8.8 Determination of PAC, Benzoic Acid, Benzaldehyde and Benzyl Alcohol

Concentrations ...................................................................................................76

2.8.9 Determination of Acetoin Concentration ...................................................77

2.8.10 Determination of Soluble Octanol Concentration.....................................78

2.8.11 Determination of PDC Enzyme Carboligase Activity...............................79

2.9 Calculations Methods...............................................................................................79

2.9.1 Specific PDC Production...........................................................................79

2.9.2 Biotransformations Systems ......................................................................80

2.9.2.1 Substrate and PDC Enzyme Stock Solution Concentrations ..............80

2.9.2.2 PAC and By-Product Formation .......................................................81

2.9.3 Experimental Errors ..................................................................................83

3. YEAST PYRUVATE DECARBOXYLASES: VARIATION IN BIOCATALYTIC

CHARACTERISTICS .................................................................................................84

3.1 Introduction .............................................................................................................85

3.2 Results and Discussion ............................................................................................86

3.2.1 Specific PDC Activity ...............................................................................86

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xi

3.2.2 Pyruvate Conversion in the Absence of Benzaldehyde...............................86

3.2.3 PAC and By-Product Formation in the Aqueous (Soluble Benzaldehyde)

System................................................................................................................89

3.2.4 PAC and By-Product Formation in the Aqueous/Benzaldehyde Emulsion

System................................................................................................................91

3.2.5 PAC and By-Product Formation in the Aqueous/Octanol-Benzaldehyde

Emulsion System ...............................................................................................93

3.2.6 Efficiency of PAC Formation ....................................................................95

3.2.7 Effect of Benzaldehyde and Acetaldehyde on PAC Formation with C. utilis

and C. tropicalis PDCs.......................................................................................96

3.2.8 PDC Stability ..........................................................................................101

3.2.9 Further Characterization of C. utilis PDC Activity...................................103

3.2.9.1 Effect of Agitation Rate on PDC Deactivation ................................103

3.2.9.2 Effect of Initial Enzyme Concentration on PDC Deactivation .........104

3.3 Conclusions ...........................................................................................................105

4. FACTORS AFFECTING PDC ENZYME DEACTIVATION AND PAC

PRODUCTION IN TWO-PHASE AQUEOUS/ORGANIC SYSTEM ....................106

4.1 Introduction ...........................................................................................................107

4.2 Results and Discussion ..........................................................................................109

4.2.1 Factors Affecting PDC Deactivation........................................................109

4.2.1.1 Effect of Soluble Octanol and Benzaldehyde in the Aqueous Phase 109

4.2.1.2 Effect of Agitation Rate in the Aqueous Phase................................110

4.2.1.3 Effect of Ratio of Organic Phase Interfacial Area to Aqueous Phase

Volume ......................................................................................................111

4.2.1.4 Effect of Initial Enzyme Concentration ...........................................115

4.2.1.5 Discussion of Toxicity Effects on PDC Enzyme .............................117

4.2.1.6 Discussion of Organic-Aqueous Benzaldehyde Transfer .................118

4.2.2 Effect of Organic to Aqueous Phase Volume Ratio on PDC Deactivation

and PAC Production.........................................................................................119

4.2.2.1 PAC and By-Product Formation .....................................................119

4.2.2.2 PDC Deactivation ...........................................................................124

4.2.2.3 Discussion of the Phase Ratio Effects .............................................125

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xii

4.3 Conclusion.............................................................................................................127

5. PROCESS ENHANCEMENT AND FURTHER KINETIC EVALUATIO NS FOR

TWO-PHASE AQUEOUS/ORGANIC SYNTHESIS OF PAC ...............................128

5.1 Introduction ...........................................................................................................129

5.2 Results and Discussion ..........................................................................................130

5.2.1 Effect of Changing the Organic to Aqueous Phase Volume Ratio at 20°C on

Reaction Kinetics.............................................................................................130


5.2.1.2 PDC Deactivation ...........................................................................135

5.2.1.3 Discussion ......................................................................................136

5.2.2 Effect of Changing Organic to Aqueous Phase Volume Ratio at 20°C at

Lower MOPS Concentration (20 mM) .............................................................138


5.2.2.2 Discussion ......................................................................................141

5.2.3 Effect of Increasing Temperature at Lower MOPS Concentration (20 mM)143


5.2.3.2 Discussion ......................................................................................146

5.2.4 Effect of Dipropylene Glycol (DPG) as Additive at Lower MOPS

Concentration (20 mM) with Lowered Organic to Aqueous Phase Volume Ratio149


5.2.4.2 Discussion ......................................................................................153

5.3 Conclusion.............................................................................................................155

6. FINAL CONCLUSIONS AND FUTURE WORK ...............................................156

6.1 Yeast Pyruvate Decarboxylases: Variation in Biocatalytic Characteristics..............157

6.2 Factors Affecting PDC Enzyme Deactivation and PAC Production in Two-Phase

Aqueous/Organic System.............................................................................................158

6.3 Process Enhancement and Further Kinetic Evaluations for Two-Phase

Aqueous/Organic Synthesis of PAC ............................................................................159

6.4 Recommended Future Work ..................................................................................160

REFERENCES..........................................................................................................163

Table of Contents

xiii

APPENDIX A ............................................................................................................177

APPENDIX B .............................................................................................................184

APPENDIX C ............................................................................................................192

APPENDIX D ............................................................................................................195

List of Tables

xiv

LIST OF TABLES Table 1.1: Recently developed biocatalytic systems at several chemical companies

(adapted from Schmid et al. [2001] with modifications)............................5

Table 1.2: Effect of benzaldehyde on in vivo PAC production with S. cerevisiae [Long

and Ward, 1989b; c]. ..............................................................................19

Table 1.3: Comparison of baker’s yeast fatty acids and proteins released into the

biotransformation medium with observed biocatalytic activity [Nikolova

and Ward, 1992b]. ..................................................................................26

Table 2.1: Chemicals and enzymes..........................................................................36

Table 2.2: Buffer compositions. ..............................................................................39

Table 2.3: Stock solution concentrations and sterilization methods for the preparation

of agar and liquid media for fermentation. ..............................................41

Table 2.4: Agar media compositions for the various fermentation methods..............42

Table 2.5: Preseed and seed media compositions and operating conditions for the

various fermentation methods. ................................................................43

Table 2.6: Final fermentation media compositions for the various fermentation methods. .44

Table 2.7: Biotransformation systems employed in the selection of biocatalyst for

PAC production (Chapter 3). ..................................................................49

Table 2.8: Biotransformation systems employed in the characterization of the two-

phase aqueous/octanol-benzaldehyde system for PAC production

(Chapters 4 and 5). .................................................................................52

Table 2.9: Treatment and types of measurement performed on the biotransformation

samples. .................................................................................................58

Table 2.10.A: Aqueous-based and two-phase aqueous/organic systems employed in the

PDC enzyme deactivation studies (Chapter 4).........................................63

Table 2.10.B: Performed investigations in the PDC enzyme deactivation studies (Chapter

4)............................................................................................................65

Table 2.11: Biotransformation systems employed in the two-phase aqueous/organic

PAC synthesis with 20 mM MOPS buffer system (Chapter 5). ...............70

Table 2.12: Respiratory quotient (RQ) calculation for fermentation process. .............73

List of Tables

xv

Table 2.13: Composition of the reaction mixture in pyruvate and acetaldehyde (Section

2.8.7) assays (modified from Czok and Lamprecht 1974). ......................75

Table 2.14: Calculation method for pyruvate and acetaldehyde (Section 2.8.7)

concentrations. .......................................................................................75

Table 2.15: Component specifications and operating conditions of the HPLC system for

quantification of PAC, benzoic acid, benzaldehyde and benzyl alcohol. ......77

Table 2.16: Component specifications and operating conditions of the GC system for

quantification of acetoin concentration. ..................................................78

Table 2.17: Calculation method for PDC carboligase activity. ...................................79

Table 2.18: Method for calculating substrate and PDC enzyme stock solution

concentration in setting up the biotransformation systems.......................80

Table 2.19: Method for calculating PAC and by-product concentrations in two-phase

aqueous/octanol-benzaldehyde system....................................................81

Table 2.20.A:Calculation method for unaccounted benzaldehyde in the biotransformation

systems...................................................................................................82

Table 2.20.B:Calculation method for unaccounted pyruvate in the biotransformation

systems...................................................................................................83

Table 2.21: Calculation method for experimental error..............................................83

Table 3.1: Biotransformations with the four yeast PDCs in three different systems:

estimated yields of PAC on consumed benzaldehyde and pyruvate. ........95

Table 4.1: Effect of aqueous phase octanol and benzaldehyde on PDC deactivation at

4°C, pH 7.0. 220 rpm, 2 U/mL PDC carboligase activity (C. utilis partially

purified), 2.5 M MOPS buffer, 0.5 mM Mg2+, 1 mM TPP. Same

experiments as shown in Fig 4.4.a. .......................................................109

Table 4.2: Performance summary: effect of organic to aqueous phase volume ratio on

PAC production in the aqueous/octanol-benzaldehyde emulsion system at

4°C, initial pH 6.5 (48 h). .....................................................................126


PAC production in the aqueous/octanol-benzaldehyde emulsion system at

20°C, initial pH 6.5 (48 h). ...................................................................137


PAC production in the aqueous/octanol-benzaldehyde emulsion system

with 20 mM MOPS at 20°C, controlled pH 7.0.....................................142

List of Tables

xvi

Table 5.3: Performance summary: effect of temperature on PAC production in the

aqueous/octanol-benzaldehyde emulsion system with 20 mM MOPS,

controlled pH 7.0..................................................................................148

Table 5.4: Performance summary: PAC production in the aqueous/octanol-

benzaldehyde emulsion system with 2.5 M MOPS and 20 mM MOPS +

2.5 M DPG at 0.25:1 ratio and 20°C, pH controlled at 7.0.....................154

Table A.1: Description on the mass transfer rate equations. ....................................177

Table A.2: KA aint and KA values comparison: effects of physical parameters on

organic-aqueous benzaldehyde transfer in two-phase aqueous/octanol-

benzaldehyde system. ...........................................................................183

Table B.1: Performance summary: effect of octanol addition on PAC formation at

4°C, initial pH 7.0 (48h). ......................................................................187

Table B.2: Performance summary: effect of octanol addition on PAC formation at

20°C, initial pH 7.0 (48h). ....................................................................190

List of Figures

xvii

LIST OF FIGURES

Figure 1.1: Cumulative number of biotransformation processes that have been started on

an industrial scale [Straathof et al., 2002]. ...................................................3

Figure 1.2: The type of compounds produced using biotransformation processes (based

on 134 industrial processes) [Straathof et al., 2002]. ....................................3

Figure 1.3: Industrial sectors in which the products of industrial biotransformations are

used (based on 134 industrial processes) [Straathof et al., 2002]. .................4

Figure 1.4: Enzyme types used in industrial biotransformations (based on 134 processes)

[Straathof et al., 2002]. ................................................................................4

Figure 1.5: Two biologically active isomers of ephedrine..............................................6

Figure 1.6: Photograph of Ephedra distachya [Schoenfelder]. .......................................7

Figure 1.7: Synthesis of (1R, 2S) ephedrine and (1S, 2S) pseudoephedrine from PAC. ..7

Figure 1.8: Mechanisms of PAC formation catalyzed by pyruvate decarboxylase [Shin,

1994].........................................................................................................10

Figure 1.9: (a) α, β, and γ domains in PDC subunit [Arjunan et al., 1996] and (b) binding

site of Mg2+ demonstrating octahedral coordination with the enzyme, which

also forms a hydrogen bond with TPP [Furey et al., 1998] (Fig b was

constructed with the program CHAIN). .....................................................13

Figure 1.10:Overall structure of PDC tetramer is shown in ribbon drawing. The side

chains of Cys-221 residues, which involve in substrate activation are shown

in shaded boxes (adapted from Furey et al. [1998] with modification by

Leksawasdi [2004]). ..................................................................................14

Figure 1.11: Structure of thiamine pyrophosphate (TPP) [Campbell, 1999]...................14

Figure 1.12:Scanning electron micrographs of yeast cells isolated from biphasic media

containing: (a) hexane (26 h), (b) decane (26 h), (c) toluene (26 h), (d)

chloroform (0 h), and (e) chloroform (2 h) [Nikolova and Ward, 1992a]....27

Figure 1.13:Batch biotransformation kinetics and model fitting for determination of

overall rate constants for the formation of PAC, acetaldehyde, and acetoin

(Vp, Vq, and Vr) at (a) 50 mM benzaldehyde/60 mM sodium pyruvate with

initial PDC activity of 3.4 U/mL carboligase, (b) 150 mM benzaldehyde/180

mM sodium pyruvate with initial PDC activity of 3.4 U/mL carboligase, and

List of Figures

xviii

(c) 100 mM benzaldehyde/120 mM sodium pyruvate with initial PDC

activity of 1.1 U/mL carboligase: ( ) pyruvate, ( ) benzaldehyde, ()

acetaldehyde, () acetoin, ( ) PAC, and (x) enzyme activity. Line of best

fit through each data profile was created from the optimal value of Vp, Vq,

and Vr [Leksawasdi et al., 2004]. ..............................................................30

Figure 1.14:Diagrammatic representation of the two-phase aqueous/organic PAC

production system [Rosche et al., 2002b; Sandford et al., 2005]. ...............31

Figure 1.15:PAC production as a function of carboligase activity in the rapidly stirred

two-phase system after 40 h, and phase-separated two-phase system after

395 h (4°C, organic octanol phase contained 1500 mM benzaldehyde and

the aqueous phase contained 1430 mM pyruvate, 2.5 M MOPS, 1 mM TPP,

1 mM Mg2+, pH 6.5). A 1:1 volume ratio of organic and aqueous phases was

used...........................................................................................................32

Figure 2.1: 30 L BIOSTAT® C fermenter system used in the aerobic-partially anaerobic

two-stage fermentation method for PDC production. .................................46

Figure 2.2:5 L BIOSTAT® A (B.Braun) fermenter system used in the pH shift

fermentation method for PDC production. .................................................47

Figure 2.3: Sampling procedures for the fermentation processes. ..................................47

Figure 2.4: Freeze drier used in partially purified PDC preparation. ..............................48

Figure 2.5:Lewis Cell for experimentation on the aqueous/organic phase-separated

system. ......................................................................................................61

Figure 2.6:Temperature controlled aqueous/organic phase-separated system (Lewis

Cell). .........................................................................................................62

Figure 3.1: Comparison of specific PDC activities of six yeasts. Culturing conditions

(g/L): 90 glucose, 10 yeast extract, 10 (NH4)2SO4, 3 KH2PO4, 2

Na2HPO4.12H2O, 1 MgSO4.7H2O, 0.05 CaCl2.2H2O, 39 MES buffer, initial

pH 6, 30°C, 160 rpm. The data is shown as mean values for four

fermentation batches for S.c, C.u, C.t, K.m and two batches for S.p and C.g.

S.c: Saccharomyces cerevisiae, C.u: Candida utilis, C.t: Candida tropicalis,

S.p: Schizosaccharomyces pombe, C.g: Candida glabrata, K.m:

Kluyveromyces marxianus. The error bars show highest and lowest values

for the above experiments..........................................................................87

List of Figures

xix

Figure 3.2: Acetaldehyde and acetoin formation in the absence of benzaldehyde. Product

concentrations after 7.3 h at 22°C, initial pH 6.5. Initial agitation 220 rpm,

initial concentrations: 80 mM pyruvate, 1.5 U/ml PDC carboligase activity

(crude extract), 2.5 M MOPS buffer, 1 mM Mg2+, 1 mM TPP. Acetaldehyde

concentrations were immediately measured upon samplings. The mean

values were determined from triplicate experiments and error bars show the

highest and lowest values. Refer to Fig 3.1 for strain abbreviations. ..........88

Figure 3.3:Biotransformation results in the aqueous system (presence of soluble

benzaldehyde): (a) PAC (at 0.5 h and 7.3 h) and (b) by-product (at 7.3 h)

concentrations at 22°C, initial pH 6.5. Initial agitation 220 rpm, initial

concentrations: 80 mM benzaldehyde, 80 mM pyruvate, 1.5 U/ml PDC

carboligase activity (crude extract), 2.5 M MOPS buffer, 1 mM Mg2+, 1 mM

TPP. ..........................................................................................................90

Figure 3.4: Biotransformation results in the aqueous/benzaldehyde emulsion system: (a)

PAC (at 3 h and 24 h) and (b) by-product (at 24 h) concentrations at 22°C,

initial pH 6.5. Initial agitation 220 rpm, initial concentrations: 325 mM

benzaldehyde, 420 mM pyruvate, 1.5 U/ml PDC carboligase activity (crude

extract), 2.5 M MOPS buffer, 1 mM Mg2+, 1 mM TPP. .............................92

Figure 3.5: Biotransformation results in the aqueous/octanol-benzaldehyde emulsion

system: (a) PAC (at 3 h, 24 h and 48 h) and (b) by-product (at 48 h)

concentrations at 22°C, initial pH 6.5. Initial agitation 250 rpm, initial

concentrations: 850 mM TRV benzaldehyde, 450 mM TRV pyruvate, 1.5

U/ml TRV PDC carboligase activity (permeabilized whole cells), 2.5 M

MOPS, 1 mM Mg2+, 1 mM TPP. The organic to aqueous phase volume ratio

was 1:1 and concentrations of substrates, enzyme, product and by-products

are given per total reaction volume by combining both phases (TRV)........94

Figure 3.6: Effect of acetaldehyde on initial PAC formation with: (a) C. utilis (C.u) and

(b) C. tropicalis (C.t) PDCs at various benzaldehyde concentrations in 32

min at 22°C, initial pH 6.5. Agitation 220 rpm, initial concentrations: 250

mM pyruvate, 0 and 30 mM acetaldehyde 1.5 U/ml PDC carboligase

activity (crude extract), 2.5 M MOPS, 1 mM Mg2+ & 1 mM TPP. .............97

List of Figures

xx

Figure 3.7: Effect of acetaldehyde on initial acetoin formation with: (a) C. utilis (C.u)

and (b) C. tropicalis (C.t) PDCs at various benzaldehyde concentrations in

32 min at 22°C, initial pH 6.5. Same experiments as shown in Fig 3.6.......98

Figure 3.8: Ratio of PAC over acetoin with C. utilis (C.u) and C. tropicalis (C.t) PDCs

at various benzaldehyde concentrations in the presence of 30 mM

acetaldehyde (32 min) at 22°°°°C, initial pH 6.5. Same experiments as shown

as Fig 3.6...................................................................................................99

Figure 3.9:Ratio of PAC over acetoin for the four yeast PDCs in the different

biotransformation systems at 22°C, initial pH 6.5. Same experiments as

shown in Figs 3.3, 3.4, and 3.5. ...............................................................100

Figure 3.10:PDC stabilities in the absence and presence of soluble benzaldehyde at

22°C: (a) crude extract and (b) whole cell preparations. Concentrations: 50

mM benzaldehyde, 1.5 U/ml PDC carboligase activity, 2.5 M MOPS (pH

6.5), 1 mM Mg2+ & 1 mM TPP................................................................102

Figure 3.11: Effect of agitation rate on the deactivation of partially purified PDC from C.

utilis in the absence and presence of 48 mM soluble benzaldehyde at 4°°°°C,

pH 7. 95, 220 and 250 rpm agitation, 0 and 48 mM benzaldehyde, 3 U/mL

PDC carboligase activity, 2.5 M MOPS buffer, 0.5 mM Mg2+, 1 mM TPP.

Extensive foam formation at 250 rpm......................................................104

Figure 3.12:Effect of initial enzyme concentration on the deactivation of partially

purified PDC from C. utilis in the absence and presence of 48 mM soluble

benzaldehyde at 4°C, pH 7. 220 rpm agitation, 0 and 48 mM benzaldehyde,

3 and 7.3 U/mL PDC carboligase activity, 2.5 M MOPS buffer, 0.5 mM

Mg2+, 1 mM TPP.....................................................................................104

Figure 4.1: PAC production in various biotransformation systems. ............................108

Figure 4.2: Effect of agitation rate on PDC deactivation in the presence of soluble

octanol and benzaldehyde at 4°C, pH 7.0. 4.5 mM octanol, 48 mM

benzaldehyde, 2 U/mL PDC carboligase activity (C. utilis partially purified),

2.5 M MOPS buffer, 0.5 mM Mg2+, 1 mM TPP.......................................110

Figure 4.3:Effect of aqueous/organic interfacial area on PDC deactivation in the

aqueous/octanol-benzaldehyde phase-separated system at 4°C, pH 7.0. 1.39

M organic phase benzaldehyde, 4.5 mM aqueous phase octanol, 48 mM

aqueous phase benzaldehyde, 60 rpm and 125 rpm agitation for organic and

List of Figures

xxi

aqueous phase respectively in Lewis cell, 4 U/mL aqueous phase or 2 U/mL

TRV PDC carboligase activity, 2.5 M MOPS buffer, 0.5 mM Mg2+, 1 mM

TPP. TRV: total reaction volume by combining both phases....................112

Figure 4.4:Effect of excess octanol and benzaldehyde on PDC deactivation in the

aqueous/octanol-benzaldehyde emulsion system at 4°C, pH 7.0: (a)

aqueous-based system and (b) two-phase aqueous/organic system. 220 rpm

agitation, 2 U/mL TRV PDC carboligase activity, 2.5 M MOPS buffer, 0.5

mM Mg2+, 1 mM TPP. TRV: total reaction volume by combining both

phases. ....................................................................................................113

Figure 4.5: Effect of initial enzyme concentration on PDC deactivation in the two-phase

aqueous/octanol-benzaldehyde system at 4°C, pH 7.0: (a) phase-separated

system, 125 rpm agitation in the aqueous phase and (b) emulsion system,

220 rpm agitation. 1.46 M organic phase benzaldehyde, 4.5 mM aqueous

phase octanol, 48 mM aqueous phase benzaldehyde, 2.5 M MOPS buffer,

0.5 mM Mg2+, 1 mM TPP. The enzyme activities were expressed as

concentrations in the aqueous phase. .......................................................116

Figure 4.6: Effect of organic to aqueous phase volume ratio on PAC production in the

aqueous/octanol-benzaldehyde emulsion system at 1:1 ratio at 4°C, initial

pH 6.5: (a) organic and (b) aqueous phase substrates, PAC and by-product

concentration profiles. Initial agitation 250 rpm, initial concentrations: 1.36

M organic phase benzaldehyde, the aqueous phase contained 1.26 M

pyruvate, 5.6 U/mL PDC carboligase activity (C. utilis partially purified),

2.5 M MOPS buffer, 1 mM Mg2+, 1 mM TPP. ORG: organic phase, AQ:

aqueous phase. Approximate values for acetaldehyde concentration due to

possible evaporative loss during sampling and analysis. The mean values

were determined from triplicate analyses and error bars show the highest

and lowest values. ...................................................................................120


aqueous/octanol-benzaldehyde emulsion system at 0.67:1 ratio at 4°C, initial

pH 6.5: (a) organic and (b) aqueous phase concentration profiles. Initial

agitation 235 rpm, initial concentrations: 1.7 M organic phase benzaldehyde,

the aqueous phase contained 1.06 M pyruvate, 4.7 U/mL PDC carboligase

List of Figures

xxii

activity (C. utilis partially purified), 2.5 M MOPS buffer, 1 mM Mg2+, 1

mM TPP..................................................................................................121




agitation 220 rpm, initial concentrations: 2.26 M organic phase

benzaldehyde, the aqueous phase contained 0.93 M pyruvate, 4 U/mL PDC

carboligase activity (C. utilis partially purified), 2.5 M MOPS buffer, 1 mM

Mg2+, 1 mM TPP.....................................................................................122




agitation 205 rpm, initial concentrations: 3.48 M organic phase

benzaldehyde, the aqueous phase contained 0.8 M pyruvate, 3.5 U/mL PDC


Mg2+, 1 mM TPP.....................................................................................123

Figure 4.10:Effect of organic to aqueous phase volume ratio in emulsion

aqueous/octanol-benzaldehyde system at 4°C, initial pH 6.5: residual

enzyme activity. Same experiments as shown in Figs 4.6 – 4.9................125


aqueous/octanol-benzaldehyde emulsion system at 1:1 ratio at 20°C, initial

pH 6.5: (a) organic and (b) aqueous phase substrates, PAC and by-product

concentration profiles. Initial agitation 250 rpm, initial concentrations: 1.4

M organic phase benzaldehyde, the aqueous phase contained 1.29 M

pyruvate, 5.6 U/mL PDC carboligase activity (C. utilis partially purified),

2.5 M MOPS buffer, 1 mM Mg2+, 1 mM TPP. ORG: organic phase, AQ:

aqueous phase, Approximate values for acetaldehyde concentration due to

possible evaporative losses during sampling and analysis. The mean values

were determined from triplicate analyses and error bars show the highest

and lowest values. ...................................................................................131


aqueous/octanol-benzaldehyde emulsion system at 0.67:1 ratio at 20°C,

initial pH 6.5: (a) organic and (b) aqueous phase concentration profiles.

List of Figures

xxiii

Initial agitation 235 rpm, initial concentrations: 1.76 M organic phase

benzaldehyde, the aqueous phase contained 1.075 M pyruvate, 4.7 U/mL

PDC carboligase activity (C. utilis partially purified), 2.5 M MOPS buffer, 1

mM Mg2+, 1 mM TPP. ............................................................................132





benzaldehyde, the aqueous phase contained 0.92 M pyruvate, 4 U/mL PDC


Mg2+, 1 mM TPP.....................................................................................133





benzaldehyde, the aqueous phase contained 0.8 M pyruvate, 3.5 U/mL PDC


Mg2+, 1 mM TPP.....................................................................................134

Figure 5.5: Effect of organic to aqueous phase volume ratio on PDC deactivation in the

aqueous/octanol-benzaldehyde emulsion system at 20°C, initial pH 6.5.

Same experiments as shown in Figs 5.1 – 5.4. .........................................135


aqueous/octanol-benzaldehyde emulsion system with 20 mM MOPS at

20°C, controlled pH 7.0: organic and aqueous phase concentration profiles

are shown. Constant agitation 160 rpm, initial concentrations: 775 – 810

mM TRV benzaldehyde, 400 – 465 mM TRV pyruvate, 1 U/mL TRV PDC

carboligase activity (C. utilis whole cell), 20 mM MOPS buffer, 1 mM

Mg2+, 1 mM TPP. ORG: organic phase, AQ: aqueous phase, TRV: total

reaction volume by combining both phases. The mean values were

determined from triplicate analyses and error bars show the highest and

lowest values. ..........................................................................................139

List of Figures

xxiv

Figure 5.7: Effect of organic to aqueous phase volume ratio on by-products acetaldehyde

and acetoin formation in the aqueous/octanol-benzaldehyde emulsion

system with 20 mM MOPS at 20°C, controlled pH 7.0: (a) final organic and

(b) aqueous phase concentrations. Same experiments as shown in Fig 5.6.

Approximate values for acetaldehyde concentration due to possible

evaporative losses during sampling and analysis......................................140

Figure 5.8:Effect of temperature on PAC production in the aqueous/octanol-

benzaldehyde emulsion system with 20 mM MOPS, controlled pH 7.0.

Organic phase concentration profiles: (a) 5°C – 20°C and (b) 25°C – 35°C.

Constant agitation 160 rpm, initial concentrations: 1.6 – 1.64 M organic

phase benzaldehyde, the aqueous phase containing 0.93 – 0.98 M pyruvate,

2 U/mL PDC carboligase activity (C. utilis whole cell), 20 mM MOPS

buffer, 1 mM Mg2+, 1 mM TPP, 1:1 organic to aqueous phase volume ratio.

ORG: organic phase. The mean values were determined from triplicate

analyses and error bars show the highest and lowest values. ....................144

Figure 5.9:Effect of temperature on PAC production in the aqueous/octanol-

benzaldehyde emulsion system with 20 mM MOPS, controlled pH 7.0.

Aqueous phase concentration profiles: (a) 5°C – 20°C and (b) 25°C – 35°C.

AQ: aqueous phase. Same experiments as shown in Fig 5.8.....................145

Figure 5.10:Effect of temperature on by-products acetaldehyde and acetoin formation in

the aqueous/octanol-benzaldehyde emulsion system with 20 mM MOPS,

controlled pH 7.0: (a) final organic and (b) aqueous phase concentrations.

ORG: organic phase, AQ: aqueous phase. Same experiments as shown in

Fig 5.8. Estimated values for acetaldehyde concentrations due to evaporative

losses during sampling and analysis.........................................................147

Figure 5.11:Effect of 2.5 M dipropylene glycol (DPG) addition on PAC production in

the aqueous/octanol-benzaldehyde emulsion system with 20 mM MOPS and

partially purified PDC at 20°C, controlled pH 7.0: (a) organic and (b)

aqueous phase substrate, PAC and by-product concentration profiles.

Organic to aqueous phase volume ratio of 0.25:1. Constant agitation 160

rpm, initial concentrations: 3.6 M organic phase benzaldehyde, the aqueous

phase contained 0.785 M pyruvate, 3.5 U/mL PDC carboligase activity (C.

utilis), 20 mM MOPS buffer, 2.5 M DPG, 1 mM Mg2+, 1 mM TPP. ORG:

List of Figures

xxv

organic phase, AQ: aqueous phase. Approximate values for acetaldehyde

concentration due to possible evaporative losses during sampling and

analysis. The mean values were determined from triplicate analyses and

error bars show the highest and lowest values..........................................151

Figure 5.12:Effect of 2.5 M dipropylene glycol (DPG) addition on PAC production in

the aqueous/octanol-benzaldehyde emulsion system with 20 mM MOPS and

whole cell PDC at 20°C, controlled pH 7.0: (a) organic and (b) aqueous

phase concentration profiles. Organic to aqueous phase volume ratio of

0.25:1. Constant agitation 160 rpm, initial concentrations: 3.65 M organic

phase benzaldehyde, the aqueous phase contained 0.83 M pyruvate, 3.5

U/mL PDC carboligase activity (C. utilis), 20 mM MOPS buffer, 2.5 M

DPG, 1 mM Mg2+, 1 mM TPP. ................................................................152

Figure A.1:Concentration profiles across an aqueous/organic interface [Hines and

Maddox, 1985]. .......................................................................................178

Figure A.2:Saturation profiles: effects of physical parameters on organic-aqueous

benzaldehyde transfer in the two-phase aqueous/octanol-benzaldehyde

system: (a) ratio of organic phase contact area to aqueous phase volume,

organic phase benzaldehyde concentration and (b) temperature. 117 and 361

cm2/L organic phase contact area to aqueous phase volume ratios, 1.5 M

and 2.5 M organic phase benzaldehyde concentrations, 4°C and 20°C

temperatures, 60 rpm and 125 rpm agitation for organic and aqueous phase

respectively in Lewis cell, 2.5 M MOPS buffer (pH 7.0). ........................181

Figure A.3: Plot of ln (ABZD* / (ABZD*- A BZD)) as a function of time with slope KA aint:

effects of physical parameters on organic-aqueous benzaldehyde transfer in

the two-phase aqueous/octanol-benzaldehyde system: (a) ratio of organic

phase contact area to aqueous phase volume, organic phase benzaldehyde

concentration and (b) temperature. Calculated from data in experiments

shown in Fig A.2. ....................................................................................182

Figure B.1: Effect of octanol addition on PAC formation at 4°C, initial pH 7.0. Initial

concentrations: 0, 50, 100, 300, 500, 700 and 2600 mM octanol, 725 – 810

mM benzaldehyde, 735 – 785 mM pyruvate, 2.8 U/mL PDC carboligase

activity (C. utilis whole cells), 2.5 M MOPS, 1 mM Mg2+, 1 mM TPP. For

the 1:1 two-phase emulsion system with 2600 mM octanol, all

List of Figures

xxvi

concentrations were given per total reaction volume by combining both

phases (TRV). The mean values were determined from triplicate analyses

and error bars show the highest and lowest values. ..................................185

Figure B.2:Effect of octanol addition on by-products acetaldehyde and acetoin formation

at 4°C, initial pH 7.0 (48 h). Same experiments as shown in Fig B.1.

Approximate values for acetaldehyde concentrations due to possible



concentrations: 0, 50, 100, 300, 500, 700 and 2600 mM octanol, 725 – 760

mM benzaldehyde, 710 – 770 mM pyruvate, 2.8 U/mL PDC carboligase

activity (C. utilis whole cells), 2.5 M MOPS, 1 mM Mg2+, 1 mM TPP. For

the 1:1 two-phase emulsion system with 2600 mM octanol, all

concentrations were given per total reaction volume by combining both

phases (TRV). .........................................................................................188

Figure B.4:Effect of octanol addition on by-products acetaldehyde and acetoin formation

at 20°C, initial pH 7.0 (48 h). Same experiments as shown in Fig B.3.

Approximate values for acetaldehyde concentrations due to possible


Figure C.1: Effect of organic to aqueous phase volume ratio on PAC production in the

two-phase aqueous/octanol-benzaldehyde emulsion system at 4°C, initial pH

6.5: overall substrate, PAC and by-product concentration profiles. Same

experiments as shown in Figs 4.6 – 4.9....................................................193

Figure C.2: Effect of organic to aqueous phase volume ratio on PAC production in the

aqueous/octanol-benzaldehyde emulsion system at 20°°°°C, initial pH 6.5:

overall substrate, PAC and by-product concentration profiles. Same

experiments as shown in Figs 5.1 – 5.4....................................................194

Figure D.1: Effect of organic to aqueous phase volume ratio on PAC production in the


20°C, controlled pH 7.0: overall concentration profiles are shown. Same

experiments as shown in Fig 5.6. TRV: total reaction volume by combining

both phases. The mean values were determined from triplicate analyses and


List of Figures

xxvii

Figure D.2:Effect of organic to aqueous phase volume ratio on by-products acetaldehyde

and acetoin formation in the aqueous/octanol-benzaldehyde emulsion

system with 20 mM MOPS at 20°°°°C, controlled pH 7.0: overall

concentrations. Same experiments as shown in Fig 5.6. Approximate values

for acetaldehyde concentration due to possible evaporative losses during

sampling and analysis..............................................................................196

Figure D.3:Effect of organic to aqueous phase volume ratio in the aqueous/octanol-

benzaldehyde emulsion system with 20 mM MOPS at 20°C, controlled pH

7.0: acid addition profiles. Same experiments as shown in Fig 5.6. ..........196

Figure D.4:Effect of temperature on PAC production in the aqueous/octanol-

benzaldehyde emulsion system with 20 mM MOPS, controlled pH 7.0:

overall concentration profiles. TRV: total reaction volume by combining

both phases. Same experiments as shown in Fig 5.8. The mean values were

determined from triplicate analyses and error bars show the highest and

lowest values. ..........................................................................................197

Figure D.5:Effect of temperature on PAC production in the 1:1 two-phase

aqueous/octanol-benzaldehyde emulsion system: final overall by-product

acetaldehyde and acetoin formation. Same experiments as shown in Fig 5.8.

Approximate values for acetaldehyde concentrations presumably due to


Figure D.6:Effect of temperature on PAC production in the 1:1 two-phase

aqueous/octanol-benzaldehyde emulsion system: acid addition profiles are

shown. Same experiments as shown in Fig 5.8. .......................................198

Figure D.7: Effect of 2.5 M dipropylene glycol (DPG) addition on PAC production in the


20°C, controlled pH 7.0: overall concentration profiles of substrates, PAC

and by-products are shown: (a) partially purified PDC and (b) whole cell

PDC. Organic to aqueous phase volume ratio of 0.25:1. Same experiments

as shown in Figs 5.11 and 5.12. TRV: total reaction volumeby combining

both phases. The mean values were determined from triplicate analyses and


List of Figures

xxviii



20°C, controlled pH 7.0: acid addition profiles are shown. Organic to

aqueous phase volume ratio of 0.25:1. Same experiments as shown in Figs

5.11 and 5.12...........................................................................................200

Project Scope and Objectives

xxix

PROJECT SCOPE AND OBJECTIVES

The current project has focused on the process development of two-phase

aqueous/organic enzymatic biotransformation for (R)-phenylacetylcarbinol (PAC)

production with utilization of pyruvate and benzaldehyde as substrates and pyruvate

decarboxylase enzyme (PDC) as biocatalyst. It is a continuation from the previous

projects by our group to develop an efficient and effective enzymatic process for PAC

production. With the traditional yeast-based fermentation process concentrations of 10 –

12 g/L PAC and 70% yields on added benzaldehyde are normally achieved [Rogers et al.,

1997]. In a cell-free biotransformation process, Shin and Rogers [1996] improved the

production to 28.6 g/L PAC with pyruvate decarboxylase (PDC) enzyme from C. utilis.

Rosche et al. [2002a, b] then investigated an aqueous/benzaldehyde emulsion system

buffered with 2.5 M MOPS and achieved 50 g/L PAC using PDC from yeast and

filamentous fungi. High MOPS concentration was found to have a stabilizing effect on

PDC [Rosche et al., 2002a]. Subsequent research reported by Rosche et al. [2002b] and

Sandford et al. [2005] broadened this approach by developing an enzymatic two-phase

aqueous/octanol-benzaldehyde production system and achieved PAC concentrations in

excess of 100 g/L in the organic phase.

Further characterization and development of the two-phase aqueous/organic system has

been performed in the current project with the following specific objectives:

(1) to investigate variations in the biocatalytic characteristics of several selected yeast

PDCs with regards to PAC and by-product formation in the different biotransformation

systems,

(2) to evaluate the factors affecting PDC deactivation in the two-phase aqueous/octanol-

benzaldehyde system as a basis for designing an improved two-phase PAC production

system,

(3) to investigate the effect of changing the organic to aqueous phase volume ratio on

two-phase PAC production at high MOPS concentration with evaluation at different

temperatures,

(4) to investigate the effect of changing the phase volume ratio on two-phase PAC

production at reduced MOPS concentration and at different temperatures,

The overall objective is to further characterize the enzymatic two-phase

biotransformation and to identify key factors in developing a more cost effective process.

Chapter 1

Introduction

Cindy Gunawan 2006 PhD Thesis

1

CHAPTER 1

LITERATURE REVIEW

1. Introduction

2. Development of Biotransformation Processes

3. Ephedrine and Pseudoephedrine Synthesis

4. Biotransformation of Pyruvate and Benzaldehyde to (R)-

phenylacetylcarbinol (PAC)

5. Factors Influencing Biocatalysis for PAC Production

6. Two-Phase Aqueous/Organic Extractive Bioconversion with

Organic Solvent

7. Current Status of Two-Phase Aqueous/Organic Biotransformation

for PAC Production

8. Strategy for Two-Phase Model Development

Chapter 1

Introduction


2

1.1 Introduction The literature review presents an overview of the development of biotransformation

processes with focus on the production of (R)-phenylacetylcarbinol (PAC), a precursor

for the synthesis of pharmaceuticals (1R, 2S) ephedrine and (1S, 2S) pseudoephedrine.

The PAC is produced from pyruvate and benzaldehyde with pyruvate decarboxylase

enzyme (PDC) as biocatalyst. The review also includes details on factors affecting PAC

production and two-phase aqueous/organic extractive bioconversion.

1.2 Development of Biotransformation Processes

Biotransformation is a process involving the use of biological agents as catalysts to

conduct transformations of chemical compounds. Biotransformation processes have been

employed for thousands of years before they were recognized as having an underlying

microbial cause [Parales et al., 2002]. Louis Pasteur in 1858 identified the role of specific

microbes involved in the favorable and unfavorable grape juice fermentations [Pasteur,

1858]. In the early 1900s, many studies were conducted to reveal the properties of

enzymes and principles of biocatalysis [Michaelis and Menten, 1913]. In 1916, an

industrial-scale fermentation for acetone production was established to meet increasing

demand in wartime of Great Britain [Glazer and Kikaido, 1995]. Since then,

biotransformation technology has been developed and adapted to run on an industrial

scale for the production of fine chemicals. In a study by Straathof et al. [2002], it was

estimated that the biotransformation-based industrial process has grown from less than 10

processes in the 1960’s to 134 processes in 2002 (Fig 1.1), which indicates that

biotransformation has now become a standard technology in the fine chemicals industry.

Chapter 1

Introduction


3

Figure 1.1: Cumulative number of biotransformation processes that have been started on

an industrial scale [Straathof et al., 2002].

Most of the industrial biotransformations lead to the production of natural compounds or

their derivatives (Fig 1.2). Carbohydrates and fat derivatives are used in the food sector

with the other compounds finding applications in the pharmaceutical or agricultural

sectors. Furthermore, many products of industrial biotransformations are mostly used in

the pharma sector [Straathof et al., 2002] (Fig 1.3).

Figure 1.2: The type of compounds produced using biotransformation processes (based

on 134 industrial processes) [Straathof et al., 2002].

Chapter 1

Introduction


4

Figure 1.3: Industrial sectors in which the products of industrial biotransformations are

used (based on 134 industrial processes) [Straathof et al., 2002].

Hydrolases are the most employed biocatalyst in the industrial biotransformations

followed by transferases and lyases (Fig 1.4). High numbers of processes also involve the

use of oxidizing cells with enzymes from all classes being active, together with the

oxidoreductases [Straathof et al., 2002].

Figure 1.4: Enzyme types used in industrial biotransformations (based on 134 processes)

[Straathof et al., 2002].

Chapter 1

Introduction


5

Nowadays, biotransformation processes can be conducted in aqueous as well as in

aqueous/organic environments, therefore apolar organic compounds as well as water-

soluble compounds can be selectively and efficiently transformed with enzymes or active

cells [Schmid et al., 2001]. Table 1.1 shows the various biocatalytic processes recently

developed by chemical companies.

Table 1.1: Recently developed biocatalytic systems at several chemical companies

(adapted from Schmid et al. [2001] with modifications).

>1Addition of water

Niacin hydroxylase

Whole cellsNiacin6-Hydroxynicotinic acid

Lonza

Development product

Addition of water

Nitrilase/

hydroxylase

Whole cells2-Cyanopyrazine5-Hydroxypyrazine-carboxylic acid

Development product

Addition of water

HydroxylaseWhole cells(S)-nicotine6-Hydroxy-S-nicotine

DSM

BASF

Company

>1 to >100Selective coupling

AcylaseEnzymes6-Aminopenicillanic acid

Semisyntheticpenicillins

1000HydrolysisPenicillin acylase

EnzymesPenicillin G/V6-Aminopenicillanic acid (6-APA)

1000Addition of ammonia

Aspartic acid ammonia

lyase

EnzymesFumaricacidL-Aspartic acid

>1HydrolysisNitrilasesEnzymesRacemic mandelonitrileR-Mandelicacid

>100ResolutionLipasesEnzymesRacemic aminesR-Amide, S-amine

1000ResolutionLipasesEnzymesRacemicalcoholsEnantiopurealcohols

Scale

(tons/yr)

ReactionEnzymeBiocatalystSubstrateProduct

>1Addition of water

Niacin hydroxylase

Whole cellsNiacin6-Hydroxynicotinic acid

Lonza

Development product

Addition of water

Nitrilase/

hydroxylase

Whole cells2-Cyanopyrazine5-Hydroxypyrazine-carboxylic acid

Development product

Addition of water

HydroxylaseWhole cells(S)-nicotine6-Hydroxy-S-nicotine

DSM

BASF

Company

>1 to >100Selective coupling

AcylaseEnzymes6-Aminopenicillanic acid

Semisyntheticpenicillins

1000HydrolysisPenicillin acylase

EnzymesPenicillin G/V6-Aminopenicillanic acid (6-APA)

1000Addition of ammonia

Aspartic acid ammonia

lyase

EnzymesFumaricacidL-Aspartic acid

>1HydrolysisNitrilasesEnzymesRacemic mandelonitrileR-Mandelicacid

>100ResolutionLipasesEnzymesRacemic aminesR-Amide, S-amine

1000ResolutionLipasesEnzymesRacemicalcoholsEnantiopurealcohols

Scale

(tons/yr)

ReactionEnzymeBiocatalystSubstrateProduct

Chapter 1

Introduction


6

1.3 Ephedrine and Pseudoephedrine Synthesis

1.3.1 Pharmacological Values

Ephedrine is known chemically as 2-methylamino-1-phenyl-1-propanol. It has

biologically optically active forms: (1R, 2S) ephedrine and (1S, 2S) pseudoephedrine (Fig

1.5).

NHCH

O

CH

H

3

3

CH

NHCH

O

3

H

3

(1R,2S)-Ephedrine ( 1S,2S)-Pseudo-ephedrine

NHCH

O

CH

H

3

3

CH

NHCH

O

3

H

3

(1R,2S)-Ephedrine ( 1S,2S)-Pseudo-ephedrine

NHCH

O

CH

H

3

3NHCH

O

CH

H

3

3

CH

NHCH

O

3

H

3

CH

NHCH

O

3

H

3

(1R,2S)-Ephedrine(1R,2S)-Ephedrine ( 1S,2S)-Pseudo-ephedrine(1S,2S)-Pseudo-ephedrine

Figure 1.5: Two biologically active isomers of ephedrine.

(1R, 2S) ephedrine and (1S, 2S) pseudoephedrine are pharmaceutical alkaloid compounds

with α and β andrenergic activity: ephedrine is used in the treatment of symptoms of

asthma and hypotension, whereas pseudoephedrine is used as a nassal decongestant in

cold and influenza medications.

1.3.2 Traditional Production

Traditionally, ephedrine was extracted from dried young branches of Ephedra sp.: mainly

Ephedra sinica, Ephedra equisetina and Ephedra distachya [Reti, 1953; Boit 1961;

Tanker and Kilicer, 1978] (shown in Fig 1.6); plants with valuable pharmacological

activities. However, the total alkaloid content in Ephedra sp. is generally low, with the

highest being approx. 2.5% by weight with ephedrine and pseudoephedrine occurring as a

racemic mixture. Hence, collection of large quantity of plant materials and complex

separation processes are necessary, leading to time and labour-intensive processing

[Shukla and Kulkarni, 2000].

Chapter 1

Introduction


7

Figure 1.6: Photograph of Ephedra distachya [Schoenfelder].

1.3.3 (R)-phenylacetylcarbinol (PAC) as a Precursor

To overcome the problems associated with its traditional production, ephedrine is

produced in a one-step chemical reaction from the optically active precursor (R)-

phenylacetylcarbinol (PAC). In spite of the fact that PAC can be chemically synthesized,

biotransformations of pyruvate and benzaldehyde to PAC using various yeast species are

the most common production processes [Oliver et al., 1989]. The PAC then undergoes a

reductive amination reaction to produce (1R, 2S) ephedrine and then to (1S, 2S)

pseudoephedrine (Fig 1.7).

)-Pseudo-ephedrine

CH

NHCH

O

3

O

O

CH 3

NHCH

O

CH

H

(R)-PAC

H

(1R,2S)-Ephedrine (1S,2S

H2NCH3H2, Pt

H

3

3 3

)-Pseudo-ephedrine

CH

NHCH

O

3

O

O

CH 3

NHCH

O

CH

H

(R)-PAC

H


H2NCH3H2, Pt

H

3

3 3

)-Pseudo-ephedrine

CH

NHCH

O

3

O

O

CH 3

NHCH

O

CH

H

(R)-PAC

H


H2NCH3H2, Pt

H

3

3 3

Figure 1.7: Synthesis of (1R, 2S) ephedrine and (1S, 2S) pseudoephedrine from PAC.

Chapter 1

Introduction


8

The biotransformation process has several advantages over the chemical process for PAC

production [Oliver et al., 1999]:

(1) only requires relatively mild reaction conditions,

(2) the process is reaction-specific for R-PAC formation. PAC produced by chemical

means is a racemic mixture of R-PAC and S-PAC. The latter cannot be used to synthesize

(1R, 2S) ephedrine and hence (1S, 2S) pseudoephedrine,

(3) waste product can be directed to biological waste treatment.

Nevertheless, there are limitations associated with the biotransformation process [Rogers

et al., 1997]:

(1) toxic effects of benzaldehyde, PAC and by-products on cells and PDC enzyme,

(2) low aqueous benzaldehyde solubility.

To overcome the problems, considerable efforts have been made towards application of a

biphasic liquid-liquid extractive bioconversion process using an organic solvent (see

Section 1.6).

Chapter 1

Introduction


9

1.4 Biotransformation of Pyruvate and Benzaldehyde to PAC

The biotransformation of pyruvate and benzaldehyde to PAC is catalyzed by the cytosolic

enzyme pyruvate decarboxylase (PDC). Pyruvate is first converted to acetaldehyde via a

non-oxidative decarboxylation reaction. The resulting acetaldehyde then ligates with

benzaldehyde to form PAC. Thiamine pyrophosphate (TPP) and Mg2+ are required as

cofactors.

1.4.1 Reaction Mechanisms

1.4.1.1 Early Findings

Neuberg and co-workers in the early 1920s first demonstrated the biochemical production

of PAC by adding benzaldehyde into an actively fermenting top yeast. They proposed two

mechanisms for the biotransformation: the first one was the conversion of pyruvate to an

activated acetaldehyde via non-oxidative decarboxylation reaction catalyzed by a

carboxylase, followed by condensation of the activated acetaldehyde and added

benzaldehyde to form PAC, catalyzed by a carboligase. The second mechanism proposed

was the condensation of pyruvate and benzaldehyde by a carboligase, followed by non-

oxidative decarboxylation of the resulting complex by a carboxylase.

PAC formation by yeast was then considered to be analogous to acetoin formation as

confirmed by Green et al. in 1942 by using a crude yeast enzyme extract. In 1947, Gross

and Werkmann’s studies confirmed the role of acetaldehyde as intermediate in acetoin

formation. They found that addition of isotopically labelled acetaldehyde (13C) into a

dried yeast extract in the presence of pyruvate, resulted in the incorporation of the

labelled acetaldehyde into the acetoin formed. Furthermore, Happold and Spencer in 1952

revealed that production of acetoin might not be significant under physiological

conditions due to the low concentration of acetaldehyde present in yeast cytosol. They

discovered that addition of acetaldehyde resulted in appreciable production of acetoin.

1.4.1.2 PAC Formation

Further studies of the role of PDC in catalyzing the condensation of pyruvate and

benzaldehyde came in 1988, when Bringer-Meyer and Sahm demonstrated the production

of PAC by purified PDC from Zymomonas mobilis and Saccharomyces carlsbergensis.

Chapter 1

Introduction


10

The results were further confirmed by Cardillo et al. in 1991, who revealed that

benzaldehyde was more readily condensed with pyruvate than other substituted aldehydes

by Saccharomyces spp. Shin [1994] described the PAC formation based on the two-site

reaction mechanism proposed by Juni [1961]. As illustrated in Fig 1.8, the TPP-bound

PDC interacts with the carbonyl group of pyruvate (α-carbon), CO2 is then released via a

non-oxidative decarboxylation with the α-carbon still bound to PDC. The resulting

‘active acetaldehyde’ – TPP complex is transferred irreversibly to the other site of PDC.

The ‘active acetaldehyde’ can then undergo two fates: (1) be reversibly dissociated to

‘free acetaldehyde’ and/or (2) be condensed with other aldehydes, in this case

benzaldehyde, to form PAC. In Juni’s work [1961], it was said that the ‘active

acetaldehyde’ condensed with ‘free acetaldehyde’ (released by reversible dissociation of

the bound ‘active acetaldehyde’) to form acetoin. Hence, acetaldehyde and acetoin are

two of the major by-products from PAC formation. Formation of other by-products will

be discussed in Section 1.4.3.

PDC interacts with the carbonyl group

‘active acetaldehyde’

‘free acetaldehyde’AcyloinPAC or acetoin

PDC interacts with the carbonyl group

‘active acetaldehyde’

‘free acetaldehyde’AcyloinPAC or acetoin

Figure 1.8: Mechanisms of PAC formation catalyzed by pyruvate decarboxylase [Shin,

1994].

Chapter 1

Introduction


11

In general, the PAC, acetaldehyde, and acetoin formation can be described by the

following reactions:

(I) Acetaldehyde formation: non-oxidative decarboxylation of pyruvate.

H3C – C – C – OH+ CO2

Pyruvate ‘Active Acetaldehyde’PDC bound

O O

TPP + Mg2+

PDCC

O

H

H3CH3C – C – C – OH+ CO2

Pyruvate ‘Active Acetaldehyde’PDC bound

O O

TPP + Mg2+

PDCC

O

H

H3C

C

O

H

H3CC

O

H

H3C

‘Active Acetaldehyde’PDC bound

‘Free Acetaldehyde’

C

O

H

H3C C

O

H

H3CC

O

H

H3C C

O

H

H3C


‘Free Acetaldehyde’

(II) Acetoin formation: condensation of PDC bound ‘active acetaldehyde’ and ‘free

acetaldehyde’.

H3C – C – CH – CH3

O OH

+ C

O

H

H3CTPP + Mg2+

PDC

‘Free Acetaldehyde’ Acetoin

C

O

H

H3C


H3C – C – CH – CH3

O OH

+ C

O

H

H3CTPP + Mg2+

PDC


C

O

H

H3C H3C – C – CH – CH3

O OH

H3C – C – CH – CH3

O OH

+ C

O

H

H3C C

O

H

C

O

H

H3CTPP + Mg2+

PDC

TPP + Mg2+

PDC


C

O

H

H3C C

O

H

C

O

H

H3C


(III) PAC formation: condensation of PDC bound ‘active acetaldehyde’ and benzaldehyde.

CH C CH3

OH O

+ C

O

H

Benzaldehyde

TPP + Mg2+

PDC

R-PAC

C

O

H

H3C


CH C CH3

OH O

CH C CH3

OH O

+ C

O

H

C

O

H

C

O

HTPP + Mg2+

PDC

-

C

O

H

H3C C

O

H

C

O

H

H3C CH C CH3

OH O

+ C

O

H

Benzaldehyde

TPP + Mg2+

PDC

R-PAC

C

O

H

H3C


CH C CH3

OH O

CH C CH3

OH O

+ C

O

H

C

O

H

C

O

HTPP + Mg2+

PDC

-

C

O

H

H3C C

O

H

C

O

H

H3C

Chapter 1

Introduction


12

1.4.2 Pyruvate Decarboxylase Enzyme (PDC)

1.4.2 1 Natural Role of PDC

Pyruvate decarboxylase (PDC) is a thiamine pyrophosphate (TPP) dependent enzyme,

one of the key enzymes in glycolytic pathway of many yeasts, fungi, plants and some

bacteria [Pohl, 1997]. Pyruvate as the end product of the glycolytic pathway can undergo

many alternative fates; one of them leads to production of ethanol. PDC catalyzes the

non-oxidative decarboxylation of pyruvate to acetaldehyde with release of carbon

dioxide. For each molecule of acetaldehyde produced, one proton (H+) is consumed. The

resulting acetaldehyde is then reduced to ethanol by alcohol dehydrogenase (ADH)

isozymes and/or other unspecific oxidoreductases. For each molecule of ethanol

produced, one molecule of NADH is oxidized to NAD+.

1.4.2.2 Structure of PDC

PDC is a 240 kDa homotetrameric enzyme, the tetrameric molecule is a dimer of dimers

that binds to 4 molecules of TPP and 4 molecules of magnesium ions. PDC is generally

found as dimers or tetramers, whereby the apoenzyme exists as dimers, while the active

holoenzyme exists as tetramers. The existence of the dimer and tetramer forms is pH

dependent. In yeast PDC exists only as tetramers at pH 5.5 – 6.5, as both dimers and

tetramers at pH 6.5 – 9.5 and dimers only at pH greater than 9.5. In addition, PDC in

Zymomonas mobilis has been found to only exists as tetramers [Pohl, 1997].

The dimers are composed of monomers of which contact sites are determined mainly by

aromatic amino acids. The tetramers are composed of dimers of which contact sites are

determined mainly by electrostatic interactions. By far the greater catalytic activity of the

enzyme is related to the tetrameric species [Jabs et al., 2001].

PDC subunits are essentially identical with a slight difference in chain lengths [Hohmann

1997]. The two dimeric subunits are nearly stereochemically identical and are related by

an approximate two-fold symmetry. Each subunit consists of 3 main domains: α, β, and γ

(named accordingly to their consecutive locations in N to C terminal direction); the

domains are shown in Fig 1.9.a. Residues involved in TPP binding are associated with α

Chapter 1

Introduction


13

and γ domains [Arjunan et al., 1996] and Fig 1.9.b shows the binding of TPP and Mg2+

ion to the enzyme. The arrangement of the subunits in the overall structure can be seen in

Fig 1.10.

aa

bb

Figure 1.9: (a) α, β, and γ domains in PDC subunit [Arjunan et al., 1996] and (b) binding

site of Mg2+ demonstrating octahedral coordination with the enzyme, which also forms a

hydrogen bond with TPP [Furey et al., 1998] (Fig b was constructed with the program

CHAIN).

Chapter 1

Introduction


14

Figure 1.10: Overall structure of PDC tetramer is shown in ribbon drawing. The side

chains of Cys-221 residues, which involve in substrate activation are shown in shaded

boxes (adapted from Furey et al. [1998] with modification by Leksawasdi [2004]).

1.4.2.3 Role of Thiamine Pyrophosphate (TPP)

TPP is a biologically active form of vitamin B1. It functions as a cofactor in many

enzymatic reactions, which involve cleavages of carbon-carbon bonds adjacent to

carbonyl group. The TPP molecule is non-covalently bound in ‘V’ conformation at the

interface between two monomers, involving the α and γ domains [Jabs et al., 2001]. TPP

is bound in such a way that certain residues from the two subunits are able to interact with

the molecule [Arjunan et al., 1996]. The binding strength of TPP is pH dependent and

varies among different types of enzyme [Ullrich, 1970]. Structure of TPP is shown in Fig

1.11.

Figure 1.11: Structure of thiamine pyrophosphate (TPP) [Campbell, 1999].

Chapter 1

Introduction


15

In TPP, the carbon atom between the nitrogen and the sulfur in the thiazole ring is very

reactive. It forms a carbanion (an ion with a negative charge on a carbon atom). It attacks

the carbonyl group of pyruvate to form an adduct, and carbon dioxide splits off. The

carbon-carbon fragment left behind is covalently bonded to TPP and is sometimes called

the ‘active acetaldehyde’. There are then shifts of electrons and the ‘active acetaldehyde’

splits off as ‘free acetaldehyde’ thereby regenerating the carbanion.

1.4.2.4 PDC Isozymes

PDC exists as isozymes; the same enzyme with different subunit configuration. PDC1

and PDC5 are the only structural genes for yeast PDC (the predicted amino acid

sequences are 88% identical); with PDC1 being the major structural gene; PDC5 is only

expressed in PDC1 deletion mutants. Deletion of PDC1 stimulates the promoter activity

of both PDC1 and PDC5; a mechanism called autoregulation, which controls the

expression of PDC1 and PDC5 genes. Deletion of PDC1 gene resulted in 80% reduction

of the wild-type activity, while deletion of PDC5 gene did not result in any decrease of

activity. Mutants with both PDC1 and PDC5 genes deleted did not have any in vitro

pyruvate decarboxylase activity and were unable to consume glucose [Hoffmann and

Valencia 2004].

Other PDC genes had also been identified: PDC2 and PDC6. PDC2 has a role in PDC

synthesis at transcriptional level. PDC6 is a weakly expressed gene and is activated when

fused spontaneously under the control of the PDC1 promoter [Hoffmann and Valencia

2004].

1.4.2.5 Factors Influencing PDC Stability

PDC stability in a biotransformation system may be influenced by the chemical species

present; namely the substrates, product and by-products. Studies by Shin [1994], Chow et

al. [1995], Sandford [2002], and Leksawasdi et al. [2003] showed that there was a

significant deactivating effect of benzaldehyde on PDC. Shin [1994] further investigated

PAC formation with initial pyruvate concentration up to 600 mM and found no inhibition

on the initial reaction rates with the increasing pyruvate level. Sandford [2002] observed

increasing PDC deactivation when the enzyme was incubated with increasing levels of

Chapter 1

Introduction


16

PAC (34 – 274 mM). A similar effect was observed by the above author when incubating

the PDC with increasing acetoin concentration (5 – 60 mM) while exposure to 30 mM

acetaldehyde had no evident effect on PDC deactivation.

1.4.3 Formation of By-Products

Biotransformation of pyruvate and benzaldehyde to PAC also produces a number of by-

products: benzyl alcohol [Neuberg and Libermann, 1921] (activity of ADH and/or other

oxidoreductases), acetaldehyde and acetoin [Neuberg and Welde, 1914], benzoic acid

[Neuberg and Welde, 1914; Smith and Hendlin, 1953], optically active 1-phenyl-1,2-

propanediol [Mochizuki et al., 1995], optically inactive 2-hydroxy-1-phenyl-1-propanone

[Becvarova et al., 1963, Nikolova and Ward, 1991], acetyl benzoyl [Voets et al., 1973;

Nikolova and Ward, 1991] and transcinnamaldehyde [Voets et al., 1973].

1.4.4 Microorganisms for PAC Production

Microorganisms used for PAC production should preferably exhibit high levels of PDC

activity, low levels of ADH activity and high tolerance for benzaldehyde, PAC and by-

products [Oliver et al., 1999]. Another important criterion is the PDC affinity for

benzaldehyde. However, this is rarely measured since high PDC activity does not

necessarily mean high benzaldehyde affinity. This was demonstrated by Bringer-Meyer

and Sahm in 1988; although Zymomonas mobilis possessed five times higher PDC

activity than Saccharomyces carlsbergensis, the sugar-fermenting suspensions of the

yeast exhibited 4 – 5 times higher PAC yields due to higher affinity for benzaldehyde. In

addition, microorganisms with the highest initial PAC productivity were not necessarily

the most productive over an extended period and did not necessarily result in the highest

final concentrations [Netrval and Vojtisek, 1982; Shin and Rogers, 1996a; b].

Various yeast strains have been investigated regarding their ability to form PAC:

(1) both brewer’s (S. carlsbergensis) [Smith and Hendlin, 1953; Netrval and Vojtisek,

1982] and baker’s (S. cerevisiae) yeasts [Becvarova et al., 1963; Nikolova and Ward,

Chapter 1

Introduction


17

1992c] have been shown to possess significant PDC activity, with brewer’s yeast having

the higher ADH activity.

(2) Hansenula anomala, S. carlsbergensis and S. cerevisiae have been reported to have

higher PDC activity than Torula utilis [Becvarova and Hanc, 1963]. S. cerevisiae also had

high benzaldehyde [Mahmoud et al., 1989] and aldehyde tolerance [Seely et al., 1989a].

(3) Candida and Saccharomyces strains formed more PAC in comparison to other yeasts

[Netrval and Vojtisek, 1982; Agarwal et al., 1987].

(4) Candida utilis was reported to produce appreciable levels of PAC [Shin and Rogers,

1995; 1996a; b].

(5) In a screening of 105 yeast strains, Rosche et al. [2003b] observed very low

carboligase activities for PAC formation with Schizosacchromyces pombe but it was

associated with best resistance to pre-incubation with acetaldehyde and benzaldehyde.

Highest carboligase activities combined with medium resistance were reported with

strains of C. utilis, C. tropicalis and C. albicans.

The microorganisms can be genotypically and/or phenotypically modified to improve

PAC yields. Seely et al. [1989b] modified strains of S. cerevisiae and C. flareri for

increased acetaldehyde and ephedrine resistance. However, modifications do not

necessarily guarantee improved PAC production. Dissara and Rogers [1995] found that

an isolated C. utilis strain with reduced growth rate also had reduced productivity,

benzaldehyde tolerance and consumption rate with a lower final PAC concentration in

comparison to the parental strain.

Chapter 1

Introduction


18

1.5 Factors Influencing Biocatalysis for PAC Production

The current commercial PAC process is based on biotransformation of pyruvate and

benzaldehyde by fermenting baker’s yeast. The process consists of first growing the yeast

cells in glucose medium with optimal conditions for PDC production and accumulation of

pyruvate. Benzaldehyde is added at a later stage with sugars to initiate PAC production

[Oliver et al., 1999].

1.5.1 Enzyme Activity

Vojtisek and Netrval [1982] demonstrated that PDC enzyme activity was not a limiting

factor for PAC production with fermenting yeast cells: cells with lower PDC activity may

exhibit higher initial PAC formation rates and final yields. They further reported that

gradual addition of sodium pyruvate to the fermentation medium resulted in increase of

final PAC yield. Moreover, supplementation of the fermentation medium with sodium

pyruvate after a 6 h biotransformation caused the PAC production to re-start at the initial

rate. The authors also suggested that the activities of some enzymes involved in metabolic

pathways between the carbon source and pyruvate affected the PAC formation.

The observation that PDC activity was not a limiting factor for in vivo PAC production

was further confirmed by the work of Nikolova and Ward in 1991 on different strains of

yeast. They also suggested that a step in the glycolytic pathway was rate-limiting. As

previously mentioned, Bringer-Meyer and Sahm [1988] found that the final PAC

concentration with sugar-fermenting cells of Z. mobilis was lower in comparison to cells

of S. carlsbergensis, in spite of the fact that the bacteria contained more PDC activity. In

conclusion, high PDC activity was not always associated with high formation rate and

final yield for in vivo PAC production; they may also be functions of growth quality and

metabolism rate [Tripathi et al., 1997].

Chapter 1

Introduction


19

1.5.2 Toxicity Effect of Benzaldehyde

Long and Ward [1989b;c] studied the toxic effect of benzaldehyde on in vivo PAC

production with S. cerevisiae and sucrose as the carbon source (Table 1.2).

Table 1.2: Effect of benzaldehyde on in vivo PAC production with S. cerevisiae [Long

and Ward, 1989b; c].

Benzaldehyde level

2 g/L pulse fed once

every hour for 6 h

6 g/L fed initially and after 4

h

Initial PAC formation rate Lower Higher

Final PAC yield Higher Lower

Cell viability Increased at early stages

and decreased afterwards

Massive reduction during all

stages

Sucrose metabolism Increased when

benzaldehyde was

limited

Inhibition of metabolism

during all stages

It was revealed that at the lower benzaldehyde concentration, the cellular constituents

were protected by maintenance of the cell membrane permeability barrier towards

benzaldehyde. At the higher benzaldehyde concentration, this barrier was no longer

maintained causing the intracellular concentration of benzaldehyde to be higher than the

extracellular concentration [Long and Ward, 1989b; c].

1.5.3 Effect of Dissolved Oxygen Concentration

PDC is induced under anaerobic conditions, as observed by Sims et al. [1991] using C.

utilis with glucose as the carbon source. The glycolytic flux also increases under

anaerobic conditions thereby producing higher pyruvate levels, as reported by van Dijken

amd Scheffers [1986] by using C. utilis with glucose pulsing.

Anaerobic conditions can be achieved by control of aeration and agitation rates, or by

sparging with nitrogen gas, although as demonstrated by Sims et al. [1991], actively

Chapter 1

Introduction


20

growing biomass in the presence of oxygen also resulted in PDC synthesis. PDC appeared

to be partially deactivated under aerobic conditions [Sims et al., 1991]. Optimum

operating conditions must be experimentally found to produce sufficient biomass with

optimal PDC activity and these conditions are likely to vary among various

microorganisms. Voets et al. [1973] and Ellaiah and Krisna [1988] found that in vivo

PAC production with S. cerevisiae was affected by aeration rate. Culik et al. [1984] in

fermentation with S. coreanus increased the aeration rate when the PAC formation rate

started to decline; they found that the final PAC yield was increased.

1.5.4 Effect of pH

Protons are consumed in the conversion of pyruvate and benzaldehyde to PAC leading to

a pH rise in the biotransformation. Rosche et al. [2002a] reported that PDC carboligase

activity was optimum around pH 6.5, a pH higher than 7 caused reduction in the activity.

A pH range of 4 – 6 has generally been employed for in vivo PAC production using S.

cerevisiae [Smith and Hendlin, 1953; Gupta et al., 1979; Long and Ward, 1989b; c].

Rogers et al. [1997] employed a pH of 6.2 for their three-stage process using C. utilis and

found that PAC production was sensitive to pH.

1.5.5 Biomass Condition

In any fermentation processes for in vivo PAC production, the final concentration will be

largely dependent on the biomass condition, which is affected by medium composition

and the physicochemical conditions used throughout the process [Oliver et al., 1999].

1.5.5.1 Effect of Cell Age

Agarwal et al. [1987] observed the effect of cell age on the PAC concentration in a yeast

fermentation: younger or older biomass than that of optimum age gave lower PAC

production due to lowered PDC activity and benzaldehyde tolerance. In addition, the

freshness (ie total viability) of the yeast was considered to be an important factor in

enhancing PAC production [Voets et al., 1973].

Chapter 1

Introduction


21

1.5.5.2 Effect of Respiratory Quotient (RQ)

The respiratory quotient (RQ) of a fermentation process is defined as the ratio of carbon

dioxide evolution rate over oxygen uptake rate (both expressed as mmoles/L/h). An RQ

of 1 corresponds to full respiratory growth whereas fermentative metabolisms are

associated with an RQ greater than 1. Rogers et al. [1997] cultured C. utilis cells for in

vivo PAC production at 30°C, pH 6 and various controlled RQ values. The RQ was

maintained by adjusting the agitation rate. It was observed that at an RQ of 1, PAC was

produced at a lower specific rate in comparison to that for benzyl alcohol formation.

Increasing the RQ to 4 – 5 resulted in improved PAC production rate and yield due to

increasing fermentative conditions and greater PDC induction. Further studies reported by

Chen et al. [2005] also involved monitoring and control of the RQ value (as well the pH)

to enhance PDC activities.

In recent years, various PAC production processes have been developed for use in

enzymatic biotransformations. The traditional yeast based fermentation method is

associated with limited pyruvate availability and significant loss of benzaldehyde to

benzyl alcohol due to oxidoreductases activity. Such obstacles can be overcome by the

enzymatic process [Rosche et al., 2002a, b; Shin and Rogers, 1996]. In comparison to the

traditional fermentation with 10 – 12 g/L PAC [Rogers et al., 1997], approx. 50 g/L PAC

was produced in enzymatic processes with added pyruvate in an aqueous/benzaldehyde

emulsion system using partially purified PDC from yeast and filamentous fungi [Rosche

et al., 2002a, b; 2003a]. Full details of the enzymatic PAC processes including the effects

of various design and operational factors on product and by-product formation are

presented in the result section.

Chapter 1

Introduction


22

1.6 Two-Phase Aqueous/Organic Extractive Bioconversion with

Organic Solvent

1.6.1 Definition

Extractive bioconversion is a biotransformation process performed in the presence of

substance with extracting capability, most commonly the organic solvent. Two-phase

(biphasic) is defined as the condition in which the organic and aqueous phases are present

in excess of mutual saturation levels [Nikolova and Ward, 1992a].

Substrates with low aqueous solubility (lipophilic) are added to the organic phase,

whereas water-soluble substrates, biocatalyst and required cofactors are added to the

aqueous phase. Transfer of substrate from the organic to the aqueous phase facilitates

biotransformation in the aqueous phase. Application of high agitation rates results in the

formation of emulsion in which small organic phase droplets are suspended in the

aqueous phase thereby facilitating substrate transfer from the organic to the aqueous

phase; and also product back into the organic phase.

1.6.2 Advantages and Disadvantages of the Two-Phase Aqueous/Organic

Biotransformation

Extractive bioconversion can be very useful to processes in which lipophilic substances

are produced or when the substrates and/or products are toxic or when the substrates have

limited aqueous solubility. There are several clear advantages associated with the two-

phase aqueous/organic biotransformation [Bruce and Daugulis, 1991]:

(1) biocatalytic activity advantage – in the presence of a suitable and adequate organic

phase: (a) direct exposure of biocatalyst to toxic substrate can be avoided and (b) product

can be continuously removed into the organic phase. For a biocatalyst that is prone to

toxic substrate deactivation and/or product inhibition, this will lead to increased

productivity,

(2) reaction advantage – with introduction of a suitable and adequate solvent, the

equilibrium position of a biocatalytic reaction can be shifted towards completion.

Chapter 1

Introduction


23

Continuous product removal by the organic phase shifts the equilibrium position towards

product formation without the need to provide large excess of reactant,

(3) product recovery advantage - with employment of a suitable solvent and optimal

volume ratio of the organic to aqueous phase , it is no longer necessary to work with

dilute solutions. As a result, product recovery and waste treatment costs can be

significantly reduced.

A possible problem with two-phase aqueous/organic bioconversion is the effect of solvent

on cell viability and enzyme activity and stability. Some solvents have detrimental effects

on these parameters even at low concentrations, leading to cessation of the bioconversion.

In some cases, solvents that are biocompatible were found to have a low distribution

coefficient and selectivity for the product, leading to ineffective product extraction and

possible solvent recovery difficulties.

1.6.3 Organic Solvent Selection

Selecting the most appropriate organic solvents for biphasic extractive bioconversion is a

difficult task not only because there are abundant types of solvent available, but also there

are many criteria that have to be considered e.g. biocompatibility, toxicity, extracting

capability, and ease of solvent recovery. Solvent biocompatibility may relate to cell

viability, whereas solvent toxicity deals with its effect on the activity and stability of the

biocatalyst. The use of a solvent with high extracting capability towards product is

required for effective product extraction. Finally, a solvent which is easy to recover from

the biotransformation product is desirable as the solvent can be recycled, leading to more

economical process.

1.6.3.1 Solvent Biocompatibility

Non-biocompatible solvents can cause appreciable reductions in cell viability even at low

concentrations. In addition, a solvent should not be biodegradable so that the

microorganism does not use it as substrate [Bruce and Daugulis, 1991]. Laane et al.

[1987] discovered that there was a strong relationship between biocompatibility and the

logarithm of partition coefficient of a solvent in a standard octanol-water system: log Poct

Chapter 1

Introduction


24

which is defined as the ratio of solvent concentration in octanol to solvent concentration

in water in a standard octanol/water two-phase system [Laane et al., 1985]. It was argued

that the octanol-water system provided a sufficient description of hydrophobicity and

transport interaction of a structure when exposed into a biological system. Poct can be

either experimentally determined or predicted from the molecular structure of the solvent.

1.6.3.1.1 Effects of Solvent on Microorganisms

The presence of some solvents can be associated with possible detrimental effects on

microorganisms. Some solvents are very toxic, they greatly reduce cell viability even at

minute concentrations. The possible detrimental effects are listed below:

(1) cytoplasmic shrinkage [Nikolova and Ward, 1992a],

(2) ultrastructural changes – e.g. displacement of the chromosome towards the cell

periphery [Nikolova and Ward, 1992], loss of intracellular electron-dense materials

[Wongkongkatep, 1992],

(3) inhibition of nutrient transport – Hampe [1986] observed that the sugar uptake in yeast

was non-competitively inhibited by alcohols,

(4) loss of membrane organization [Nikolova and Ward, 1992a] – solvents that have been

found to cause modifications of membrane permeability are toluene [Jackson and de

Moss, 1965; de Smet et al., 1978], alcohols [Ingram and Buttke, 1982], and alkanes [Teh

and Lee, 1976]. The modifications can lead to escape of ions (K+, Mg2+), low molecular

weight metabolites (NAD+, NADH), and large molecules (proteins, DNA, RNA) from the

cells [Wongkongkatep, 1992].

1.6.3.1.2 Cell Adaptation to Organic Solvents

In spite of the fact that the mechanisms of solvent tolerance in living microorganisms are

not fully understood, it has been proposed that membrane adaptation can occur,

particularly changes in lipid compositions to recover the membrane fluidity

(homeoviscous adaptation) [Heipieper and de Bont, 1994]. Several membrane

modifications have been recorded in response to solvent addition:

Chapter 1

Introduction


25

(1) changes in saturation index of the membrane lipids. For examples, cells of E. coli

when incubated with benzene and octanol, react to recover increasing membrane fluidity

by increasing the fraction of the saturated membrane fatty acids. Stronger interactions

between saturated chains have been reported to reduce membrane fluidity [Ingram 1977;

Keweloh et al., 1990;],

(2) increase in fatty acid acyl chain length and protein to lipid ratio [Heipieper et al.,

1994].

It is suggested that these modifications can only be carried out by de novo synthesis of

membrane lipids during growth; as most bacteria are not able to alter their membrane

fluidity by post-biosynthetic modifications [Heipieper and de Bont, 1994].

1.6.3.2 Solvent Toxicity

Solvent toxicity is related to the effect of the solvent on the activity and stability of the

biocatalyst. The degree of toxicity is related to the type and concentration of solvent used

and its uptake by the cell membrane. Bar [1987] classified solvent toxicity into two

categories:

(1) dissolved (molecular) toxicity which is essentially the effect of solvent at levels below

saturation in the aqueous phase. It has been argued that there is solvent absorption by the

cell membrane resulting in the modification of membrane permeability, which leads to

enzyme inhibition, deactivation and possibly, breakdown of the transport mechanisms

[Lilly et al., 1987],

(2) physical (phase) toxicity which describes the effect of solvent in excess of saturation

its level. It has been argued that there is direct cell-solvent contact, extraction of nutrients

from the aqueous phase, or limited access to nutrients caused by interfacial cell adherence

or entrapment in an emulsion [Bar, 1988]. At this level of toxicity, a cell may be

surrounded by a solvent coat, causing cell wall disruption and extraction of inner cellular

components [Bar, 1987].

Chapter 1

Introduction


26

Nikolova and Ward [1992a; b] investigated the effects on toxicity and biocompatibility of

various organic solvents in the two-phase aqueous/organic synthesis of PAC at 10%

aqueous phase content with whole cells of S. cerevisiae and pyruvate and benzaldehyde

as substrates. The results of these studies can be summarized as follows:

(1) solvents with the highest biotransformation rates were hexane, dodecane, and

hexadecane, intermediate rates were observed with ethylacetate and butylacetate, and

lowest production were observed with toluene and chloroform,

(2) solvent biocompatibility was evaluated by observing the surface structure of the cells

isolated from the biotransformation systems using scanning electron microscopy.

Solvents with log Poct ≥ 2.5: hexane, decane, and toluene caused no apparent damage to

the cell surface after 26 h (Fig 1.12). More hydrophilic solvents with log Poct < 2:

ethylacetate, butylacetate, and chloroform (Fig 1.12) caused cell puncturing after a shorter

reaction period. The observations were further confirmed by evaluating the amount of

fatty acids and proteins released into the organic and aqueous phase due to cell damage

(Table 1.3).

Table 1.3: Comparison of baker’s yeast fatty acids and proteins released into the

biotransformation medium with observed biocatalytic activity [Nikolova and Ward,

1992b].

Solvent Total fatty acids

(µg/mL)

Total proteins

(µg/mL)

Activity

(mmol PAC / h / mg dry

cells)

Hexane 140 45 60

Butylacetate 164 50 31

Chloroform 170 115 10

Toluene 178 80 7

Chapter 1

Introduction


27

a b c

d e

a b c

dd ee

Figure 1.12: Scanning electron micrographs of yeast cells isolated from biphasic media

containing: (a) hexane (26 h), (b) decane (26 h), (c) toluene (26 h), (d) chloroform (0 h),

and (e) chloroform (2 h) [Nikolova and Ward, 1992a].

Solvents associated with high PAC productivity would certainly cause little or no damage

on the cells after a certain time period (in this case hexane). However, biocompatible

solvents do not necessarily have little toxic effects on the biocatalyst (in this case

toluene). Hence, there was no perfect correlation between the biocatalytic activity and cell

resistance to solvent damage.

Chapter 1

Introduction


28

1.6.3.3 Extraction Efficiency

Based on the nature of their extraction mechanisms [Leon et al., 1998], solvents can be

classified as physical and chemical extractive solvents. Examples of physical extractive

solvents are hydrocarbons, ketones, and esters. In this type of solvents, the interactions

are based on the solvation of products by weak, unspecific donor bonds of different types.

With the chemical extractive solvents, such as trioctyl-phosphine oxide, there are

formations of stable adducts or even new compounds and the interactions are specific and

relatively stable.

Parameters used to define extraction capacity of a solvent with respect to a product are

distribution coefficient (KD) and separation factor (α).KD is defined as the ratio of product

concentration in the organic phase to product concentration in the aqueous culture

medium, at equilibrium [Bruce and Daugulis, 1991]. α is defined as the ratio of the

distribution coefficient of the product to distribution coefficient of any other contaminant

from which the product is isolated (e.g. remaining substrate, by-products) [Leon et al.,

1998]. KD determines the extraction efficiency of a solvent whereas a solvent with a high

α value exhibits high selectivity towards the product in preference to any other substances

[Bruce and Daugulis, 1991]. Daugulis et al. [1987] and Roffer et al. [1988] had shown

that cycling a water-immiscible solvent through the culture medium could result in

simultaneous reaction and product extraction in a single processing unit.

1.6.3.4 Ease of Solvent Recovery

Solvent density, viscosity, and boiling point affect the ease of solvent recovery from the

biotransformation product [Bruce and Daugulis, 1991]. Solvents of which boiling points

are close to those of the product are difficult to recover (with consequent difficult product

isolation from the solvent). Furthermore, for long or continuous biotransformation

processes, chemical and thermal stability of the solvents are necessary since the processes

may involve numerous recycling of the solvents [Bruce and Daugulis, 1991].

Chapter 1

Introduction


29

1.7 Current Status of Two-Phase Aqueous/Organic Biotransformation

for PAC Production

Bioprocess development for PAC production has been maintained in our group for more

than 15 years. Shin and Rogers in 1995 reported production of 15.2 g/L PAC in a fed-

batch fermentation process with immobilized Candida utilis, which demonstrated

improvements compared to the traditional yeast-based fermentation with 10 – 12 g/L

PAC. In 1996, Shin and Rogers improved the production to 28.6 g/L PAC with added

pyruvate and benzaldehyde in 40 mM potassium phosphate buffer by using PDC enzyme

from C. utilis, thereby adopting an enzymatic biotransformation process.

Leksawasdi et al. [2004] developed and validated a mathematical modelling for a batch

production of PAC from pyruvate and benzaldehyde using C. utilis PDC. The

biotransformation model was used to determine the overall rate constants for the

formation of PAC and by-products acetaldehyde and acetoin. These values were

determined from three batches of biotransformation data with concentration ranges of 50

– 150 mM benzaldehyde, 60 – 180 mM pyruvate, and 1.1 – 3.4 U/mL enzyme activity.

The model was validated in biotransformation experiments (initial concentrations of

substrates and enzyme are given in the Figure legend) giving an acceptable fitting with R2

value of 0.9963 (Fig 1.13).

Rosche et al. [2002a, b] developed an aqueous/benzaldehyde emulsion system buffered

with 2.5 M MOPS and achieved 50 g/L PAC using PDC from yeast and filamentous

fungi. PDC carboligase activity was optimum at pH 6.5 and the use of high buffering

capacity was therefore essential as proton uptake in the biotransformation process

increases the pH to above 7 [Rosche et al., 2002a]. Moreover, high MOPS concentration

was found to have an additional stabilizing effect on PDC [Rosche et al., 2002a].

However, the aqueous/benzaldehyde emulsion process was limited by increased PDC

deactivation presumably due to increased benzaldehyde droplet/enzyme interaction in the

emulsion system [Sandford et al., 2005; Rosche et al., 2005b].

Chapter 1

Introduction


30

Figure 1.13: Batch biotransformation kinetics and model fitting for determination of

overall rate constants for the formation of PAC, acetaldehyde, and acetoin (Vp, Vq, and

Vr) at (a) 50 mM benzaldehyde/60 mM sodium pyruvate with initial PDC activity of 3.4

U/mL carboligase, (b) 150 mM benzaldehyde/180 mM sodium pyruvate with initial PDC

activity of 3.4 U/mL carboligase, and (c) 100 mM benzaldehyde/120 mM sodium

pyruvate with initial PDC activity of 1.1 U/mL carboligase: () pyruvate, ( )

benzaldehyde, () acetaldehyde, () acetoin, ( ) PAC, and (x) enzyme activity. Line of

best fit through each data profile was created from the optimal value of Vp, Vq, and Vr

[Leksawasdi et al., 2004].

Chapter 1

Introduction


31

To overcome the restriction, Sandford et al. [2005] developed a two-phase

aqueous/organic enzymatic biotransformation process for PAC production. They

performed a solvent screening study at a 1:1 organic to aqueous phase volume ratio with

C. utilis PDC and observed highest PAC production with 1-octanol and 1-nonanol as

organic phase solvents. The use of 1-octanol as a suitable solvent for two-phase PAC

production was further confirmed by Rosche et al. [2005] with whole cells of C. utilis.

Sandford et al. [2005] investigated PAC formation as a function of enzyme concentration

in two extreme operations of the two-phase system: the slowly stirred phase-separated

and the rapidly stirred emulsion systems (Fig 1.14).

ORGANIC PHASE

AQUEOUS PHASE

BENZALDEHYDE PAC

PYRUVATE + BENZALDEHYDE PAC + CO2

PDC

ORGANIC PHASE

AQUEOUS PHASE

BENZALDEHYDE PAC

PYRUVATE + BENZALDEHYDE PAC + CO2

PDC

Figure 1.14: Diagrammatic representation of the two-phase aqueous/organic PAC

production system [Rosche et al., 2002b; Sandford et al., 2005].

Phase-Separated Emulsion

Chapter 1

Introduction


32

The phase-separated system was associated with high specific PAC production per unit

enzyme, however productivities were low. In contrast, productivities were high in the

emulsion system, however the specific production was reduced. Fig 1.15 shows PAC

production as a function of initial enzyme concentration in the two systems. Detailed

explanation on the profiles in the context of the present investigation will be presented in

Sections 4.2.1.5 and 4.2.1.6.

Figure 1.15: PAC production as a function of carboligase activity in the rapidly stirred

two-phase system after 40 h, and phase-separated two-phase system after 395 h (4°C,

organic octanol phase contained 1500 mM benzaldehyde and the aqueous phase

contained 1430 mM pyruvate, 2.5 M MOPS, 1 mM TPP, 1 mM Mg2+, pH 6.5). A 1:1

volume ratio of organic and aqueous phases was used.

Leksawasdi et al. [2005] implemented a pH control system in a two-phase PAC

production with 2.5 M MOPS and C. utilis PDC and reported a specific reaction rate of

0.60 mg/U/h, a 1.6 times improvement in comparison to the same biotransformation

without pH control. Lowering the expensive MOPS concentration to 20 mM MOPS with

controlled pH resulted in three times decreased PAC production in comparison to the 2.5

Chapter 1

Introduction


33

M MOPS system. Further addition of low cost 2.5 M dipropylene glycol into the 20 mM

MOPS system resulted in comparable overall PAC production of 92.1 g/L. In the search

of a low cost biocatalyst for PAC production, Satianegara et al. [2006] observed higher

stability of PDC in the form of C. utilis whole cells towards benzaldehyde and

temperature in comparison to the partially purified preparations.

1.8 Strategy for Two-Phase Model Development

Development of a process model for the two-phase PAC production would serve as a tool

to predict optimum parameters for favorable reaction conditions and to allow a

description of the macroscopic reaction [Goetz et al., 2001]. Conceptually, a two-phase

process model would include a description of the reaction kinetics in the aqueous phase

with influence by the organic phase, mass transfer kinetics and determination of changes

in mass balances for both organic and aqueous phase [Willeman et al., 2002]. In addition,

modeling of the rate of PDC enzyme deactivation in the presence of the organic phase

(octanol) would be needed to facilitate overall process optimization.

Reaction Kinetics in Aqueous Phase

Experiments would need to be conducted to plot the time profiles of PAC formation over

a relatively short period (e.g. 30 mins) for a range of enzyme, pyruvate and benzaldehyde

concentrations. The initial rate of PAC formation would be determined by a tangential

method determined at time zero and plotted against the concentration; a mathematical

equation would then be fitted with determination of the kinetic constants for each plot.

Each of these equations would be combined to develop an overall rate equation for PAC

formation. Other rate equations regarding pyruvate and benzaldehyde consumption and

formation of the by-products acetaldehyde and acetoin would also need to be derived.

Batch biotransformation experiments would then be performed for determination of the

overall rate constants. Finally, the completed mathematical model would be validated by

using the model and the calculated rate constants to predict profiles of substrate

consumption, product and by-product formation for certain initial conditions.

Chapter 1

Introduction


34

PDC Enzyme Deactivation Rate in Aqueous Phase

The model should include terms for PDC deactivation rate by soluble benzaldehyde and

octanol in the aqueous phase, presence of aqueous/organic interfacial area. The slowly

stirred phase-separated system would be associated with contact of enzyme with defined

interfacial area while the effect of enzyme-droplet interaction would be evaluated in the

rapidly stirred emulsion system.

Mass Transfer Kinetics

The following mass transfer equation would be included:

φx = kLx a (Corg,x / mx – Caq,x) (mol Laq-1 s-1)

φx is the mass transfer rate from the aqueous to the organic phase (mol Laq-1 s-1)

kLx is the lumped mass transfer coefficient (m s-1)

a is the interfacial area per volume of aqueous phase (m-1)

Cx is the molar concentration of species x. (mol Laq-1)

mx is the ratio of equilibrium concentrations (organic over aqueous phase)

Measurements of kLx a could be conducted during biotransformation when the reaction

rate was much faster than the mass transfer rate, leading to Caq,x ≈ 0. This would enable

the determination of kLx a to be independent of the reaction kinetics.

Mass Balance Equations

PAC synthesis would be considered to only occur in the aqueous phase and by assuming

that no changes in volume would take place during the biotransformation, the following

equations would apply:

Rate of change in the aqueous phase: Vaq dCaq,x / dt = rx Vaq – φx Vaq (mol s-1)

Rate of change in the organic phase: Vorg dCorg,x / dt = φx Vaq (mol s-1)

rx is the reaction rate (mol Laq-1 s-1)

V is the volume of the aqueous or the organic phase (L)

The present research project aims at further developing the two-phase system for an

improved and efficient PAC production process with particular focus on the key factors

likely to influence this particular enzymatic biotransformation. The thesis provides an

experimental basis for subsequent two-phase model development.

Chapter 2

Materials and Methods


35

CHAPTER 2

MATERIALS AND METHODS

1. Microorganisms

2. Chemicals, Enzymes and Sources

3. Buffer Compositions

4. PDC Enzyme Production

5. Biotransformation Systems for PA) Production

6. PDC Enzyme Deactivation and Organic-Aqueous Benzaldehyde

Transfer Studies in the Two-Phase Aqueous/Octanol-

Benzaldehyde System

7. Two-Phase Aqueous/Organic PAC Synthesis at Lower Buffer

Concentration (20 mM MOPS, Larger Scale)

8. Analytical Methods

9. Calculation Methods

Chapter 2



36

2.1 Microorganisms

Saccharomyces cerevisiae strain UNSW 102200, Candida utilis strain UNSW 70940,

and Kluyveromyces marxianus strain UNSW 510700 were obtained from the Culture

Collection of the School of Biotechnology and Biomolecular Sciences, University of

New South Wales (World Directory of Culture Collections No. 248), Sydney, Australia.

Candida tropicalis strain 57, Candida glabrata strain LU 10336 and

Schizosaccharomyces pombe strain LU 311 were provided by BASF (Germany). The

stock cultures of the microorganisms were stored in glycerol at –20°C.

2.2 Chemicals, Enzymes and Sources

Table 2.1: Chemicals and enzymes

Name Formula Supplier Cat. No.

Chemicals

2-[N-morpholino]ethanesulfonic acid (MES)

C6H13NO4S Sigma M8250

3-[N-morpholino]propanesulfonic acid (MOPS)

C7H15NO4S Sigma M1254

Acetaldehyde C2H4O Fluka 00071

Acetic acid, glacial CH3COOH Allied Signal 33209

Acetoin C4H8O2 Fluka 00540

Acetone CH3COCH3 LAB-SCAN A3501

Acetonitrile CH3CN APS 2315

Ammonium sulphate (NH4)2SO4 APS 56

Antifoam (propylene glycol) - Fluka 81380

Bacteriological agar - Oxoid Code L11

Benzaldehyde C6H5CHO Crown Scientific

H3C080

Benzoic acid C7H6O2 Sigma B7521

Buffer standard pH 4 - Merck 19239.5W

Buffer standard pH 7 - Merck 19240.5H

Chapter 2



37

Table 2.1 (Continued): Chemicals and enzymes.

Name Formula Supplier Cat. No.

Chemicals

Calcium chloride dihydrate CaCl2.2H2O APS 127

Citric acid, anhydrous C6H8O7 Sigma C0759

Copper (II) sulphate, hydrated CuSO4.5H2O APS 10091

Dipropylene glycol (DPG) C6H14O3 Merck 8.03265

Di-sodium hydrogen orthophosphate dodecahydrate

Na2HPO4.12H2O APS 10248

Electrode cleaner (pepsin/HCl) - Ingold Order No.209891250

Electrolyte (1M LiCl in acetic acid) - Mettler Toledo

Order No. 51340051

Ethanol, absolute C2H5OH APS 214

Ethylenediaminetetra-acetic acid (EDTA) di-sodium salt

EDTA-Na2H2.2H2O APS 180

Glucose (D-) anhydrous C6H12O6 APS 783

Hydrochloric acid HCl FSE H/1100/PB17AU

Iron (II) sulphate heptahydrate FeSO4.7H2O APS 226

Magnesium sulphate heptahydrate MgSO4.7H2O APS 302

Manganese chloride heptahydrate MnCl2.7H2O BDH 10152

Methanol (HPLC grade) CH3OH APS 2314

Nicotinamide adenine dinucleotide disodium salt (NADH)

C21H27N7O14P2Na2 Roche 92464233

Octanol C8H18O APS 2370

Phosphoric acid H3PO4 APS 371

Potassium chloride KCl APS 383

Potassium dihydrogen orthophosphate KH2PO4 APS 391

Potassium hydroxyde KOH APS 10210

Reactivation solution for glass electrodes (diluted HF/HCl)

- Ingold Order No. 9895

R-phenylacetylcarbinol (R-PAC) C9H10O2 BASF -

Sodium hydrogen carbonate NaHCO3 APS 475

Chapter 2



38

Table 2.1 (Continued): Chemicals and enzymes.

Name Formula Supplier Cat.No.

Chemicals

Sodium hydroxyde NaOH APS 482

Sodium pyruvate (pyruvic acid sodium salt)

C3H3NaO3 Fluka 15990

Sulfuric acid H2SO4 Allied Signal

30743

Thiamine pyrophosphate (TPP) C12H19ClN4O7P2S Sigma C8754

Trichloroacetic acid (TCA) - Sigma T4396

Triethanolamine hydrochloride C6H15NO3HCl Sigma T1502

Yeast Extract - Oxoid Code L21

Zinc sulphate heptahydrate ZnSO4.7H2O BDH 10299

Proteins

Alcohol dehydrogenase (ADH) - Sigma A7011

Lactate dehydrogenase (LDH)

(from rabbit muscle)

- Roche 127876

Chapter 2



39

2.3 Buffer Compositions

Table 2.2: Buffer compositions.

Breakage Buffer

Citric acid 200 mM

MgSO4.7H2O 20 mM

TPP 0.5 mM

pH adjusted to 6.5 at 6°C.

Collection Buffer

Citric acid 400 mM

MgSO4.7H2O 40 mM

TPP 4 mM

pH adjusted to 6.0 at 25°C.

Triethanolamine Buffer

Triethanolamine-HCl 250 mM

EDTA-disodium salt 2.5 mM

pH adjusted to 7.6 with 5 M NaOH at 25°C.

NADH Buffer

NADH-disodium salt 7 mM

NaHCO3 120 mM

pH adjustment not necessary.

Carboligase Buffer

Citric acid 200 mM

MgSO4.7H2O 20 mM

TPP 2 mM

Sodium pyruvate 200 mM

Benzaldehyde 80 mM

Ethanol 3 M

pH adjusted to 6.4 at 25°C. The buffer was

stored in aliquots at –20°C for a maximum of

two weeks.

Biotransformation / PDC Enzyme

Deactivation Study Buffer

MOPS 2.5 M or 20 mM / 2.5 M

MgSO4.7H2O 1 mM

TPP 1 mM / 0.5 mM

pH adjusted to 6.5 or 7 with 10 M KOH for

2.5M MOPS and 5 M KOH for 20 mM

MOPS at the temperature of interest.

Chapter 2



40

2.4 PDC Enzyme Production PDC enzyme was produced via a yeast fermentation of glucose-based media. Various

scales and methods of fermentation were employed: (I) 2 x 0.22 L shake flask

fermentations to culture 6 yeast strains simultaneously (S. cerevisiae, C. utilis, C.

tropicalis, S. pombe, C. glabrata, and K. marxianus), (II) 20 L aerobic-partially anaerobic

two-stage fermentation for C. utilis partially purified PDC production and (III) 2 x 3 L

pH shift fermentations for C. utilis whole cell production. All fermenters, shake flasks

(Erlenmeyer), apparatus and media used were sterilized by autoclaving at 121°C and 125

kPa for 20 mins (Atherton). Media mixing and yeast culturing processes were performed

in a sterile environment.

2.4.1 General Steps in the Fermentation Processes

Each of the fermentation methods was associated with different operating conditions;

however each process comprised four culturing steps: (I) growth on agar media, (II)

preseed, (III) seed, and (IV) final fermentation (for the shake flask fermentation method

there was no preseed stage).

2.4.1.1 Media Preparation

Three types of media were prepared: (I) solid agar media, (II) liquid preseed and seed

media and (III) liquid final fermentation media. Concentrated stock solutions for each

compound were prepared, autoclaved or filtered, mixed and adjusted to the required

concentrations with sterile RO water. The concentrations of the stock solutions are shown

in Table 2.3.

To prepare solid media, the mixed and volume-adjusted agar solution was poured while

above 60°C into sterile Petri dishes to prevent early solidification. The agar media were

stored at 4°C prior to usage.

Chapter 2



41

Table 2.3: Stock solution concentrations and sterilization methods for the preparation of

agar and liquid media for fermentation.

Compound Stock Solution Sterilization

Method

Glucose 400 g/L Autoclaving

Yeast extract 200 g/L Autoclaving

Agar 30 g/L Autoclaving

(NH4)2SO4 5x concentrated 1 Autoclaving

Other Salts

KH2PO4, Na2HPO4.12H2O, MgSO4.7H2O 5x concentrated 1 Autoclaving

CaCl2.2H2O 3 10x and 100x concentrated 1,2 Autoclaving

CuSO4.5H2O, ZnSO4.7H2O,

FeSO4.7H2O 4, MnCl2.4H2O

100x concentrated 1 Autoclaving

MES adjusted to pH 6.0 5 4x concentrated 1 Filtration

(1) With respect to the final concentrations in media

(2) 10X for the shake flask and aerobic-partially anaerobic two-stage fermentation methods 100X for the pH shift fermentation method

(3) Prepared separately due to precipitation, had limited solubility in water

(4) Acid addition prior to autoclaving to prevent oxidation

(5) pH adjustment prior to autoclaving

The liquid preseed and seed media were prepared immediately or maximum 1 – 2 days

prior to the culturing process to minimize the risk of contamination. The mixed and

volume-adjusted media were aliquoted into baffled Erlenmeyer flasks then covered with

cotton bungs to allow release of CO2 during the culturing process. The media were stored

at 4°C prior to usage.

The final fermentation media were prepared 1 – 2 days prior to the culturing process. The

mixed and volume-adjusted media was transferred into 5 L Erlenmeyer flask, which has a

connector to the fermenter. The media were stored at 4°C prior to usage. For the shake

flask fermentation method, in which there was no preseed stage, the final fermentation

medium composition was the same as the seed medium. The mixed and volume-adjusted

Chapter 2



42

medium was aliquoted into non-baffled Erlenmeyer flasks to prevent extensive aeration,

thereby achieving a fermentative condition.

2.4.1.2 Growth on Agar Media

Yeast colonies from stock culture were aseptically inoculated onto agar plates. The plates

were sealed with parafilm and incubated at 30°C. Further subculturing was performed

every 2 – 3 days. Outlined below in Table 2.4 are agar media compositions used in the

various fermentation methods.

Table 2.4: Agar media compositions for the various fermentation methods.

Compound Fermentation method

Shake flask (g/L) Aerobic-partially anaerobic

two-stage (g/L)

pH shift (g/L)

Glucose 30 30 20

Yeast extract 5 5 -

Peptone - - 10

(NH4)2SO4 10 10 -

KH2PO4 3 3 1

Na2HPO4.12H2O 2 2 -

MgSO4.7H2O 1 1 0.5

CaCl2.2H2O 0.05 0.05 -

Agar 15 15 15

2.4.1.3 Preseed and Seed

The process involved growing yeast in fresh liquid media in two subsequent steps after

the growth on agar medium. Yeast from an agar plate was inoculated into preseed

medium. Inoculations of the preseed culture to the seed medium and of the seed culture

to the final fermentation medium were performed when OD660 (optical density at 660

nm) was 8 – 10. The preseed and seed steps were conducted in Erlenmeyer flasks with

the media being 10% of the flask volume. The inoculations were performed aseptically

Chapter 2



43

and the yeast was cultivated at 30°C in a shaker. Outlined below in Table 2.5 are preseed

and seed media compositions and operating conditions used in the various fermentation

methods.

Table 2.5: Preseed and seed media compositions and operating conditions for the various

fermentation methods.


Shake flask (g/L)* Aerobic-partially

anaerobic two-stage

(g/L)

pH shift (g/L)

Glucose 90.0 10.0 20.0

Yeast extract 10.0 5.0 -

(NH4)2SO4 10.0 10.0 6.0

KH2PO4 3.0 3.0 1.0

Na2HPO4.12H2O 2.0 2.0 -

MgSO4.7H2O 1.0 1.0 0.5

CaCl2.2H2O 0.05 0.05 0.02

CuSO4.5H2O - - 0.0005

ZnSO4.7H2O - - 0.0106

FeSO4.7H2O - - 0.02

MnCl2.4H2O - - 0.002

MES 39.0 39.0 39.0

Operating conditions Fermentation method

Shake flask * Two-stage pH shift

Initial pH 6.0 6.0 6.0

Shaker Speed 160 rpm 250 rpm 250 rpm

* Same compositions for the seed and final fermentation media

2.4.1.4 Final Fermentation

Seed culture was inoculated into the final fermentation medium aseptically. Adjustment

of glucose concentration was necessary prior to inoculation. Table 2.6 lists the final

fermentation media compositions used in the various fermentation methods.

Chapter 2



44

Table 2.6: Final fermentation media compositions for the various fermentation methods.


Shake flask (g/L) Aerobic-partially

anaerobic two-stage

(g/L)

pH shift (g/L)

Glucose 90.0 90.0 100.0

Yeast extract 10.0 10.0 -

(NH4)2SO4 10.0 10.0 10.0

KH2PO4 3.0 3.0 1.0

Na2HPO4.12H2O 2.0 2.0 -

MgSO4.7H2O 1.0 1.0 0.5

CaCl2.2H2O 0.05 0.05 0.02

CuSO4.5H2O - - 0.0005

ZnSO4.7H2O - - 0.0106

FeSO4.7H2O - - 0.02

MnCl2.4H2O - - 0.002

MES 39.0 - -

2.4.2 Fermentation Processes

2.4.2.1 Shake Flask Fermentation

The shake flask fermentations were performed to culture six yeast strains simultaneously.

The method involved growing yeast in seed medium; the grown yeast was then

inoculated to final fermentation medium (no preseed stage). The harvested culture was

processed for whole cell and crude extract production of PDC. The fermentation method

was described by Chen [2005b].

The final fermentation was conducted with initial pH 6 in two 1 L non-baffled

Erlenmeyer flasks for each yeast strain; each flask accommodating 0.22 L working

volume (0.02 L seed + 0.2 L media). The flasks were fitted in a 30°C shaker, rotating at

160 rpm. The seed culture was prepared by inoculating a single yeast colony from agar

medium into 50 mL medium in each non-baffled Erlenmeyer flask.

Chapter 2



45

The cultures were harvested by centrifugation (Hettrich Zentrifugen, Model Universal

32R, Rotor Type 1617) at 5,000 rpm for 15 mins at 4°C when glucose concentration fell

to below 10 g/l, washed with 0.9% NaCl and stored at –20°C. The specific PDC activities

of the yeast strains are shown in Section 3.2.1.

2.4.2.2 Aerobic-Partially Anaerobic Two–Stage Fermentation

The two-stage fermentation was performed to culture C. utilis for partially purified PDC

production. The method involved yeast culturing in two steps in the final fermentation:

first step was to grow the yeast aerobically (RQ = 1), second step was to switch to partial

anaerobic phase (RQ = 4). The respiratory quotient (RQ) was controlled by manually

adjusting the stirrer speed. The harvested culture was processed for partially purified

PDC production. The fermentation method was described by Sandford [2002].

The final fermentation was conducted in a 30 L BIOSTAT® C fermenter system (with

system controller) accommodating 20 L working volume (Fig 2.1). 1.5 L seed culture

aged 8.5 h was inoculated into 18.5 L final fermentation medium. The seed culture was

prepared by inoculating 2.5 mL preseed culture aged 14 h into 250 mL seed medium in

each baffled Erlenmeyer flask. The preseed culture was prepared by inoculating a single

yeast colony from agar medium into 50 mL preseed medium in each baffled Erlenmeyer

flask.

The final fermentation was controlled at 30°C, pH 6 (controlled by 4 M NaOH and 20%

(v/v) H3PO4 addition) and air flow rate was 0.5 vvm. The culture was maintained at RQ =

1 for the first 9 h by keeping the stirrer speed at 500 rpm; the speed was then raised to

750 – 780 rpm until RQ = 1 could not be maintained. The culture was switched to partial

anaerobic phase with RQ = 4±1 for the next 4 h by decreasing the stirrer speed to 275 –

350 rpm to induce PDC production. Dissolved oxygen was above 90% air saturation

initially, it then dropped to 0% at 9 h until the end of fermentation course at 13 h.

The culture was harvested by centrifugation (Sorvall RC-5B, Du-Pont Instruments) at

5,000 rpm for 15 mins at 6°C when glucose concentration fell to 18 g/L, washed with RO

Chapter 2



46

water, suspended in breakage buffer and stored at –20°C. The specific PDC activity was

115 U/g dry biomass.

Figure 2.1: 30 L BIOSTAT® C fermenter system used in the aerobic-partially anaerobic

two-stage fermentation method for PDC production.

2.4.2.3 pH Shift Fermentation

In the pH shift fermentation process, the pH was lowered from 6 to 3 in the final

fermentation when 20 g/L glucose had been consumed. The pH was automatically

controlled with acid and base addition. This method had been proven by Chen [2005a] to

dramatically increase the specific PDC production by C. utilis. The harvested culture was

stored as cell pellets.

The final fermentation was conducted in two 5 L BIOSTAT® A (B.Braun) fermenters

systems (with system controllers) accommodating 3 L working volume for each batch

(Fig 2.2). 0.3 L seed culture aged 8 h was inoculated into 2.7 L final fermentation

medium. The seed culture was prepared by inoculating 5 mL preseed culture aged 16 h

into 50 mL seed medium in each baffled Erlenmeyer flask. The preseed culture was

prepared by inoculating a single yeast colony from agar medium into 50 mL preseed

medium in each baffled Erlenmeyer flask. The final fermentation was controlled at 30°C,

initially at pH 6 (controlled by 5 M NaOH and 5 M H2SO4 addition). The stirrer speed

and air flow rate was set at 300 rpm and 0.1 vvm respectively, dissolved oxygen was 0 –

3% air saturation for the two batches.

Chapter 2



47

When approx. 20 g/L glucose had been consumed, the pH was shifted to 3 and

maintained until the end of fermentation course at 40 h. The culture was harvested by

centrifugation (Beckman, Model AvantiTM J-20, Rotor Type JLA-8.1000) at 5,000 rpm

for 15 mins at 4°C when glucose concentration dropped to below 10 g/L, washed with

RO water and stored at –20°C. The specific PDC activity was 390 U/g dry biomass.

Figure 2.2: 5 L BIOSTAT® A (B.Braun) fermenter system used in the pH shift

fermentation method for PDC production.

2.4.2.4 Sampling Procedure

The protocol used for sampling from the various fermentations is shown in Fig 2.3.

Cell culture sample

Preseed / seed / final fermentation final fermentation final fermentation

2 x 1 mL 2 x 5 mL 2 x 10 mL

OD660measurement

Cell pellet Supernatant

Centrifugation

Dry biomassmeasurement

Glucosemeasurement

Centrifugation

Cell pelletstored at –20°C

Cell pelletsuspended in breakage buffer

Crude extractpreparation

PDC carboligase activitymeasurement

Cell culture sample

Preseed / seed / final fermentation final fermentation final fermentation

2 x 1 mL 2 x 5 mL 2 x 10 mL

OD660measurement

Cell pellet Supernatant

Centrifugation

Dry biomassmeasurement

Glucosemeasurement

Centrifugation

Cell pelletstored at –20°C

Cell pelletsuspended in breakage buffer

Crude extractpreparation

PDC carboligase activitymeasurement

Figure 2.3: Sampling procedures for the fermentation processes.

Chapter 2



48

2.4.3 PDC Enzyme Preparations

Three types of PDC preparation were employed as biocatalysts for PAC production: (I)

whole cells, (II) crude extract and (III) partially purified PDC.

2.4.3.1 Whole Cell PDC

The wet frozen cell pellets were thawed in a 25°C water bath. The suspension was then

subjected to treatment specify in Section 2.5.3.4.1.

2.4.3.2 Crude Extract PDC

The frozen cell pellets were thawed in a 25°C water bath and suspended in breakage

buffer. The suspension was frozen with liquid nitrogen and thawed five times and further

blended (Hamilton Beach/Proctor-Silex, Inc., Model 908-220) with 0.5 mm glass beads

(Biospec, Cat. No. 11079105) to release the PDC. Crude extract was isolated after

clarification with centrifugation (Hettrich Zentrifugen, Model Universal 32R, Rotor Type

1617) at 5,000 rpm for 5 mins at 4°C and stored at –20°C.

2.4.3.3 Partially Purified PDC

The partially purified PDC was prepared in a one-step precipitation of the crude extract

with 40 – 50% (v/v) acetone at -10°C. The precipitated protein was isolated by

centrifugation at 5,000 rpm for 5 mins at 0°C. The recovered paste was freeze-dried

(Dynavac, Model FD3) at –50°C and 0.1 mbar for 24 h (Fig 2.4), then ground to powder

and stored at –20°C.

Figure 2.4: Freeze drier used in partially purified PDC preparation.

Chapter 2



49

2.5 Biotransformation Systems for PAC Production Biotransformations for PAC production were performed in various systems in the current

project.

2.5.1 Yeast Pyruvate Decarboxylases: Variation in Biocatalytic

Characteristics

As described in Chapter 3, a study was designed to select the best biocatalyst for PAC

production: four yeast PDCs with different properties (S. cerevisiae, C. utilis, C.

tropicalis, and K. marxianus) were tested for the production of PAC and by-products

acetaldehyde and acetoin. The yeast PDCs were tested in three biotransformation systems

with increasing benzaldehyde concentrations: (I) aqueous (soluble benzaldehyde), (II)

aqueous/benzaldehyde emulsion and (III) two-phase aqueous/octanol-benzaldehyde

emulsion systems. There were also investigations of the effects of benzaldehyde and

acetaldehyde on initial PAC formation. Table 2.7 lists the types of biotransformation

system employed and investigations performed in the studies.

Table 2.7: Biotransformation systems employed in the selection of biocatalyst for PAC

production (Chapter 3).

Biotransformation system [Benzaldehyde]

(mM)

[Pyruvate]

(mM)

[Acetaldehyde]

(mM)

Type of PDC

Investigations on PAC and by-products acetaldehyde and acetoin formation at 22(±2)°C

Aqueous (soluble benzaldehyde) 80 80 - Crude extract

Aqueous/benzaldehyde emulsion 325 420 - Crude extract

Two-phase

aqueous/benzaldehyde-octanol

850

(TRV)*

450

(TRV)*

- Whole cells

Chapter 2



50

Table 2.7 (continued): Biotransformation systems employed in the selection of

biocatalyst for PAC production (Chapter 3).

Biotransformation system [Benzaldehyde]

(mM)

[Pyruvate]

(mM)

[Acetaldehyde]

(mM)

Type of PDC

Investigations on the effect of benzaldehyde on initial PAC formation at 22(±2)°C

Aqueous (soluble benzaldehyde) 45 250 - Crude extract

80 250 - Crude extract

Aqueous/benzaldehyde emulsion 120 250 - Crude extract

175 250 - Crude extract

Investigations on the effect of acetaldehyde on initial PAC formation at 22(±2)°C

Aqueous (soluble benzaldehyde) 45 250 30 Crude extract

80 250 30 Crude extract

Aqueous/benzaldehyde emulsion 125 250 30 Crude extract

185 250 30 Crude extract

* Total reaction volume (TRV) is determined by combining both phases.

The studies were carried out at 2 mL scale in 4 mL glass vials (inner diameter 12.5 mm,

height 35 mm) at 22(±2)°C and stirred magnetically (Bibby, Model B292) at 220 – 190

rpm for the systems I and II and 250 – 190 rpm for system III (the systems were stirred at

the higher speed initially; the speed was then decreased following volume reduction after

sampling).

In all biotransformations, 2.5 M MOPS buffer system was used with initial pH of 6.5 and

0.5 mM Mg2+ and 1 mM TPP as cofactors. Initial PDC activities were 1.5 U/mL

carboligase for all systems (the two-phase system was associated with 3 U/mL activities

in the aqueous phase or 1.5 U/mL TRV (total reaction volume calculated by combining

the volumes of both phases)). Experiments on each system (with each biocatalyst) were

performed in triplicate and three to four analyses were repeated for each sample; the

mean values were determined and error bars show the highest and lowest values.

Chapter 2



51

2.5.2 Effect of Organic to Aqueous Phase Volume Ratio on Two-Phase

Aqueous/Organic PAC Synthesis (2.5 M MOPS)

The selected yeast PDC from the first study was employed as the biocatalyst to improve

PAC production in the two-phase aqueous/octanol-benzaldehyde emulsion system by

changing the organic to aqueous phase volume ratio as detailed in Chapters 4 and 5.

Table 2.8 lists the types of biotransformation system employed and investigations

performed in the studies.

The studies were carried out at 10 mL scale in 20 mL glass vials (inner dia. 24 mm,

height 47 mm) at 4(±1)°C and 20(±1)°C and stirred magnetically (IKA®-Werke, Model

RO 5 power). All biotransformations employed 2.5 M MOPS buffer system with initial

pH of 6.5 and 1 mM Mg2+ and 1 mM TPP as cofactors. Three to four times analyses were

repeated for each sample; the mean values were determined and error bars show the

highest and lowest values.

2.5.3 Biotransformation Systems

For the aqueous (soluble benzaldehyde) and aqueous/benzaldehyde emulsion systems,

pyruvate and benzaldehyde were added into the buffer solution. For the two-phase

aqueous/octanol-benzaldehyde system, the phases were independently prepared: pyruvate

was dissolved into the buffer solution (aqueous phase) whilst benzaldehyde was

dissolved in octanol (organic phase). In all systems, PDC enzyme solution was prepared

separately and added at the start of biotransformations.

2.5.3.1 MOPS Buffer System

All biotransformations in the current studies employed MOPS buffer. High buffering

capacity was required as there was proton consumption during the biotransformation of

pyruvate and benzaldehyde to PAC (optimum pH for PDC was 6.5 – 7.5) [Rosche et al.

2002a]. Furthermore, Leksawasdi et al. [2005] reported that employment of 2.5 M MOPS

in the two-phase aqueous/octanol-benzaldehyde system was associated with higher

aqueous phase benzaldehyde levels in comparison to lower MOPS concentrations.

Chapter 2 Materials and Methods


52

Table 2.8: Biotransformation systems employed in the characterization of the two-phase aqueous/octanol-benzaldehyde system for PAC

production (Chapters 4 and 5).

Biotransformation

system

Organic to

aqueous phase

volume ratio

[Octanol]

(mM)

[Benzaldehyde]

(mM)

[Pyruvate]

(mM)

PDC Enzyme

Carboligase Activity

(U/mL)

Stirrer Speed (rpm)**

Organic

(4°C/20°C)

TRV*

(4°C/20°C)

Aqueous

(4°C/20°C)

TRV*

(4°C/20°C)

Aqueous TRV*

Type

Investigation on the effect of organic to aqueous phase volume ratio in the two-phase aqueous/octanol-benzaldehyde emulsion system

for PAC production (4(±1)°C and 20(±1)°C)

Two-phase

aqueous/octanol

-benzaldehyde

1:1 Benzaldehyde and

octanol creating a

second phase

1360/1400 680/700 1260/1290 630/645 5.6 2.8 Partially purified 250 – 235

0.67:1 As above 1700/1760 680/705 1060/1075 635/645 4.7 2.8 Partially purified 235 – 220



* Total reaction volume (TRV) by combining both phases. ** The systems were stirred at the higher speed initially; the speed was then decreased following volume reduction after sampling.



53

Despite the advantages offered by the high MOPS concentration, MOPS is an expensive

material. Studies were also performed to evaluate PAC formation at a much lower MOPS

concentration of 20 mM (Chapter 5).

The MOPS buffer was prepared by dissolving the powder in RO water at room

temperature. The solution was adjusted to the desired working pH (6.5 – 7) with 10 M

KOH for 2.5 M MOPS and 5 M KOH for 20 mM MOPS. The buffer solution was stored

at 4°C prior to usage.

2.5.3.2 Aqueous (Soluble Benzaldehyde) and Aqueous/Benzaldehyde Emulsion

Systems

2.5.3.2.1 Substrate and PDC Enzyme Concentrations

The aqueous soluble system contained less than 100 mM benzaldehyde, whereas the

aqueous/benzaldehyde emulsion system contained more than 100 mM benzaldehyde.

Pyruvate concentration was usually determined to be similar or 1.5 times of the

benzaldehyde concentration to minimize the possibility of pyruvate limitation. PDC

activities were in the range of 1.5 – 2.8 U/mL carboligase. The substrate to enzyme stock

solution volume ratio was 4 parts of substrate to 1 part of enzyme making up the

biotransformation system.

2.5.3.2.2 Substrate Stock Solution Preparation

Pyruvate was dissolved in the buffer solution at room temperature. The pH was adjusted

at the biotransformation temperature to 6.5 – 7 with 6.4 % HCl (pyruvate addition tends

to increase the pH). Pyruvate concentration was measured by enzymatic assay and any

insufficiency was corrected (pyruvate addition might have required further pH

adjustment). The pyruvate solution was stored at 4°C prior to usage.

Benzaldehyde (97 – 99% purity) was transferred into the pyruvate solution at room

temperature: fully dissolved for the aqueous system and suspended for the

aqueous/benzaldehyde emulsion system (any dilution on the pyruvate solution was



54

insignificant since pure benzaldehyde was used). The benzaldehyde was added on the day

of biotransformation to avoid evaporative losses. The cofactors Mg2+ and TPP were

added prior to benzaldehyde addition. The container was then wrapped in foil, purged

with nitrogen gas and stored at 4°C prior to usage. Benzaldehyde concentration was

measured by HPLC and any insufficiency corrected.

In the case of acetaldehyde addition, a 5 µL microsyringe was used to add pure

acetaldehyde on the day of biotransformation in a 5°C constant temperature room since

acetaldehyde is very volatile (boiling point 20°C at 1 atm). Enzymatic assay was

performed immediately to determine the acetaldehyde concentration.

2.5.3.3 Two-Phase Aqueous/Octanol-Benzaldehyde System

2.5.3.3.1 Substrate and PDC Enzyme Concentrations

For the organic phase, the concentration of the stock substrate solution was determined

by taking into account the phase volume ratio. For the aqueous phase, the concentrations

of the stock substrate and enzyme solutions were determined by taking into account both

the phase volume ratio and substrate to enzyme stock solution volume ratio. The substrate

(pyruvate only) to enzyme stock solution volume ratio was similar to that in Section

2.5.3.2.1.

2.5.3.3.2 Substrate Stock Solution Preparation

To prepare the organic phase, benzaldehyde was dissolved in octanol on the day of

biotransformation at room temperature. The container was wrapped in foil, purged with

nitrogen gas and stored at 4°C prior to usage. Benzaldehyde concentration was measured

by HPLC and any insufficiency corrected. For the aqueous phase, the same method as in

Section 2.5.3.2.2 was used to dissolve the pyruvate. The cofactors 1 mM Mg2+ and 1 mM

TPP were added into the aqueous phase on the day of biotransformation.



55

2.5.3.4 PDC Enzyme Stock Solution Preparation

2.5.3.4.1 Whole Cell PDC Solution

Frozen wet cell pellets from the fermentation processes were thawed in a 25°C water

bath. The pellet was then suspended in the corresponding MOPS buffer with added 1 mM

Mg2+ and 1 mM TPP cofactors. The suspension was diluted and subjected to carboligase

assay to determine the PDC carboligase activity. The suspension was stored at –20°C

prior to usage and adjusted to the relevant PDC activity.

2.5.3.4.2 Crude Extract PDC Solution

The crude extract was diluted and subjected to carboligase assay, then adjusted to the

relevant PDC activity.

2.5.3.4.3 Partially Purified PDC Solution

Partially purified PDC powder was suspended in the corresponding MOPS buffer with

added 1 mM Mg2+ and 1 mM TPP cofactors, stirred overnight at 125 rpm (Bibby, Model

B292) in a 5°C constant temperature room. The enzyme solution was isolated by

centrifugation (Hettrich Zentrifugen, Model Universal 32R) at 5,000 rpm for 5 mins at

4°C. The solution was diluted and subjected to carboligase assay. The enzyme solution

was stored at -20°C prior to usage and adjusted to the relevant PDC activity.

2.5.4 Biotransformation Experiments

2.5.4.1 Set Up of Biotransformation Systems

The substrate stock solution was incubated in a 25°C water bath for biotransformations at

20°C and 22°C or stored in ice for biotransformations at 4°C. The substrate stock solution

was transferred into vials with magnetic bars and stirred in a constant temperature room.

For the aqueous and aqueous/benzaldehyde emulsion systems, relatively high stirring

speed was immediately employed as to achieve homogeneity. For the two-phase



56

aqueous/octanol-benzaldehyde system, low stirring speed was first employed as to keep

the two phases separate and to allow certain degree of organic-aqueous benzaldehyde

transfer.

After approx. 30 min of stirring, enzyme stock solution was added. For the aqueous and

aqueous/benzaldehyde emulsion systems, the enzyme stock solution was directly added

to the stirring substrate stock solution. For the two-phase system, the enzyme was added

to the aqueous phase by deeply immersing the pipette tip. For all systems, the stirring

speed was increased to the operating speed after enzyme addition.

2.5.4.2 Controls

For each type of biotransformation system, a control experiment was established. A

control contained the same concentrations of substrates without the enzyme. The roles of

control were: (I) confirmation of initial benzaldehyde and pyruvate concentrations, (II)

determination of benzaldehyde and pyruvate loss in the absence of reaction and (III)

determination of benzoic acid build up (from oxidation of benzaldehyde). These values

were determined for material balance purposes.

2.5.4.3 Sampling

Sampling was carried out for both control and reaction vials. For the control, samples

were taken at the beginning and end of reaction. Samples from reaction vials were treated

with tricholoroacetic acid (TCA) (100% w/v) to stop the biotransformation. For the two-

phase aqueous/octanol-benzaldehyde system, all samples were centrifuged (Hettrich

Zentrifugen, Model Universal 32R) at 13,000 rpm for 5 mins at 4°C to separate the

phases and TCA was then added to the aqueous phase.

All samples were stored at –20°C prior to further treatment and measurements. PAC,

benzoic acid and benzaldehyde concentrations were measured by HPLC (Section 2.8.8).

Pyruvate and by-product acetaldehyde concentrations were quantified enzymatically

(Sections 2.8.6 and 2.8.7). By-product acetoin concentration was measured by GC



57

(Section 2.8.9). Table 2.9 lists the treatment and measurements performed on the samples

from different types of biotransformation system.

2.5.5 Determination of Residual PDC Enzyme Activities in Biotransformation

Systems

To determine the residual enzyme activities from a biotransformation system, filtration of

the sample through a gel column (Micro Bio-Spin® 6 Chromatography Column, Bio-

Rad, Cat. No. 732-6200) was necessary for PAC removal. The PDC containing solution

was recovered by centrifugation (Hettrich Zentrifugen, Model Universal 32R) at 1,000 g

for 2 mins at 4°C, trapped in collection buffer and incubated in ice for 20 mins refolding

time. The solution was diluted and subjected to carboligase assay. The residual enzyme

activities were determined from the formed PAC and with respect to 100% activity at

time zero. In the case of a two-phase aqueous/octanol-benzaldehyde system, the enzyme

was contained in the aqueous phase.



58

Table 2.9: Treatment and types of measurement performed on the biotransformation samples. System Measurement Treatment

Substance Dilution Factor (approx.)

Aqueous Phase Control 1 Benzaldehyde, Benzoic

Acid 5 Sampled from stirring vials into tubes filled with RO water for the

dilution factor

2 Pyruvate 20 Further dilution from (1) with RO water

Reaction 3 PAC, Benzaldehyde, Benzoic acid

5 Sampled from stirring vials into tubes filled with 2.5% (v/v) TCA for the dilution factor

4 Pyruvate 20 Further dilution from (3) with RO water

5 Acetaldehyde * Further dilution from (3) with RO water

6 Acetoin * Further dilution from (3) with RO water

Benzaldehyde Emulsion

Control 7 Benzaldehyde, Benzoic Acid

10 – 50 Same method as (1)

8 Pyruvate 100 – 300 Further dilution form (7) with RO water

Reaction 9 PAC, Benzaldehyde, Benzoic acid

10 – 50 Same method as (3)

10 Pyruvate 100 – 300 Further dilution from (9) with RO water

11 Acetaldehyde

* Further dilution from (9) with RO water

12 Acetoin * Further dilution from (9) with RO water



59

Table 2.9 (Continued): Treatment and types of measurement performed on the biotransformation samples. System Measurement Treatment

Substance Dilution Factor (approx.)

Aqueous/Octanol Two-Phase

Control

Octanol Phase 13 Benzaldehyde, Benzoic Acid 100 – 200 Dilution with RO water

Aqueous Phase 14 Pyruvate 300 Same method as (13)

Reaction

Octanol Phase 15 PAC, Benzaldehyde, Benzoic acid 100 – 200 Same method as (13)

16 Acetaldehyde * Same method as (13)

17 Acetoin * Same method as (13)

Aqueous Phase 18 PAC, Benzaldehyde

11

Addition of 10% (v/v) of 100% (w/v) TCA into the aqueous phase Diluted to the dilution factor with RO water

19 Pyruvate

110 – 330 Diluted to the dilution factor with RO water from the TCA added sample

20 Acetaldehyde * Same method as (19)

21 Acetoin

* Same method as (19)

* Dilution factor was determined from the material balance on pyruvate utilization



60

2.6 PDC Enzyme Deactivation and Organic-Aqueous Benzaldehyde

Transfer Studies in the Two-Phase Aqueous/Octanol-Benzaldehyde

System

Intensive studies were performed on the two types of the two-phase aqueous/octanol-

benzaldehyde: the slowly stirred phase-separated and the rapidly stirred emulsion

systems (Chapter 4) regarding factors influencing PDC enzyme deactivation and organic-

aqueous benzaldehyde transfer. A Lewis cell phase-separated system was constructed and

equipped with a temperature control system. The emulsion system was constructed using

a magnetically stirred glass vial in a constant temperature room.

2.6.1 Construction of the Aqueous/Organic Phase-Separated System – A

Temperature Controlled Lewis Cell System

2.6.1.1 Lewis Cell Construction

A Lewis cell in its original version was a device employed to study the interfacial

interaction in a gas-liquid system [Lewis, 1954] and the current investigation adopted a

modified design of the cell [Baldascini et al., 2000].

The Lewis cell used in the present experiment was a glass cylinder with 75 mm inner

diameter and height, which consisted of two compartments: top and bottom parts to

contain 92 – 94 mL of organic and aqueous phase respectively. In the middle, a movable

plate with different hole-sizes was inserted allowing a range of aqueous/organic contact

areas. The phases were independently stirred to achieve homogeneity within each phase

The cell was equipped with a top metal plate lined with rubber O-ring. A middle metal

plate with different openings and attached baffles (to enhance mixing) was fitted into the

cylinder. Two holes were drilled through the top and middle plates to the bottom part of

the cell (aqueous phase) and one hole through the top plate to the top part of the cell

(organic phase) for syringes (3 ml, Becton Dickinson, Cat. No. 639461) (with needles

18G x 1½ inches, Terumo) insertions. The functions of the holes were different



61

depending on the nature of the studies. In the PDC deactivation study, the two aqueous

phase holes were used to transfer the enzyme solution and for sampling purposes. In the

benzaldehyde transfer study, the organic phase hole was used to transfer pure

benzaldehyde and the two aqueous phase holes were used for sampling purposes.

Another hole was drilled through the top plate to accomodate a metal stirrer with a 4

bladed impeller to the organic phase. The Lewis cell was fitted into a transparent

rectangular container filled with water and the container was placed on a magnetic stirrer

(Bibby, Model HC1202). A magnetic bar was placed in the aqueous phase. A diagram of

the Lewis cell is shown in Fig 2.5.

Middle Plate

Baffle

Magnetic Bar

Syringe

Top Plate

Overhead Stirrer Shaft

Middle Plate

Baffle

Magnetic Bar

Syringe

Top Plate

Overhead Stirrer Shaft

Figure 2.5: Lewis Cell for experimentation on the aqueous/organic phase-separated

system.

2.6.1.2 Temperature Control System

A temperature control system was necessary as a relatively large working volume was

employed. Additionally, the system could not be placed inside a constant temperature

water bath since it must be visible for easier maintenance of the defined interfacial area.

The temperature control system comprised of a metal sensor inserted into the Lewis cell.



62

The sensor sent signals to a controller (BABS, UNSW, Australia), to which a small pump

(Nikkiso Magpon®, Model CP08-PPRV-24) was connected. Water was pumped from the

water-filled container (in which the cell was positioned) to a metal coil placed inside a

constant temperature water bath (Thermoline, Model TBC/TU4) and back to the

container. Fig 2.6 shows the temperature controlled Lewis cell system.

Temperature Controller

Pump Lewis CellWater Bath Magnetic Stirrer

Temperature Controller

Pump Lewis CellWater Bath Magnetic Stirrer

Figure 2.6: Temperature controlled aqueous/organic phase-separated system (Lewis Cell).

2.6.2 PDC Enzyme Deactivation

2.6.2.1 Experimental Details

In a two-phase aqueous/octanol-benzaldehyde system, PDC enzyme in the aqueous phase

was exposed to the soluble octanol and benzaldehyde and the effects of changing the

organic/aqueous interfacial area on PDC deactivation were investigated. Moreover, there

were possible effects of agitation rate and enzyme concentration. Tables 2.10.A and

2.10.B list the types of system employed and investigations performed in the deactivation

studies.

The PDC stability studies were performed at 4(±1)°C in 2.5 M MOPS buffer system (pH

7) with 0.5 mM Mg2+ and 1 mM TPP as cofactors and no pyruvate; no biotransformation



63

taking place under these conditions. All samples were diluted and subjected to

carboligase assay. The residual enzyme activities were determined from the PAC formed

and with respect to 100% activity at time zero. Triplicate analyses were conducted for

each sample and except for the Lewis cell study, experiments were performed in

triplicate; the mean values were determined and error bars show the highest and lowest

values.

Aqueous-based and two-phase aqueous/organic systems were employed in the study. In

the latter case, the enzyme was contained in the aqueous phase, centrifugation (Hettrich

Zentrifugen, Model Universal 32R) at 13,000 rpm for 5 mins at 4°C was necessary for

aqueous phase isolation.

Table 2.10.A: Aqueous-based and two-phase aqueous/organic systems employed in the

PDC enzyme deactivation studies (Chapter 4).

Aqueous-based system Two-phase aqueous/organic system

I MOPS as control

II MOPS + 4.5 mM octanol

III MOPS + 48 mM benzaldehyde

IV MOPS + 4.5 mM octanol

+ 48 mM benzaldehyde

V Phase-separated system

Organic phase: 1.39 M benzaldehyde in

octanol

Aqueous phase: MOPS + 4.5 mM octanol +

48 mM benzaldehyde

VI Emulsion system

Organic phase: octanol

Aqueous phase: MOPS + 4.5 mM octanol



64

Table 2.10.A (continued): Aqueous-based and two-phase aqueous/organic systems

employed in the PDC enzyme deactivation studies (Chapter 4).

Aqueous-based system Two-phase aqueous/organic system

VII Emulsion system


octanol


48 mM benzaldehyde

VIII Phase-separated system


octanol


48 mM benzaldehyde

IX Emulsion system


octanol


48 mM benzaldehyde



65

Table 2.10.B: Performed investigations in the PDC enzyme deactivation studies (Chapter 4).

* Total reaction volume (TRV) by combining both phases. Investigation System PDC enzyme

carboligase activity

(U/mL)

Stirrer

speed

(rpm)

Specific

interfacial

area

(cm2/L)

Effect of soluble octanol I and II 2 220 -

Effect of soluble benzaldehyde

I and III 2 220 -

Effect of soluble octanol and

benzaldehyde

I and IV 2 220 -

Effect of agitation rate and initial enzyme concentration

In the presence of MOPS (Chapter 3) I 3 and 7.3 95 and

220

-

In the presence of soluble benzaldehyde

(Chapter 3)

III 3 and 7.3 95,220

and 250

-

In the presence of soluble octanol and

benzaldehyde

IV 2 125 and

220

-

Investigation System PDC enzyme

carboligase activity

(U/mL)

Stirrer

speed

(rpm)

Specific

interfacial

area

(cm2/L)

Aqueous TRV*

Effect of interfacial area

In the two-phase phase-separated

system

V 4 2 Organic:

60

Aqueous:

125

117,361,475

In the two-phase emulsion system

I,II,IV

- 2 220 -

V I and

VII

4 2 220 Undefined

Effect of initial enzyme concentration

In the two-phase phase-separated

system

VIII 3.1,5.5,8.6

and 11.9

1.5,2.7,4.3

and 5.9

125 475

In the two-phase emulsion system IX 1.6,4.1,7.1

and 11.6

0.8,2.0,3.5

and 5.8

220 Undefined



66

2.6.2.2 Effects of Soluble Octanol and Benzaldehyde in the Aqueous Phase and

Agitation Rate on PDC Deactivation

The investigations were carried out at 2 mL scale in 4 mL glass vials (inner dia. 12.5 mm,

height 35 mm) and magnetically stirred (Bibby, Model B292). To investigate the effects

of soluble octanol and benzaldehyde, systems (I) to (IV) were employed (Tables 2.10.A

and 2.10.B). All systems were prepared by first dissolving 2.5 M MOPS into RO water

with pH adjusted to 7 with 10 M KOH. System II was prepared by saturating the MOPS

solution with octanol at 4(±1)°C. System IV was prepared by saturating the MOPS

solution with 1.5 M benzaldehyde in octanol in 1:1 phase volume ratio at 4(±1)°C.

System III was prepared by dissolving the same concentration of benzaldehyde as system

IV into the MOPS solution at room temperature. To investigate the effect of agitation

rate, systems I, III, and IV (Tables 2.10.A and 2.10.B) were employed.

PDC enzyme powder was suspended into the systems by wheel rotation at 35 rpm for 1 h

at 4(±1)°C. The resulting suspension was centrifuged (Hettrich Zentrifugen, Model

Universal 32R) at 2,800 g for 5 mins at 4°C to isolate the supernatant.

2.6.2.3 Effect of Ratio of Organic Phase Interfacial Area to Aqueous Phase Volume

on PDC Deactivation

The PDC stability studies were performed in two-phase aqueous/octanol-benzaldehyde

system, comparing the phase-separated and emulsion systems.

2.6.2.3.1 Studies with a Phase-Separated System

The investigations were performed in system V (Tables 2.10.A and 2.10.B) in

temperature controlled Lewis cell system with defined changes in the organic phase

contact area to aqueous phase volume ratio. A relatively small top metal stirrer was used

(BABS, UNSW, Australia) (stirrer dia. 11 mm, shaft dia. 5 mm, shaft length 23 mm) with

stirring in counter clockwise direction. A magnetic bar was placed in the aqueous phase,

stirring in clockwise direction. The aqueous phase comprised of octanol and



67

benzaldehyde saturated MOPS buffer system and concentrated enzyme solution was

added at the start of experiment.

For every volume of enzyme solution transferred into the aqueous phase, the same

volume of the MOPS solution must be withdrawn to maintain a defined interfacial area at

the hole. Later on, for each sample withdrawn, the same volume of the MOPS solution

was transferred into the aqueous phase. A dilution factor was hence included in the

residual enzyme activity calculations at each time point.

2.6.2.3.2 Studies with an Emulsion System

The investigations were carried out at 2 mL scale in the 4 mL glass vials and

magnetically stirred (Bibby, Model B292). Three types of aqueous-based system were

involved: system (I), (II), (IV) with the addition of two types of aqueous/organic system:

system (VI): a 1:1 two-phase system with system (II) as the aqueous phase and octanol as

the organic phase, and system (VII): a 1:1 two-phase system with system (IV) as the

aqueous phase and 1.33 M benzaldehyde in octanol as the organic phase (Tables 2.10.A

and 2.10.B). The PDC enzyme was added in similar manner as in Section 2.6.2.2. For the

two-phase system, the organic phase was added above the enzyme containing aqueous

phase.

2.6.2.4 Effect of Initial Enzyme Concentration on PDC Deactivation

Similar to the experiments on the effect of interfacial area, the PDC stability studies were

conducted in the phase-separated and emulsion two-phase systems. Both systems were

constructed by the 4 mL glass vials and magnetically stirred (Bibby, Model B292). The

investigations were performed in systems VIII and IX (Tables 2.10.A and 2.10.B). The

aqueous phase comprised of octanol and benzaldehyde saturated MOPS buffer system

and enzyme solution. PDC enzyme was added in similar manner as in Section 2.6.2.2.

The organic phase was added above the enzyme containing aqueous phase.



68

2.6.3 Estimation of Organic-Aqueous Benzaldehyde Transfer in Two-Phase

System

2.6.3.1 Experimental Details

In a two-phase aqueous/octanol-benzaldehyde system, PAC formation was affected by

benzaldehyde mass transfer rate from the organic to the aqueous phase. In the current

studies, there were investigations on the effects of organic phase contact area to aqueous

phase volume ratio, organic phase benzaldehyde concentration and temperature on the

mass transfer rate. The investigations were conducted in a similar temperature controlled

aqueous/organic phase-separated (Lewis cell) system as that described in Section 2.6.1.

The only difference was that in the benzaldehyde transfer study, a relatively large top

metal stirrer was used (IKA, Model RW 20n) with an R-1342 impeller (stirrer dia. 5 cm,

shaft dia. 0.8 cm, shaft length 35 cm) with stirring in clockwise direction. A magnetic bar

was also placed in the aqueous phase, stirring in clockwise direction.

The investigations were carried out at 4(±1)°C and 20(±1)°C in 2.5 M MOPS buffer

system with pH 7 and no cofactors, PDC enzyme and pyruvate. Samples were withdrawn

from the aqueous phase, diluted and measured by HPLC for benzaldehyde. Triplicate

analyses were conducted for each sample; the mean values were determined and error

bars show the highest and lowest values.

2.6.3.2 Organic-Aqueous Benzaldehyde Transfer Experiments

When the temperature of the system was maintained constant, pure benzaldehyde was

added into the octanol with a syringe. Samples were withdrawn from the aqueous phase

every min for the first 10 min, every 2 – 10 min for the next 80 min, and every 0.5 h for

the next 3.5 h. For every volume of sample withdrawn, the same volume of the MOPS

solution was added into the aqueous phase to maintain a flat interfacial area. A dilution

factor [((V initial at a previous time point + V MOPS added) / V initial at a previous time

point) x dilution factor at two previous time point] was therefore included in the

benzaldehyde concentration calculations at each time point.



69

2.7 Two-Phase Aqueous/Organic PAC Synthesis at Lower Buffer

Concentration (20 mM MOPS, Larger Scale)

Efforts were made to evaluate process parameters for the two-phase aqueous/octanol-

benzaldehyde synthesis of PAC at lower MOPS concentration of 20 mM (Chapter 5).

Whole cell biotransformations in emulsion system were conducted at various organic to

aqueous phase volume ratios, temperatures and there were evaluations on the effect of 2.5

M dipropylene glycol (DPG) as low cost additive [Leksawasdi et al., 2005]. Table 2.12

lists the types of biotransformation system employed and investigations performed in the

present studies. All biotransformations were temperature and pH controlled.

2.7.1 Experimental Details

The studies were carried out in 180 mL total volume of the organic and aqueous phases in

rapidly stirred emulsion with temperature and pH control. The same glass cylinder with

top metal plate (without the middle plate) and temperature control system as in Sections

2.6.1.1 and 2.6.1.2 were used; the construction of the pH control system was described

below.

Two extra holes were drilled through the top metal plate for insertion of the pH probe and

autoburette delivery tip. A relatively large overhead stirrer (IKA, Model RW 20n) with

R-1342 impeller (stirrer dia. 5 cm, shaft dia. 0.8 cm, shaft length 35 cm) was inserted

through the top plate and stirring at constant speed of 160 rpm. The biotransformations

were performed in a fume cupboard (Conditionaire International, Model 2000 series)

with the whole system shown in Fig 2.7.

In all biotransformations, 20 mM MOPS or 20 mM MOPS + 2.5 M DPG buffer systems

were used, pH was controlled at 7; cofactors were 1 mM Mg2+ and 1 mM TPP. The

organic and aqueous phases were prepared as in Section 2.5.3.3.2. Triplicate analyses

were conducted for each sample; the mean values were determined and error bars show

the highest and lowest values.



70

Table 2.11: Biotransformation systems employed in the two-phase aqueous/organic PAC synthesis with 20 mM MOPS buffer system (Chapter 5).

* Total reaction volume (TRV) by combining both phases. Organic to aqueous phase volume ratio [Benzaldehyde] (mM) [Pyruvate] (mM) PDC enzyme Temperature (°C)

Carboligase activity

(U/mL)

Organic TRV* Aqueous TRV* Aqueous TRV*

Type

Investigation on optimum operating temperature (20 mM MOPS buffer)

1:1 1600 800 980 490 2.0 1.0 Whole cell 4(±1)

1:1 1640 820 960 480 2.0 1.0 Whole cell 10(±1)

1:1 1640 820 950 475 2.0 1.0 Whole cell 15(±1)

1:1 1620 810 930 465 2.0 1.0 Whole cell 20(±1)

1:1 1620 810 950 475 2.0 1.0 Whole cell 25(±1)

1:1 1600 800 950 475 2.0 1.0 Whole cell 30(±1)

1:1 1610 805 940 470 2.0 1.0 Whole cell 35(±1)

Investigation on the effect of organic to aqueous phase volume ratio (20 mM MOPS buffer)

0.67:1 1950 780 715 430 1.7 1.0 Whole cell 20(±1)

0.43:1 2585 775 615 430 1.4 1.0 Whole cell 20(±1)

0.25:1 3900 780 510 410 1.2 1.0 Whole cell 20(±1)

Investigation on the effect of 2.5 M DPG addition (20 mM MOPS + 2.5 M DPG buffer)

Whole cells and partially purified PDC

0.25:1 3650 730 830 665 3.5 2.8 Whole cell 20(±1)

0.25:1 3600 720 785 630 3.5 2.8 Partially purified 20(±1)



71

The pH probe used was a combination pH electrode (Mettler Toledo, Cat. No. 10 465 4505)

with 1 M LiCl in acetic acid as electrolyte. The pH probe was inserted through the top metal

plate to the reaction system. The probe sent signals to a digital pH-stat controller

(Radiometer, Model PHM 290), which was connected to a 20 mL autoburette (Radiometer,

Model ABU901) with an antidiffusion delivery tip (Radiometer, Cat. No. 956-309) inserted

through the metal plate to the reaction system. The autoburette was able to deliver a

minimum dosing volume of 2.5 µL. The pH was controlled at 7 by 5 M acetic acid addition.

The horizon of the adaptive addition algorithm (AAA) of the pH-stat controller was set at 80.

Default time constant of 2 s was used.

2.7.2 Biotransformation Experiments

The substrate and enzyme stock solutions were incubated in ice for biotransformations at

4°C to 15°C or in 25°C water bath for biotransformations at 20°C to 35°C. When the

temperature of the system was maintained constant at the biotransformation temperature, the

enzyme solution was transferred into the reactor with a syringe. For each type of

biotransformation, there was one set of control. Samplings for the reaction systems were

performed using a syringe. Treatments and types of measurement performed on the samples

were listed in Table 2.9.



72

2.8 Analytical Methods

2.8.1 Determination of Cell Culture Optical Density (OD660)

The cell density in the fermentation culture was determined by measuring the absorbance of

the culture at 660 nm using a spectrophotometer (Pharmacia Biotech, Model Ultrospec 2000)

and a 4.5 mL disposable cuvette (Kartell, Cat. No. 1940). The linear range of the ratio

absorbance : biomass concentration was at an absorbance of 0.2 – 0.3; the culture was at

least 20 times diluted prior to measurements in this linear range.

2.8.2 Determination of Glucose concentration

The glucose concentration in the fermentation culture was determined by employing a

glucose and lactate analyser (YSI Model 2300 STAT PLUS). The analysis is based on the

following principle: the glucose is converted to glucono-δ-lactone and hydrogen peroxide

(H2O2); the reaction is catalyzed by an immobilized glucose oxidase enzyme; H2O2 then

diffuses across a membrane. H2O2 is reduced and a Pt anode is oxidized; the two half-

reactions generate a current, which is correlated to the glucose concentration. The samples

were contained in 1.5 mL eppendorf tubes and centrifuged at 13,000 rpm for 5 mins prior to

measurements to remove any impurities. The linear concentration range of glucose was up to

27.8 mM; samples with higher concentrations were diluted.

2.8.3 Determination of Dissolved Oxygen Concentration

The dissolved oxygen (DO) concentration in the fermentation culture was determined as

percentage air saturation using galvanic (BABS, UNSW, Australia) and polarographic

oxygen electrodes (Ingold Cat. No. 341003047). For both electrodes, the oxygen first

diffuses through a gas-permeable membrane; the oxygen is then reduced at the cathode. For

the galvanic electrode, a current generated from the reactions at the cathode and anode gives

rise to a detectable voltage, which is then correlated to DO level. By comparison, for the

polarographic electrode, a constant voltage is applied across the cathode and anode, which



73

gives rise to a detectable current [Bailey and Ollis 1986]. The electrodes were calibrated

against nitrogen then air prior to autoclaving.

2.8.4 Determination of Respiratory Quotient

The respiratory quotient (RQ) in the fermentation culture is described as the ratio of carbon

dioxide evolution rate (CER, mmol/L/h) over oxygen uptake rate (OUR, mmol/L/h). The

exhaust gas leaving the 30 L and 5 L fermenters was passed through a dehumidifier

(Komatsu Electronics Inc., Model DH 1052 G) before entering the online gas analysers,

which measured the O2 (Servomix type 1400A) and CO2 (Servomix-R type 1410)

concentrations in the exhaust gas. The RQ was calculated from these values and the gas flow

rates as shown in Table 2.13.

Table 2.12: Respiratory quotient (RQ) calculation for fermentation process.

Variables Descriptions Values

Fi Inlet flow (L / min)

Oi Concentration of inlet O2 (%) 20.9

Ci Concentration of inlet CO2 (%) 0.03

Ni Concentration of inlet inert gas (%) = 100 – Oi – Ci 79.07

Oe Concentration of exhaust O2 (%) (gas analyser 1400A)

Ce Concentration of exhaust CO2 (%) (gas analyser 1410)

Ne Concentration of exhaust inert gas (%) (100 – Oe – Ce)

Fe Exhaust flow (L / min) = Fi Ni / Ne

Vf Fermentor volume (L)

*K OUR Constant for OUR calculation (min mmol / (L h)) 26.44

*K CER Constant for CER calculation (min mmol / (L h)) 26.59

OUR Oxygen uptake rate (mmol / (L h)) = (Fi Oi – Fe Oe) x (KOUR / Vf)

CER Carbon dioxide evolution rate (mmol / (L h))

= (Fe Ce – Fi Ci) x (KCER / Vf)

RQ Respiratory quotient = CER / OUR

* [Sandford 2002]



74

2.8.5 Determination of Dry Biomass

Fermentation samples of 5 mL volume were transferred into a pre-weighed dry biomass tube

and centrifuged at 5,000 rpm for 5 mins. The supernatant was removed for glucose analysis;

the cell pellet was washed twice with RO water. The tube was then placed in a 108°C oven

overnight. The tube was weighed to determine the dry biomass.

2.8.6 Determination of Pyruvate Concentration

The pyruvate concentration was determined by an enzymatic lactate dehydrogenase (LDH)

assay. The analysis is based on the following principle: the pyruvate is reduced to L-lactate

with NADH oxidized to NAD+; NADH consumption is determined by difference in

absorbance at 340 nm before and after LDH addition.

Pyruvate L-lactate

NADH + H+ NAD+

Pyruvate L-lactate

NADH + H+ NAD+

The reaction was conducted for 8 mins in a buffered system in a 2.5 mL UV disposable

cuvette (Kartell, Cat. No. 1941) at room temperature. The mixture was mixed with a spatula.

The 340 nm absorbance of the mixture was recorded twice using a spectrophotometer

(Pharmacia Biotech, Model Ultrospec 2000): first, before the LDH addition and second, 8

mins after the addition. The linear concentration range of pyruvate was 0.5 – 5 mM; samples

with higher concentrations were diluted prior to measurements. Tables 2.14.A and 2.14.B

show the composition of the reaction mixture and the method of calculating the pyruvate

concentration respectively.



75

Table 2.13: Composition of the reaction mixture in pyruvate and acetaldehyde (Section

2.8.7) assays (modified from Czok and Lamprecht 1974).

Order of addition Volume (µL) Incubation during assay

Triethanolamine buffer 750 25°C water bath

NADH buffer 25 ice

Sample or RO water for blank 25 ice

LDH (550 U / mg) 5 ice

Table 2.14: Calculation method for pyruvate and acetaldehyde (Section 2.8.7)

concentrations.

Variables Descriptions

Ablank,i 340 nm absorbance reading of the blank before LDH addition

Asamp,i 340 nm absorbance reading of the sample before LDH addition

Ablank,8min 340 nm absorbance reading of the blank after 8 min of LDH addition

Asamp,8min 340 nm absorbance reading of the sample after 8 min of LDH addition

Asamp Asamp,8min – Asamp,,i

Ablank Ablank,8min – Ablank,i

A Asamp – Ablank

Vassay Final assay volume = 750 + 25 + 25 + 5 = 805 µL

Vsam Volume of blank or sample = 25 µL

λ Light path length = 1 cm

ε Extinction coefficient of NADH at 340 nm = 6300 L / (mol cm)

[Pyruvate]

[Acetaldehyde)

Pyruvate or acetaldehyde concentration (mM)

= (Vassay / Vsam) x A x (1 / (ελ)) x (1000 mmol / mol) = 5.111 x A

2.8.7 Determination of Acetaldehyde Concentration

The acetaldehyde concentration was determined by an enzymatic alcohol dehydrogenase

(ADH) assay. The principle was similar to the pyruvate assay: the acetaldehyde is reduced to

ethanol with NADH oxidized to NAD+; NADH consumption is determined by difference in

absorbance at 340 nm before and after ADH addition.



76

Acetaldehyde Ethanol

NADH + H+ NAD+

Acetaldehyde Ethanol

NADH + H+ NAD+

The reaction was conducted in similar fashion as Section 2.8.6. The linear concentration

range of acetaldehyde was 1 – 2.5 mM. RO water was used to extract acetaldehyde from the

organic phase with at least 20 times dilution. For the composition of the reaction mixture and

the method of calculating the acetaldehyde concentration, refer to Table 2.14.A and 2.14.B

previously. The acetaldehyde detected was only an approximation with regards to its real

value in the biotransformation system as acetaldehyde is very volatile with boiling point of

20°C at 1 atm.

2.8.8 Determination of PAC, Benzoic Acid, Benzaldehyde and Benzyl Alcohol

Concentrations

The concentrations of PAC, benzoic acid, benzaldehyde and benzyl alcohol were measured

using a High Pressure Liquid Chromatography (HPLC) system (Fig 2.8) as described by

Rosche et al [2001]. PAC, benzoic acid and benzaldehyde were detected at 283 nm and

benzyl alcohol at 263 nm. The samples were centrifuged at 13,000 rpm for 5 mins at 4°C

prior to measurements to remove any impurities. A standard curve was constructed for each

component to correlate the peak areas on the chromatogram to known concentrations. The

concentration ranges of the standards were 0.6 – 19 mM for PAC, 1.5 – 7 mM for benzoic

acid and 6 – 25 mM for benzaldehyde (benzyl alcohol was not detected in any of the

samples). Samples with higher concentrations were diluted. RO water was used to extract the

components from the organic phase with at least 100 times dilution. The mobile phase was

composed of 32% (v/v) acetonitrile and 0.5% (v/v) acetic acid and was prepared by using

milli-Q water and a detergent free glass cylinder. Component specifications and operating

conditions are listed in Table 2.15.



77

Table 2.15: Component specifications and operating conditions of the HPLC system for

quantification of PAC, benzoic acid, benzaldehyde and benzyl alcohol.

C8 Column (Alltech, AlltimaTM C8)

Particle size 5 µm

Column length 15 cm

Column internal diameter 4.6 mm

Guard column (Alltech, Alltima All-guardTM )

Particle size 5 µm

Column length 7.5 mm


Oven type Column oven (Shimadzu, CTO-10AS VP)

Oven temperature Room temperature

Injector type Autoinjector (Shimadzu, SIL-10AD VP)

Pump type Liquid chromatograph (Shimadzu, LC-10AT

VP)

Pump operation Isocratic flow of 1 ml / min

Detector type Diode array detector (Shimadzu, SPD-M10A

VP)

System pressure 1000 – 2000 psi, column threshold 3000 psi

Injection volume 5 µL

Running time 20 min (retention times were 4.5 – 5.5 min, 5.5 – 6.5 min, 7.5 – 8.5 min for PAC, benzoic acid, benzaldehyde respectively.

2.8.9 Determination of Acetoin Concentration

Acetoin concentration was measured using a Gas Chromatography (GC) system (Packard,

Model 427). The samples were centrifuged at 13,000 rpm for 5 mins prior to measurements

to remove any impurities. A standard curve was constructed to correlate the peak areas on

the chromatogram to known concentrations. The concentration range of the standard was 0.1

– 15 mM; samples with higher concentrations were diluted. RO water was used to extract

acetoin from the organic phase with at least 20 times dilution. The mobile phase was



78

nitrogen at 25 psig with mixture of air and hydrogen as ignition source. Component

specifications and operating conditions are listed in Table 2.16.

Table 2.16: Component specifications and operating conditions of the GC system for

quantification of acetoin concentration.

Column (Alltech)

Packing material 10% carbowax® on chromsorb® W-AW

Column mesh range 80 – 100 µm

Column length 3.6 m


Oven temperature 150°C

Injector temperature 175°C

Detector temperature 175°C

Detector type Flame ionisation detector (FID) with hydrogen (15 psig) and air (15

psig)

Operation Isotherm

Injection volume 3 µL

Running time 20 min (retention time was 2 – 2.5 min).

2.8.10 Determination of Soluble Octanol Concentration

The soluble octanol concentration (in the aqueous phase of two-phase aqueous/octanol-

benzaldehyde system) was determined by using a capillary GC with CP-SIL 5 CB column

(chrompack, 47 m x 0.25 µm thin film) operating at 100°C for 5 mins then ramped up to

240°C at 40°C/min, detector and injector temperature of 250°C, mobile phase was nitrogen at

25 psig with mixture of air and hydrogen as ignition source.

The soluble octanol was extracted from the 2.5 M MOPS buffer system with chloroform (n-

decane as internal standard). Octanol standard solutions were prepared with isopropanol,

mixed with the 2.5 M MOPS buffer and extracted with chloroform; the concentration range of

the standard was 2.3 – 20 mM octanol.



79

2.8.11 Determination of PDC Enzyme Carboligase Activity

The PDC carboligase activity was determined by a method described by Rosche et al. [2002]

as carboligase assay. Into the PDC containing sample, an equal volume of carboligase buffer

was added; the mixture was vortexed for 0.5 s and incubated for 20 min in 25°C water bath.

Biotransformation of benzaldehyde and pyruvate to PAC would take place. The reaction was

stopped by addition of 10% volume (10 – 100 µL depending on the volumes of the samples)

of 100% (w/v) trichloroacetic acid (TCA). The mixture was incubated in ice for 5 min prior

to centrifugation at 13,000 rpm for 5 mins at 4°C to remove precipitated proteins. The PAC

formed was measured by HPLC. One unit of PDC carboligase activity was defined as the

amount of enzyme needed to form 1.0 µmol of PAC from benzaldehyde and pyruvate in one

minute at 25°C and pH 6.4 [Rosche et al., 2002]. The linear range of carboligase activity was

0.2 – 0.6 U/mL; samples with higher concentrations were diluted. Table 2.17 shows the

method of calculating the PDC carboligase activity.

Table 2.17: Calculation method for PDC carboligase activity.

Variable Description

Vsamp Sample volume = 100 µL

Vbuff Carboligase buffer volume = 100 µL

VTCA 100% (v/v) trichloroacetic acid volume = 20 µL

DFassay Dilution factor for the assay = total volume / sample volume

= (100 µL + 100 µL + 20 µL) / 100 µL = 2.2

[PAC] PAC concentration formed (mM)

t Assay time = 20 min

Ecarboligase PDC carboligase activity = [PAC] x DFassay / t

= [PAC] x 0.11 (U carboligase / mL)

2.9 Calculations Methods

2.9.1 Specific PDC Production

Specific PDC production=PDC activity in the fermentation sample (U/mL)

Dry biomass (g/mL)Specific PDC production=

PDC activity in the fermentation sample (U/mL)

Dry biomass (g/mL)

2.1



80

2.9.2 Biotransformations Systems

2.9.2.1 Substrate and PDC Enzyme Stock Solution Concentrations

Table 2.18: Method for calculating substrate and PDC enzyme stock solution concentration

in setting up the biotransformation systems.


[Pyruvate]sys [Benzaldehyde]sys

[PDC]sys

Pyruvate, benzaldehyde, PDC concentrations in the biotransformation system

Rsubs/enz Substrate : enzyme stock solution volume ratio = 4

Aqueous (soluble benzaldehyde) and aqueous/benzaldehyde emulsion systems

[Pyruvate]stock Pyruvate concentration in the substrate stock solution = [Pyruvate]sys x (Rsubs/enz + 1) / Rsubs/enz = [Pyruvate]sys x 1.25

[Benzaldehyde]stock Benzaldehyde concentration in the substrate stock solution = [Benzaldehyde]sys x (Rsubs/enz + 1) / Rsubs/enz = [Benzaldehyde]sys x 1.25

[PDC]stock PDC concentration in the enzyme stock solution = [PDC]sys x (Rsubs/enz + 1) = [PDC]sys x 5

Two-phase aqueous/octanol-benzaldehyde system

Rorg/aq Organic : aqueous phase volume ratio

[Pyruvate]TRV [Benzaldehyde]TRV

[PDC]TRV

Pyruvate, benzaldehyde, PDC concentrations in the biotransformation system by combining both phases (total reaction volume)

[Pyruvate]stock = [Pyruvate]TRV x (Rorg/aq + 1) x (Rsubs/enz + 1) / Rsubs/enz = [Pyruvate]TRV x (Rorg/aq + 1) x 1.25

[Benzaldehyde]stock = [Benzaldehyde]TRV x (Rorg/aq + 1) / Rrgt/aq

[PDC]stock

= [PDC]TRV x (Rorg/aq + 1) x (Rsubs/enz + 1) = [PDC]TRV x (Rorg/aq + 1) x 5



81

2.9.2.2 PAC and By-Product Formation

2.9.2.2.1 Concentrations

In the aqueous (soluble benzaldehyde) and aqueous/benzaldehyde emulsion systems, the

calculation on the concentrations of PAC and by-products formed did not include a different

phase factor. In the two-phase aqueous/octanol-benzaldehyde system, the concentrations

could be expressed as the aqueous phase, organic phase and the total reaction volume

concentrations by combining volumes of both phases (Table 2.19).

Table 2.19: Method for calculating PAC and by-product concentrations in two-phase

aqueous/octanol-benzaldehyde system.


[X] aq

[X] org

[X] TRV

PAC or by-product concentration in the aqueous phase, organic phase and the total

reaction volume by combining both phases

[X] TRV = ([X]aq + [X]org Rorg/aq) / (Rorg/aq + 1)

2.9.2.2.2 PAC Productivity

PAC Productivity =PAC concentration (mM or g/L)

Biotransformation period (h)PAC Productivity =

PAC concentration (mM or g/L)

Biotransformation period (h)

2.9.2.2.3 PAC Enzyme Efficiency (Specific PAC Production)

PAC Enzyme Efficiency =PAC concentration (mg/mL)

PDC enzyme activity (U/mL)PAC Enzyme Efficiency =

PAC concentration (mg/mL)

PDC enzyme activity (U/mL)

2.9.2.2.4 Yield on substrates

YPAC/Benzaldehyde(ini) or

YPAC/Benzaldehyde(cons)

=PAC concentration (mM)

Initial or consumed benzaldehyde concentration (mM)

YPAC/Benzaldehyde(ini) or

YPAC/Benzaldehyde(cons)


Initial or consumed benzaldehyde concentration (mM)

2.2

2.3

2.4



82

YPAC/Pyruvate(ini) or

YPAC/Pyruvate(cons)


Initial or consumed pyruvate concentration (mM)

YPAC/Pyruvate(ini) or

YPAC/Pyruvate(cons)


Initial or consumed pyruvate concentration (mM)

2.9.2.2.5 Material Balances

The material balance was performed on the substrate benzaldehyde and pyruvate. Tables

2.20.A and 2.20.B demonstrate the calculation method for unaccounted benzaldehyde and

pyruvate respectively.

Table 2.20.A: Calculation method for unaccounted benzaldehyde in the biotransformation

systems.


[Benzaldehyde]sys,i Initial benzaldehyde concentration in the biotransformation system

= [Benzaldehyde]control,i

[Benzaldehyde]sys,e Benzaldehyde concentration at the end of biotransformation

[Benzaldehyde]control,i Initial benzaldehyde concentration in the control

[Benzaldehyde]control,e Benzaldehyde concentration at the end of biotransformation in the control

[Benzaldehyde]lost Benzaldehyde lost (possibly through evaporation)

= [Benzaldehyde]control,i - [Benzaldehyde]control,e

[Benzoic acid]control,i Initial benzoic acid concentration in the control

[Benzoic acid]sys,e Benzoic acid concentration at the end of biotransformation

[Benzoic acid]prod Benzoic acid produced = [Benzoic acid]sys,e - [Benzoic acid]control,i

[PAC]sys PAC produced

[Benzaldehyde]utilized Benzaldehyde utilized = [PAC]sys + [Benzoic acid]prod

[Benzaldehyde]unacc Unaccounted benzaldehyde

= ([Benzaldehyde]sys,i - [Benzaldehyde]sys,e) - [Benzaldehyde]utilized -

[Benzaldehyde]lost

% Benzaldehydeunacc % Unaccounted benzaldehyde

= [Benzaldehyde]unacc / [Benzaldehyde]sys,i x 100%

2.5



83

Table 2.20.B: Calculation method for unaccounted pyruvate in the biotransformation

systems.


[Pyruvate]sys,i Initial pyruvate concentration in the biotransformation system

= [Benzaldehyde]control,i

[Pyruvate]sys,e Pyruvate concentration at the end of biotransformation

[Pyruvate]control,i Initial pyruvate concentration in the control

[Pyruvate]control,e Pyruvate concentration at the end of biotransformation in the control

[Pyruvate]lost Pyruvate lost = [Pyruvate]control,i - [Pyruvate]control,e

[PAC]sys PAC produced

[Acetaldehyde]sys Acetaldehyde produced

[Acetoin]sys Acetoin produced

[Pyruvate]utilized Pyruvate utilized = [PAC]sys + [Acetaldehyde]sys + 2 x [Acetoin]sys

(1 mol of acetoin was formed from 2 moles of pyruvate)

[Pyruvate]unacc Unaccounted pyruvate

= ([Pyruvate]sys,i - [Pyruvate]sys,e) - [Pyruvate]utilized - [Pyruvate]lost

% Pyruvateunacc % Unaccounted pyruvate

= [Pyruvate]unacc / [Pyruvate]sys,i x 100%

2.9.3 Experimental Errors

Table 2.21: Calculation method for experimental error.


Xmean Mean value from the replicates

Emax Maximum error from the mean value = Maximum value from the replicates – Xmean

Emin Minimum error from the mean value = Xmean – minimum value from the replicates

Chapter 3 Yeast Pyruvate Decarboxylases: Variation in Biocatalytic Characteristics


84

CHAPTER 3

YEAST PYRUVATE DECARBOXYLASES:

VARIATION IN BIOCATALYTIC

CHARACTERISTICS

Selection of productive and stable biocatalyst for PAC

production



85

3.1 Introduction

In spite of the fact that PAC can be chemically synthesized, the current commercial process

is based on biotransformation of pyruvate and benzaldehyde by fermenting baker’s yeast.

Numerous attempts have been made to improve PAC production: reported values are in the

range of 4.5 – 15 g/L PAC for various yeast-based fermentation processes [Becvarova and

Hanc, 1963; Long et al., 1989; Seely et al., 1994; Mochizuki et al., 1995; Shin and Rogers,

1995]. Significant progress has been reported when employing enzyme-based processes with

added substrates pyruvate and benzaldehyde. In an aqueous/benzaldehyde emulsion system

at 4°C, more than 50 g/L PAC with 97% molar yields on consumed benzaldehyde were

achieved when using partially purified PDCs from yeasts and filamentous fungi [Rosche et

al., 2002a; Rosche et al., 2003a]. Furthermore, PAC levels in excess of 100 g/L in the organic

phase with similar molar yields were produced in a two-phase aqueous/octanol-benzaldehyde

system at 4°C [Rosche et al., 2002b; Sandford et al., 2005]. The use of whole cells in such a

system at 21°C resulted in similar PAC concentrations and increased specific productivity

[Rosche et al., 2005].

In a screening of 105 yeast strains for enzymatic PAC production [Rosche et al., 2003b],

three species of Candida were identified as the most interesting candidates since their PDCs

showed the highest PAC formation together with low inactivation and/or inhibition by

benzaldehyde and acetaldehyde. In the present study, six yeast PDCs were considered for a

more detailed study for enzymatic PAC production: PDC from Saccharomyces cerevisiae

(yeast used in commercial PAC production); PDC from three species in which the enzyme

showed resistance towards aldehydes: Candida utilis, Candida tropicalis and

Schizosaccharomyces pombe [Rosche et al., 2003b]; PDC from the pyruvate producer

Candida glabrata [Yonehara and Yomoto, 1987; Yonehara and Miyata, 1994] and PDC from

the thermotolerant Kluyveromyces marxianus [Banat and Marchant, 1995]. Three

biotransformation systems were employed to cover a wide range of benzaldehyde

concentrations (in the order of increasing concentration): (I) aqueous (soluble benzaldehyde)

with crude extract PDC, (II) aqueous/benzaldehyde emulsion with crude extract PDC and

(III) aqueous/octanol benzaldehyde emulsion system with whole cell PDC. Production of



86

PAC and by-products acetaldehyde and acetoin were compared as well as stability of the

various yeast PDCs (refer to Section 2.5 for details of the biotransformation experiments).

3.2 Results and Discussion

3.2.1 Specific PDC Activity

Fig 3.1 demonstrates that the six yeast PDCs were characterized by different specific

activities for PAC formation under similar shake flask culturing conditions (Section 2.4.2.1

of Materials and Methods): C. tropicalis demonstrated the highest specific activity with 255

U/g dry biomass while C. glabrata and S. pombe were the lowest with 65 and 20 U/g dry

biomass respectively. Under these conditions, the PDC activity of C. utilis was 145 U/g dry

biomass. However, it has been reported that up to 392 U/g dry biomass PDC production

could be achieved for C. utilis through employment of a pH shift fermentation method with

controlled pH shifted from 6.0 to 3.0 after consumption of approx. 20 g/L glucose [Chen et

al., 2005], indicating that PDC production could be influenced appreciably by changing the

environmental conditions. C. glabrata and S. pombe were therefore excluded from further

investigations due to their low specific PDC activities.

3.2.2 Pyruvate Conversion in the Absence of Benzaldehyde

PDC catalyses the non-oxidative decarboxylation of pyruvate to acetaldehyde (with release

of CO2) and the PDC bound ‘active acetaldehyde’ is either released or undergoes

carboligation with ‘free acetaldehyde’ to form acetoin. With initial pyruvate of 80 mM and in

the absence of benzaldehyde, experiments with the four PDCs resulted in levels of

acetaldehyde within 7 – 14 mM (highest for C. tropicalis PDC) and acetoin within 22 – 32

mM (highest for K. marxianus PDC) after 7.3 h when the reactions were completed (Fig 3.2).

The results demonstrate that all PDCs are capable of both decarboxylation and carboligation

reactions.



87

0 50 100 150 200 250 300

K.m

C.g

S.p

C.t

C.u

S.c

Str

ain

Specific PDC Activity (U/g dry biomass)

Figure 3.1: Comparison of specific PDC activities of six yeasts. Culturing conditions (g/L):

90 glucose, 10 yeast extract, 10 (NH4)2SO4, 3 KH2PO4, 2 Na2HPO4.12H2O, 1 MgSO4.7H2O,

0.05 CaCl2.2H2O, 39 MES buffer, initial pH 6, 30°C, 160 rpm. The data is shown as mean

values for four fermentation batches for S.c, C.u, C.t, K.m (two batches were grown by Allen

Chen) and two batches for S.p and C.g (grown by Allen Chen). S.c: Saccharomyces

cerevisiae, C.u: Candida utilis, C.t: Candida tropicalis, S.p: Schizosaccharomyces pombe,

C.g: Candida glabrata, K.m: Kluyveromyces marxianus. The error bars show highest and

lowest values for the above experiments.



88

0

5

10

15

20

25

30

35

S.c C.u C.t K.mStrain

[ace

tald

ehyd

e], [

acet

oin]

(m

M)

acetaldehyde acetoin

Figure 3.2: Acetaldehyde and acetoin formation in the absence of benzaldehyde. Product

concentrations after 7.3 h at 22°C, initial pH 6.5. Initial agitation 220 rpm, initial

concentrations: 80 mM pyruvate, 1.5 U/ml PDC carboligase activity (crude extract), 2.5 M

MOPS buffer, 1 mM Mg2+, 1 mM TPP. Acetaldehyde concentrations were immediately

measured upon samplings. The mean values were determined from triplicate experiments and

error bars show the highest and lowest values. Refer to Fig 3.1 for strain abbreviations.



89

3.2.3 PAC and By-Product Formation in the Aqueous (Soluble Benzaldehyde)

System

At a soluble concentration of 80 mM benzaldehyde and with initial 80 mM pyruvate,

pyruvate and benzaldehyde were converted into PAC, acetaldehyde and acetoin at varying

concentrations with the different sources of PDC. As demonstrated in Fig 3.3.a, in the first

0.5 h the biotransformation with C. tropicalis and K. marxianus PDCs produced the highest

level of PAC of approx. 50 mM. At 7.3 h, the most PAC of approx. 60 mM was formed when

using C. tropicalis and K. marxianus PDCs while experiments with C. utilis and S. cerevisiae

PDCs resulted in less PAC formation.

As shown in Fig 3.3.b, biotransformation with C. tropicalis PDC was associated with the

least by-product acetoin formation of approx. 2.5 mM whereas higher levels of 7 – 8 mM

were formed with the other enzymes. The accumulation of acetaldehyde was highest for S.

cerevisiae PDC of approx. 11 mM. At the end of the reaction period (7.3 h), pyruvate was

fully utilized in all biotransformations while some benzaldehyde remained. The substrate

molar balances closed to within ± 5% and ± 10% for benzaldehyde and pyruvate respectively

for all biotransformations.

Comparison of Figs 3.2 and 3.3.b illustrates that in the presence of 80 mM benzaldehyde,

acetoin and acetaldehyde formation were decreased for all enzymes, except for S. cerevisiae

PDC with unchanged acetaldehyde formation. The relatively high acetaldehyde accumulation

with S. cerevisiae PDC might have been responsible for the lowest PAC formation with this

enzyme as acetaldehyde has been previously reported to inhibit PAC formation [Shin and

Rogers, 1995].



90

0

10

20

30

40

50

60

70


[PA

C] (

mM

)

0.5h 7.3h

a

0

2

4

6

8

10

12


[ace

tald

ehyd

e], [

acet

oin]

(m

M)


b

Figure 3.3: Biotransformation results in the aqueous system (presence of soluble

benzaldehyde): (a) PAC (at 0.5 h and 7.3 h) and (b) by-product (at 7.3 h) concentrations at

22°C, initial pH 6.5. Initial agitation 220 rpm, initial concentrations: 80 mM benzaldehyde,

80 mM pyruvate, 1.5 U/ml PDC carboligase activity (crude extract), 2.5 M MOPS buffer, 1

mM Mg2+, 1 mM TPP.



91

3.2.4 PAC and By-Product Formation in the Aqueous/Benzaldehyde Emulsion

System

To avoid any substrate limitation effects, the four PDCs were tested in an

aqueous/benzaldehyde emulsion system with initial benzaldehyde and pyruvate concentration

of 325 mM and 420 mM respectively. As shown in Fig 3.4.a, different catalytic

characteristics were demonstrated when compared to the aqueous phase system and it was

found that the reactions were completed after 24 h without substrate limitation. At 24 h with

C. utilis PDC, the highest PAC level of 240 mM was formed; this was followed by lower

concentrations with S. cerevisiae and K. marxianus PDCs; while C. tropicalis PDC was

associated with the least amount (its activity was zero after 3 h).

Acetoin formation was similar to that in the aqueous system with the biotransformation with

C. tropicalis PDC again resulting in the least acetoin (approx. 1.5 mM) and higher

concentrations of 22 – 41 mM being formed with the other three PDCs (Fig 3.4.b). As

shown, S. cerevisiae PDC was associated with the highest concentrations of acetoin (41 mM)

and acetaldehyde (14 mM) (Fig 3.4.b).



92

0

50

100

150

200

250

300


[PA

C] (

mM

)

3h 24h

a

0

10

20

30

40

50


[ace

tald

ehyd

e], [

acet

oin]

(m

M)


b

Figure 3.4: Biotransformation results in the aqueous/benzaldehyde emulsion system: (a)

PAC (at 3 h and 24 h) and (b) by-product (at 24 h) concentrations at 22°C, initial pH 6.5.

Initial agitation 220 rpm, initial concentrations: 325 mM benzaldehyde, 420 mM pyruvate,

1.5 U/ml PDC carboligase activity (crude extract), 2.5 M MOPS buffer, 1 mM Mg2+, 1 mM

TPP.



93

3.2.5 PAC and By-Product Formation in the Aqueous/Octanol-Benzaldehyde

Emulsion System

The four PDCs were tested at increasing substrate concentrations in an aqueous/octanol-

benzaldehyde emulsion system with initial benzaldehyde and pyruvate concentrations of 850

mM and 450 mM in the total reaction volume by combining both phases (TRV) respectively.

An organic to aqueous phase volume ratio of 1:1 was used together with whole cell PDC. As

shown in a previous study [Sandford et al. 2005], benzaldehyde and PAC partitioned strongly

into the organic phase whilst pyruvate fully partitioned into the aqueous phase. Results were

obtained after 48 h when neither substrate was limiting. As illustrated in Fig 3.5.a, the trends

were similar to those in the aqueous/benzaldehyde emulsion system although PAC and by-

product concentrations were higher. At 48 h, the most PAC of approx. 310 mM TRV was

produced when using C. utilis PDC; this was followed by lower concentrations with S.

cerevisiae and C. tropicalis PDCs, while K. marxianus PDC was associated with the least

amount.

As for the previous two systems, the biotransformation with C. tropicalis PDC resulted in the

lowest acetoin level of approx 6 mM TRV whereas higher levels of 30 – 45 mM TRV were

formed for the other PDCs (Fig 3.5.b). As shown, S. cerevisiae PDC was associated with the

highest concentrations of acetoin (45 mM TRV) and acetaldehyde (20 mM TRV) (Fig 3.5.b).



94

0

50

100

150

200

250

300

350


[PA

C] (

TR

V)

(mM

)

3h 24h 48h

a

0

10

20

30

40

50


[ace

tald

ehyd

e]T

RV

, [ac

etoi

n]T

RV

(m

M)


b

Figure 3.5: Biotransformation results in the aqueous/octanol-benzaldehyde emulsion system: (a)

PAC (at 3 h, 24 h and 48 h) and (b) by-product (at 48 h) concentrations at 22°C, initial pH 6.5.

Initial agitation 250 rpm, initial concentrations: 850 mM TRV benzaldehyde, 450 mM TRV

pyruvate, 1.5 U/ml TRV PDC carboligase activity (permeabilized whole cells), 2.5 M MOPS, 1

mM Mg2+, 1 mM TPP. The organic to aqueous phase volume ratio was 1:1 and concentrations of

substrates, enzyme, product and by-products are given per total reaction volume by combining

both phases (TRV).



95

3.2.6 Efficiency of PAC Formation

The yeast PDCs were associated with varying efficiencies regarding benzaldehyde and

pyruvate conversion to PAC in the different systems (Table 3.1). Overall experiments with C.

utilis PDC resulted in consistent 80 – 85% yields of PAC on consumed benzaldehyde in all

systems. Based on pyruvate consumed, C. tropicalis PDC with the lowest by-product acetoin

formation resulted in consistent 70 – 80% yields of PAC. The yields in these studies are only

indicative values as they were calculated from small-scale data, much more accurate yields

were calculated later in larger scale experiments.

Table 3.1: Biotransformations with the four yeast PDCs in three different systems: estimated

yields of PAC on consumed benzaldehyde and pyruvate.

Strain

Aqueous

(soluble benzaldehyde)

Aqueous/benzaldehyde

emulsion

Aqueous/octanol-

benzaldehyde emulsion

Y PAC / bzdc

Y PAC/pyrc

Y PAC / bzdc

Y PAC/pyrc

Y PAC / bzdc

Y PAC/pyrc

S.c

0.77

0.47 **

0.72

0.58 **

0.74

0.55 **

C.u

0.82

0.59 **

0.87

0.80

0.82

0.70

C.t

0.86

0.71

0.47 *

0.80

0.77

0.81

K.m

0.86

0.72

0.62

0.62

0.77

0.51 **

Calculated from experimental data shown in Figs 3.3, 3.4 and 3.5. Y PAC / bzdc: yield of PAC on

consumed benzaldehyde. Consumed benzaldehyde = PAC formation + evaporative loss +

unaccounted benzaldehyde (conversion to other substance(s)). Y PAC/pyrc: yield of PAC on

consumed pyruvate. Consumed pyruvate = PAC, acetaldehyde and acetoin formation + pyruvate

degradation + unaccounted pyruvate (acetaldehyde evaporation + conversion to other substance(s)). *

Relatively high evaporative losses of benzaldehyde. ** Relatively high pyruvate degradation and

presumably certain degree of evaporative losses of acetaldehyde due to sampling and analysis.



96

3.2.7 Effect of Benzaldehyde and Acetaldehyde on PAC Formation with C. utilis

and C. tropicalis PDCs

Based on the evaluation of the four PDCs in the various biotransformation systems for PAC

and by-product formation, it was evident that C. utilis and C. tropicalis PDCs had

demonstrated the most useful properties, viz. in the systems with the higher benzaldehyde

concentrations, the highest PAC concentrations were produced when using C. utilis PDC

while C. tropicalis PDC was associated with the lowest by-product formation (particularly

acetoin). As a result, the biotransformation characteristics of these two enzymes were further

investigated.

The two PDCs were tested for initial PAC formation (32 min) with and without addition of

30 mM acetaldehyde and with increasing benzaldehyde concentrations: from soluble

concentrations (45 mM and 80 mM) to emulsions (125 mM and 185 mM). At soluble

benzaldehyde concentrations without acetaldehyde addition, C. tropicalis PDC was

associated with a higher initial PAC formation than C. utilis PDC (Figs 3.6.a and b). The

initial PAC formation with C. utilis PDC increased when more benzaldehyde was added (Fig

3.6.a) while this resulted in reduced values with C. tropicalis PDC (Fig 3.6.b).

Only low concentrations of ‘free acetaldehyde’ were formed from pyruvate in these

experiments. In order to investigate the competition of benzaldehyde and ‘free acetaldehyde’

for carboligation with the enzyme bound ‘active acetaldehyde’, 30 mM acetaldehyde was

added to the reaction mixture. The presence of acetaldehyde resulted not only in much lower

initial PAC formation (Figs 3.6.a and c) but also in increased acetoin formation for C. utilis

and C. tropicalis PDCs (Figs 3.7.a and b). In the absence of 30 mM acetaldehyde, no acetoin

was formed with C. tropicalis PDC for all tested benzaldehyde concentrations, while C. utilis

PDC was associated with trace levels of acetoin at the soluble benzaldehyde concentrations

and none at the higher benzaldehyde concentrations (Figs 3.7.a and b).



97

0

5

10

15

20

25

30

35

40

45 80 125 185

[PA

C] (

mM

)

a

0

5

10

15

20

25

30

35

40

45 80 125 185

[Benzaldehyde] (mM)

[PAC

] (m

M)

no acetaldehyde 30 mM acetaldehyde

b

Figure 3.6: Effect of acetaldehyde on initial PAC formation with: (a) C. utilis (C.u) and (b)

C. tropicalis (C.t) PDCs at various benzaldehyde concentrations in 32 min at 22°C, initial pH

6.5. Agitation 220 rpm, initial concentrations: 250 mM pyruvate, 0 and 30 mM acetaldehyde

1.5 U/ml PDC carboligase activity (crude extract), 2.5 M MOPS, 1 mM Mg2+ & 1 mM TPP.



98

0.0

0.2

0.4

0.6

0.8

1.0

45 80 125 185

[Ace

toin

] (m

M)

a

0.0

0.2

0.4

0.6

0.8

1.0

45 80 125 185

[Benzaldehyde] (mM)

[Ace

toin

] (m

M)

no acetaldehyde 30 mM acetaldehyde

b

Figure 3.7: Effect of acetaldehyde on initial acetoin formation with: (a) C. utilis (C.u) and

(b) C. tropicalis (C.t) PDCs at various benzaldehyde concentrations in 32 min at 22°C, initial

pH 6.5. Same experiments as shown in Fig 3.6.



99

The PAC to acetoin ratio is demonstrated in Fig 3.8. Increasing the benzaldehyde

concentration resulted in a general increase in PAC to acetoin ratio for both enzymes;

however the ratio was appreciably higher for C. tropicalis PDC at all benzaldehyde

concentrations.

0

5

10

15

20

25

30

35

45 80 125 185[Benzaldehyde] (mM)

PA

C/A

ceto

in (

mM

/mM

)

C.u C.t

Figure 3.8: Ratio of PAC over acetoin with C. utilis (C.u) and C. tropicalis (C.t) PDCs at

various benzaldehyde concentrations in the presence of 30 mM acetaldehyde (32 min) at

22°C, initial pH 6.5. Same experiments as shown as Fig 3.6.

The lowest acetoin formation was observed with C. tropicalis PDC in the different

biotransformation systems (Figs 3.3.b, 3.4.b, and 3.5.b), and the ratios of PAC to acetoin

were highest for this enzyme (Fig 3.9). The reduced acetoin production was further

confirmed in the presence of added 30 mM acetaldehyde (Figs 3.7 and 3.8). The results

demonstrate that C. tropicalis PDC had a higher preference for benzaldehyde (leading to

PAC formation) over ‘free acetaldehyde’ (leading to acetoin formation) under these

experimental conditions.



100

0

10

20

30

40

50

60

70


PA

C/A

ceto

in (

mM

/mM

)

aqueous (soluble benzaldehyde) aqueous/benzaldehyde emulsion

aqueous/octanol-benzaldehyde emulsion

Figure 3.9: Ratio of PAC over acetoin for the four yeast PDCs in the different

biotransformation systems at 22°C, initial pH 6.5. Same experiments as shown in Figs 3.3,

3.4, and 3.5.



101

3.2.8 PDC Stability

The stability of the four PDCs were evaluated to further investigate their potential in the

different biotransformation systems. The half-life values of crude extract and whole cell PDC

in the absence and presence of 50 mM benzaldehyde at 22°C are shown in Figs 3.10.a and b.

In the absence of benzaldehyde, S. cerevisiae and C. utilis PDCs were very stable with half-

life values of nearly two weeks for crude extract and slightly longer for whole cells. By

comparison, C. tropicalis PDC was much less stable with half life of 3 days for crude extract

and less than 1 day for whole cells.

In the presence of 50 mM benzaldehyde, C. utilis PDC was the most stable with a half-life

value of 7 days for both crude extract and whole cell preparations. This was followed by S.

cerevisiae and K. marxianus PDCs. C. tropicalis PDC was the least stable with half-life

values of less than 1 day for crude extract and whole cells.

Considering the differences in stability, PDC is a homomeric enzyme, which exists as

tetramers and dimers at physiological conditions [Jabs et al., 2001]. The dimers are

composed of monomers of which contact sites are mainly determined by aromatic amino

acids. The contact sites of dimers forming the tetramers are mainly determined by

electrostatic interactions. The catalytic activity of PDC is mainly related to the tetrameric

species [Jabs et al. 2001]. PDC deactivation occurs when the native tetrameric structure

dissociates into its dimeric halves, TPP and Mg2+ are released from the PDC and there is loss

of biocatalytic activity [Hübner et al. 1990]. The most stable C. utilis PDC might have the

capability to maintain its tetrameric structure more strongly under stress. Other possible

explanation might relate to unfolding phenomenon and covalent modification by

benzaldehyde. In the present studies, the highest PDC stability is associated with the highest

PAC formation when employing C. utilis PDC in systems with relatively high benzaldehyde

concentrations



102

0

2

4

6

8

10

12

14

16

S.c C.u C.t K.m

Hal

f-lif

e (d

ay)

a

0

2

4

6

8

10

12

14

16


Hal

f-lif

e (d

ay)

No benzaldehyde 50 mM benzaldehyde

b

Figure 3.10: PDC stabilities in the absence and presence of soluble benzaldehyde at 22°C:

(a) crude extract and (b) whole cell preparations. Concentrations: 50 mM benzaldehyde, 1.5

U/ml PDC carboligase activity, 2.5 M MOPS (pH 6.5), 1 mM Mg2+ & 1 mM TPP.

Experiments were performed by Gernalia Satianegara.



103

Other researchers have reported comparisons between PDC characteristics of yeast and

bacteria. For example, Zymomonas mobilis PDC exhibited higher stability than S.

carlsbergensis PDC upon removal of the cofactors thiamine diphosphate and Mg2+ under

slightly alkaline conditions [König et al., 1992; Pohl et al., 1994]. However, S. carlsbergensis

PDC exhibited a 20-fold higher carboligase activity in the presence of 46 – 48 mM

benzaldehyde compared to Z. mobilis PDC [Bringer-Meyer and Sahm, 1988].

3.2.9 Further Characterization of C. utilis PDC Activity

C. utilis PDC (partially purified) was further studied for the effect on deactivation rate of

agitation and initial enzyme concentration in the aqueous system with soluble benzaldehyde.

3.2.9.1 Effect of Agitation Rate on PDC Deactivation

Experiment results in Fig 3.11 show no evident effect of agitation rate on the deactivation

rates of C. utilis PDC in the absence and presence of 48 mM soluble benzaldehyde at 4°C as

long as foam formation was prevented. In the case of extensive foam formation, the half-life

value decreased to approx. 50% in system with 48 mM benzaldehyde. Other researchers have

found that agitation rate of 600 rpm with 200 mL working volume decreased the half-life

value of papain from longer than 50 h to 32 h in system with 0.73 M acetate buffer (pH 5.15)

at 40°C [Feliu et al. 1994].

3.2.9.2 Effect of Initial Enzyme Concentration on PDC Deactivation

As shown in Fig 3.12, the deactivation rates of C. utilis PDC were relatively unaffected by

the initial enzyme concentration (3 and 7.3 U/mL carboligase) in the absence and presence of

48 mM benzaldehyde at 4°C.



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220 rpm, 0 mM BZD 220 rpm, 48 mM BZD 95 rpm, 0 mM BZD95 rpm, 48 mM BZD 250 rpm, 48 mM BZD

Figure 3.11: Effect of agitation rate on the deactivation of partially purified PDC from C. utilis in the absence and presence of 48 mM soluble benzaldehyde at 4°C, pH 7. 95, 220 and 250 rpm agitation, 0 and 48 mM benzaldehyde, 3 U/mL PDC carboligase activity, 2.5 M MOPS buffer, 0.5 mM Mg2+, 1 mM TPP. Extensive foam formation at 250 rpm.

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48 mM BZD, 3 U/mL 48 mM BZD, 7.3 U/mL

Figure 3.12: Effect of initial enzyme concentration on the deactivation of partially purified PDC from C. utilis in the absence and presence of 48 mM soluble benzaldehyde at 4°C, pH 7. 220 rpm agitation, 0 and 48 mM benzaldehyde, 3 and 7.3 U/mL PDC carboligase activity, 2.5 M MOPS buffer, 0.5 mM Mg2+, 1 mM TPP.



105

3.3 Conclusions

From the present study it was evident that C. utilis PDC had the highest stability compared to

the other yeast PDCs, and this ultimately resulted in the highest PAC production in systems

with relatively high benzaldehyde concentrations. Interestingly, C. tropicalis PDC exhibited

a substantially lower acetoin formation under these conditions. In the present study, C. utilis

PDC was the biocatalyst of choice for further process development to improve two-phase

aqueous/organic PAC production.

Chapter 4 Factors Affecting PDC Enzyme Deactivation and PAC Production in Two-Phase Aqueous/Organic System


106

CHAPTER 4

FACTORS AFFECTING PDC ENZYME

DEACTIVATION AND PAC PRODUCTION IN

TWO-PHASE AQUEOUS/ORGANIC SYSTEM

Identification of key factors in two-phase aqueous/organic

PAC synthesis



107

4.1 Introduction

Previous studies by our group have shown that the enzymatic biotransformation process for

PAC production could be restricted by the toxicity and low aqueous solubility of

benzaldehyde. An aqueous system could usually accommodate up to 100 mM soluble

benzaldehyde (depending on the buffer system), thereby resulting in relatively low PAC

production (Fig 4.1). PAC production was improved in the aqueous/benzaldehyde emulsion

system (Fig 4.1), however the process was restricted by increased PDC deactivation

presumably resulting from benzaldehyde droplet/enzyme interaction in the emulsion system

[Sandford et al., 2005; Rosche et al., 2005b].

To overcome this problem, a two-phase aqueous/organic system with 1-octanol as solvent

has been developed [Rosche et al., 2002b; Sandford et al., 2005]. In this system both PAC

and benzaldehyde strongly partitioned into the octanol phase and thereby the enzyme in the

aqueous phase was protected from high interfacial benzaldehyde concentrations. Organic

phase PAC concentrations in excess of 100 g/L were achieved [Sandford et al., 2005]. Two

aqueous/organic phase systems were evaluated in these studies (Fig 4.1): (1) a rapidly stirred

emulsion system and (2) a slowly stirred phase-separated system. In the first, a relatively

high overall volumetric productivity was achieved (39.1 g PAC/L/day), however the overall

specific PAC production was low (9.4 mg PAC/U). In the latter system, the productivity was

reduced (3.6 g PAC/L/day), however the PDC activity was maintained resulting in an

appreciable improvement in specific PAC production (64 mg PAC/U).

The current study evaluates the key factors involved in PDC deactivation in the two-phase

phase-separated and emulsion systems as means to further improve PAC production. PDC

deactivation in an aqueous/octanol-benzaldehyde system is likely to be influenced by the

soluble octanol and benzaldehyde concentrations in the aqueous phase, aqueous/organic

interfacial area as well as agitation rate and the enzyme concentration (refer to Section 2.6 for

details of the PDC deactivation experiments). The effects of these variables as well as phase

volume ratio have been evaluated as a basis for designing a two-phase process with improved



108

specific PAC production and productivities (refer to Section 2.5 for details of the phase

volume ratio biotransformation experiments).

SaccharomycesSaccharomyces cerevisiaecerevisiae,, Industrial fermentation Industrial fermentation

0 40 80 120 160

PAC (g/l)Candida Candida utilisutilis PDCPDC

Aqueous phase, 7 U/Aqueous phase, 7 U/ mLmL carboligasecarboligase [Shin [Shin and Rogers, 1996]and Rogers, 1996]

C. C. utilisutilis PDCPDC

Aqueous/Aqueous/ benzaldehydebenzaldehyde emulsion, 8.4 U/emulsion, 8.4 U/ mLmLcarboligasecarboligase [[RoscheRosche et al., 2002b]et al., 2002b]

RhizopusRhizopus javanicusjavanicus PDC PDC



TwoTwo --phase phasephase phase --separated, 0.45 separated, 0.45 U/U/mLmL carboligasecarboligase [[SandfordSandford et al., et al., 2005]*2005]*


TwoTwo --phase emulsion, 4.25 U/phase emulsion, 4.25 U/ mLmLcarboligasecarboligase [[SandfordSandford et al., 2005]*et al., 2005]*

Aqueous phase

Organic phase

PAC (g/L)SaccharomycesSaccharomyces cerevisiaecerevisiae,, Industrial fermentation Industrial fermentation

0 40 80 120 160

PAC (g/l)Candida Candida utilisutilis PDCPDC

Aqueous phase, 7 U/Aqueous phase, 7 U/ mLmL carboligasecarboligase [Shin [Shin and Rogers, 1996]and Rogers, 1996]



RhizopusRhizopus javanicusjavanicus PDC PDC



TwoTwo --phase phasephase phase --separated, 0.45 separated, 0.45 U/U/mLmL carboligasecarboligase [[SandfordSandford et al., et al., 2005]*2005]*


TwoTwo --phase emulsion, 4.25 U/phase emulsion, 4.25 U/ mLmLcarboligasecarboligase [[SandfordSandford et al., 2005]*et al., 2005]*

Aqueous phase

Organic phase

PAC (g/L)

Figure 4.1: PAC production in various biotransformation systems.

* PDC activities in the two-phase system are based on total reaction volume by combining

both phases.



109


4.2.1 Factors Affecting PDC Deactivation

4.2.1.1 Effect of Soluble Octanol and Benzaldehyde in the Aqueous Phase

To further understand the characteristics of a two-phase aqueous/octanol-benzaldehyde

system, the effects on PDC deactivation of soluble octanol (4.5 mM) and soluble

benzaldehyde (48 mM) in the aqueous phase were studied. The data in Table 4.1 shows that

soluble octanol had a minor deactivating effect on PDC after 45 h and confirms the strong

deactivating effect of soluble benzaldehyde. In the presence of both soluble octanol and

benzaldehyde, deactivation was faster and the effects were approximately additive.

The effects of organic solvent molecules of 2-octanone and butylbenzene dissolved in the

aqueous phase on increasing urease deactivation have been reported previously [Ghatorae et

al., 1993], as well as the deactivating effect of soluble benzaldehyde on PDC [Chow et al.,

1995; Leksawasdi et al., 2003]. In general, the mechanism of PDC deactivation by an

aldehyde might involve covalent protein modification or non-covalent interaction. It is not

known though how benzaldehyde inactivates PDC.

Table 4.1: Effect of aqueous phase octanol and benzaldehyde on PDC deactivation at 4°C,

pH 7.0. 220 rpm, 2 U/mL PDC carboligase activity (C. utilis partially purified), 2.5 M MOPS

buffer, 0.5 mM Mg2+, 1 mM TPP. Same experiments as shown in Fig 4.4.a.

Aqueous-based system

Residual activity after 45 h (%)

Buffer

95

4.5 mM octanol

90

48 mM benzaldehyde

63

4.5 mM octanol + 48 mM benzaldehyde

50



110

4.2.1.2 Effect of Agitation Rate in the Aqueous Phase

Comparing the aqueous/organic phase-separated process to the emulsion system, the first

system was associated with a lower agitation rate than the latter. To study the impact of the

different agitation rates, experiments were designed with agitation rates for the aqueous

phase of 125 rpm and 220 rpm, typical of those for the phase-separated and emulsion system

respectively. The profiles in Fig 4.2 illustrate that application of the higher agitation resulted

in a greater PDC deactivation rate in the presence of both soluble octanol (4.5 mM) and

benzaldehyde (48 mM). This indicates that the higher agitation rate will have an additional

impact on PDC deactivation in the higher productivity emulsion system.

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125 rpm 220 rpm

Figure 4.2: Effect of agitation rate on PDC deactivation in the presence of soluble octanol

and benzaldehyde at 4°C, pH 7.0. 4.5 mM octanol, 48 mM benzaldehyde, 2 U/mL PDC

carboligase activity (C. utilis partially purified), 2.5 M MOPS buffer, 0.5 mM Mg2+, 1 mM

TPP.



111

Referring to previous results in Fig 3.11, agitation had no effect on PDC deactivation rate in

the presence of soluble benzaldehyde (48 mM) as long as there was no foam formation (the

half-life value was drastically reduced in the event of extensive foam formation at 250 rpm).

The disparity in results might be due to possible entrainment of a small amount of gas in the

system with soluble octanol and benzaldehyde at 220 rpm resulting in increased enzyme

deactivation.

4.2.1.3 Effect of Ratio of Organic Phase Interfacial Area to Aqueous Phase Volume

In addition to exposure to soluble octanol and benzaldehyde in the aqueous phase, PDC in

the two-phase aqueous/octanol-benzaldehyde system is exposed to the aqueous/organic

interface. Interfacial phenomena are likely to have caused some PDC deactivation as there

was presence of a white to yellowish interfacial layer evident in the two-phase synthesis of

PAC (following phase separation with centrifugation), which would have involved close

contact between the PDC and octanol/benzaldehyde interfaces.

It has been reported that protein molecules can gather at gas-liquid interfaces and be

deactivated by interfacial tension effects [Thomas et al. 1979, Thomas and Dunnill 1979,

Harrington et al. 1991]. Feliu et al. [1994] argued also that enzyme molecules could

accumulate at liquid-liquid interfaces and be deactivated by such interfacial effects.

4.2.1.3.1 Studies with a Phase-Separated System

A Lewis cell with aqueous and organic phase layers (each 90 mL) was used to study the

effect of defined changes in the ratio of organic phase contact area to aqueous phase volume

on PDC stability. The aqueous phase containing 4 U/mL carboligase PDC was saturated with

4.5 mM octanol and 48 mM benzaldehyde prior to the experiments and the organic phase

contained 1.39 M benzaldehyde. The agitation rate (125 rpm) was selected to maintain

defined separation between both phases.



112

As illustrated in Fig 4.3, a change from 117 to 475 cm2/L had no effect on PDC deactivation.

Similar half-life values of approx. 80 h were determined for all conditions. The observed

deactivation was due to effects of soluble octanol and benzaldehyde in the aqueous phase at

the mild agitation rate of 125 rpm.

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117 cm2/L 361 cm2/L 475 cm2/L

Figure 4.3: Effect of aqueous/organic interfacial area on PDC deactivation in the

aqueous/octanol-benzaldehyde phase-separated system at 4°C, pH 7.0. 1.39 M organic phase

benzaldehyde, 4.5 mM aqueous phase octanol, 48 mM aqueous phase benzaldehyde, 60 rpm

and 125 rpm agitation for organic and aqueous phase respectively in Lewis cell, 4 U/mL

aqueous phase or 2 U/mL TRV PDC carboligase activity, 2.5 M MOPS buffer, 0.5 mM

Mg2+, 1 mM TPP. TRV: total reaction volume by combining both phases.

4.2.1.3.2 Studies with an Emulsion System

Since it was not possible to further increase the interfacial contact area in the Lewis cell, a

rapidly stirred emulsion (220 rpm) with a much higher, but undefined interfacial area to

volume ratio was investigated. Some PDC deactivation would occur solely in the aqueous



113

phase without interfacial contact, and in Fig 4.4.a, the effects of soluble octanol (4.5 mM)

and soluble benzaldehyde (48 mM) on PDC deactivation in this aqueous phase are shown. In

Fig 4.4.b, the results of PDC deactivation are shown in an emulsion system with excess

concentrations of both octanol and benzaldehyde (1.33 M) and a 1:1 volume ratio for each

phase. The resultant benzaldehyde concentration in the aqueous phase under these conditions

was 48 mM. As shown in Fig 4.4.b, in the emulsion system, a greater degree of PDC

deactivation resulted from both effects in the aqueous phase as well those from interfacial

contacts with the organic phase.

The combined effects of octanol and benzaldehyde on PDC deactivation are evident from

Figs 4.4.a and b. As shown in Fig 4.4.a for the aqueous phase, PDC was relatively stable in

the MOPS buffer over 70 h (A). When the buffer was saturated with octanol, the stability

decreased slightly (B). Saturating the buffer with octanol and benzaldehyde resulted in

significantly faster deactivation (C). As shown in Fig 4.4.b, for the aqueous/organic emulsion

system, PDC deactivation increased with octanol saturated aqueous phase and only octanol in

the organic phase (D). The addition of high concentration of benzaldehyde (1.33 M) into the

octanol phase caused significantly higher deactivation (E). In analyzing the various factors

affecting PDC deactivation, since A-D is greater than A-B, this demonstrates the additional

effect of aqueous/octanol interface in a benzaldehyde-free emulsion system. Further, since D-

E is much greater than B-C, this indicates the appreciable PDC deactivation, which occurred

as a result of the high benzaldehyde concentration in the excess of octanol, presumably at the

aqueous/organic droplet interfaces in the emulsion.

Other researchers have found that smaller organic droplets (i.e. larger surface contact area) in

aqueous/organic (carbon tetrachloride, trichloroethylene, benzene, toluene, and n-heptane)

emulsion systems lowered the half-life values of an enzyme such as papain [Feliu et al.,

1995]. Furthermore, Ghatorae et al. [1994] reported that the degree of enzyme deactivation

was proportional to the total interfacial area of the solvents hexane and tridecane to which the

enzyme urease was exposed in a liquid-liquid bubble column reactor.



114

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(A ) M O P S B u ffer (B ) 4 .5 m M so lu b le o c ta n o l in M O P S

(C ) 4 .5 m M so lu b le o c ta n o l + 4 8 m M so lu b le b en za ld eh y d e in M O P S

(D ) a s (B ) p lu s ex cess o c ta n o l (tw o -p h a se )

(E ) a s (C ) p lu s ex cess o c ta n o l w ith 1 .3 3 M b en za ld eh y d e (tw o -p h a se )









Figure 4.4: Effect of excess octanol and benzaldehyde on PDC deactivation in the aqueous/octanol-benzaldehyde emulsion system at 4°C, pH 7.0: (a) aqueous-based system and (b) two-phase aqueous/organic system. 220 rpm agitation, 2 U/mL TRV PDC carboligase activity, 2.5 M MOPS buffer, 0.5 mM Mg2+, 1 mM TPP. TRV: total reaction volume by combining both phases.



115

Rosche et al. [2005] reported strong deactivation of 8 U/mL carboligase PDC in octanol-free

benzaldehyde emulsion (400 mM) with half-life value of 0.7 h (2.5 M MOPS, 6°C). The

present study employed 2 U/mL TRV (total reaction volume) carboligase PDC and 1.33 M

benzaldehyde in excess of octanol, yet the PDC was approx. 10 times more stable. Despite

some deactivation effect of the excess octanol, its major advantage is in maintaining the high

benzaldehyde concentration in the organic phase, such that PDC in the aqueous phase is not

directly exposed to the toxic benzaldehyde. This efficient protecting effect of the octanol

phase was observed as well by Sandford et al. [2005].

4.2.1.4 Effect of Initial Enzyme Concentration

The effects of enzyme concentration on PDC deactivation are compared in Fig 4.5 for

systems with low interfacial area (phase-separated) and high interfacial area (emulsion

system). While deactivation was relatively unaffected by enzyme concentration in the phase-

separated system (Fig 4.5.a), the rates of PDC deactivation were greater at all enzyme

concentrations studied (1.6 – 11.9 U/mL in the aqueous phase) in the emulsion system and

the deactivation was faster at the lower initial enzyme activities (Fig 4.5.b). For example, a

half-life value of 16 h was estimated at 4.1 U/mL PDC activity while the value was 49 h at

11.6 U/mL indicating a much greater PDC stability at the higher initial enzyme activity.

Other researchers have reported a similar effect with immobilized creatine amidinohydrolase

on polyurethane polymer support [Berberich et al., 2004].

Enzyme degradation by phase toxicity can be described by: (1) an immediate one-off

sequestration effect and (2) inactivation of the enzyme at the aqueous/organic interface,

followed by replacement of the inactive enzyme molecules by the active ones, leading to

continual inactivation [Feliu et al., 1995]. Observing the effect of initial enzyme

concentration on deactivation rates in the emulsion system (Fig 4.5.b), it was suggested that

the first aspect is more important leading to high deactivation rates at low enzyme loading

and an insignificant effect at high loading. Moreover in comparison to the phase-separated



116

system, the higher agitation rate in the emulsion system with increased exposure of the PDC

to interfacial benzaldehyde presumably resulted in more rapid enzyme deactivation.

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b

Figure 4.5: Effect of initial enzyme concentration on PDC deactivation in the two-phase

aqueous/octanol-benzaldehyde system at 4°C, pH 7.0: (a) phase-separated system, 125 rpm agitation

in the aqueous phase and (b) emulsion system, 220 rpm agitation. 1.46 M organic phase

benzaldehyde, 4.5 mM aqueous phase octanol, 48 mM aqueous phase benzaldehyde, 2.5 M MOPS

buffer, 0.5 mM Mg2+, 1 mM TPP. The enzyme activities were expressed as concentrations in the

aqueous phase.



117

The increased rate of deactivation with lower enzyme concentrations was not found in

aqueous system. Leksawasdi [2004] reported no evident effect of enzyme concentration (7 –

27 U/mL decarboxylase) on deactivation rate of crude extract PDC from Rhizopus javanicus

with no benzaldehyde (0.6 M MOPS, 6°C). Similar characteristics were observed in the

present study for partially purified PDC from C. utilis (3 and 7.3 U/mL carboligase) with 0

and 48 mM benzaldehyde (2.5 M MOPS, 4°C) (Fig 3.12).

4.2.1.5 Discussion of Toxicity Effects on PDC Enzyme

An enzyme in a two-phase aqueous/organic system may be deactivated by molecules of

organic species dissolved in the aqueous phase, termed molecular toxicity, and by contact of

the enzyme with the bulk organic liquid at the interface, termed phase toxicity [Bar, 1988].

In the present two-phase aqueous/octanol-benzaldehyde systems, the molecular toxicity is

likely to be similar for both phase-separated and emulsion systems (aqueous phase saturated

with benzaldehyde and octanol). However, the phase toxicity will be greater in the emulsion

system due to larger surface area of contact between enzyme molecules and emulsion

droplets. The degree of agitation has also been shown to have a separate deactivation effect

with soluble benzaldehyde and octanol (Fig 4.2) and the effect will be greater in the two-

phase emulsion system. The effect of excess octanol and benzaldehyde concentrations on

PDC deactivation has also been studied and is likely to be evident in both phase-separated

and emulsion systems but greater in the latter system due to higher frequency of contact

between enzyme molecules and excess organics (Figs 4.3 and 4.4.b).

Results from the present PDC deactivation study explained the relatively low organic phase

PAC with concentrations up to 27 g/L formed in the emulsion system at initial PDC activities

in the aqueous phase of 0.5 – 3 U/mL, in comparison to the phase-separated system with high

organic phase PAC concentrations in excess of 100 g/L for similar initial PDC activities

[Sandford et al., 2005]. The former results were consistent with the more severe PDC

deactivation in the emulsion system and increased rates of PDC deactivation at lower enzyme



118

concentrations. The phase-separated system on the other hand, offered relatively high

stability for the PDC and the deactivation rate was unaffected by enzyme concentration. The

specific productivity (PAC/U/h) of the phase-separated system was therefore better; however

the volumetric productivity (PAC/L/h) of the emulsion system was superior presumably due

to higher benzaldehyde transfer rate from the organic into the aqueous phase [Sandford et al.

2005].

4.2.1.6 Discussion of Organic-Aqueous Benzaldehyde Transfer

The present study also investigated the effects of aqueous/organic contact area, organic phase

benzaldehyde concentration, and temperature on the organic-aqueous benzaldehyde transfer

rate. Higher organic phase contact area to aqueous phase volume ratios, higher organic phase

benzaldehyde concentrations, and higher temperatures resulted in increased benzaldehyde

transfer rates (see Appendix A). Equilibrium aqueous phase saturation concentrations of

approx. 50 mM benzaldehyde were achieved in all experimental conditions.

Woodley et al. [1991] reported that combination of a Lewis cell possessing a relatively high

organic-aqueous substrate transfer coefficient (KA aint of 1.8 h-1) together with a lower

enzyme concentration (0.005 g/L aqueous phase) resulted in sufficiently high steady state

aqueous phase substrate concentration to kinetically control the reaction. Conversely, a

Lewis cell experiment designed with a lower organic-aqueous substrate transfer coefficient

(KA aint of 0.64 h-1) together with a higher enzyme concentration (0.01 g/L aqueous phase)

resulted in a lower steady-state aqueous phase substrate concentration at which the reaction

rate was mass transfer limited.

Two-phase synthesis of PAC is likely to show evidence of mass transfer limitation when the

enzyme in the aqueous phase converts a high proportion of pyruvate to acetaldehyde and

acetoin due to low organic-aqueous benzaldehyde transfer rate. Increased by-products with

reduced PAC formation was observed by Sandford et al. [2005] in the biphasic phase-

separated system at initial PDC activities in the aqueous phase higher than 4 U/mL

carboligase, indicating that benzaldehyde mass transfer limitation may have been occuring.



119

By comparison, the biphasic emulsion system with higher agitation was associated with

increased PAC formation for similar initial PDC activities presumably due to higher organic-

aqueous benzaldehyde transfer rates. In addition, lower PDC deactivation rates at the higher

enzyme concentrations were likely to have occured.

4.2.2 Effect of Organic to Aqueous Phase Volume Ratio on PDC Deactivation

and PAC Production

To find a balance between maintaining enzyme stability while enhancing PAC productivity,

a two-phase system was designed in the present investigation to reduce the interfacial contact

by decreasing the organic to aqueous phase volume ratio. In such a system, sufficiently high

organic-aqueous substrate transfer might be attained while achieving reduced deactivating

conditions for the enzyme. Experiments were designed to lower the organic to aqueous phase

volume ratios from 1:1 to 0.25:1 in the emulsion aqueous/octanol-benzaldehyde system at

4°C to determine whether or not this would influence PDC stability and PAC productivity.


In Figs 4.6 – 4.9 the profiles are shown for the concentrations of substrates, PAC, and by-

products for each phase for these ratios (see Appendix C for the overall concentration

profiles calculated by combining the volumes of both phases). Results were obtained after 48

h when neither benzaldehyde nor pyruvate was limiting. Pyruvate was consumed at generally

higher rates than benzaldehyde, resulting from additional conversion to acetaldehyde and

acetoin. The rates of PAC formation declined over time, although it was expected that the

biotransformations could proceed beyond 48 h.



120

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G (

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[Ben

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0

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[Ace

tald

ehyd

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]AQ

(m

M)

b

Figure 4.6: Effect of organic to aqueous phase volume ratio on PAC production in the aqueous/octanol-benzaldehyde emulsion system at 1:1 ratio at 4°C, initial pH 6.5: (a) organic and (b) aqueous phase substrates, PAC, and by-product concentration profiles. Initial agitation 250 rpm, initial concentrations: 1.36 M organic phase benzaldehyde, the aqueous phase contained 1.26 M pyruvate, 5.6 U/mL PDC carboligase activity (C. utilis partially purified), 2.5 M MOPS buffer, 1 mM Mg2+, 1 mM TPP. ORG: organic phase, AQ: aqueous phase. Approximate values for acetaldehyde concentration due to possible evaporative loss during sampling and analysis. The mean values were determined from triplicate analyses and error bars show the highest and lowest values.



121

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aqueous/octanol-benzaldehyde emulsion system at 0.67:1 ratio at 4°C, initial pH 6.5: (a)

organic and (b) aqueous phase concentration profiles. Initial agitation 235 rpm, initial

concentrations: 1.7 M organic phase benzaldehyde, the aqueous phase contained 1.06 M

pyruvate, 4.7 U/mL PDC carboligase activity (C. utilis partially purified), 2.5 M MOPS

buffer, 1 mM Mg2+, 1 mM TPP.



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[Ace

tald

ehyd

e]A

Q, [

Ace

toin

]AQ

(mM

)

b





pyruvate, 4 U/mL PDC carboligase activity (C. utilis partially purified), 2.5 M MOPS buffer,

1 mM Mg2+, 1 mM TPP.



123

0

500

1000

1500

2000

2500

3000

3500

0 10 20 30 40 50

[Ben

zald

ehyd

e]O

RG

, [P

AC

]OR

G (

mM

)

0

5

10

15

20

25

[Ace

tald

ehyd

e]O

RG

, [A

ceto

in]O

RG

(m

M)a

0

200

400

600

800

1000

1200

1400

0 10 20 30 40 50

T im e (h )

[Ben

zald

ehyd

e]A

Q,

[Pyr

uvat

e]A

Q, [

PA

C]A

Q (

mM

)

0

10

20

30

40

[Ace

tald

ehyd

e]A

Q, [

Ace

toin

]AQ

(m

M)

b









124

Decreasing the phase volume ratio resulted in higher PAC concentrations in both organic and

aqueous phases (Figs 4.6 – 4.9). At a 0.43:1 ratio for example, 1220 mM organic phase PAC

was produced in association with 180 mM PAC in the aqueous phase. These compared with

concentrations at a 1:1 ratio of 745 mM and 130 mM organic and aqueous phase PAC

respectively. As shown from data analysis reported in the Appendix, the overall PAC

concentration based on total reaction volume (TRV) was 490 mM in the former case, and 435

mM for the higher volume ratio.

Lowering the ratio to 0.25:1 resulted in a significantly higher organic and aqueous phase

PAC concentrations of 2220 mM and 225 mM respectively (625 mM TRV), however there

were increased discrepancies (10 – 20%) in the substrate molar balances (Table 4.2) in this

latter case and difficulties in separating the organic phase from the interfacial layer. Increases

in PAC were also found to be accompanied by increases in acetoin formation with overall

concentrations ranging from 18 mM to 30 mM for 1:1 to 0.25:1 ratio respectively (Figs 4.6 –

4.9).

4.2.2.2 PDC Deactivation

The rate of PDC deactivation was not affected by the reduced phase volume ratio with 50 –

60% residual PDC activity after 48 h for an initial overall activity of 2.8 U/mL based on the

total reaction volume (Fig 4.10).

It is possible that the similar deactivation rates resulted from the reduced deactivation

associated with lower interfacial contact being counteracted by the effect of higher organic

phase benzaldehyde concentrations at the lower ratios. Moreover, it appears that the higher

organic phase benzaldehyde concentrations at the lower ratios had a greater effect in

enhancing the organic-aqueous benzaldehyde tranfer rates than reducing interfacial area,

which resulted in enhanced PAC productivities at the lower ratios. The results imply a lesser

interfacial area reduction when decreasing the phase volume ratio from 1:1 (1.36 M organic

benzaldehyde) to 0.25:1 (3.48 M organic benzaldehyde) which corresponds to less than 2.6-



125

fold reduction in interfacial area. Additionally, the relatively high initial enzyme

concentrations (3.5 – 5.6 U/mL in the aqueous phase) might cause the phase toxicity to be

less pronounced.

0

20

40

60

80

100

120

0 10 20 30 40 50

% R

esid

ual E

nzym

e A

ctiv

ity

Time (h)

0

20

40

60

80

100

120

0 10 20 30 40 50

% R

esid

ual E

nzym

e A

ctiv

ity

Time (h)

1:1 0.67:1 0.43:1 0.25:11:11:1 0.67:10.67:1 0.43:10.43:1 0.25:10.25:1

Figure 4.10: Effect of organic to aqueous phase volume ratio in emulsion aqueous/octanol-

benzaldehyde system at 4°C, initial pH 6.5: residual enzyme activity. Same experiments as

shown in Figs 4.6 – 4.9.

4.2.2.3 Discussion of the Phase Ratio Effects

The results are summarized in Table 4.2, which shows increasing overall specific PAC

production and volumetric productivities as the volume ratios were decreased. The PAC

yields on consumed benzaldehyde were close to theoretical, while those on consumed

pyruvate were 90 – 95% theoretical. Good substrate molar balance closure was achieved in



126

all experiments except for the lower ratio of 0.25:1 where sampling problems occurred due to

phase separation difficulties.

Table 4.2: Performance summary: effect of organic to aqueous phase volume ratio on PAC

production in the aqueous/octanol-benzaldehyde emulsion system at 4°C, initial pH 6.5 (48

h).

Organic : aqueous

1:1

0.67:1

0.43:1

0.25:1

Overall PAC (g/L)

65.7

71.9

73.8

93.9

Organic PAC (g/L)

111.8

143.4

182.9

333.1

Aqueous PAC (g/L)

19.6

24.2

27.0

34.1

Overall specific PAC production

(mg/U initial PDC carboligase activity)

23.5

25.7

26.3

33.5

Overall volumetric productivity (g/L/day)

32.9

35.9

36.9

46.9

Overall by-product acetoin (g/L)

1.6

2.1

2.5

2.6

Overall by-product acetaldehyde (g/L)*

0.2

0.2

0.3

0.2

Yield of PAC on consumed benzaldehyde (mol/mol)

1.1

1.0

0.99

1.1

Yield of PAC on consumed pyruvate (mol/mol)

0.94

0.92

0.95

1.1

Benzaldehyde balance (%)

106

104

100

111

Pyruvate balance (%)

103

102

107

122

Calculated from data in experiments shown in Figs 4.6 – 4.9. *Approximate values for

acetaldehyde concentration due to possible evaporative loss during sampling and analysis.

Other researchers have reported that reducing the organic to aqueous phase volume ratio can

have a negative effect on a biocatalysis reaction. In a study reported by Panintrarux et al.

[1995], equilibrium yields of the β-glucosidase catalyzed biphasic production of n-alkyl-β-D



127

glucosides from glucose and n-alcohols were lower when the organic (n-alcohol only) to

aqueous phase volume ratio was decreased. This was associated with higher equilibrium

yields of by-product β-glucobioses. However, Yi et al. (1998) observed increasing reaction

rate of hexyl β-D glucoside production with a decreasing hexanol to water volume ratio.

4.3 Conclusion

The organic to aqueous phase volume ratio has been identified as a key factor in enhancing

the potential of a two-phase enzymatic process for PAC production with results

demonstrating that a more concentrated product stream can be produced by lowering this

ratio. These results were achieved with increasing specific production and productivities of

PAC while maintaining enzyme activity.

Chapter 5 Process Enhancement and Further Kinetic Evaluations for Two-Phase Aqueous/Organic Synthesis of PAC


128

CHAPTER 5

__________________________________________________

PROCESS ENHANCEMENT AND FURTHER

KINETIC EVALUATIONS FOR TWO-PHASE

AQUEOUS/ORGANIC SYNTHESIS OF PAC

Optimization of two-phase aqueous/organic PAC

production



129

5.1 Introduction

Identification of the organic to aqueous phase volume ratio in the present investigation as a

key factor in the enhancement of two-phase aqueous/organic PAC synthesis has encouraged

further development of a cost effective two-phase process. To achieve this objective, in the

current investigation the possibility is evaluated of operating the two-phase biotransformation

at lowered organic to aqueous phase volume ratio, increased temperatures, reduced MOPS

concentration, with utilization of whole cell PDC, and addition of low cost solute into the

aqueous phase.

The investigation was started by studying the effect of changing the organic to aqueous phase

volume ratio at the increased temperature of 20°C with partially purified PDC and 2.5 M

MOPS system. Further potential process simplification was evaluated by lowering the MOPS

concentration to 20 mM (MOPS is a relatively expensive material) with employment of

whole cell as biocatalyst to study the effects of changing the phase volume ratio and

temperature on two-phase PAC production. Finally, addition of 2.5 M dipropylene glycol

(DPG) into the aqueous phase as a potential substitute for MOPS [Leksawasdi et al., 2005]

was studied with comparison of whole cell and partially purified PDC (refer to Section 2.7

for details of the biotransformation experiments).



130


5.2.1 Effect of Changing the Organic to Aqueous Phase Volume Ratio at 20°°°°C on

Reaction Kinetics

5.2.1.1 PAC and By-Product Formation To illustrate the effect of increasing temperature on two-phase aqueous/organic PAC

synthesis, the results for experiments with decreasing organic to aqueous phase volume ratio

from 1:1 to 0.25:1 at 20°C in the aqueous/octanol-benzaldehyde emulsion system are

presented in Figs 5.1 – 5.4. These figures show the profiles for the concentrations of

substrates, PAC, and by-products for each phase (the overall concentration profiles

calculated by combining the volumes of both phases are shown in Appendix C). Pyruvate

was fully consumed in systems with 0.43:1 and 0.25:1 ratios with some benzaldehyde

remaining. Rates of pyruvate consumption were higher than benzaldehyde consumption due

to by-product formation and the rates of PAC formation were significantly faster in

comparison to biotransformations at 4°C (see Section 4.2.1).

From the data, it is evident that an increase in PAC concentration occurred in both organic

and aqueous phases. For example, biotransformation with 0.43:1 ratio was associated with

1415 mM and 170 mM organic and aqueous phase PAC respectively, in comparison to lower

levels of 740 mM and 100 mM organic and aqueous phase PAC at 1:1 ratio. The overall PAC

concentration based on total reaction volume (TRV) was 545 mM in the former case, and 420

mM for the higher volume to volume ratio (Appendix C). Lowering the ratio to 0.25:1

resulted in higher organic and aqueous phase PAC of 1810 mM and 195 mM, however the

overall PAC concentration based on TRV was reduced slightly (520 mM).



131

0

500

1000

1500

2000

2500

3000

3500

4000

0 10 20 30 40 50

[Ben

zald

ehyd

e]O

RG

, [P

AC

]OR

G

(mM

)

0

10

20

30

40

50

60

70

[Ace

tald

ehyd

e]O

RG

, [A

ceto

in]O

RG

(mM

)

a

0

500

1000

1500

2000

2500

3000

3500

4000

0 10 20 30 40 50

[Ben

zald

ehyd

e]O

RG

, [P

AC

]OR

G

(mM

)

0

10

20

30

40

50

60

70

[Ace

tald

ehyd

e]O

RG

, [A

ceto

in]O

RG

(mM

)

0

500

1000

1500

2000

2500

3000

3500

4000

0

500

1000

1500

2000

2500

3000

3500

4000

0 10 20 30 40 500 10 20 30 40 50

[Ben

zald

ehyd

e]O

RG

, [P

AC

]OR

G

(mM

)

[Ben

zald

ehyd

e]O

RG

, [P

AC

]OR

G

(mM

)

0

10

20

30

40

50

60

70

0

10

20

30

40

50

60

70

[Ace

tald

ehyd

e]O

RG

, [A

ceto

in]O

RG

(mM

)

[Ace

tald

ehyd

e]O

RG

, [A

ceto

in]O

RG

(mM

)

a

0

200

400

600

800

1000

1200

1400

0 10 20 30 40 50

[Ben

zald

ehyd

e]A

Q,[P

yruv

ate]

AQ

,

[PA

C]A

Q (

mM

)

0

10

20

30

40

50

60

70

80

[Ace

tald

ehyd

e]A

Q, [

Ace

toin

]AQ

(mM

)

T im e (h)

b

0

200

400

600

800

1000

1200

1400

0 10 20 30 40 50

[Ben

zald

ehyd

e]A

Q,[P

yruv

ate]

AQ

,

[PA

C]A

Q (

mM

)

0

10

20

30

40

50

60

70

80

[Ace

tald

ehyd

e]A

Q, [

Ace

toin

]AQ

(mM

)

0

200

400

600

800

1000

1200

1400

0

200

400

600

800

1000

1200

1400

0 10 20 30 40 500 10 20 30 40 500 10 20 30 40 50

[Ben

zald

ehyd

e]A

Q,[P

yruv

ate]

AQ

,

[PA

C]A

Q (

mM

)

[Ben

zald

ehyd

e]A

Q,[P

yruv

ate]

AQ

,

[PA

C]A

Q (

mM

)

0

10

20

30

40

50

60

70

80

0

10

20

30

40

50

60

70

80

[Ace

tald

ehyd

e]A

Q, [

Ace

toin

]AQ

(mM

)

[Ace

tald

ehyd

e]A

Q, [

Ace

toin

]AQ

(mM

)

T im e (h)

b


aqueous/octanol-benzaldehyde emulsion system at 1:1 ratio at 20°C, initial pH 6.5: (a) organic and

(b) aqueous phase substrates, PAC, and by-product concentration profiles. Initial agitation 250 rpm,

initial concentrations: 1.4 M organic phase benzaldehyde, the aqueous phase contained 1.29 M

pyruvate, 5.6 U/mL PDC carboligase activity (C. utilis partially purified), 2.5 M MOPS buffer, 1 mM

Mg2+, 1 mM TPP. ORG: organic phase, AQ: aqueous phase, Approximate values for acetaldehyde

concentration due to possible evaporative losses during sampling and analysis. The mean values were

determined from triplicate analyses and error bars show the highest and lowest values.



132

0

5 00

1 00 0

1 50 0

2 00 0

2 50 0

3 00 0

3 50 0

4 00 0

0 10 2 0 3 0 4 0 5 0

[Ben

zald

ehyd

e]O

RG

, [P

AC

]OR

G

(mM

)

0

10

20

30

40

50

60

70

[Ace

tald

ehyd

e]O

RG

, [A

ceto

in]O

RG

(mM

)

a

0

5 00

1 00 0

1 50 0

2 00 0

2 50 0

3 00 0

3 50 0

4 00 0

0

5 00

1 00 0

1 50 0

2 00 0

2 50 0

3 00 0

3 50 0

4 00 0

0 10 2 0 3 0 4 0 5 00 10 2 0 3 0 4 0 5 0

[Ben

zald

ehyd

e]O

RG

, [P

AC

]OR

G

(mM

)

[Ben

zald

ehyd

e]O

RG

, [P

AC

]OR

G

(mM

)

0

10

20

30

40

50

60

70

0

10

20

30

40

50

60

70

[Ace

tald

ehyd

e]O

RG

, [A

ceto

in]O

RG

(mM

)

[Ace

tald

ehyd

e]O

RG

, [A

ceto

in]O

RG

(mM

)

a

0

200

400

600

800

100 0

120 0

140 0

0 10 20 30 4 0 5 0

[Ben

zald

ehyd

e]A

Q,[P

yruv

ate]

AQ

,

[PA

C]A

Q (

mM

)

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

[Ace

tald

ehyd

e]A

Q, [

Ace

toin

]AQ

(mM

)

b

T im e (h )

0

200

400

600

800

100 0

120 0

140 0

0 10 20 30 4 0 5 0

[Ben

zald

ehyd

e]A

Q,[P

yruv

ate]

AQ

,

[PA

C]A

Q (

mM

)

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

[Ace

tald

ehyd

e]A

Q, [

Ace

toin

]AQ

(mM

)

0

200

400

600

800

100 0

120 0

140 0

0

200

400

600

800

100 0

0

200

400

600

800

100 0

120 0

140 0

0 10 20 30 4 0 5 00 10 20 30 4 0 5 0

[Ben

zald

ehyd

e]A

Q,[P

yruv

ate]

AQ

,

[PA

C]A

Q (

mM

)

[Ben

zald

ehyd

e]A

Q,[P

yruv

ate]

AQ

,

[PA

C]A

Q (

mM

)

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

[Ace

tald

ehyd

e]A

Q, [

Ace

toin

]AQ

(mM

)

[Ace

tald

ehyd

e]A

Q, [

Ace

toin

]AQ

(mM

)

b

T im e (h )









133

0

500

1000

1500

2000

2500

3000

3500

4000

0 10 20 30 40 50

[Ben

zald

ehyd

e]O

RG

, [P

AC

]OR

G

(mM

)

0

10

20

30

40

50

60

70

[Ace

tald

ehyd

e]O

RG

, [A

ceto

in]O

RG

(mM

)

a

0

500

1000

1500

2000

2500

3000

3500

4000

0 10 20 30 40 50

[Ben

zald

ehyd

e]O

RG

, [P

AC

]OR

G

(mM

)

0

10

20

30

40

50

60

70

[Ace

tald

ehyd

e]O

RG

, [A

ceto

in]O

RG

(mM

)

0

500

1000

1500

2000

2500

3000

3500

4000

0

500

1000

1500

2000

2500

3000

3500

4000

0 10 20 30 40 500 10 20 30 40 50

[Ben

zald

ehyd

e]O

RG

, [P

AC

]OR

G

(mM

)

[Ben

zald

ehyd

e]O

RG

, [P

AC

]OR

G

(mM

)

0

10

20

30

40

50

60

70

0

10

20

30

40

50

60

70

[Ace

tald

ehyd

e]O

RG

, [A

ceto

in]O

RG

(mM

)

[Ace

tald

ehyd

e]O

RG

, [A

ceto

in]O

RG

(mM

)

a

0

200

400

600

800

1000

1200

1400

0 10 20 30 40 50

[Ben

zald

ehyd

e]A

Q,[P

yruv

ate]

AQ

,

[PA

C]A

Q (

mM

)

0

10

20

30

40

50

60

70

80

[Ace

tald

ehyd

e]A

Q, [

Ace

toin

]AQ

(mM

)

b

T im e (h )

0

200

400

600

800

1000

1200

1400

0 10 20 30 40 50

[Ben

zald

ehyd

e]A

Q,[P

yruv

ate]

AQ

,

[PA

C]A

Q (

mM

)

0

10

20

30

40

50

60

70

80

[Ace

tald

ehyd

e]A

Q, [

Ace

toin

]AQ

(mM

)

0

200

400

600

800

1000

1200

1400

0

200

400

600

800

1000

1200

1400

0 10 20 30 40 500 10 20 30 40 50

[Ben

zald

ehyd

e]A

Q,[P

yruv

ate]

AQ

,

[PA

C]A

Q (

mM

)

[Ben

zald

ehyd

e]A

Q,[P

yruv

ate]

AQ

,

[PA

C]A

Q (

mM

)

0

10

20

30

40

50

60

70

80

0

10

20

30

40

50

60

70

80

[Ace

tald

ehyd

e]A

Q, [

Ace

toin

]AQ

(mM

)

[Ace

tald

ehyd

e]A

Q, [

Ace

toin

]AQ

(mM

)

b

T im e (h )





pyruvate, 4 U/mL PDC carboligase activity (C. utilis partially purified), 2.5 M MOPS buffer,

1 mM Mg2+, 1 mM TPP.



134

0

500

1000

1500

2000

2500

3000

3500

4000

0 10 20 30 40 50

[Ben

zald

ehyd

e]O

RG

, [P

AC

]OR

G

(mM

)

0

10

20

30

40

50

60

70

[Ace

tald

ehyd

e]O

RG

, [A

ceto

in]O

RG

(mM

)

a

0

500

1000

1500

2000

2500

3000

3500

4000

0 10 20 30 40 50

[Ben

zald

ehyd

e]O

RG

, [P

AC

]OR

G

(mM

)

0

10

20

30

40

50

60

70

[Ace

tald

ehyd

e]O

RG

, [A

ceto

in]O

RG

(mM

)

0

500

1000

1500

2000

2500

3000

3500

4000

0

500

1000

1500

2000

2500

3000

3500

4000

0 10 20 30 40 500 10 20 30 40 50

[Ben

zald

ehyd

e]O

RG

, [P

AC

]OR

G

(mM

)

[Ben

zald

ehyd

e]O

RG

, [P

AC

]OR

G

(mM

)

0

10

20

30

40

50

60

70

0

10

20

30

40

50

60

70

[Ace

tald

ehyd

e]O

RG

, [A

ceto

in]O

RG

(mM

)

[Ace

tald

ehyd

e]O

RG

, [A

ceto

in]O

RG

(mM

)

a

0

20 0

40 0

60 0

80 0

1 00 0

1 20 0

1 40 0

0 10 20 3 0 40 50

[Ben

zald

ehyd

e]A

Q,[P

yruv

ate]

AQ

,

[PA

C]A

Q (

mM

)

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

[Ace

tald

ehyd

e]A

Q, [

Ace

toin

]AQ

(mM

)

b

T im e (h )

0

20 0

40 0

60 0

80 0

1 00 0

1 20 0

1 40 0

0 10 20 3 0 40 50

[Ben

zald

ehyd

e]A

Q,[P

yruv

ate]

AQ

,

[PA

C]A

Q (

mM

)

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

[Ace

tald

ehyd

e]A

Q, [

Ace

toin

]AQ

(mM

)

0

20 0

40 0

60 0

80 0

1 00 0

1 20 0

1 40 0

0

20 0

40 0

60 0

80 0

1 00 0

1 20 0

1 40 0

0 10 20 3 0 40 500 10 20 3 0 40 50

[Ben

zald

ehyd

e]A

Q,[P

yruv

ate]

AQ

,

[PA

C]A

Q (

mM

)

[Ben

zald

ehyd

e]A

Q,[P

yruv

ate]

AQ

,

[PA

C]A

Q (

mM

)

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

[Ace

tald

ehyd

e]A

Q, [

Ace

toin

]AQ

(mM

)

[Ace

tald

ehyd

e]A

Q, [

Ace

toin

]AQ

(mM

)

b

T im e (h )









135

Increases in PAC production were accompanied by increases in acetoin formation with

overall concentrations between 43 – 63 mM. Acetoin formation was greater at 20°C than

4°C. Increased acetoin production at higher temperatures has been reported previously by

Shin and Rogers [1996] for PAC production using partially purified PDC from C. utilis.

Additionally, the initial benzaldehyde concentration in the aqueous phase was generally

higher at 20°C than 4°C, which is likely to increase the reaction rates.

5.2.1.2 PDC Deactivation

The rates of deactivation for partially purified PDC were similar for all phase ratios in the

two-phase system, being significantly faster at 20°C than 4°C (see Section 4.2.1) with

residual activity of 10 – 20% after 20 h for all ratios at 20°C (Fig 5.5).

T im e (h )

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

0 1 0 2 0 3 0 4 0 5 0

% R

esid

ual E

nzym

e A

ctiv

ity

T im e (h )

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

0 1 0 2 0 3 0 4 0 5 0

% R

esid

ual E

nzym

e A

ctiv

ity

1:1 0.67:1 0.43:1 0.25:11:11:1 0.67:10.67:1 0.43:10.43:1 0.25:10.25:1

Figure 5.5: Effect of organic to aqueous phase volume ratio on PDC deactivation in the

aqueous/octanol-benzaldehyde emulsion system at 20°C, initial pH 6.5. Same experiments as

shown in Figs 5.1 – 5.4.



136

The effect of temperature on the stability of partially purified PDC at 4°C and 20°C was

confirmed by Satianegara et al. [2006] who reported faster deactivation rates at the higher

temperature. However, these latter studies were obtained only in the absence and presence of

50 mM benzaldehyde and not in the two-phase system.

5.2.1.3 Discussion

The results of these phase ratio studies are summarized in Table 5.1, which shows that

biotransformations at 20°C resulted in higher overall specific PAC production and increased

overall volumetric productivities as the volume ratios were decreased from 1:1 to 0.43:1. No

further improvement occurred at 0.25:1 ratio (in fact a small decline was evident). Substrate

molar balance closures within 8% were achieved in all experiments. The PAC yields on

consumed benzaldehyde were close to theoretical, while those on pyruvate were lower with

values of 80 – 84% due to significant acetoin formation.

Compared to the previous results at 4°C (Table 4.2), the data at 20°C show significantly

higher overall PAC volumetric productivities at all ratios. The PAC yields on consumed

pyruvate were lower at 20°C due to increased acetoin formation.

In comparison to earlier results for PAC production using partially purified PDC in the two-

phase aqueous/octanol-benzaldehyde emulsion system, Sandford et al. [2005] reported PAC

levels of 141 g/L and 19 g/L in the organic and aqueous phase respectively in 49 h at 4°C

with initial PDC carboligase activity of 8.5 U/mL in the aqueous phase and 1:1 organic to

aqueous phase volume ratio. At 21°C and 1:1 ratio with whole cell PDC, Rosche et al. [2005]

reported 103 g/L and 12.8 g/L organic and aqueous phase PAC in 15 h with 5 U/mL initial

PDC carboligase activity in the aqueous phase, indicating that whole cell PDC at the higher

temperature resulted in higher productivity and specific PAC production than with partially

purified PDC at the lower temperature.



137

In the present study, lowering the ratio to 0.43:1 resulted in an appreciable improvement in

PAC production with 212 g/L and 25.8 g/L organic and aqueous phase PAC in 20 h at 20°C

with a relatively low 4 U/mL initial PDC carboligase activity in the aqueous phase. The

strategy created conditions which favored both reduced PDC enzyme deactivation rates while

maintaining adequate organic-aqueous benzaldehyde transfer.


production in the aqueous/octanol-benzaldehyde emulsion system at 20°C, initial pH 6.5 (48

h).

Organic : aqueous

1:1

0.67:1

0.43:1

0.25:1

Overall PAC (g/L)

63.1

72.4

81.7

77.6

Organic PAC (g/L)

111.1

152.2

212.4

271.2

Aqueous PAC (g/L)

15.1

19.2

25.8

29.2



22.5

25.9

29.2

27.7

Overall volumetric productivity (g/L/day)*

75.7

86.9

98

93.1


3.8

4.9

5.5

5.0

Overall by-product acetaldehyde (g/L)**

0.3

0.34

0.32

0.27


1.0

1.1

0.97

0.91


0.81

0.84

0.84

0.81


105

108

100

95


100

105

106

100

Calculated from data in experiments shown in Figs 5.1 – 5.4. *The productivity was calculated

based on 20 h time point. **Approximate values for acetaldehyde concentration due to possible

evaporative losses during sampling and analysis.



138

5.2.2 Effect of Changing Organic to Aqueous Phase Volume Ratio at 20°°°°C at

Lower MOPS Concentration (20 mM)

The effect of organic to aqueous phase volume ratio on two-phase PAC synthesis was studied

with 20 mM MOPS buffer system with the ratio lowered from 1:1 to 0.25:1 in the

aqueous/octanol-benzaldehyde emulsion system at 20°C with whole cell PDC. The pH was

controlled at 7.0 through acetic acid addition (see Appendix D for the acid addition profiles).

The lower MOPS concentration and whole cell PDC conditions were studied as possible

means of improving the overall process economics.


The PAC concentration profiles for each phase at the different phase ratios are presented in

Fig 5.6 (the overall concentration profiles calculated by combining the volumes of both

phases are shown in Appendix). Lowering the organic to aqueous phase volume ratio resulted

in faster reaction completion and decreased PAC formation rates (based on total reaction

volume). Neither substrate was limiting in any experiment.

Lowering the ratio resulted in slightly increasing final organic phase PAC with

concentrations of 220 mM at 1:1, followed by 230 mM and 260 mM at 0.67:1 and 0.43:1

respectively, and finally 270 mM at 0.25:1. Lower final PAC concentrations of 12 – 16 mM

partitioned into the aqueous phase. Lowering the ratio was associated with reduced overall

PAC concentration when account is taken of decreasing organic phase volumes. Reductions

in overall PAC concentration were accompanied by decreases in acetoin formation with final

concentrations decreasing from 4.3 mM to 3 mM and 19 mM to 6.5 mM in the organic and

aqueous phases respectively (Fig 5.7).



139

0

50

100

150

200

250

300

0 5 10 15 20 25 30 35 40

Time (h)

[PA

C]O

RG

(m

M)

0

10

20

30

40

[PA

C]A

Q (

mM

)

1: 1 0.67 : 1 0.43 : 1 0.25 : 1 Organic phaseAqueous phase1: 1 0.67 : 1 0.43 : 1 0.25 : 1 Organic phaseAqueous phase1: 1 0.67 : 1 0.43 : 1 0.25 : 1 1: 1 0.67 : 1 0.43 : 1 0.25 : 1 Organic phaseAqueous phase


aqueous/octanol-benzaldehyde emulsion system with 20 mM MOPS at 20°C, controlled pH

7.0: organic and aqueous phase concentration profiles are shown. Constant agitation 160

rpm, initial concentrations: 775 – 810 mM TRV benzaldehyde, 400 – 465 mM TRV

pyruvate, 1 U/mL TRV PDC carboligase activity (C. utilis whole cell), 20 mM MOPS buffer,

1 mM Mg2+, 1 mM TPP. ORG: organic phase, AQ: aqueous phase, TRV: total reaction

volume by combining both phases. The mean values were determined from triplicate

analyses and error bars show the highest and lowest values.



140

0

1

2

3

4

5

'1:1' '0.67:1' '0.43:1' '0.25:1'

[Ace

tald

ehyd

e]O

RG

, [A

ceto

in]O

RG

(m

M) a

0

5

10

15

20

'1:1' '0.67:1' '0.43:1' '0.25:1'

Organic : Aqueous

[Ace

tald

ehyd

e]A

Q, [

Ace

toin

]AQ

(m

M)


b

Figure 5.7: Effect of organic to aqueous phase volume ratio on by-products acetaldehyde

and acetoin formation in the aqueous/octanol-benzaldehyde emulsion system with 20 mM

MOPS at 20°C, controlled pH 7.0: (a) final organic and (b) aqueous phase concentrations.

Same experiments as shown in Fig 5.6. Approximate values for acetaldehyde concentration

due to possible evaporative losses during sampling and analysis.



141

5.2.2.2 Discussion The results in Figs 5.6 and 5.7 summarized in Table 5.2 show that lowering the organic to

aqueous phase volume ratio in two-phase emulsion PAC synthesis with 20 mM MOPS at

20°C resulted in: (1) slightly increasing organic phase PAC concentration although with

much lower values than with 2.5 M MOPS (Table 5.1), (2) decreasing overall specific PAC

production and volumetric productivities, and (3) decreasing acetoin formation. The low

yields of PAC on consumed benzaldehyde and pyruvate were atypical; high benzaldehyde

concentrations in the organic phase (up to 3.9 M) and operations at 20°C gave rise to a higher

degree of evaporative losses of benzaldehyde and presumably acetaldehyde, as well as

appreciable pyruvate loss (a phenomenon also noticed by Rosche et al. [2002a]).

Benzaldehyde losses were measured using controls at all phase ratios and in some cases up to

45% losses occurred in these larger and more open systems (180 mL).

As opposed to the two-phase biotransformations in 2.5 M MOPS buffer systems, decreasing

the organic to aqueous phase volume ratio in 20 mM MOPS buffer system resulted in

reduction in overall PAC concentration. This might have been caused by reduction in PDC

stability at the lowered phase ratios due insufficient stabilizing effect on PDC by 20 mM

concentration of MOPS [Rosche et al., 2002a].



142


production in the aqueous/octanol-benzaldehyde emulsion system with 20 mM MOPS at

20°C, controlled pH 7.0.

Organic : aqueous

1:1

0.67:1

0.43:1

0.25:1

Reaction period (h)

38

35

32

27

Overall PAC (g/L)

17.7

15.0

13.3

9.5

Organic PAC (g/L)

33.1

34.6

38.9

40.1

Aqueous PAC (g/L)

2.3

2.0

2.4

1.8



17.7

15.0

13.3

9.4

Overall volumetric productivity (g/L/day)

11.2

10.3

10.0

8.4


1.0

0.8

0.7

0.5

Overall by-product acetaldehyde (g/L)*

0.02

0.02

0.01

0.02


0.73

0.51

0.45

0.6


0.72

0.79

0.64

0.8

Calculated from data in experiments as shown in Fig 5.6. *Approximate values for acetaldehyde

concentration due to possible losses during sampling and analysis.



143

5.2.3 Effect of Increasing Temperature at Lower MOPS Concentration (20 mM)

To investigate the effect of temperature on two-phase aqueous/organic PAC synthesis with

20 mM MOPS, experiments were designed with increasing temperatures from 5°C to 35°C in

1:1 aqueous/octanol-benzaldehyde emulsion system with employment of whole cell PDC to

determine the potential for process simplification and cost reductions. The pH was controlled

at 7.0 through acetic acid addition (see Appendix for the acid addition profiles).


The profiles of PAC concentration for each phase at the different temperatures are shown in

Figs 5.8 and 5.9 (the overall concentration profiles calculated by combining the volumes of

both phases are shown in Appendix D). Operation at increasing temperatures resulted in

shortened reaction period and increasing PAC formation rates. Neither substrate was limiting

in any experiments.

As illustrated in Fig 5.8, increasing the temperature from 5°C to 20°C resulted in increased

final organic phase PAC concentrations with values of 155 mM and 190 mM at 5°C and

10°C respectively and 220 mM at 15°C and 20°C. Further increasing the temperature

decreased the final PAC concentrations to 145 mM at 25°C and 30°C, with further reduction

to 75 mM at 35°C. The lower concentrations of final PAC which partitioned into the aqueous

phase (5 – 17 mM) corresponded to the changes in values in the organic phase (Fig 5.9).



144

0

50

100

150

200

250

0 10 20 30 40 50 60 70

[PA

C]O

RG

(m

M)

a

0

50

100

150

200

250

0 10 20 30 40 50 60 70

[PA

C]O

RG

(m

M)

a

0

50

100

150

200

250

0 10 20 30 40 50 60 70

[PA

C]O

RG

(m

M)

b

Tim e (h )

0

50

100

150

200

250

0 10 20 30 40 50 60 70

[PA

C]O

RG

(m

M)

b

0

50

100

150

200

250

0 10 20 30 40 50 60 70

[PA

C]O

RG

(m

M)

b

Tim e (h )

4 °°°°C 10 °°°°C 15°°°°C 20 °°°°C 25°°°°C 30 °°°°C 35°°°°C4 °°°°C4 °°°°C 10 °°°°C10 °°°°C 15°°°°C15°°°°C 20 °°°°C20 °°°°C 25°°°°C25°°°°C 30 °°°°C30 °°°°C 35°°°°C35°°°°C

Figure 5.8: Effect of temperature on PAC production in the aqueous/octanol-benzaldehyde emulsion

system with 20 mM MOPS, controlled pH 7.0. Organic phase concentration profiles: (a) 5°C – 20°C

and (b) 25°C – 35°C. Constant agitation 160 rpm, initial concentrations: 1.6 – 1.64 M organic phase

benzaldehyde, the aqueous phase containing 0.93 – 0.98 M pyruvate, 2 U/mL PDC carboligase

activity (C. utilis whole cell), 20 mM MOPS buffer, 1 mM Mg2+, 1 mM TPP, 1:1 organic to aqueous

phase volume ratio. ORG: organic phase. The mean values were determined from triplicate analyses

and error bars show the highest and lowest values.



145

0

4

8

12

16

20

0 10 20 30 40 50 60 70

[PA

C]A

Q (

mM

)

a

0

4

8

12

16

20

0 10 20 30 40 50 60 70

[PA

C]A

Q (

mM

)

a

0

4

8

12

16

20

0 10 20 30 40 50 60 70Tim e (h)

[PA

C]A

Q (

mM

)

b

0

4

8

12

16

20

0 10 20 30 40 50 60 70Tim e (h)

[PA

C]A

Q (

mM

)

b

4 °°°°C 10 °°°°C 15°°°°C 20 °°°°C 25°°°°C 30 °°°°C 35°°°°C4 °°°°C4 °°°°C 10 °°°°C10 °°°°C 15°°°°C15°°°°C 20 °°°°C20 °°°°C 25°°°°C25°°°°C 30 °°°°C30 °°°°C 35°°°°C35°°°°C

Figure 5.9: Effect of temperature on PAC production in the aqueous/octanol-benzaldehyde

emulsion system with 20 mM MOPS, controlled pH 7.0. Aqueous phase concentration

profiles: (a) 5°C – 20°C and (b) 25°C – 35°C. AQ: aqueous phase. Same experiments as

shown in Fig 5.8.



146

Fig 5.10 shows the final acetaldehyde and acetoin concentrations for each phase (the overall

concentrations are shown in Appendix). Increases in PAC concentration at 5°C to 20°C were

accompanied by increases in acetoin formation with aqueous phase concentrations of

between 10 – 19 mM in association with 0.5 – 4.5 mM in the organic phase. Reductions in

PAC concentration at 25°C to 35°C were associated with decreases in acetoin formation.

Concentrations of 15 mM and 3.5 mM were estimated in the aqueous and organic phase

respectively at 25°C and 30°C, while 7.5 mM and 1.8 mM were determined in the aqueous

and organic phase respectively at 35°C. The reduction in acetaldehyde concentrations at the

higher temperatures may be an artefact, as increasing evaporation of this volatile by-product

will occur as temperature increases.

5.2.3.2 Discussion

The results in Figs 5.8, 5.9, and 5.10 summarized in Table 5.3 show that performing the

aqueous/organic emulsion synthesis of PAC with 20 mM MOPS concentration from 5°C to

35°C resulted in: (1) highest organic phase PAC concentrations at 15°C and 20°C, (2) highest

overall specific PAC production at 15°C and 20°C, (3) overall volumetric productivity

increases with temperature, however the final PAC concentrations are low at the highest

temperature, and (4) highest acetoin concentration at 20°C. As found previously, the PAC

yields on consumed benzaldehyde and pyruvate were relatively low; longer

biotransformation periods at the lower temperatures and operation at higher temperatures

gave rise to increased evaporative losses of benzaldehyde and presumably acetaldehyde, as

well as appreciable pyruvate loss. Benzaldehyde losses were measured using controls at all

temperatures and in some cases up to 40% losses occurred in these larger and more open

systems (180 mL).



147

0

1

2

3

4

5

4 10 15 20 25 30 35

[ace

tald

ehyd

e]O

RG

, [ac

etoi

n]O

RG

(m

M)

a

0

5

10

15

20

4 10 15 20 25 30 35

T (oC)

[ace

tald

ehyd

e]A

Q, [

acet

oin]

AQ

(m

M)


b

Figure 5.10: Effect of temperature on by-products acetaldehyde and acetoin formation in the

aqueous/octanol-benzaldehyde emulsion system with 20 mM MOPS, controlled pH 7.0: (a)

final organic and (b) aqueous phase concentrations. ORG: organic phase, AQ: aqueous

phase. Same experiments as shown in Fig 5.8. Estimated values for acetaldehyde

concentrations due to evaporative losses during sampling and analysis.



148

Table 5.3: Performance summary: effect of temperature on PAC production in the

aqueous/octanol-benzaldehyde emulsion system with 20 mM MOPS, controlled pH 7.0.

Temperature (°C)

4

10

15

20

25

30

35

Reaction period (h)

60

58

52

38

23

18

6

Initial PAC rate (g/L/h)*

1.3

1.7

2.4

2.9

3.6

4.1

3.6

Overall PAC (g/L)

12.5

15.6

17.6

17.7

11.5

11.7

5.9

Organic PAC (g/L)

23.0

28.8

32.7

33.1

21.5

22.0

11.1

Aqueous PAC (g/L)

2.0

2.5

2.6

2.3

1.6

1.4

0.8



12.5

15.6

17.6

17.7

11.5

11.7

5.9

Overall volumetric productivity

(g/L/day)

5.0

6.5

8.3

11.2

11.9

15.5

23.8


0.48

0.71

0.87

1.0

0.84

0.8

0.41

Overall by-product acetaldehyde

(g/L)**

0.13

0.09

0.02

0.02

0.03

0.02

0.0

Yield of PAC on consumed

benzaldehyde (mol/mol)

0.52

0.65

0.72

0.73

0.52

0.73

0.4

Yield of PAC on consumed pyruvate

(mol/mol)

0.72

0.68

0.7

0.72

0.55

0.61

0.4

Calculated from data in experiments as shown in Fig 5.8. *Measured over the first hour.

**Estimated values for acetaldehyde concentration due to evaporative losses during sampling and

analysis.



149

The effect of temperature up to 30°C on increasing initial PAC formation rates (Table 5.3,

the formation rate was reduced at 35°C) in the aqueous/octanol-benzaldehyde emulsion

system with whole cell PDC has been shown in the present investigation. A similar increase

was reported by Shin and Rogers [1996a] and Satianegara et al. [2005] in the aqueous

(soluble benzaldehyde) and aqueous/benzaldehyde emulsion system respectively over a more

limited temperature range (up to 20°C) with partially purified PDC. Furthermore as shown in

Chapter 4, two-phase aqueous/organic PAC production was influenced by PDC enzyme

stability as well as the rate of organic-aqueous benzaldehyde transfer, with likely

enhancement of the transfer rate at higher temperatures. In the current study, the highest total

PAC formation was obtained at 15°C and 20°C regardless of the increasing reaction rates at

the higher temperatures. It appeared that the effects of enhanced organic-aqueous

benzaldehyde transfer rates were counteracted by reduction in PDC stability at the higher

temperatures.

5.2.4 Effect of Dipropylene Glycol (DPG) as Additive at Lower MOPS

Concentration (20 mM) with Lowered Organic to Aqueous Phase Volume Ratio

As previously shown, two-phase aqueous/organic PAC production with 20 mM MOPS (pH

controlled at 7.0) and whole cell PDC was relatively low in comparison to biotransformations

with 2.5 M MOPS (see Sections 4.2.2 and 5.2.1). The relatively low PAC production with 20

mM MOPS was observed earlier by Leksawasdi et al. [2005] with partially purified PDC.

The initial benzaldehyde concentrations in the aqueous phase were lower in the 20 mM

MOPS systems of approx. 20 mM in comparison to approx. 40 – 50 mM in systems with 2.5

M MOPS, which indicated a decreased concentration driving force in the lower MOPS

system for PAC formation in the aqueous phase (reported also by Leksawasdi et al. [2005]).

For the viewpoint of PDC stability, deactivation rates would be slower at lower benzaldehyde

concentrations in the aqueous phase, however the decreased PDC stability at lower MOPS

concentration would be likely to counteract this effect [Rosche et al. 2002a].



150

From the present study, it was apparent that maintenance of high MOPS concentration was

essential to achieve relatively high specific PAC production and productivities by stabilizing

the PDC enzyme and enhancing benzaldehyde partitioning into the aqueous phase.

Leksawasdi et al. [2005] evaluated the potential of several compounds to serve as possible

substitutes for the expensive MOPS and reported that similar specific PAC production could

be achieved with partially purified PDC in a two-phase emulsion system by replacing 2.5 M

MOPS with 20 mM MOPS and 2.5 M dipropylene glycol (DPG) in the aqueous phase.

The current investigation evaluates the effect on PAC production in the aqueous/octanol-

benzaldehyde emulsion system of 2.5 M DPG and 20 mM MOPS at 20°C. A 0.25:1 organic

to aqueous phase volume ratio was selected to enhance the more concentrated organic phase

product stream. The investigation also compares the use of whole cell and partially purified

PDC for PAC production in the larger scale system. The pH was controlled at 7.0 through

acetic acid addition (see Appendix D for the acid addition profiles).


Figs 5.11 and 5.12 show the profiles for the concentrations of substrates, PAC, and by-

products for each phase (the overall concentration profiles calculated by combining the

volumes of both phases are shown in Appendix D). The biotransformations were completed

at 26 h for both whole cell and partially purified PDC. Neither substrate was limiting in

either experiment.

Addition of 2.5 M DPG had a positive effect in enhancing two-phase PAC production in 20

mM MOPS system with organic phase PAC concentration of 420 mM with partially purified

PDC (Fig 5.11.a) and a higher concentration of 630 mM organic phase PAC with whole cell

PDC (Fig 5.12.a). Lower concentrations of 40 mM and 60 mM final PAC partitioned into the

aqueous phase for whole cell and partially purified PDC respectively (Figs 5.11.b and

5.12.b).



151

0

500

1000

1500

2000

2500

3000

3500

0 5 10 15 20 25 30

[Ben

zald

ehyd

e]O

RG

(m

M)

0

100

200

300

400

500

600

700

[PA

C]O

RG

, [A

ceta

ldeh

yde]

OR

G,

[Ace

toin

]OR

G (

mM

)

a

0

100

200

300

400

500

600

700

800

900

0 5 10 15 20 25 30

Tim e (h)

[Ben

zald

ehyd

e]A

Q, [

Pyr

uvat

e]A

Q

(mM

)

0

10

20

30

40

50

60

70

[PA

C]A

Q, [

Ace

tald

ehyd

e]A

Q,

[Ace

toin

]AQ

(m

M)

b

Figure 5.11: Effect of 2.5 M dipropylene glycol (DPG) addition on PAC production in the

aqueous/octanol-benzaldehyde emulsion system with 20 mM MOPS and partially purified PDC at

20°C, controlled pH 7.0: (a) organic and (b) aqueous phase substrate, PAC, and by-product

concentration profiles. Organic to aqueous phase volume ratio of 0.25:1. Constant agitation 160 rpm,

initial concentrations: 3.6 M organic phase benzaldehyde, the aqueous phase contained 0.785 M

pyruvate, 3.5 U/mL PDC carboligase activity (C. utilis), 20 mM MOPS buffer, 2.5 M DPG, 1 mM

Mg2+, 1 mM TPP. ORG: organic phase, AQ: aqueous phase. Approximate values for acetaldehyde

concentration due to possible evaporative losses during sampling and analysis. The mean values were

determined from triplicate analyses and error bars show the highest and lowest values.



152

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

3 0 0 0

3 5 0 0

0 5 1 0 1 5 2 0 2 5 3 0

[Ben

zald

ehyd

e]O

RG

(m

M)

0

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

6 0 0

7 0 0

[PA

C]O

RG

, [A

ceta

ldeh

yde]

OR

G,

[Ace

toin

]OR

G (

mM

)

a

0

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

6 0 0

7 0 0

8 0 0

9 0 0

0 5 1 0 1 5 2 0 2 5 3 0

T im e (h )

[Ben

zald

ehyd

e]A

Q, [

Pyr

uvat

e]A

Q

(mM

)

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

[PA

C]A

Q, [

Ace

tald

ehyd

e]A

Q,

[Ace

toin

]AQ

(m

M)

b

Figure 5.12: Effect of 2.5 M dipropylene glycol (DPG) addition on PAC production in the

aqueous/octanol-benzaldehyde emulsion system with 20 mM MOPS and whole cell PDC at

20°C, controlled pH 7.0: (a) organic and (b) aqueous phase concentration profiles. Organic to

aqueous phase volume ratio of 0.25:1. Constant agitation 160 rpm, initial concentrations:

3.65 M organic phase benzaldehyde, the aqueous phase contained 0.83 M pyruvate, 3.5

U/mL PDC carboligase activity (C. utilis), 20 mM MOPS buffer, 2.5 M DPG, 1 mM Mg2+, 1

mM TPP.



153

Biotransformation with whole cell PDC was associated with higher final acetoin

concentrations of 13 mM and 17 mM in the organic and aqueous phase respectively, while

lower organic and aqueous phase concentrations of 3 mM and 4 mM acetoin were formed

with partially purified PDC (Figs 5.11 and 5.12).

5.2.4.2 Discussion

The summarized results for two-phase PAC production in the aqueous/octanol-benzaldehyde

emulsion system with 2.5 M MOPS (Section 5.2.1) are compared to biotransformations with

20 mM MOPS + 2.5 M DPG (Figs 5.11 and 5.12) at 0.25:1 organic to aqueous phase volume

ratio and 20°C in Table 5.4. In comparison to 2.5 M MOPS system with 270 g/L organic

phase PAC, biotransformation with 20 mM MOPS + 2.5 M DPG produced less concentrated

PAC of 63 g/L in the organic phase with much less overall specific PAC production and

productivity when using partially purified PDC. Employment of whole cell PDC in the latter

system resulted in enhanced PAC production with an organic phase concentration of 95 g/L

and approx. 45% increase in overall specific PAC production and productivity compared to

the results with partially purified PDC.

As shown in Figs 5.11 and 5.12, two-phase systems with 20 mM MOPS + 2.5 M DPG in the

aqueous phase and 0.25:1 ratio (20°C) were associated with relatively high initial

benzaldehyde concentrations in the aqueous phase with levels exceeding 100 mM. This

compares to lower initial aqueous phase benzaldehyde concentrations of approx. 50 mM in

system with 2.5 M MOPS at the same phase ratio (20°C). This might have been one of the

factors responsible for the faster PDC deactivation rates in system containing 20 mM MOPS

+ 2.5 M DPG with no remaining enzyme activity after 26 h (partially purified PDC) (Table

5.4) in comparison to retention of approx. 20% residual activity in the 2.5 M MOPS system

(partially purified PDC) (Fig 5.5). Furthermore, Leksawasdi et al. [2005] observed less

protective effect of 2.5 M DPG on the enzyme stability compared to 2.5 M MOPS.



154

Table 5.4: Performance summary: PAC production in the aqueous/octanol-benzaldehyde emulsion

system with 2.5 M MOPS and 20 mM MOPS + 2.5 M DPG at 0.25:1 ratio and 20°C, pH controlled at

7.0.

Two-phase emulsion system

2.5 M MOPS

20 mM MOPS

+ 2.5 M DPG

20 mM MOPS

+ 2.5 M DPG

Reaction period (h)

48

26

26

Type of biocatalyst

Partially Purified

Partially Purified

Whole cell

Initial overall PDC activity

(U/mL carboligase)

2.8

2.8

2.8

Initial aqueous phase PDC activity

(U/mL carboligase)

3.5

3.5

3.5

Final residual PDC activity (%)

20

0

Not determined

Overall PAC (g/L)

77.6

17.6

25.9

Organic PAC (g/L)

271.2

63

94.5

Aqueous PAC (g/L)

29.2

6.3

8.8



27.7

6.3

9.3

Overall volumetric productivity

(g/L/day)

93.1*

16.2

23.9


5.0

0.3

1.5

Overall by-product acetaldehyde

(g/L)**

0.27

0.03

0.02

Y PAC / benz cons (mol/mol)

0.91

0.4***

0.62***

Y PAC / pyr cons (mol/mol)

0.81

0.87

0.7

*The productivity was calculated based on 20 h time point. **Approximate values for acetaldehyde

concentration due to possible losses during sampling and analysis. ***Evaporative benzaldehyde losses of

30 – 40% in these larger and more open system (180 mL).



155

Less acetoin was produced in system with 20 mM MOPS + 2.5 M DPG compared to system

with 2.5 M MOPS. This might due to the relatively high aqueous phase benzaldehyde

concentration and reduction in PDC stability in the former system.

5.3 Conclusion

When compared to a low buffer concentration process (20 mM MOPS), an improved two-

phase aqueous/organic PAC synthesis was achieved by operating the biotransformation with

(1) 20 mM MOPS + 2.5 M DPG in the aqueous phase, (2) lowered organic to aqueous phase

volume ratio of 0.25:1, (3) 20°C temperature, (4) whole cell PDC as biocatalyst, and (5) pH

controlled at 7.0. A product stream containing 95 g/L PAC in the organic phase was

produced in 26 h with an initial enzyme activity of 3.5 U/mL carboligase PDC in the aqueous

phase.

Chapter 6 Final Conclusions and Future Work


156

CHAPTER 6

_____________________________________

FINAL CONCLUSIONS AND FUTURE WORK



157

6.1 Yeast Pyruvate Decarboxylases: Variation in Biocatalytic

Characteristics

Strains of four yeasts Saccharomyces cerevisiae, Candida utilis, Candida tropicalis and

Kluyveromyces marxianus were investigated for their pyruvate decarboxylase (PDC) enzyme

properties with regards to PAC production. In the presence of pyruvate only, the PDCs were

associated with similar decarboxylation and carboligation activities based on acetaldehyde

and acetoin formation. Introduction of benzaldehyde (aqueous system with soluble

benzaldehyde) resulted in appreciable PAC formation and reduced acetaldehyde and acetoin

formation, indicating redirection of carboligation activity towards PAC production for all

enzymes.

Evaluating the enzymes for PAC production with increasing benzaldehyde concentrations in

the three different systems, it was found for relatively high benzaldehyde concentrations that

the highest levels of PAC were formed when using C. utilis PDC. This result is consistent

with the highest stability of C. utilis PDC in the absence and presence of 50 mM of the toxic

benzaldehyde. In terms of enantioselectivity, it has been reported that C. utilis PDC is highly

selective with e.e.values of over 90% for R-PAC formation [Rosche et al., 2001]. C.

tropicalis PDC had the advantage that it was associated with the lowest levels of by-product

acetoin in all tested systems. The trend was further confirmed in the presence of added

acetaldehyde (30 mM) and various benzaldehyde concentrations with the PAC to acetoin

ratio higher for C. tropicalis than C. utilis PDC. The commercially employed S. cerevisiae

PDC was associated with medium PAC levels and the highest acetaldehyde and acetoin

accumulation in all tested systems. On present evidence, S. cerevisiae would not appear to be

the most efficient yeast for enzymatic PAC production.

Based on the investigation, it was evident that C. utilis and C. tropicalis PDCs had the most

valuable properties and this observation may open up future opportunities in PDC protein

engineering for construction of a modified enzyme possessing both desirable properties. In

the present study, C. utilis PDC was selected as the biocatalyst for process development to

enhance PAC production in the two-phase aqueous/organic system.



158

6.2 Factors Affecting PDC Enzyme Deactivation and PAC Production in

Two-Phase Aqueous/Organic System

The present investigation was designed to identify key factors in PDC deactivation and PAC

production in a two-phase aqueous/octanol-benzaldehyde system. It follows earlier reported

studies by Sandford et al. [2005] that while high PAC productivities can be achieved in an

emulsion system, PDC activities could be sustained only in a more slowly stirred phase-

separated process with a lower degree of interphase contact.

It was shown in the present investigation that some degree of PDC deactivation in the two-

phase system was caused by presence of soluble octanol and benzaldehyde in the aqueous

phase. In a further analysis, the effect of aqueous/organic contact area was studied using a

Lewis cell for depiction of the phase-separated system, and it was demonstrated that changes

in interfacial area over a limited range (117 – 475 cm2/L) had no effect on the rate of PDC

deactivation. However, extension to a more rapidly stirred emulsion system with presumably

much greater interfacial area resulted in faster decline of PDC activity. Additionally, a

decreased rate of PDC deactivation was evident at higher PDC concentrations in the

emulsion system.

To enhance both enzyme efficiency and productivity, a two-phase emulsion system was then

developed with reduced deactivating conditions for PDC while maintaining non-limiting

organic-aqueous substrate transfer. Lowering the organic to aqueous phase volume ratio from

1:1 to 0.43:1 at 4°C (2.5 M MOPS buffer system with partially purified PDC) resulted in

12% higher overall PAC (based on the total reaction volume) while maintaining the PDC

activity. The PAC was highly concentrated in the organic phase with 183 g/L in octanol in

comparison to 112 g/L when using the 1:1 ratio. Under these conditions, both overall specific

PAC production and volumetric productivity were increased while enzyme activity was

maintained. The main advantage of this organic phase volume reduction and higher PAC

concentration is that it greatly facilitates the downstream processing and recovery of the

product.



159

6.3 Process Enhancement and Further Kinetic Evaluations for Two-Phase

Aqueous/Organic Synthesis of PAC

Further studies were carried out to improve the two-phase aqueous/organic PAC production

by developing a cost effective process. Lowering the organic to aqueous phase volume ratio

at increased temperature of 20°C (2.5 M MOPS buffer system with partially purifed PDC)

resulted in similar biotransformation patterns as those at 4°C although the higher temperature

was associated with faster reaction rates, increased PDC deactivation, and reduced yields on

pyruvate due to increased acetoin production. Lowering the ratio from 1:1 to 0.43:1 at 20°C

resulted in 29% increase in overall PAC production with an organic phase PAC

concentration of 212 g/L at 0.43:1 in comparison to 111 g/L at 1:1 ratio.

The potential of further two-phase process simplification was evaluated by reducing the

expensive MOPS concentration to 20 mM with pH controlled at 7.0 and employment of

whole cell PDC. It was found that 20°C was the optimum temperature for PAC production in

this system; however at the lower MOPS concentration, lowering the organic to aqueous

phase volume ratio resulted in decreased overall PAC production. Two-phase PAC

production was relatively low in 20 mM MOPS system compared to biotransformations in

2.5 M MOPS system. Addition of 2.5 M dipropylene glycol (DPG) into the aqueous phase in

the former system at 0.25:1 ratio and 20°C improved the PAC production with product

stream containing 95 g/L PAC. Despite its reduced PAC productivity, the system may have

the benefit of a reduction in the production cost as: (1) DPG is ten times cheaper than MOPS

[Sigma catalogue, 2006], (2) utilization of whole cell PDC would mean elimination of the

costly enzyme purification process, (3) operation at 20°C eliminates the necessity for

cooling, and (4) the lower organic phase volume would lead to reduction in the downstream

processing cost.



160

6.4 Recommended Future Work

(a) PDC Enzyme Engineering

Following the characterization of the selected yeast PDCs in the present study (Chapter 3),

protein engineering/PDC mutation techniques could be used to develop a modified PDC with

the following properties: (1) increased activity and stability for PAC production, (2)

reduction of by-product formation particularly acetoin, and (3) increased resistance to

benzaldehyde, PAC, and by-products. With regards to reduced acetoin formation, the studies

with C. tropicalis PDC may suggest ways in which the amino acid sequence of the PDC may

be modified. Furthermore, studies on the amino acid sequence of Zymomonas mobilis PDC,

which exhibits high carboligase activity may indicate how activity of PDC could be

improved.

(b) Evaluation of Different Bioreactor Designs for Two-Phase Aqueous/Organic PAC

Production

It has been shown in the present study (Chapter 4) that PDC deactivation in the two-phase

aqueous/octanol-benzaldehyde system was faster in the emulsion system with high

aqueous/organic interfacial area and agitation rate. To minimize the effect on PDC

deactivation at the aqueous/organic interface, a two-phase membrane reactor could be

designed with the enzyme protected from the organic phase. Alternatively, to decrease the

effect of agitation rate on enzyme deactivation, a liquid-liquid bubble column reactor could

be designed with milder shearing effects on the enzyme while maintaining sufficient

aqueous/organic phase contact.

(c) Mathematical Modelling of the Two-Phase Aqueous/Organic PAC Production

Leksawasdi et al. [2004] has developed and validated a mathematical model to determine the

overall rate constants for PAC, acetaldehyde, and acetoin formation in biotransformation



161

systems with up to 150 mM benzaldehyde, which also includes a term for PDC deactivation

by benzaldehyde. Further mathematical modeling could be carried out for two-phase

aqueous/octanol-benzaldehyde PAC production in the emulsion system in order to identify

optimal operating conditions and ultimately achieve overall process optimization. The

following are several approaches to facilitate the two-phase system modelling:

(1) evaluate the effects of initial benzaldehyde, pyruvate, and enzyme concentrations on the

reaction rate for whole cell PDC as previous studies have been carried out on partially

purified PDC,

(2) determine the rate of PDC deactivation by soluble benzaldehyde in the presence of

octanol in the aqueous phase and also at the aqueous/organic interface, as experiments

reported in Chapter 4 indicated that soluble octanol (and possibly also interfacial

interactions) affect PDC deactivation kinetics,

(3) include a term for the rate of mass transfer of benzaldehyde from the organic to the

aqueous phase and determine the effects of agitation and organic phase benzaldehyde

concentration on this rate in scale-up studies,

(4) include terms for mass transfer of PAC and possibly by-products from the aqueous to the

organic phase and also determine the effects of agitation and product concentrations on mass

transfer rates.

(d) Economic Analysis on Two-Phase Aqueous/Organic PAC Production

Following the characterization and development of the two-phase process in the present

study, an economic evaluation could be perfomed, including a sensitivity analysis, to

determine the economic feasibility of PAC production by the present two-phase process. The

study would take account of the cost of input materials, PAC production and product

recovery costs as well as yields of product on the two substrates. The following are several



162

suggested experiments which could be carried out in association with this economic

evaluation:

(1) investigate possible employment of other organic solvents for reducing cost of product

recovery and solvent recycling,

(2) determine optimum buffer concentration to reduce the costs of addition of expensive

buffers (such as MOPS) while maintaining PDC activity and stability,

(3) determine the optimum organic to aqueous phase volume ratio and temperature to further

minimize production costs,

(4) determine the economic benefit of using an enhanced yeast PDC (via protein engineering)

with lowered by-product formation (particularly acetoin) to ease product recovery and

increase yields on pyruvate.

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Appendix A


177

APPENDIX A

Factors Affecting Organic-Aqueous Benzaldehyde Transfer in the Two-

Phase Aqueous/Octanol-Benzaldehyde System

An investigation was conducted to identify the key factors affecting benzaldehyde transfer

from the organic to the aqueous phase in the aqueous/octanol-benzaldehyde system.

Abbreviations

Table A.1: Description on the mass transfer rate equations.

Term Description

dBZD/dt Organic-aqueous benzaldehyde transfer rate

OBZD Benzaldehyde concentration in the bulk organic phase

ABZD Benzaldehyde concentration in the bulk aqueous phase

OBZDint Benzaldehyde concentration at the interface in the organic phase

ABZDint Benzaldehyde concentration at the interface in the aqueous phase

OBZD* Benzaldehyde concentration in the organic phase in equilibrium with

the concentration of benzaldehyde in the bulk aqueous phase

ABZD* Benzaldehyde concentration in the aqueous phase in equilibrium with

the concentration of benzaldehyde in the bulk organic phase

kO (Local) organic phase mass transfer coefficient

kA (Local) aqueous phase mass transfer coefficient

K O Overall mass transfer coefficient related to the organic phase driving

force

K A Overall mass transfer coefficient related to the aqueous phase driving

force

aint Specific interfacial area (organic phase contact area to aqueous phase

volume ratio)

t Time

Appendix A


178

A.1 Benzaldehyde Transfer across a Phase Boundary

The transfer of benzaldehyde from the organic to the aqueous phase involves transport of

solute across a phase boundary. The transfer rate depends on several factors such as the

organic phase benzaldehyde concentration, the aqueous phase benzaldehyde solubility, and

the design of the mass transfer equipment which accounts for the effects of the

aqueous/organic interfacial area, temperature, and pressure of the process [Hines and

Maddox, 1985]. The route of organic-aqueous phase benzaldehyde transfer is illustrated in

Fig A.1.

Organic phase Aqueous phase

Interface

OBZD

Bulk Thin film BulkThin film

OBZDint

A BZDint

A BZD

Organic phase Aqueous phase

Interface

OBZD

Bulk Thin film BulkThin film

OBZDint

A BZDint

A BZD

Figure A.1: Concentration profiles across an aqueous/organic interface [Hines and Maddox,

1985].

The following are the mass transfer rate equations with the description on the abbreviations

shown in Table A.1.

Appendix A


179

The mass transfer rate is directly proportional to the concentration gradient by means of an

empirical mass transfer coefficient. At steady-state, the benzaldehyde transfer rate through

the organic phase is equal to the rate in the aqueous phase. The organic-aqueous

benzaldehyde transfer rate can be described by the following equation:

Equation A.1 describes the transfer rate in terms of local mass transfer coefficient; the

drawback of such correlation is that the concentration gradient is expressed in terms of

interfacial compositions, which are not usually possible to determine in an experimental

apparatus [Hines and Maddox, 1985]. This is encountered by introducing an overall mass

transfer coefficient such that the concentration gradient is now expressed in terms of

equilibrium compositions (marked by *) (Equation A.2).

The aqueous phase mass transfer rate equation was chosen as the basis for this particular

study on benzaldehyde transfer since PAC biosynthesis takes place in the aqueous phase.

Integrating Equation A.2 (aqueous phase) and setting initial condition at t = 0, ABZD(0) = 0,

resulted in Equation A.3:

By using the saturation profiles and the saturation concentration of benzaldehyde (the

equilirium composition is equal to the saturation composition), the term KA aint was the slope

obtained by plotting ln (ABZD* / (ABZD* - ABZD)) as a function of time. The term KA aint

o rg a n ic p h a s e a q u e o u s p h a s e

o rg a n ic p h a s e a q u e o u s p h a s e

d B Z D (t)

d t= K O ain t (O B Z D ( t) – OB Z D * ) = K A ain t (A B Z D * – A B Z D ( t))

d B Z D (t)

d t= K O ain t (O B Z D ( t) – OB Z D * ) = K A ain t (A B Z D * – A B Z D ( t))

l n (A B Z D * / (A B Z D * - A B Z D ( t ) ) ) = K A a in t t

A.1

A.2

A.3

d B Z D ( t)

d t= kO ain t (O B Z D ( t) – OB Z D in t) = kA ain t (A B Z D in t – A B Z D ( t) )

d B Z D ( t)

d t= kO ain t (O B Z D ( t) – OB Z D in t) = kA ain t (A B Z D in t – A B Z D ( t) )

Appendix A


180

allowed evaluation on the organic-aqueous benzaldehyde transfer characteristics among

various systems.

A.2 Effects of Aqueous/Organic Interfacial Area, Organic Phase Benzaldehyde

Concentration and Temperature

A Lewis cell with an organic and aqueous phase layers (each 90 mL) was employed to

investigate the effects of several physical parameters on organic-aqueous benzaldehyde

transfer: (1) ratio of organic phase contact area to aqueous phase volume, (2) organic phase

benzaldehyde concentration, and (3) temperature.

Figs A.2.a and b present the benzaldehyde saturation profiles for the investigated parameters.

In spite of the fact that similar saturation concentrations of aqueous phase benzaldehyde of

approx. 50 mM were achieved under the experimental conditions, there were distinguishable

differences in the transfer rates: higher ratio of organic phase contact area to aqueous phase

volume, higher organic phase benzaldehyde concentration, and higher temperature resulted in

increased transfer rates.

The effects were quantified by comparing the slopes (KA aint) on Figs A.3.a and b, of which

values were determined for the first 15 mins to depict maximum transfer rates. Referring to

Table A.2, increasing the ratio of organic phase contact area to aqueous phase volume from

117 to 361 cm2/L, the organic phase benzaldehyde concentration from 1.5 M to 2.5 M, and

the temperature from 4°C to 20°C resulted in approximately twice the KA aint values.

Appendix A


181

AB

ZD

(mM

)

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

0 6 0 1 2 0 1 8 0 2 4 0 3 0 0 3 6 0

aA

BZ

D(m

M)

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

0 6 0 1 2 0 1 8 0 2 4 0 3 0 0 3 6 0

AB

ZD

(mM

)

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

0 6 0 1 2 0 1 8 0 2 4 0 3 0 0 3 6 0

a

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

0 6 0 1 2 0 1 8 0 2 4 0 3 0 0 3 6 0

T im e (m in )

AB

ZD

(mM

)

b

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

0 6 0 1 2 0 1 8 0 2 4 0 3 0 0 3 6 0

T im e (m in )

AB

ZD

(mM

)

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

0 6 0 1 2 0 1 8 0 2 4 0 3 0 0 3 6 0

T im e (m in )

AB

ZD

(mM

)

b

Figure A.2: Saturation profiles: effects of physical parameters on organic-aqueous benzaldehyde

transfer in the two-phase aqueous/octanol-benzaldehyde system: (a) ratio of organic phase contact

area to aqueous phase volume, organic phase benzaldehyde concentration and (b) temperature. 117

and 361 cm2/L organic phase contact area to aqueous phase volume ratios, 1.5 M and 2.5 M organic

phase benzaldehyde concentrations, 4°C and 20°C temperatures, 60 rpm and 125 rpm agitation for

organic and aqueous phase respectively in Lewis cell, 2.5 M MOPS buffer (pH 7.0).

Appendix A


182

0

1

2

3

4

0 5 10 15 20

ln (

[AB

ZD]*

/ ([A

BZ

D]*

- [A

BZ

D]))

a

0

1

2

3

4

0 5 10 15 20

Time (m in)

ln (

[AB

ZD]*

/ ([

AB

ZD]*

- [A

BZ

D])

)

b

Figure A.3: Plot of ln (ABZD* / (ABZD*- A BZD)) as a function of time with slope KA aint:

effects of physical parameters on organic-aqueous benzaldehyde transfer in the two-phase

aqueous/octanol-benzaldehyde system: (a) ratio of organic phase contact area to aqueous

Appendix A


183

phase volume, organic phase benzaldehyde concentration and (b) temperature. Calculated

from data in experiments shown in Fig A.2.

Table A.2: KA aint and KA values comparison: effects of physical parameters on organic-

aqueous benzaldehyde transfer in two-phase aqueous/octanol-benzaldehyde system.

System

Temperature

(°C)

aint

[Benzaldehyde]ORG

(M)

KA aint

(h-1)

KA

(m/h)

cm2/L

m2/m3

I

20

361

36.1

2.5

11.45

0.32

II

20

361

36.1

1.5

5.11

0.14

III

20

117

11.7

2.5

5.32

0.46

IV

20

117

11.7

1.5

2.66

0.23

V

5

361

36.1

2.5

4.73

0.13

* Maximum transfer rates (first 15 mins)

A.3 Conclusions

The present study confirms the increased rates of organic-aqueous benzaldehyde transfer in the

two-phase aqueous/octanol-benzaldehyde system with increased ratio of organic phase contact

area to aqueous phase volume, organic phase benzaldehyde concentration, and temperature.

Appendix B


184

APPENDIX B

Effect of Low Octanol : Aqueous Phase Volumes on Whole Cell

Biotransformation for PAC Production

It was evident from the present study (Chapters 4 and 5) that employment of a reduced

octanol phase volume (0.25:1 to 1:1) resulted in improved PAC production in the two-phase

aqueous/octanol-benzaldehyde emulsion system at 4°C and 20°C. Prior to this investigation,

a preliminary study was conducted on the effect of octanol on PAC formation at 4°C and

20°C by creating systems, which are between those of the aqueous/benzaldehyde emulsion

and the two-phase aqueous/octanol-benzaldehyde emulsion system. In addition, employment

of whole cell preparation provides an alternative low cost and efficient biocatalyst for PAC

production [Satianegara et al., 2006; Rosche et al., 2005].

B.1 PAC and By-Product Formation at 4°°°°C

The effect of increasing octanol concentration on PAC formation at 4°C is shown in Fig B.1.

The rates of PAC formation were faster when adding 50 – 700 mM octanol into the octanol-

free aqueous/benzaldehyde emulsion system, however the formation rates dropped when

further increasing the octanol concentration to 2600 mM, creating a two phase

aqueous/octanol-benzaldehyde emulsion system with 1:1 phase volume ratio. The rates of

PAC formation declined overtime, although it was expected that the biotransformations could

proceed beyond 48 h. Neither substrate was limiting in any experiments.

After 48 h, systems with 300 mM, 500 mM and 700 mM octanol were associated with the

highest PAC concentrations of 340 – 350 mM followed by systems with 50 mM and 100 mM

octanol with 290 mM and 310 mM respectively. The octanol-free aqueous/benzaldehyde

emulsion system along with the 1:1 two-phase emulsion system with 2600 mM octanol

Appendix B


185

formed the least concentration of 225 mM (per total reaction volume by combining both

phases (TRV) for the 1:1 two-phase system) (Fig B.1). [P

AC

] (m

M)

0

50

100

150

200

250

300

350

400

0 10 20 30 40 50 60

0 mM

2600 mM

50 – 700 mM

Time (h)

[PA

C] (

mM

)

0

50

100

150

200

250

300

350

400

0 10 20 30 40 50 600 10 20 30 40 50 60

0 mM

2600 mM

50 – 700 mM

Time (h)


concentrations: 0, 50, 100, 300, 500, 700 and 2600 mM octanol, 725 – 810 mM

benzaldehyde, 735 – 785 mM pyruvate, 2.8 U/mL PDC carboligase activity (C. utilis whole

cells), 2.5 M MOPS, 1 mM Mg2+, 1 mM TPP. For the 1:1 two-phase emulsion system with

2600 mM octanol, all concentrations were given per total reaction volume by combining both

phases (TRV). The mean values were determined from triplicate analyses and error bars

show the highest and lowest values.

The trends of by-products acetaldehyde and acetoin formation were similar to those for PAC

formation: increasing in systems with 0 – 700 mM octanol with concentrations between 1 –

6.7 mM and 1.8 – 5.5 mM for acetaldehyde and acetoin respectively; then lower in the 1:1

two-phase emulsion system with 2600 mM octanol with 2 mM and 2.7 mM TRV for

acetaldehyde and acetoin respectively (Fig B.2).

Appendix B


186

0

1

2

3

4

5

6

7

8

0 50 100 300 500 700 2600

[Octanol] (mM)

[ace

tald

ehyd

e], [

acet

oin]

(m

M)


Figure B.2: Effect of octanol addition on by-products acetaldehyde and acetoin formation at

4°C, initial pH 7.0 (48 h). Same experiments as shown in Fig B.1. Approximate values for

acetaldehyde concentrations due to possible evaporative losses during sampling and analysis.

The results in Fig B.1 summarized in Table B.1 show that adding up to 700 mM octanol into

the aqueous/benzaldehyde emulsion system at 4°C resulted in: (1) increasing PAC formation,

(2) higher specific PAC production and volumetric productivities, and (3) evidence of

increase in acetaldehyde and acetoin concentrations. Increasing the octanol concentration to

2600 mM (1:1 two-phase emulsion) resulted in lower overall PAC and by-product formation.

The substrate molar balances closed within 9% for all systems.

Appendix B


187

Table B.1: Performance summary: effect of octanol addition on PAC formation at 4°C,

initial pH 7.0 (48 h).

[Octanol] (mM)

0

50

100

300

500

700

2600*

Specific PAC production


12

15.6

16.7

18.5

18.7

18.3

11.9

Volumetric productivity (g/L/day)

16.8

21.9

23.3

25.9

26.2

25.6

16.7



0.69

0.89

0.89

0.9

0.9

0.9

0.73


(mol/mol)

0.87

0.74

0.75

0.77

0.77

0.77

0.66


91

98

98

98

98

98

94


98

94

94

98

98

97

92

Calculated from data in experiments shown in Fig B.1. *1:1 two-phase emulsion system: all

results were given per total reaction volume by combining both phases (TRV).

B.2 PAC and By-Product Formation at 20°°°°C

The effect of increasing octanol concentration on PAC formation at 20°C is shown in Fig

B.3. The reactions were completed after 20 – 30 h with neither substrate was limiting. The

rates of PAC formation were faster at 20°C than 4°C with higher rates for systems with 50 –

700 mM octanol for the first 10 h, while the octanol-free aqueous/benzaldehyde emulsion

system and the 1:1 two-phase aqueous/octanol-benzaldehyde emulsion system with 2600

mM octanol were associated with higher formation rates at 10 – 20 h.

At 48 h, systems with 500 mM and 700 mM octanol produced the highest PAC

concentrations of 350 mM followed by systems with 0 – 300 mM octanol with 310 – 320

mM and the 1:1 two-phase emulsion system with 2600 mM octanol was associated with the

Appendix B


188

least formation of 290 mM (per total reaction volume by combining both phases (TRV) (Fig

B.3).

Increase in PAC formation was associated with increase in acetaldehyde and acetoin

formation: increasing in systems with 0 – 700 mM octanol with levels between 2 – 8.3 mM

and 11 – 24 mM for acetaldehyde and acetoin respectively; then lower in the 1:1 two-phase

system with 2600 mM octanol with 4.5 mM and 15.5 mM TRV for acetaldehyde and acetoin

respectively (Fig B.4). Furthermore, acetaldehyde and acetoin concentrations were

appreciably higher at 20°C than at 4°C.

[PA

C] (

mM

)

0

50

100

150

200

250

300

350

400

0 10 20 30 40 50 60Time (h)

2600 mM

0 mM

50 - 700 mM

[PA

C] (

mM

)

0

50

100

150

200

250

300

350

400

0 10 20 30 40 50 600 10 20 30 40 50 60Time (h)

2600 mM

0 mM

50 - 700 mM


concentrations: 0, 50, 100, 300, 500, 700 and 2600 mM octanol, 725 – 760 mM

benzaldehyde, 710 – 770 mM pyruvate, 2.8 U/mL PDC carboligase activity (C. utilis whole

cells), 2.5 M MOPS, 1 mM Mg2+, 1 mM TPP. For the 1:1 two-phase emulsion system with

2600 mM octanol, all concentrations were given per total reaction volume by combining both

phases (TRV).

Appendix B


189

0

5

10

15

20

25

30

0 50 100 300 500 700 2600

[Octanol] (mM)

[ace

tald

ehyd

e], [

acet

oin]

(m

M)


Figure B.4: Effect of octanol addition on by-products acetaldehyde and acetoin formation at

20°C, initial pH 7.0 (48 h). Same experiments as shown in Fig B.3. Approximate values for

acetaldehyde concentrations due to possible evaporative losses during sampling and analysis.

The results in Fig B.3 summarized in Table B.2 show that adding up to 700 mM octanol into

the aqueous/benzaldehyde emulsion system at 20°C resulted in: (1) increasing PAC

formation, (2) higher specific PAC production and volumetric productivities, and (3)

evidence of increase in acetaldehyde and acetoin concentrations. Increasing the octanol

concentration to 2600 mM (1:1 two-phase emulsion) resulted in lower overall PAC and by-

product formation. The substrate molar balances closed within 15% for all systems.

In comparison to 4°C, biotransformations at 20°C resulted in significantly higher PAC

volumetric productivities at all octanol concentrations from 0 – 2600 mM. The PAC yields

on consumed benzaldehyde were 70 – 90% and 60 – 70% theoretical at 4°C and 20°C

respectively due to increasingly high evaporative losses of benzaldehyde at 20°C, while those

Appendix B


190

on pyruvate were 70 – 85% theoretical at 4°C and lower at 20°C due to increased acetoin

formation (Tables B.1 and B.2).

Table B.2: Performance summary: effect of octanol addition on PAC formation at 20°C,

initial pH 7.0 (48 h).

[Octanol] (mM)

0

50

100

300

500

700

2600*

Specific PAC production


16.5

16.8

17.1

16.6

19

18.8

15.4

Volumteric productivity (g/L/day)**

44.4

45.1

45.9

44.6

51

50.5

41.4



0.64

0.61

0.59

0.57

0.69

0.7

0.6


(mol/mol)

0.75

0.62

0.65

0.6

0.66

0.64

0.57


90

87

85

82

92

94

88


90

87

88

86

85

87

85

Calculated from data in experiments shown in Fig B.3. * 1:1 two-phase emulsion system: all

results were given per total reaction volume by combining both phases (TRV). ** Based on

approximate reaction completion at 25 h.

B.3 Discussion and Conclussion

At both 4°C and 20°C, adding octanol up to 700 mM into the octanol-free

aqueous/benzaldehyde emulsion system with employment of whole cell preparation of PDC

resulted in improved PAC production, with the increase being more pronounced at the lower

temperature. Further increasing the octanol concentration to 2600 mM by creating a 1:1 two-

phase aqueous/octanol-benzaldehyde emulsion system resulted in less PAC production at

Appendix B


191

4°C and 20°C. Increases in acetoin formation were observed with increases in PAC, with the

increase being greater at 20°C.

With respect to the aqueous/benzaldehyde emulsion system, octanol addition might have

lowered the degree of benzaldehyde droplet/ PDC enzyme interaction, which would give rise

to less deactivation and thereby increased PAC formation. Sandford et al. [2005] observed

that containment of the high benzaldehyde concentration in octanol in the 1:1

aqueous/organic two-phase systems resulted in a lower degree of PDC deactivation in

comparison to the octanol-free aqueous/benzaldehyde emulsion system. Sandford et al.

[2005] also studied the effect of octanol on PAC production in the aqueous (soluble

benzaldehyde) system with 50 mM benzaldehyde and found that the rate of PAC formation

in the octanol-saturated biotransformation buffer was 46% higher at 50 mM PAC/h than

without octanol at 34 mM PAC/h.

In analysing the systems with less octanol against the 1:1 two-phase emulsion system with

2600 mM octanol, the lower octanol systems were expected to have maintained higher

benzaldehyde concentrations in the aqueous phase due to enhanced transfer of benzaldehyde

into the aqueous phase, which would therefore result in higher PAC production.

Finally, it was demonstrated from the present investigation that addition of octanol resulted

in improved PAC production in the aqueous/benzaldehyde emulsion system. The amount of

octanol added can be seen as a balance between the necessity to provide the PDC enzyme

with sufficient protective effect from direct exposure to toxic benzaldehyde while

maintaining adequate benzaldehyde transfer to the aqueous phase.

Appendix C


192

APPENDIX C Effect of Organic to Aqueous Phase Volume Ratio on PAC Production in

the Aqueous/Octanol-Benzaldehyde System (2.5 M MOPS)

In addition to the concentration profiles of substrates, PAC, and by-products in the organic

and aqueous phases shown in Chapters 4 and 5, Appendix C presents the overall

concentration profiles based on total reaction volume by combining both phases for

comparative purposes (Figs C.1 and C.2). The results demonstrate that lowering the organic

to aqueous phase volume ratio from 1:1 to 0.43:1 in the 2.5 M MOPS system increased the

PAC formation at 4°C and 20°C. Further reducing the phase volume ratio to 0.25:1 at 4°C

resulted in even higher PAC concentration, however the production was slightly reduced at

20°C with this ratio.

Appendix C


193

Figure C.1: Effect of organic to aqueous phase volume ratio on PAC production in the two-phase aqueous/octanol-benzaldehyde

emulsion system at 4°C, initial pH 6.5: overall substrate, PAC and by-product concentration profiles. Same experiments as shown in Figs

4.6 – 4.9.

0

1 00

2 00

3 00

4 00

5 00

6 00

7 00

8 00

0 10 2 0 30 40 50

[Ben

zald

ehyd

e]T

RV

, [P

yruv

ate]

TR

V,

[PA

C]T

RV

(m

M)

0

5

10

15

20

25

30

35

[Ace

tald

ehyd

e]T

RV

, [A

ceto

in]T

RV

(m

M)

0

100

200

300

400

500

600

700

800

0 10 20 30 40 50

[Ben

zald

ehyd

e]T

RV

, [P

yruv

ate]

TR

V,

[PA

C]T

RV

(m

M)

0

5

10

15

20

25

30

35

[Ace

tald

ehyd

e]T

RV

, [A

ceto

in]T

RV

(m

M)

0

100

200

300

400

500

600

700

800

0 10 20 30 40 50

[Ben

zald

ehyd

e]T

RV

, [P

yruv

ate]

TR

V,

[PA

C]T

RV

(m

M)

0

5

10

15

20

25

30

35

[Ace

tald

ehyd

e]T

RV

, [A

ceto

in]T

RV

(m

M)

0

100

200

300

400

500

600

700

800

0 10 20 3 0 40 50

[Ben

zald

ehyd

e]T

RV

, [P

yruv

ate]

TR

V,

[PA

C]T

RV

(m

M)

0

5

10

15

20

25

30

35

[Ace

tald

ehyd

e]T

RV

, [A

ceto

in]T

RV

(m

M)

I. 1:1

III. 0.43:1

II. 0.67:1

IV. 0.25:1

Time (h) Time (h)

Appendix C


194

Figure C.2: Effect of organic to aqueous phase volume ratio on PAC production in the aqueous/octanol-benzaldehyde emulsion system at

20°C, initial pH 6.5: overall substrate, PAC and by-product concentration profiles. Same experiments as shown in Figs 5.1 – 5.4.

0

100

200

300

400

500

600

700

800

0 10 20 30 40 50

[Ben

zald

ehyd

e]T

RV

,

[Pyr

uvat

e]T

RV

, [P

AC

]TR

V (

mM

)

0

10

20

30

40

50

60

70

80

[Ace

tald

ehyd

e]T

RV

, [A

ceto

in]T

RV

(mM

)

0

100

200

300

400

500

600

700

800

0

100

200

300

400

500

600

700

800

0 10 20 30 40 500 10 20 30 40 50

[Ben

zald

ehyd

e]T

RV

,

[Pyr

uvat

e]T

RV

, [P

AC

]TR

V (

mM

)

[Ben

zald

ehyd

e]T

RV

,

[Pyr

uvat

e]T

RV

, [P

AC

]TR

V (

mM

)

0

10

20

30

40

50

60

70

80

0

10

20

30

40

50

60

70

80

[Ace

tald

ehyd

e]T

RV

, [A

ceto

in]T

RV

(mM

)

[Ace

tald

ehyd

e]T

RV

, [A

ceto

in]T

RV

(mM

)

0

100

200

300

400

500

600

700

800

0 10 20 30 40 50

[Ben

zald

ehyd

e]TR

V,

[Pyr

uvat

e]T

RV

, [P

AC

]TR

V (

mM

)

0

10

20

30

40

50

60

70

80

[Ace

tald

ehyd

e]T

RV

, [A

ceto

in]T

RV

(mM

)

0

100

200

300

400

500

600

700

800

0

100

200

300

400

500

600

700

800

0 10 20 30 40 500 10 20 30 40 50

[Ben

zald

ehyd

e]TR

V,

[Pyr

uvat

e]T

RV

, [P

AC

]TR

V (

mM

)

[Ben

zald

ehyd

e]TR

V,

[Pyr

uvat

e]T

RV

, [P

AC

]TR

V (

mM

)

0

10

20

30

40

50

60

70

80

0

10

20

30

40

50

60

70

80

[Ace

tald

ehyd

e]T

RV

, [A

ceto

in]T

RV

(mM

)

[Ace

tald

ehyd

e]T

RV

, [A

ceto

in]T

RV

(mM

)

0

100

200

300

400

500

600

700

800

0 10 20 30 40 50

[Ben

zald

ehyd

e]T

RV

,

[Pyr

uvat

e]T

RV

, [P

AC

]TR

V (

mM

)

0

10

20

30

40

50

60

70

80

[Ace

tald

ehyd

e]T

RV

, [A

ceto

in]T

RV

(mM

)0

100

200

300

400

500

600

700

800

0 10 20 30 40 500 10 20 30 40 50

[Ben

zald

ehyd

e]T

RV

,

[Pyr

uvat

e]T

RV

, [P

AC

]TR

V (

mM

)

[Ben

zald

ehyd

e]T

RV

,

[Pyr

uvat

e]T

RV

, [P

AC

]TR

V (

mM

)

0

10

20

30

40

50

60

70

80

[Ace

tald

ehyd

e]T

RV

, [A

ceto

in]T

RV

(mM

)

[Ace

tald

ehyd

e]T

RV

, [A

ceto

in]T

RV

(mM

)0

100

200

300

400

500

600

700

800

0 10 20 30 40 50

[Ben

zald

ehyd

e]T

RV

, [P

yruv

ate]

TR

V, [

PA

C]T

RV

(m

M)

0

10

20

30

40

50

60

70

80

[Ace

tald

ehyd

e]T

RV

, [A

ceto

in]T

RV

(m

M)

0

100

200

300

400

500

600

700

800

0

100

200

300

400

500

600

700

800

0 10 20 30 40 500 10 20 30 40 50

[Ben

zald

ehyd

e]T

RV

, [P

yruv

ate]

TR

V, [

PA

C]T

RV

(m

M)

[Ben

zald

ehyd

e]T

RV

, [P

yruv

ate]

TR

V, [

PA

C]T

RV

(m

M)

0

10

20

30

40

50

60

70

80

0

10

20

30

40

50

60

70

80

[Ace

tald

ehyd

e]T

RV

, [A

ceto

in]T

RV

(m

M)

[Ace

tald

ehyd

e]T

RV

, [A

ceto

in]T

RV

(m

M)

Time (h) Time (h)

III. 0.43:1 IV. 0.25:1

I. 1:1 II. 0.67:1

Appendix D


195

APPENDIX D

The experimental data given in Appendix D are additional to that given in Chapter 5 on two-

phase aqueous/organic PAC synthesis with reduced MOPS concentration of 20 mM MOPS

and pH controlled at 7.0. The data shows the concentration profiles of substrates, PAC, and

by-products per total reaction volume by combining both phases.

D.1 Effect of Changing the Organic to Aqueous Phase Volume Ratio on

Two-Phase Aqueous/Organic PAC Production with 20 mM MOPS at 20°°°°C

The results illustrate that reducing the organic to aqueous phase volume ratio is not

advantageous for PAC formation in the low MOPS buffer system at 20°C presumably due to

higher rate of PDC deactivation (Figs D.1 and D.2).

0

20

40

60

80

100

120

140

0 10 20 30 40

Time (h)

[PA

C]T

RV

(m

M)

1 to 1 0.667 to 1 0.428 to 1 0.25 to 1

Figure D.1: Effect of organic to aqueous phase volume ratio on PAC production in the

aqueous/octanol-benzaldehyde emulsion system with 20 mM MOPS at 20°C, controlled pH 7.0:

overall concentration profiles are shown. Same experiments as shown in Fig 5.6. TRV: total

reaction volume by combining both phases. The mean values were determined from triplicate

analyses and error bars show the highest and lowest values.

Appendix D


196

0

3

6

9

12

15

'1:1' '0.67:1' '0.43:1' '0.25:1'Organic : Aqueous

[Ace

tald

ehyd

e]T

RV

, [A

ceto

in]T

RV

(m

M)


Figure D.2: Effect of organic to aqueous phase volume ratio on by-products acetaldehyde and acetoin formation in the aqueous/octanol-benzaldehyde emulsion system with 20 mM MOPS at 20°C, controlled pH 7.0: overall concentrations. Same experiments as shown in Fig 5.6. Approximate values for acetaldehyde concentration due to possible evaporative losses during sampling and analysis.

0

1

2

3

4

5

6

0 10 20 30 40

T im e (h )

Aci

d (m

L)

Figure D.3: Effect of organic to aqueous phase volume ratio in the aqueous/octanol-benzaldehyde emulsion system with 20 mM MOPS at 20°C, controlled pH 7.0: acid addition profiles. Same experiments as shown in Fig 5.6.

1 : 1 0 .6 7 : 1 0 .4 3 : 1 0 .2 5 : 11 : 1 0 .6 7 : 10 .6 7 : 1 0 .4 3 : 10 .4 3 : 1 0 .2 5 : 10 .2 5 : 1

Appendix D


197

D.2 Effect of Increasing Temperature on Two-Phase Aqueous/Organic

PAC Production with 20 mM MOPS

It was observed that 20°C was the optimum operating temperature for two-phase PAC

production in the 20 mM MOPS system as it was associated with the highest final PAC

concentration and volumetric productivity.

Time (h)

[PA

C]T

RV

(m

M)

0

20

40

60

80

100

120

140

0 10 20 30 40 50 60 70Time (h)

[PA

C]T

RV

(m

M)

0

20

40

60

80

100

120

140

0

20

40

60

80

100

120

140

0 10 20 30 40 50 60 70

4 °°°°C 10 °°°°C 1 5 °°°°C 2 0 °°°°C 25 °°°°C 30 °°°°C 35 °°°°C4 °°°°C4 °°°°C 10 °°°°C10 °°°°C 1 5 °°°°C1 5 °°°°C 2 0 °°°°C2 0 °°°°C 25 °°°°C25 °°°°C 30 °°°°C30 °°°°C 35 °°°°C35 °°°°C

Figure D.4: Effect of temperature on PAC production in the aqueous/octanol-benzaldehyde

emulsion system with 20 mM MOPS, controlled pH 7.0: overall concentration profiles.

TRV: total reaction volume by combining both phases. Same experiments as shown in Fig

5.8. The mean values were determined from triplicate analyses and error bars show the

highest and lowest values.

Appendix D


198

0

3

6

9

12

15

5 10 15 20 25 30 35

T (C)

[ace

tald

ehyd

e]T

RV

, [ac

etoi

n]T

RV

(m

M)


Figure D.5: Effect of temperature on PAC production in the 1:1 two-phase aqueous/octanol-benzaldehyde emulsion system: final overall by-product acetaldehyde and acetoin formation. Same experiments as shown in Fig 5.8. Approximate values for acetaldehyde concentrations presumably due to evaporative losses during sampling and analysis.

0

1

2

3

4

5

6

0 10 20 30 40 50 60 70

Time (h)

Aci

d (m

L)

Figure D.6: Effect of temperature on PAC production in the 1:1 two-phase aqueous/octanol-benzaldehyde emulsion system: acid addition profiles are shown. Same experiments as shown in Fig 5.8.

4°°°°C 10°°°°C 15°°°°C 20°°°°C 25°°°°C 30°°°°C 35°°°°C4°°°°C 10°°°°C 15°°°°C 20°°°°C 25°°°°C 30°°°°C 35°°°°C

Appendix D


199

D.3 Effect of Dipropylene Glycol (DPG) as Additive on Two-Phase Aqueous/Organic PAC Production with 20 mM MOPS at 20°°°°C and 0.25:1 Organic to Aqueous Phase Volume Ratio Addition of 2.5 M DPG into the aqueous phase in the 20 mM MOPS system at 20°C and 0.25:1 phase volume ratio enhanced the PAC production. Moreover, employment of whole cell PDC resulted in higher formation.

0

100

200

300

400

500

600

700

800

0 5 10 15 20 25 30

[Ben

zald

ehyd

e]TR

V, [

Pyr

uvat

e]TRV (m

M)

0

50

100

150

200

[PAC

]TR

V, [

acet

alde

hyde

]TR

V, [

acet

oin]

TR

V

(mM

)

a

0

100

200

300

400

500

600

700

800

0 5 10 15 20 25 30

Time (h)

[Ben

zald

ehyd

e]TRV,

[Pyr

uvat

e]TR

V (m

M)

0

50

100

150

200

[PAC

]TRV, [

acet

alde

hyde

]TRV,

[ace

toin

]TRV (m

M)

b

Figure D.7: Effect of 2.5 M dipropylene glycol (DPG) addition on PAC production in the aqueous/octanol-benzaldehyde emulsion system with 20 mM MOPS at 20°C, controlled pH 7.0: overall concentration profiles of substrates, PAC and by-products are shown: (a) partially purified PDC and (b) whole cell PDC. Organic to aqueous phase volume ratio of 0.25:1. Same experiments as shown in Figs 5.11 and 5.12. TRV: total reaction volumeby combining both phases. The mean values were determined from triplicate analyses and error bars show the highest and lowest values.

Appendix D


200

0

1

2

3

4

5

6

0 5 10 15 20 25 30

Time (h)

Aci

d (m

L)

partially purified PDC whole cell PDC


aqueous/octanol-benzaldehyde emulsion system with 20 mM MOPS at 20°C, controlled pH

7.0: acid addition profiles are shown. Organic to aqueous phase volume ratio of 0.25:1. Same

experiments as shown in Figs 5.11 and 5.12.

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