ARTICLE

Bubble-Free Oxygenation of a Bi-EnzymaticSystem: Effect on Biocatalyst Stability

Wouter Van Hecke,1 Roland Ludwig,2,3 Jo Dewulf,1 Markus Auly,2 Tom Messiaen,1

Dietmar Haltrich,1 Herman Van Langenhove1

1Research Group Environmental Organic Chemistry and Technology (ENVOC),

Faculty of Bioscience Engineering, Department of Organic Chemistry, Ghent University,

Coupure Links 653, B-9000 Ghent, Belgium; telephone: þ32-9-264-59-00;

fax: þ32-9-264-62-45; e-mail: herman.vanlangenhove@ugent.be2Division of Food Biotechnology, Department of Food Science and Technology, BOKU,

University of Natural Resources and Applied Life Sciences Vienna, Wien, Austria3Research Centre Applied Biocatalysis, Petersgasse, Graz, Austria

Received 29 January 2008; revision received 6 May 2008; accepted 24 June 2008

Published online 18 July 2008 in Wiley InterScience (www.interscience.wiley.com). D

OI 10.1002/bit.22042

ABSTRACT: The effect of bubble-free oxygenation on thestability of a bi-enzymatic system with redox mediatorregeneration for the conversion of lactose to lactobionicacid was investigated in a miniaturized reactor with bubble-less oxygenation. Earlier investigations of this biocatalyticoxidation have shown that the dispersive addition of oxygencan cause significant enzyme inactivation. In the processstudied, the enzyme cellobiose dehydrogenase (CDH) oxi-dizes lactose at the C-1 position of the reducing sugar moietyto lactobionolactone, which spontaneously hydrolyzes tolactobionic acid. 2,20-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt was used as electron accep-tor for CDH and was continuously regenerated (reoxidized)by laccase, a blue multi-copper oxidase. Oxygen served as theterminal electron acceptor of the reaction and was fullyreduced to water by laccase. The overall mass transfercoefficient of the miniaturized reactor was determined at30 and 458C; conversions were conducted both in thereaction-limited and diffusion-limited regime to study cat-alyst inactivation. The bubbleless oxygenation was successfulin avoiding gas/liquid interface inactivation. It was alsoshown that the oxidized redox mediator plays a key rolein the inactivation mechanism of the biocatalysts unob-served during previous studies.

Biotechnol. Bioeng. 2009;102: 122–131.

� 2008 Wiley Periodicals, Inc.

Abbreviations used: ABTS, 2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)

diammonium salt; CDH, cellobiose dehydrogenase; Da, Damkohler number; DCIP,

2,6-dichloroindophenol; e.c.n., enzyme consumption number; MtCDH, cellobiose

dehydrogenase originating from Myriococcum thermophilum; PDMS, polydimethylsi-

loxane; PVDF, polyvinylidenefluoride; SrCDH, cellobiose dehydrogenase originating

from Sclerotium rolfsii.

Correspondence to: Herman Van Langenhove

Contract grant sponsor: IWT Flanders; Austrian Science Fund

Contract grant number: L395-B11

122 Biotechnology and Bioengineering, Vol. 102, No. 1, January 1, 2009

KEYWORDS: bubble-free oxygenation; laccase-mediatorsystem; lactobionic acid; composite membrane; cellobiosedehydrogenase

Introduction

Enzymatic activation of molecular oxygen is an interestingbiotechnological reaction in the conversion of varioussubstrates into their more valuable oxidized products.Chemical oxidation methodology is affected by severaldisadvantages, that is, it is extremely hard to performoxidations in a regio- and stereoselective fashion. Further-more, the most inexpensive and harmless oxidant molecularoxygen cannot be used efficiently (Faber, 2004).

In contrast, oxygen-dependent enzymes are active undermild pH and temperature, and are not limited by thepreceding disadvantages. These enzymes can be divided intwo broad categories: oxygenases catalyze the incorporationof oxygen atoms into their substrates while oxidases solelyuse molecular oxygen as an electron acceptor with theconcomitant formation of hydrogen peroxide or water(Mathews et al., 1999). These oxygen-dependent enzymesare studied for different applications in the food industry,environmental technology and fine chemistry (Abadullaet al., 2000; Baminger et al., 2001a; Schwarz-Linek et al.,2001).

Even though the numerous advantages mentioned above,a critical factor for the use of these oxygen-dependentbiocatalysts has proved to be the mass transfer of oxygen

� 2008 Wiley Periodicals, Inc.

from the gas to the liquid phase. Dispersive methods foroxygen delivery potentially inactivate the enzymes at thegas–liquid interface, especially when high velocity gradientsare present (Maa and Hsu, 1997; Thomas, 1990). Thisinactivation is a complex phenomenon and is a combinedeffect of the hydrodynamic shear stress and the gas/liquidinterface surface (Elias and Joshi, 1997; Ghadge et al., 2003;Mohanty et al., 2001).

A possible solution to prevent the inactivation of enzymesat the gas/liquid interface is the use of bubble-freeoxygenation to dissolve oxygen in the liquid containingthe oxygen-dependent biocatalysts. Bubble-free oxygenationreduces protein denaturation since bursting bubbles aregreatly reduced or completely avoided at the liquid/proteininterface. For instance Rissom et al. (1997) and laterSchwarz-Linek et al. (2001) prevented the inactivation ofcyclohexanone monooxygenase by using a thin silicon tubefor aeration. Lutz et al. (2006) have reviewed the use ofmembranes in biotechnology and devoted a section on thesubject of bubble-free oxygen gassing. However, under theprevailing cases mentioned, experimental studies describingthe explicit relation between bubbleless oxygenation andenzyme stability under different reaction conditions arelacking.

The enzymatic conversion of lactose to lactobionic acidwas studied by Baminger et al. (2001a) and later by Ludwiget al. (2004). Cellobiose dehydrogenase (CDH; EC 1.1.99.18)from Sclerotium rolfsii was coupled to different redoxmediators [e.g., benzoquinones, catechol, 2,6-dichloroin-dophenol (DCIP), and 2,20-azinobis(3-ethylbenzothiazo-line-6-sulfonic acid) (ABTS)] and laccase (EC 1.10.3.2).Laccases are characterized by their ability to oxidize a widerange of various compounds, including phenols, amines orcomplexed metal ions, while utilizing oxygen as terminalelectron acceptor (Riva, 2006). Laccases exhibit thereforeactivity on several redox mediators in their reduced form,and can be employed for their efficient regeneration(Baminger et al., 2001a).

During one of the conversions of lactose to lactobionicacid, the dissolved oxygen tension in the reaction mixturewas regulated at a constant value of 10% (air saturation)using a sparger system. When using pure oxygen for aerationthe volumetric flow rate was 27.2 L h�1, while the flow ratehad to be increased to 163 L h�1 when using air. The specificproductivity of the biocatalytic conversion was comparablein these two experiments, but aeration by air resulted insignificantly higher inactivation of the enzymes. Theaeration rate was the process parameter that affected thestability of CDH and laccase most (Ludwig et al., 2004).

This prerequisite made the described coupled enzymesystem an ideal model system to investigate the biocatalyststability. Furthermore, the studied redox mediator regen-eration system is of greater importance since it can becoupled to other flavo-enzymes, for example, pyranoseoxidase which catalyzes the oxidation of several aldopyr-anoses at position C-2 to yield the corresponding 2-ketoaldoses. Some redox mediators have proven to be better

electron acceptors in comparison to oxygen, improving thelimiting rates of pyranose oxidase up to 11-fold (Bamingeret al., 2001a; Leitner et al., 2001).

A composite membrane consisting of a microporoussupport layer and a dense top layer served as a physicalseparation between the gas phase and the liquid phasecontaining both enzymes, and allows the permeation ofoxygen (Fig. 1). The overall mass transfer coefficient KLa ofthe reaction system was measured for different temperaturesand a modified Damkohler number (Calik et al., 2004) wasused to compare oxygen transfer and consumption. Theconversion was performed at different temperatures andreaction regimes to study its influence on biocatalyststability.

Furthermore, we compared CDH from two differentorganisms, SrCDH from the mesophilic basidiomyceteS. rolfsii (Baminger et al., 2001b) and MtCDH from thethermophilic ascomycete Myriococcum thermophilum (Har-reither et al., 2006) for better performance.

Materials and Methods

Experimental Set-Up

The experimental set-up to measure the mass transfercoefficient KLa and to conduct the bioconversion experi-ments was based on an in-house developed miniaturizedreactor made of acrylic resin (Perspex, ERIKS NV, Hoboken,Belgium) with a working volume of 200 mL (Fig. 2). Thereactor was double-jacketed for temperature control, and amagnetic stirring bar ensured adequate mixing of the liquidinside the reactor. The dissolved oxygen tension was mea-sured with an LDO1 (Hach-Lange, Dusseldorf, Germany)sensor, and the pH was monitored with a Sension gel-filledpH electrode (Hach-Lange, Dusseldorf, Germany). TheLDO1 sensor was calibrated for different temperatureswhere 100% dissolved oxygen (DO) corresponds to satura-tion with air throughout all the tests. Both sensors wereconnected to a HQ20 analyzer (Hach-Lange). The analyzerwas interfaced with a computer, which acquired dissolvedoxygen and temperature data as a function of time. Anoxygen-permeable membrane was clasped between twoslitted sheets at the bottom of the reactor; this membranehad a total exchange surface of 14.50 cm2. Gas was suppliedthrough channels at the bottom of the reactor.

KLa Measurements

The volumetric mass transfer coefficient KLa was deter-mined for the composite material that consisted of a 2-mmdense top layer of polydimethylsiloxane and a microporoussupport of polyvinylidene fluoride (GKSS-Forschungszen-trum, Geesthacht, Germany). A dynamic method was usedfor the determination of the KLa of the miniaturized reactorat different temperatures. The procedure was as follows: the

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Biotechnology and Bioengineering

Figure 1. Bubble free oxygenation of a bi-enzymatic system for the production of lactobionic acid with redox mediator regeneration. [Color figure can be seen in the online

version of this article, available at www.interscience.wiley.com.]

minireactor was completely filled with water. Then, oxygenwas stripped by purging pure nitrogen (Air Liquide, Aalter,Belgium) at a flow rate of 200 mL min�1 through the gaschannel at the bottom of the reactor. Gas streams werecontrolled by mass flow controllers (Brooks Instruments5850S, Veenendaal, The Netherlands). Subsequently, when

Figure 2. Miniaturized membrane contactor. a: pH electrode inlet; b: LDO1

sensor inlet; c: sample point; d: reactor vessel; e: heating jacket; f: gas inlet; g: gas

outlet; h: composite membrane. [Color figure can be seen in the online version of this

article, available at www.interscience.wiley.com.]

124 Biotechnology and Bioengineering, Vol. 102, No. 1, January 1, 2009

the DO was lower than 30%, air was flown through the gaschannels and the DO was continuously measured until itreached 95%. Here, the importance of removing residual gasbubbles was revealed. Since entrapped gas bubbles act as asink during oxygen absorption, they may cause an under-estimation of the volumetric mass transfer coefficients.

The probe response time (90% response time of 30 s) wasnot taken into account, given that the rate of dissolvedoxygen concentration change was negligible compared tothe probe response time. Due to the small membrane area(14.50 cm2) and the large gas flow (200 mL min�1), thedriving force ðC�

O2� CO2Þ was considered to be constant in

the gas channels at a given time.Under these conditions, the increase of the oxygen

concentration in the liquid CL over time can be described as:

dCO2

dt¼ KLaðC�

O2� CO2Þ (1)

The overall mass transfer coefficient KL is the resultant of theresistance to mass transfer in the gas phase boundary layer,the membrane and the liquid phase boundary layer. Drioliet al. (2006) provides more information to the interestedreader on this resistance-in-series model since it was not ourgoal to determine these different resistances. After all, it isthe overall mass transfer coefficient that will determine thetransfer rate.

Integration of Equation (1) gives:

CO2;t ¼ C�O2

� ðC�O2

� CO2;t¼0Þ e�KLat (2)

CO2;t¼0 is a known parameter. C�O2

and KLa were estimatedby fitting the experimental data between 30% and 95%dissolved oxygen concentration to this model. To this end,the generalized reduced gradient algorithm (GRG2) fornonlinear programming (Lason et al., 1978) was used with atolerance level set at 5% and convergence at 0.001.

Chemicals

Sodium fluoride, 2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, lactobionic acid, sodiumhydroxide and calcium chloride were obtained from Sigma–Aldrich (Schnelldorf, Germany) at the highest gradeavailable and lactose and acetic acid were obtained fromFluka (Buchs, Switzerland) at the highest grade available.

Enzymes

Cellobiose dehydrogenase (EC 1.1.99.18) from the basidio-mycete S. rolfsii CBS 191.62 (SrCDH) was produced inaccordance with the optimized procedure described byLudwig and Haltrich (2003). The basidiomycete wascultivated at 308C in a fermentor with a working volumeof 30 L using a medium based on 30 g L�1 cellulose. After9 days, 7.9 U mL�1 of CDH activity was produced. Thepreparation of SrCDH used throughout this study waspartially purified by a single anion exchange chromato-graphy step using DEAE-Sepharose fast flow (Amersham-Pharmacia, Uppsala, Sweden) with 50 mM sodium acetatebuffer (pH 5.5) and elution with a linear NaCl gradient(0–0.5 M). This preparation had a specific activity of15 U mg�1 [homogeneous enzyme: 63 U mg�1 (Bamingeret al., 2001b)].

CDH from the ascomycete M. thermophilum (MtCDH)was heterologously expressed in the methylotrophic yeastPichia pastoris, using the ‘‘Invitrogen Easy selectTM Pichiaexpression kit’’ with P. pastoris strain X-33 and theexpression vector pPICZa according to the instructionsof the manufacturer (Invitrogen Life Technologies, Carls-bad, CA). P. pastoris was cultivated according to themanufacturer’s instructions. Heterologous protein produc-tion was induced by the feed of methanol. Routinely, 2.15 Uof CDH activity was produced per mL of culture liquid bythis procedure. Since the heterologously expressed protein isthe major extracellular protein obtained by this procedure,the MtCDH preparation used throughout this study wasobtained by concentrating the fermentation supernatantusing centrifugal ultrafiltration devices (Millipore, Billerica,MA) with a cutoff of 10 kDa. The MtCDH preparationobtained this way had a specific activity of 3.7 U mg�1

[homogeneous enzyme: 3.8 U mg�1 (Harreither et al.,2006)].

Laccase (EC 1.10.3.2) was produced by the white-rotfungus Trametes pubescens MB 89 based on the proceduredescribed by Galhaup and Haltrich (2001), using theaddition of millimolar amounts of Cu(II) to activelygrowing cultures of T. pubescens MB 89 to stimulate laccaseformation. The white-rot fungus was cultivated at 308C in a30 L fermentor using a medium based on 48 g L�1 glucoseand produced 45 U mL�1 of laccase activity after 16 days.The laccase preparation used was partially purified by anionexchange chromatography using a Q-Sepharose fast flow(Amersham-Pharmacia) column previously equilibratedwith 50 mM sodium acetate buffer pH 5.5. The laccaseisoenzyme of interest (LAP2) was eluted with a lineargradient of 0–1 M NaCl in the same buffer. The specificactivity of this partially purified enzyme was 17 U mg�1

[homogeneous enzyme: 350 U mg�1 at pH 4.5 (Galhaupet al., 2002)].

Enzyme Activity Assays

Cellobiose dehydrogenase activity was assayed by followingthe decrease in absorbance of 2,6-dichloroindophenol(DCIP) with a UV-1650PC spectrophotometer (Shimadzu,Kyoto, Japan) at 520 nm (e520 ¼ 6,800 M�1 cm�1) at pH 4.0and at the same temperature as used in the minireactor(30 and 458C). The reaction mixture contained DCIP(0.15 mM) and lactose (30 mM) in 81 mM sodium acetatebuffer. One unit of enzyme activity is defined as the amountof enzyme reducing 1 mmol of DCIP per minute underthe above reaction conditions. To avoid laccase interferencewith the determination of CDH activity by rapidly reoxi-dizing the reduced form of DCIP, sodium fluoride (4 mM)was added to the assay to selectively inhibit laccase activity(Baminger et al., 1999).

The reaction mixture for assaying laccase activitycontained 1 mM 2,20-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) in 20 mM acetate buffer, pH 3.5(Galhaup et al., 2002), and the oxidation of ABTS to thebluish-green cation radical was followed spectrophotome-trically at 436 nm (e436 ¼ 29,300 M�1 cm�1) and at 30 or458C. To avoid CDH interference with the determination oflaccase activity by rapidly reducing the ABTS cation radicalin the presence of lactose, the assay mixture (enzyme sampleplus buffer) was incubated with 1.8 U mL�1 (final activity)of b-galactosidase from Aspergillus oryzae (Sigma G 5160)for 10 min, and then the reaction was started by the additionof the ABTS reagent. One unit of enzyme activity is definedas the amount of enzyme oxidizing 1 mmol of ABTS perminute under the above reaction conditions.

High Performance Liquid Chromatography Analysis

Lactose (retention time¼ 7.7 min) and lactobionic acid(retention time¼ 17.1 min) were separated and quantified

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by ion-moderated partition chromatography using a resin-based Aminex1 HPX 87C column (Bio-Rad, Hercules, CA)at 858C with 5 mM CaCl2 as eluent (0.7 mL min�1) andrefractive index detection.

Bioconversion Experiments With Bubble-FreeOxygenation

The conversion was studied both at 30 and 458C in adiffusion- and reaction-limited regime. For the diffusion-limited experiments the initial lactose concentration was50 mM, and for the reaction-limited regime the initiallactose concentration was 25 mM. CDH and laccase wereadded in various amounts to ensure the selected reactionregimes, with laccase activity always added in excess toensure rapid reoxidation of the spent redox mediator ABTS.The conversion was started by the addition of ABTS (0.18 mM).As soon as the oxygen was depleted from the liquid, pureoxygen (Air Liquide) at a flow rate of 200 mL min�1 waspassed through the gas channels of the minireactor. The pHof the reaction mixture was kept constant at 4.0 by usingpotassium carbonate buffer (400 mM).

Table I. Estimation of the apparent limiting CDH activities VappCDH in the

reaction mixture where Da¼ 1 based on oxygen mass transfer using air or,

as used in the conversion experiments, pure oxygen as feed gas.

T(8C) KLa (s�1) Feed gas C�O2

(mol m�3) VappCDH (U mL�1)

30 1.01 10�3 Air 0.24 0.03

O2 1.19 0.14

45 1.58 10�3 Air 0.20 0.04

O2 0.99 0.19

Results and Discussion

Oxygen Mass Transfer Characteristics

The volumetric mass transfer coefficient KLa based on liquidconcentrations was determined for the composite mem-brane. The used model fits well with the experimentalresults. The KLa values for 30 and 458C were calculatedas (1.01� 0.014) 10�3 s�1 (n¼ 3) and (1.58� 0.08)10�3 s�1 (n¼ 5), respectively.

The mass transfer rate of oxygen from the gas to the liquidphase can be written according to the film theory:

NO2 ¼ KLaðC�O2

� CO2Þ (3)

The maximum transfer rate occurs when CO2 ¼ 0 mol m�3,leading to Equation (4):

NO2;max ¼ KLaC�O2

(4)

The measured mass transfer coefficients were subse-quently used to estimate the minimum activity of CDHrequired in the reaction system to utilize the maximumtransfer rate of oxygen for the biochemical conversion. Forthis, a modified Damkohler number (Da) was developed.This modified Damkohler number (Calik et al., 2004;Gomez et al., 2006) was defined as the ratio between themaximum oxygen uptake (consumption through thebioconversion) and maximum oxygen transfer rate:

Da ¼ maximum oxygen uptake

maximum oxygen transfer rate(5)

126 Biotechnology and Bioengineering, Vol. 102, No. 1, January 1, 2009

From Equation (5) it follows that when Da> 1 the conver-sion is diffusion-limited, and when Da< 1 the conversionis reaction-limited with an intermediary reaction regimewhere Da 1.

The global reaction of the conversion considered in thisstudy can be written as:

2 lactose þ O2 ! 2 lactobionic acid (6)

When laccase activity is present in excess and hence does nothamper the final electron transfer step (Fig. 1), and whenneglecting a possible mass transfer enhancement because ofthe reaction, Da can be written as:

Da ¼ VappCDH

2KLaC�O2

(7)

Solving Equation (7) for different temperatures using air orpure oxygen as feed gas gives the apparent limiting activitiesof CDH in the reaction mixture where Da¼ 1 (Table I).

If more CDH than the estimated apparent limitingactivity is added to the minireactor, the limiting step of theoverall reaction will be the diffusion process (O2 transportinto the reaction mixture). If on the other hand less CDHactivity than this limiting value is added to the reactionsystem, the limiting step will be the biochemical reaction.In this latter case, the full oxygenation capacity of the reactorwill not be utilized.

Bioconversion Experiments With Bubble-FreeOxygenation

In order to investigate the bubble-free oxygenation and itseffect on the biocatalysts we conducted a series ofexperiments for the conversion of lactose to lactobionicacid in the thermostated miniaturized reactor equipped withpH electrode and oxygen sensor. For the diffusion-limitedregime (Da> 1) at 308C (Fig. 4a and b) three reactionphases could be distinguished with respect to oxygenconsumption. First, a very sharp decrease in dissolvedoxygen concentration with the concomitant generation ofthe ABTS cation radical (clearly visible by the formation ofan intense bluish-green color) was observed. This effectoriginates from the high laccase activity, which is in highexcess over the CDH activity (4.4-fold in the SrCDH-,

Table II. Characteristics for the bioconversion experiments.

T(8C) Da

Measured initial

activity (U mL�1)Activity ratio

laccase/CDH

Reaction rate in

reactor (mmol mL�1 min�1)

Conversion

time (h)

e.c.n.

(U mmol�1)CDH laccase

SrCDH

30 >1 0.45 2.0 4.4 0.14 �6.0 0

<1 0.06 2.0 33 0.06 �7.0 0.024

45 >1 0.76 3.0 3.9 0.15 �5.5 0.15

<1 0.10 2.5 25 0.10 �7.0 0.42

MtCDH

30 >1 0.26 2.0 7.7 0.14 �6.0 0

<1 0.08 2.1 26 0.08 >7.0 0.16

45 >1 0.17 2.6 15 0.14 �6.0 0.10

<1 0.03 2.8 93 0.04 n.d. 0.84

Data are shown for the applied enzymatic activities, the recalculated reaction rate from HPLC data, conversion time and the enzyme consumption number(e.c.n.) based on inactivated CDH activity per mmol product formed.

7.7-fold in the MtCDH experiment, Table II) to preventthe limitation of the conversion rate by the regenerationsystem.

In the second reaction phase, the dissolved oxygenconcentration stayed close to depletion even though theoxygen supply was switched on by the laccase catalyzedoxidation of ABTS, indicating that the oxygen transportedinto the system was immediately consumed. The color of thereaction mixture became nearly colorless indicating adominance of the reduced redox mediator due to oxygenlimitation. The reaction phase showed with respect to theoxygen consumption a steady-state regime and correspondsto the largest time range in the experiment. Here, theenzymatically driven process follows zero-order ratekinetics. The space-time yield for the experiment employingSrCDH was calculated as 3.0 g L�1 h�1 and for MtCDH3.3 g L�1 h�1. Specific productivities for the formation oflactobionic acid were calculated over the whole reactiontime and are 6.7 g kU�1 h�1 for SrCDH and 12.7 g kU�1 h�1

for MtCDH by using the measured initial activities (SrCDH,0.45 U mL�1; MtCDH, 0.26 U mL�1, Table II). Thisdifference in the specific productivities is, however,misleading and results from the CDH activity applied insignificant excess under diffusion limiting conditions. Arecalculation from the actual conversion rate shows that 0.14mmol mL�1 min�1 lactose was converted in both reactions,corresponding to a volumetric activity of 0.14 U mL�1.Under the given conditions, this is the limiting activity ofCDH in the reaction mixture where Da¼ 1 (Table I) and theexcess activity is limited by the oxygen transport. Whenusing this limiting activity, the theoretical specific produc-tivity is 21.4 g kU�1 h�1, which is very close to the resultobtained by Ludwig et al. (2004) for a batch reaction withdispersive oxygen supply (19.3 g kU�1 h�1).

The third reaction phase was the final phase of thereaction with lactose conversion being close to complete.While the residual lactose was converted into lactobionicacid, the oxygen tension and ABTS cation radicalconcentration were increasing. This rise of the dissolvedoxygen concentration in the reactor is a good indication for

the end of the biotransformation reaction. Lactose wascompletely converted to lactobionic acid within approxi-mately 6 h and without the formation of by-products inthese experiments as shown by HPLC analysis.

In contrast to the results obtained by Ludwig et al. (2004)where, depending on the dissolved oxygen tension andthe air flow rate, up to 2.34 units of SrCDH activity wereinactivated per mmol of lactobionic acid formed, no enzymeinactivation was observed during the course of the reactionat 308C. Hence, both CDHs and laccase were stabilized inthe diffusion-limited regime by avoiding gas/liquid inter-faces in the reactor when using the bubble-free oxygenationsystem.

For the reaction-limited regime (Da< 1) at 308C(Fig. 4c and d) the initial sharp decrease in oxygenconcentration is again clearly visible. However, when theoxygen supply was switched on, the dissolved oxygenconcentration in the reaction mixture increased rapidly to alevel that was out of range for the oxygen sensor (>16mg L�1 DO), indicating that oxygen supply was in fact muchfaster than its consumption by the biocatalytic reaction.Hence, the third and final phase of the reaction when lactoseis depleted and the oxygen consumption decreases, cannotbe judged from the dissolved oxygen tension. Oxidation oflactose to lactobionic acid was complete for the reactionusing SrCDH, while this reaction was slower when MtCDHwas used, because a lower volumetric activity was applied.Additionally, the MtCDH activity started to decreasesignificantly after 3–4 h. Again a space-time yield can becalculated and was found 1.3 g L�1 h�1 (SrCDH) and1.7 g L�1 h�1 (MtCDH, only considering the first 3 h ofconversion). Obviously, the specific productivities equalthe above calculated theoretical value with 21.3 g kU�1 h�1

(SrCDH) and 21.7 g kU�1 h�1 (MtCDH, only the first 3 h)under reaction-limited conditions when using the measuredinitial activities from Table II for this calculation.

SrCDH and laccase were stable under these reactionconditions and their activities stayed constant during theentire reaction time of 7 h, while MtCDH activity decreasedconsiderably after 3 h of conversion (Fig. 3d). This

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Figure 3. Conversion of lactose to lactobionic acid using CDH and laccase at 308C in a miniaturized reactor with bubble-free oxygenation. One hundred percent DO

corresponds to air saturation. a: SrCDH and laccase in the diffusion-limited regime. b: MtCDH and laccase in the diffusion-limited regime. c: SrCDH and laccase in the reaction-

limited regime. d: MtCDH and laccase in the reaction-limited regime. [Color figure can be seen in the online version of this article, available at www.interscience.wiley.com.]

observation is in some respect surprising, because MtCDH isderived from a thermophilic fungus and stable for 1 week at508C and shows a half-life time of 2 h at 608C (Kittl et al.,unpublished results), while in contrast, SrCDH has a half-life time of only 12 h at 468C (Baminger et al., 2001b).Certainly one should not confuse thermodynamic (resting)stability with the biocatalyst stability under turnoverconditions. Due to the higher thermostability of MtCDH,thermal deactivation was most probably not responsible forthe higher losses of CDH activity in this experiment as thisenzyme was also perfectly stable under diffusion-limitedconditions. It was therefore of interest to investigate theprocess at higher temperatures to find out the influence onthe reaction rates, and the stability of the biocatalysts. Itshould be noted that the enzyme assays were also measuredat increased temperatures (458C).

The same three reaction phases as observed in thebioconversion at 308C were also observed at 458C (Fig. 4).Under diffusion-limited conditions (Fig. 4a and b), theinitial sharp decrease in DO was followed by the long phaseof the steady state, characterized by low DO in the reactor.

128 Biotechnology and Bioengineering, Vol. 102, No. 1, January 1, 2009

As the applied volumetric SrCDH activity was higher(Table II) than the apparent limiting CDH activity (Table I)the calculated specific productivity is not relevant fordescribing the process. When using the apparent limitingCDH activity (Da¼ 1) at 458C (0.19 U mL�1) the theo-retically calculated specific productivity is 17.1 g kU�1 h�1

for SrCDH. MtCDH was applied with a volumetric activityof 0.17 U mL�1, which is near the limiting CDH activityhence the specific productivity can be calculated with thisvalue and was found to be 17.6 g kU�1 h�1 for MtCDH.These specific activities are similar to the ones obtainedat 308C, but it should be pointed out, that the activityassays for these experiments were performed at 458C wherethe specific activity is approximately 2 times higher. Thiswas the base for calculating the applied enzyme quantity (inmg protein), which was therefore 2 times less than in theexperiments performed at 308C. When considering thisfact, the specific productivities obtained in this experimentcompare well with the value obtained by Ludwig et al. (2004)for a batch conversion using the similar conditions in areactor equipped with a sparger (32 g kU�1 h�1).

Figure 4. Conversion of lactose to lactobionic acid using CDH and laccase at 458C in a miniaturized reactor with bubble-free oxygenation. One hundred percent DO

corresponds to air saturation. a: SrCDH and laccase in the diffusion-limited regime. b: MtCDH and laccase in the diffusion-limited regime. c: SrCDH and laccase in the reaction-

limited regime. d: MtCDH and laccase in the reaction-limited regime. [Color figure can be seen in the online version of this article, available at www.interscience.wiley.com.]

Table III. Apparent kinetic constants of the enzymes; CDH and laccase LAP2 from Trametes pubescens (Baminger et al., 2001b; Galhaup et al., 2002).

Stoichiometrya KM (mM) kcat (s�1) kcat/KM (M�1 s�1)

SrCDH

Lactose (pH 4.0)b 2.4 26 0.011 106

ABTS cation radical (pH 4.0)c 2 0.0004 54e 67.5 106

DCIP (pH 4.0)c 1 0.015 30 2 106

MtCDH

Lactose (pH 4.5)b 0.095 12.6 0.13 106

ABTS cation radical (pH 4.5) 2 0.0137 37.4 2.73 106

DCIP (pH 4.5) 1 0.032 16.9 0.5 106

Laccase LAP2 from T. pubescens

ABTS (pH 3.5)d 0.033 490 14 106

Oxygen 0.25 0.410 2900 7 106

aConstants were determined at 258C using 2 mM cellobiose as the substrate and at the pH optimum of the respective acceptor. Protein was determinedusing the bicinchoninic acid method.

bKinetics was measured using the standard DCIP assay at pH 4.0 and 258C. Protein was determined using the Bradford method.cUsing oxygen (air-saturated solution) as electron acceptor.dMolecules of electron acceptor reduced per molecule of electron donor.eRecalculated from the published data.

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During this reaction phase under diffusion-limitedconditions CDH (both SrCDH and MtCDH) as well aslaccase were stable. The third reaction phase was againstarting when the oxygen concentration increased sharplyafter 5–6 h, indicating that lactose was almost completelyconverted. Interestingly, this third reaction phase, which ischaracterized by high oxygen concentrations in the reactionmixture, showed a considerable decrease of both SrCDH andespecially MtCDH activity. This inactivation also startedwhen the dissolved oxygen concentration in the reactionmixture increased. In contrast to the CDHs, laccase activitywas not affected significantly.

When the bioconversion at 458C was conducted in thereaction-limited regime (Fig. 4c and d), the second reactionphase after turning on the oxygen supply was againcharacterized by continuous lactose conversion and highDO in the aqueous phase. Both SrCDH and MtCDHactivities decreased continuously during this reaction phase,therefore no specific productivities were calculated. Lactoseconversion was complete after 7 h when SrCDH was used.The conversion is very fast during the first 3 h, and thenslows down considerably due to the loss of activity (about50% after 6 h). MtCDH, which is the more thermostable ofboth CDHs when compared under resting conditions, wasinactivated more severely (30% residual activity after 6 h),and only 20% of the substrate was oxidized (Fig. 4d). Thebetter thermostability of MtCDH did not provide anyadvantage to the process under reaction-limited conditions.The observed inactivation is clearly not caused by theincreased temperature, but the reason of other inactivationmechanisms. It is interesting that the usually very stablelaccase was also inactivated by approx. 15% under the givenconditions in both reaction-limited experiments.

Enzyme Inactivation

The enzyme inactivation observed during turnover and alsoin the third reaction phase after complete substrateconversion might be caused by reactive molecules foundunder these conditions. The generation of hydrogenperoxide by CDH in a two-electron reduction of oxygenwas investigated as a possible inactivation reagent. Eventhough oxygen is a poor electron acceptor for cellobiosedehydrogenase as reported by Baminger et al. (2001a), thisreaction seemed likely to occur as a side-reaction of thefavored ABTS cation radical reduction at high oxygenconcentrations. In the diffusion-limited experiments,the oxygen concentration was always very low and thereforethe H2O2 producing side-reaction of CDH had to beinsignificant as expressed by the Michaelis–Menten equa-tion. In the reaction-limited experiments the oxygenconcentration was always very high and thus could resultin H2O2 production and accumulation. However, this couldnot explain the inactivation seen in Figure 4a and b after thediffusion-limited conversion since CDH is lacking thesubstrate lactose. To check the hypothesis of H2O2

inactivation we conducted experiments in the minireactor

130 Biotechnology and Bioengineering, Vol. 102, No. 1, January 1, 2009

with SrCDH and MtCDH (0.6 U mL�1) in the presence oflactose (50 mM) at a dissolved oxygen concentrationcorresponding with air saturation at 458C (no laccase orABTS present). Inactivation of the biocatalysts was notobserved during four hours of monitoring. The dissolvedoxygen concentration did not decrease, thus indicating thatoxygen is indeed a very poor electron acceptor and that thisside-reaction is too slow to affect biocatalyst stability. Thesame experiment was repeated at oxygen saturation and itwas found that cellobiose dehydrogenase was inactivatedbetween 5% and 10% after 2 h. However, this low rate ofinactivation did not correspond to the high inactivationrates observed during turnover and thus, the hypothesis ofhydrogen peroxide generation as a major cause ofinactivation was abandoned.

The second compound that might be responsible forenzyme inactivation is the redox mediator ABTS. ABTS isoxidized by laccase to the green ABTS cation radical,especially under conditions where sufficient oxygen isavailable in the reaction system. This compound is relativelystable and might result in enzyme inactivation. This canpossibly explain the much higher enzyme consumptionnumbers under reaction-limited (Da< 1) conditions andafter full lactose conversion in diffusion-limited reactions.Both conditions are characterized by an intense dark-greencolor and favor the formation of the oxidized form of theredox mediator. To support this hypothesis we conductedexperiments in the minireactor containing SrCDH (0.6U mL�1), laccase (1.15 U mL�1), and ABTS (180 mM). Theabsence of lactose favored the formation of the ABTS cationradical. A very strong inactivation of SrCDH under airsaturation (62% inactivation after 4 h) and even strongerinactivation under oxygen saturation (68% inactivationafter 4 h) was observed. The experiment was repeated with adecreased concentration of ABTS (18 mM) and under theseconditions an inactivation of only 15% of SrCDH at airsaturation after 4 h occurred.

Another observation, made only in the third reactionphase when lactose was fully converted, was a slow colorchange of the reaction mixture from bluish-green to purple.This color change indicates the overoxidation of the greenABTS radical cation to the ABTS dication with acharacteristic absorbance at 518 nm (Kadnikova and Kostic,2002). Presumably the dication radical also plays a role inthe inactivation of CDH and laccase, but its effect could notbe properly quantified.

Conclusions

It was demonstrated that the bi-enzymatic system consistingof cellobiose dehydrogenase and laccase for the productionof lactobionic acid is efficiently protected from inactivationby a bubble-free oxygenation system in the diffusion-limitedregime both at 30 and 458C, with the enzymes being stableduring turnover, even at elevated temperatures, which is incontrast to previous results obtained using a conventionalsparger (Ludwig et al., 2004) for oxygen supply.

However, when the dispersive addition of oxygen iseliminated as the main mechanism of biocatalyst inactiva-tion, another effect is dominating during turnover whenhigh oxygen concentrations prevail (especially at 458C). Theoxidized redox mediator (the ABTS cation radical) wasfound to be the dominating factor and plays a key role in theinactivation mechanism of SrCDH and MtCDH.

The bioreactor, primarily designed to increase thebiocatalyst stability, performed also well in terms of specificproductivity and space-time yield (up to 3.3 g L�1 h�1

product) although the volumetric activities of CDH appliedwere 2–20 times lower than in comparable batch reactionswith a conventional sparger-based aeration system.

The limiting factor for a higher catalyst density was thespecific membrane area of the prototype reactor with only72.5 cm2/L reactor volume, but an increase of this ratio is astraightforward engineering task.

Nomenclature

a

ratio of membrane surface to liquid volume (m2 m�3)

Cassay

concentration of chemicals used in assay (M)

CO2

oxygen concentration in liquid phase (mol m�3)

CO2 ;t

oxygen concentration in liquid phase at time t (mol m�3)

CO2 ;t¼0

oxygen concentration in liquid phase at time t¼ 0 (mol m�3)

C�O2

oxygen saturation concentration in liquid phase (mol m�3)

kcat

turnover number (s�1)

kcat=KM

specificity constant (M�1 s�1)

KL

overall mass transfer coefficient based on liquid concentrations

(m s�1)

KLa

volumetric mass transfer coefficient (s�1)

KappM

apparent Michaelis–Menten constant (M)

n

number of reiterations

NO2

oxygen mass transfer rate (mol m�3 s�1)

Vapp

apparent limiting rate (U mL�1)

VR

reactor volume (m3)

The authors acknowledge the financial support provided by the

Institute for the Promotion of Innovation through Science and

Technology in Flanders and the Austrian Science Fund (project

L395-B11). Klaus Ohlrogge of the Department of Chemistry of

GKSS-Forschungszentrum Geesthacht GmbH is acknowledged for

providing the composite membranes. The authors also express their

gratitude to Marc Troch for first-class handcraftsmanship in making

the minireactor and Marianne Bailleul for her kind assistance with

HPLC analysis.

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