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J. of Supercritical Fluids 38 (2006) 373–382

Assessment of two immobilized lipases activity treated in compressed fluids

D. Oliveira a, A.C. Feihrmann a, A.F. Rubira b, M.H. Kunita b, C. Dariva a, J. Vladimir Oliveira a,∗a Department of Food Engineering, URI, Campus de Erechim, Av. Sete de Setembro, 1621, Erechim 99700-000, RS, Brazil

b Department of Chemistry, UEM, Av. Colombo 5790, Maringa 87020-900, PR, Brazil

Received 22 June 2005; received in revised form 21 November 2005; accepted 19 December 2005

bstract

This work investigates the influence of temperature, pressure, exposure times and depressurization rate on the activity of two commercialmmobilized lipases submitted to compressed carbon dioxide, propane and n-butane. A high-pressure cell was employed in the experiments, varyinghe temperature from 35 to 75 ◦C, in the pressure range of 10–280 bar, exposure times from 1 to 6 h and adopting distinct decompression rates. Resultshowed that activity losses were verified for Lipozyme IM in all compressed solvents, markedly in carbon dioxide. For Novozym 435, treatmentn carbon dioxide though in less extent also led to activity losses, while the use of propane and n-butane promoted enhancements of the enzymectivity. In general, within the range studied, temperature and exposure times affected positively enzyme activity while the decompression ratesid not demonstrate to be a relevant variable. Additionally, an exploratory study was conducted to investigate the effect of compression/expansion

ycles on enzyme activity in all solvents. Thermogravimetric analyses show that the thermal profiles of Novozym 435 treated in n-butane and inarbon dioxide are similar to the untreated enzyme. Scanning electron micrographs of Novozym 435 illustrate that the material submitted to carbonioxide presented morphological alterations when compared to the untreated enzyme.

2006 Elsevier B.V. All rights reserved.

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eywords: Lipases; Enzyme activity; Compressed gases; High pressure; Super

. Introduction

Biocatalysis through the use of enzymes, especially lipases,ither immobilized or non-immobilized has become an increas-ngly important research field in recent years, and has beenonsidered as the most efficient way of producing fine chemicalsn a near future [1,2]. Lipases are generally effective biocata-ysts due to high-substrate specific activity, low impact on thenvironment, functional group and stereo specificities. Besides,hemical reactions can be conducted directly using lipases inrganic medium [3–6].

Though at present the high cost of enzyme production is theajor obstacle to commercialization of enzyme-catalyzed pro-

esses, recent advances in enzyme technology, such as the usef solvent-tolerant lipases and immobilized lipases—makingossible the catalyst re-utilization, have been made to develop

ost-effective systems [7].

Since the first reports on enzyme-catalyzed reactions in super-ritical fluids [8–11], much attention has been paid to the use

∗ Corresponding author. Fax: +55 54 35209090.E-mail address: vladimir@uricer.edu.br (J.V. Oliveira).

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896-8446/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.supflu.2005.12.007

l fluids

f dense gases, mainly supercritical carbon dioxide, as poten-ial alternatives to conventional organic solvents [12–20]. Thedvantages of using compressed fluids over organic liquid sol-ents as reaction media are well discussed in the literature1,16,21,22].

Numerous studies have shown that many reactions can beonducted in liquid or supercritical carbon dioxide and, in someases, rates and selectivities achieved are greater than thosebtained in normal liquid or gas phase reactions [23–25]. Com-ared to other gases, carbon dioxide has been the most widelytudied fluid as solvent medium for enzyme-catalyzed reactions23–38].

However, a serious drawback of those applications may ariserom the non-polarity of carbon dioxide, which means non-roper dissolution of both hydrophobic and hydrophilic com-ounds [1]. Furthermore, the hydrophilic characteristics of car-on dioxide over wide pressure ranges may affect negativelyhe activity of the enzyme, as the water partitioning between thenzyme and reaction mixture may be a key factor for conducting

nzymatic reactions [1,3,39].

Nevertheless, carbon dioxide is not the only gas whose prop-rties seem to be adequate for biocatalysis [22]. For instance,he comparable dielectric constant of propane and n-butane to

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artetsprof 2–50 bar min−1 for propane and n-butane, the system wasdepressurized and the lipase activity was measured. The changein the lipase activity was defined as the difference between theactivity at the beginning and at the end of the process.

74 D. Oliveira et al. / J. of Super

arbon dioxide [40,41], and the fact that much higher pressurehase transition values are generally found in systems formed byarbon dioxide with high molecular weight (e.g. triglycerides)ompounds when compared to the use of propane and n-butaneupport a firm belief that propane and n-butane may also beuitable as reaction media for enzyme-catalyzed bioconversions42–44].

It may also be opportune to mention that the recent advancesn surfactant design will certainly open new frontiers to proceedipase-catalyzed reactions in microemulsions, especially in theo-called water-in-oil microemulsions or reverse micelles, stabi-ized by surfactant molecules dispersed in compressed solvents45].

To conduct lipase-catalyzed reactions at high pressures, thenzyme behavior in compressed fluids is of primary importances the loss of enzyme activity may lead to undesirable poor reac-ion rates and low yields of target products. Enzyme stability andctivity may depend on the enzyme species, characteristics ofompressed fluid, water content of the enzyme/support/reactionixture and process variables manipulated. Inspection of the lit-

rature cited reveals that, while there is relatively abundant dataegarding activity and stability of enzymes in carbon dioxide,here is a lack of corresponding experimental information forropane and n-butane.

Thus, with the aim at understanding the potential of pressurepplication to enzyme processes, to help elucidating the reactionechanism as well as for a rational reactor design, this work

nvestigates the influence of temperature, pressure, exposureimes and decompression rates on the activity of two commercialmmobilized lipases (Lipozyme IM and Novozym 435) in super-ritical carbon dioxide and compressed propane and n-butaneedia. Latter, the analysis of these materials included studies

n the chemical modifications, thermal properties and morphol-gy. Infrared spectroscopy (FT-IR), thermogravimetric analysisTGA) and scanning electron microscopy (SEM) were used tovaluate possible alterations in the materials with treatment.dditionally, it is reported the effect of compression/expansion

ycles on enzyme activity in all solvents.

. Experimental

.1. Materials

.1.1. EnzymesTwo commercial immobilized lipases were kindly supplied

y Novozymes Brazil (Araucaria, PR, Brazil): Mucor mieheiLipozyme IM) immobilized on a macroporous anion exchangeesin and Candida antarctica (Novozym 435) immobilized onmacroporous anionic resin. According to the manufacturer,

he optimum activity is achieved at 40 ◦C for Lipozyme IM and0 ◦C for Novozym 435 [46].

.1.2. Lipase activity

The enzyme activity was determined as the initial rates in

sterification reactions between lauric acid and propanol at aolar ratio of 3:1, temperature of 60 ◦C and enzyme concen-

ration of 5 wt.% in relation to the substrates. At the begin-

Ftw(

al Fluids 38 (2006) 373–382

ing of the reaction, samples containing the mixture of lauriccid and propanol were collected and the lauric acid contentas determined by titration with NaOH 0.04N. After the addi-

ion of the enzyme to the substrates, the mixture was kept at0 ◦C for 15 min. Then, the lauric acid consumption was deter-ined. One unit of activity (U) was defined as the amount of

nzyme necessary to consume 1 �mol of lauric acid/min athe established experimental conditions presented previously.ll enzymatic activity determinations were replicate at least

hree times.

.1.3. ChemicalsLauric acid, ethanol, acetone and other chemicals (analyti-

al grade) were from Merck. Carbon dioxide (99.9%), propane99.5%) and n-butane (99.5%) were purchased from White Mar-ins S.A.

.2. Apparatus and experimental procedure

The equipment used in all experiments consists basically ofsolvent reservoir, a 20 mL view cell with three sapphire win-ows for visual observations, an absolute pressure transducerSmar LD 301), with a precision of ±0.30 bar, a portable pro-rammer (Smar, HT 201) for the pressure data acquisition andsyringe pump (ISCO 260D). The equilibrium cell contains aovable piston, which permits the pressure control inside the

ell. The experimental apparatus, built to conduct the experi-ents up to 300 bar and 80 ◦C, is schematically presented inig. 1.

The lipases (approximately 1.0 g) were charged into the cellnd the temperature established in the experimental design waseached. Afterwards, the system was pressurized and main-ained at constant temperature and pressure for a pre-establishedxposure time. Typically, the pressure come up time was lesshan 0.5 min and accordingly was not included in the pres-ure holding time because of its relatively small time com-ared to longer holding times. Then, at the decompressionates defined—10–200 kg m−3 min−1 for CO2 and in the range

ig. 1. Schematic diagram of the high-pressure apparatus for enzyme activityests: (A) solvent reservoir; (B) syringe pump; (C) equilibrium cell; (D) sapphireindows; (E) magnetic stirrer; (F) white light source; (G) pressure transducer;

H) ball valve; (I) micrometering valve; (J) relief valve.

D. Oliveira et al. / J. of Supercritical Fluids 38 (2006) 373–382 375

Table 1Results for Lipozyme IM activity treated in pressurized CO2

Experiment T (◦C) t (h) R (kg m−3 min−1) RD Enzyme activity (U/g) Activity loss (%)

Initial Final

1 35(−1) 1(−1) 10(−1) 0.5(−1) 92 91 0.5 ± 0.171.5a 71 71

2 35(−1) 1(−1) 200(+1) 1.6(+1) 94 94 0.4 ± 0.1110.4 106 105

3 35(−1) 6(+1) 10(−1) 1.6(+1) 91 82 10.4 ± 0.1110.4 100 90

4 35(−1) 6(+1) 200(+1) 0.5(−1) 83 73 13.7 ± 2.571.5 97 82

5 75(+1) 1(−1) 10(−1) 1.6(+1) 110 101 9.4 ± 1.9276.4 102 91

6 75(+1) 1(−1) 200(+1) 0.5(−1) 92 91 1.0 ± 0.1100 94 93

7 75(+1) 6(+1) 10(−1) 0.5(−1) 93 80 12.5 ± 1.8100 107 95

8 75(+1) 6(+1) 200(+1) 1.6(+1) 109 95 11.7 ± 1.2276.4 93 83

9 55(0) 3.5(0) 105(0) 1.05(0) 117 107 9.3 ± 0.4117.

Ang

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2

3

4

5

6

7

8

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a Second line along this column provides pressure values (bar) estimated from

A Plackett–Burman semi-factorial experimental planning

ith two levels and four variables (temperature, exposure

ime, decompression rate and reduced density or pressure) wasdopted to establish the experimental conditions. The exper-mental planning was conceived to cover the variable ranges

flcm“

able 2esults for Novozym 435 activity treated in pressurized CO2

xperiment T (◦C) t (h) R (kg m−3 min−1) R

35(−1) 1(−1) 10(−1)

35(−1) 1(−1) 200(+1)1

35(−1) 6(+1) 10(−1)1

35(−1) 6(+1) 200(+1)

75(+1) 1(−1) 10(−1)2

75(+1) 1(−1) 200(+1)1

75(+1) 6(+1) 10(−1)1

75(+1) 6(+1) 200(+1)2

55(0) 3.5(0) 105(0)1

a Second line along this column provides pressure values (bar) estimated from Ang

9 109 98

us et al.[49] equation.

ommonly used for enzyme-catalyzed reactions in compressed

uids. The experiments were accomplished randomly, and dupli-ate runs were carried out at the central point of the experi-ental design to allow estimation of standard deviation. The

−1” level represents the lower limit, while the “+1” level

D Enzyme activity (U/g) Activity loss (%)

Initial Final

0.5(−1) 873 861 1.3 ± 0.171.5a 821 810

1.6(+1) 907 880 2.5 ± 0.610.4 870 852

1.6(+1) 839 781 7.0 ± 0.110.4 926 860

0.5(−1) 874 820 6.4 ± 0.471.5 937 874

1.6(+1) 817 771 4.9 ± 0.976.4 860 823

0.5(−1) 875 830 4.9 ± 0.300 812 774

0.5(−1) 847 771 8.3 ± 0.900 890 822

1.6(+1) 917 829 8.9 ± 0.976.4 870 798

1.05(0) 835 808 3.1 ± 0.117.9 890 862

us et al. [49] equation.

376 D. Oliveira et al. / J. of Supercritical Fluids 38 (2006) 373–382

Table 3Results for Lipozyme IM submitted to compressed propane

Experiment T (◦C) P (bar) t (h) R (bar min−1) Enzyme activity (U/g) Activity loss (%)

Initial Final

1 35(−1) 30(−1) 1(−1) 2(−1) 97 95 2.0 ± 0.199 97

2 35(−1) 250(+1) 1(−1) 50(+1) 87 84 3.4 ± 0.685 82

3 35(−1) 250(+1) 6(+1) 2(−1) 83 81 1.7 ± 0.587 86

4 35(−1) 30(−1) 6(+1) 50(+1) 83 81 2.3 ± 0.689 87

5 75(+1) 250(+1) 1(−1) 2(−1) 87 86 1.1 ± 0.188 87

6 75(+1) 30(−1) 1(−1) 50(+1) 89 86 2.8 ± 0.983 81

7 75(+1) 30(−1) 6(+1) 2(−1) 89 81 8.9 ± 0.289 81

8 75(+1) 250(+1) 6(+1) 50(+1) 83 79 4.7 ± 0.787 83

9 (0)

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1

2

3

4

5

6

7

8

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55(0) 140(0) 3.5(0) 26

epresents the upper limit of each variable. A statistical mod-ling technique was used to obtain an empirical model able

o reproduce the experimental data. Empirical models wereuilt by assuming that all variable interactions were signifi-ant, the parameters related to each variable interaction andain variable effects were estimated, and the meaningless

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able 4esults for Novozym 435 submitted to compressed propane

xperiment T (◦C) P (bar) t (h) R (bar m

35(−1) 30(−1) 1(−1) 2(−1)

35(−1) 250(+1) 1(−1) 50(+1)

35(−1) 250(+1) 6(+1) 2(−1)

35(−1) 30(−1) 6(+1) 50(+1)

75(+1) 250(+1) 1(−1) 2(−1)

75(+1) 30(−1) 1(−1) 50(+1)

75(+1) 30(−1) 6(+1) 2(−1)

75(+1) 250(+1) 6(+1) 50(+1)

55(0) 140(0) 3.5(0) 26(0)

87 81 6.5 ± 0.782 77

arameters were discarded considering a confidence level of5%, by using the Student’s “t” test. This test takes into

ccount the standard error of each parameter according to aell-known procedure available in many textbooks [47]. Thearameters were estimated through the Statistica® 5.5 software48].

in−1) Enzyme activity (U/g) Activity gain (%)

Initial Final

883 899 1.7 ± 0.1897 913

831 848 2.0 ± 0.4873 891

891 928 4.1 ± 0.9863 898

865 898 3.8 ± 0.1850 883

818 898 9.3 ± 0.5825 899

879 968 10.2 ± 0.1831 917

866 958 10.4 ± 0.1851 939

813 931 14.0 ± 0.5878 998

838 918 10.1 ± 0.7852 943

D. Oliveira et al. / J. of Supercritical Fluids 38 (2006) 373–382 377

Table 5Results for Lipozyme IM submitted to compressed n-butane

Experiment T (◦C) P (bar) t (h) R (bar min−1) Enzyme activity (U/g) Activity loss (%)

Initial Final

1 35(−1) 10(−1) 1(−1) 2(−1) 98 98 097 97

2 35(−1) 250(+1) 1(−1) 50(+1) 98 98 0112 112

3 35(−1) 250(+1) 6(+1) 2(−1) 87 86 0.5 ± 0.196 96

4 35(−1) 10(−1) 6(+1) 50(+1) 150 147 3.0 ± 1.497 93

5 75(+1) 250(+1) 1(−1) 2(−1) 90 89 1.5 ± 0.5103 101

6 75(+1) 10(−1) 1(−1) 50(+1) 98 97 0.5 ± 0.183 83

7 75(+1) 10(−1) 6(+1) 2(−1) 87 83 3.6 ± 0.8153 149

8 75(+1) 250(+1) 6(+1) 50(+1) 95 92 3.2 ± 0.1153 148

9 26

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2

3

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6

7

8

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.3. Instrumentation

Infrared spectroscopy analyses using photoacustic acces-ory were made to follow possible chemical modifications asfunction of the treatment of the enzymes. FT-IR spectra were

s

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able 6esults for Novozym 435 submitted to compressed n-butane

xperiment T (◦C) P (bar) t (h) R

35(−1) 10(−1) 1(−1) 2(

35(−1) 250(+1) 1(−1) 50

35(−1) 250(+1) 6(+1) 2(

35(−1) 10(−1) 6(+1) 50

75(+1) 250(+1) 1(−1) 2(

75(+1) 10(−1) 1(−1) 50

75(+1) 10(−1) 6(+1) 2(

75(+1) 250(+1) 6(+1) 50

55(0) 130(0) 3.5(0) 26

(0) 81 80 1.7 ± 0.590 88

btained in the 4000–400 cm−1 range using a Bomem (MB-100)

pectrophotometer.

Scanning electron microscopy (SEM) and thermogravimetricnalysis (TGA) were also performed to check possible changesn the morphological and thermal properties. Thermogravimetric

(bar min−1) Enzyme activity (U/g) Activity gain (%)

Initial Final

−1) 871 880 1.8 ± 1.1835 857

(+1) 763 792 3.3 ± 0.5875 901

−1) 891 951 6.3 ± 0.6850 900

(+1) 813 850 4.5 ± 0.1891 931

−1) 851 985 15.2 ± 0.6778 893

(+1) 811 890 10.1 ± 0.5803 888

−1) 801 899 11.6 ± 0.7793 881

(+1) 803 993 21.5 ± 2.9785 938

(0) 808 882 9.9 ± 1.1835 925

3 critical Fluids 38 (2006) 373–382

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78 D. Oliveira et al. / J. of Super

nalyses were carried out in a Shimadzu, model 50 equipments,ith N2 flux at 20 mL min−1 and at a heating rate of 10 ◦C min−1.icrographs of the immobilized enzymes were obtained usingscanning electron microscope Shimadzu (SS-550) Superscan

quipment.

. Results and discussion

The experimental results obtained from the application of theactorial design to both enzymes, Lipozyme IM and Novozym35, treated in supercritical carbon dioxide and compressedropane and n-butane are presented in Tables 1–6. In these tables,eal and coded levels (±1) are given on columns 2–5, while theast three columns show the enzyme activity values before andfter treatment along with the computed percent change (lossr gain) on enzyme activity. Additional runs (last rows of theseables) were carried out at the central point, coded 0, to estimatehe overall curvature effect.

The experiments were accomplished in the temperature (T)ange of 35–75 ◦C and exposure times (t) from 1 to 6 h for allnzymes and solvents. For carbon dioxide, the decompressionates (R) covered the range 10–200 kg m−3 min−1 with reducedensity (RD) varying from 0.5 to 1.6. For this solvent, densitiesere estimated from Angus et al. [49] equation. For propane and-butane, due to their practically incompressible nature withinhe temperature and pressure ranges investigated, it was adoptedhe low (slow) and upper (fast) depressurization (R) levels of 2nd 50 bar min−1, and the system pressure (P) (30–250 bar forropane and 10–250 bar for n-butane) as process variables.

Results for Lipozyme IM can be better visualized in Fig. 2a,here one can see that, in general, treatment in carbon dioxide

ed to the highest activity losses (up to ≈14%), then followedy propane (maximum of 8.9 ± 0.2%) and in a much lesserxtent by n-butane (maximum of 3.6 ± 0.8%). For Novozym35, Fig. 2b shows that carbon dioxide also had a deleteriousffect on the activity of this enzyme, though in a less degreeompared to Lipozyme IM. On the other hand, it is worth notic-ng that treatment of Novozym 435 in compressed propane and-butane improved enzyme activity for all experimental con-itions, with resulting activity gains as high as 14% and 22%,espectively.

Tables 7 and 8 present the results obtained in the statisti-al modeling for Lipozyme IM and Novozym 435, respectively.n general, one can see from these tables that temperaturend exposure time had a pronounced effect on enzyme activ-ty, both showing a positive effect. The system pressure had aegative influence in the case Lipozyme IM/propane but influ-nced positively for Novozym 435/n-butane. Note also thatome cross-interaction effects like temperature-pressure andressure-exposure time should not be neglected. In the rangenvestigated (10–200 kg m−3 min−1, for carbon dioxide, and–250 bar min−1 for propane and n-butane) the decompressionate (R) had a weak, non-significant, effect on enzyme activity.

he same was observed for the reduced density.

From the results obtained, it can be observed that, for allxperimental conditions, the enzyme activity loss was lowerhan 15%. When compared to the work presented by Habulin

ltv

ig. 2. Enzyme activity change after treatment in compressed carbon dioxide,ropane and n-butane: (a) Lipozyme IM and (b) Novozym 435.

nd Knez [41], who studied the activity of some lipases throughhe esterification reaction of butyric acid and ethanol in super-ritical carbon dioxide and propane at 40 ◦C and 100 bar, thisctivity loss can be considered low. It is interesting to mentionhat these authors have also verified that the enzyme activity wasmproved in propane, which corroborates the results obtainedere for Novozym 435.

Results obtained in this work for both enzymes treated inarbon dioxide are in complete agreement with those reportedy some authors [1,21,22,33,35,36,38,41,50], but opposite to thenes reported by Gießauf and Gamse when dealing with porcineancreatic lipase [34]. Besides, a series of enzyme-catalyzedeactions conducted in both conventional and supercritical fluidedium has shown that while no loss of enzyme activity was

xperimentally observed for the conventional medium [51,52],he same was no longer valid for supercritical carbon dioxideontaining systems [23,24].

It has been argued that the decrease of lipase activity inupercritical carbon dioxide may be attributed to interactionsnzyme-solvent, leading to the formation of covalent complexesith the free amino groups on the surface of the enzyme to form

arbamates, which would result in charge removal at histidineesidues, and thus could contribute to the loss of enzyme activity1,15,41,53].

According to the log P hydrophobicity sensor, defined as theogarithm of the partition coefficient in a standard octanol–waterwo-phase system, activities are low in relatively hydrophilic sol-ents having log P < 2, is quite variable for solvents having log P

D. Oliveira et al. / J. of Supercritical Fluids 38 (2006) 373–382 379

Table 7Regression results for the activity of Lipozyme IM in compressed CO2, propaneand n-butane

Effect Parameter value Parameter uncertainty

Carbon dioxidea

Independent a0 = 9.5 1.18*

Temperature (T) a1 = 1.2 0.42†Exposure time (t) a2 = 4.5 0.42*

Decompression rate (R) a3 = −0.7 0.42†Tt a4 = −1.0 0.42†TRD a5 = 1.5 0.42†tRD a6 = −1.5 0.42†Quadratic a7 = −2.0 1.26†

Propaneb

Independent a0 = 6.5 0.18*

Temperature (T) a1 = 1.0 0.06*

Pressure (P) a2 = −0.7 0.06*

Exposure time (t) a3 = 1.1 0.06*

TP a4 = −0.7 0.06*

Pt a5 = −0.5 0.06*

PR a6 = 1.4 0.06*

Quadratic a7 = −3.3 0.19†

n-Butanec

Independent a0 = 1.5 0.17*

Temperature (T) a1 = 0.5 0.18*

Exposure time (t) a2 = 0.9 0.18*

TP a3 = 0.5 0.18*

Pt a4 = −0.5 0.18*

a Activity loss (%) = a0 + a1T + a2t + a3R + a4Tt + a5TRD + a6tRD + a7quadratic,correlation coefficient = 0.997.

b Activity loss (%) = a0 + a1T + a2P + a3t + a4TP + a5Pt + a6PR + a7quadratic,correlation coefficient = 0.999.

c Activity loss (%) = a0 + a1T + a2t + a3TP + a4Pt, correlation coeffi-c

bldt

Table 8Regression results for the activity of Novozym 435 in compressed CO2, propaneand n-butane

Effect Parameter value Parameter uncertainty

Carbon dioxidea

Independent a0 = 1.7 0.09*

Temperature (T) a1 = 0.7 0.03†Exposure time (t) a2 = 1.0 0.03*

Decompression rate (R) a3 = 0.1 0.03†RD a4 = 0.1 0.03†Quadratic a5 = 0.9 0.09†

Propaneb

Independent a0 = 10.0 0.82*

Temperature (T) a1 = 4.0 0.29*

Exposure time (t) a2 = 1.1 0.29*

Decompression rate (R) a3 = 0.5 0.29†Pt a4 = 0.5 0.29†Quadratic a5 = −3.0 0.87†

n-Butanec

Independent a0 = 9.4 0.42*

Temperature (T) a1 = 5.3 0.45*

Pressure (P) a2 = 2.3 0.45*

Exposure time (t) a3 = 1.6 0.45*

tP a4 = 1.4 0.45*

a Activity loss (%) = a0 + a1T + a2t + a3R + a4RD + a5quadratic, correlationcoefficient = 0.999.

b Activity gain (%) = a0 + a1T + a2t + a3R + a4Pt + a5quadratic, correlationcoefficient = 0.993.

c Activity gain (%) = a0 + a1T + a2P + a3t + a4tP, correlation coeffi-c

hr

TR

S

C

P

n

E

ient = 0.965.* Significant parameter at a 95% confidence level.† Non-significant parameter at a 95% confidence level.

etween 2 and 4, and is high in hydrophobic solvents for whichog P > 4 [3]. The reason is that solvents having log P < 2 stronglyistort the essential water–biocatalyst interactions, thereby inac-ivating or denaturing the biocatalyst [3].

sAte

able 9esults for the activity of Novozym 435 submitted to pressurization/depressurization

olvent Cycle number Initial activity (U/g)

O2 1 8832345

ropane 1 8712345

-Butane 1 8442345

xperimental conditions equal to those of experiment 5 in Tables 2, 4 and 6.

ient = 0.989.* Significant parameter at a 95% confidence level.† Non-significant parameter at a 95% confidence level.

Recent results show that pressurized carbon dioxide is ratherydrophilic, with log P values smaller than 2 in a wide pressureange and hydrophilicity increases with a decrease in pres-

ure, though the water solubility decreases simultaneously [39].ccordingly, supercritical carbon dioxide could strip the essen-

ial water from the enzyme microenvironment, thus, causingnzyme deactivation with the unfavorable water partitioning

cycles in CO2, propane and n-butane

Final activity (U/g) Activity change (%)

847 −4.0828 −6.2817 −7.4806 −8.7790 −10.5

950 9.0951 9.1951 9.1952 9.2953 9.4

980 16.1980 16.1981 16.2981 16.2982 16.3

380 D. Oliveira et al. / J. of Supercritical Fluids 38 (2006) 373–382

Fis

Fos

ig. 3. FT-IR spectra of (a) untreated Novozym 435, (b) Novozym 435 treatedn n-butane and (c) Novozym 435 treated in carbon dioxide. In the last two cases,amples from experiment 8 of Tables 2 and 6.

Ftt

ig. 5. SEM micrographs of Novozym 435 samples: (a) amplification of 50 times, (b)f 10,000 times. From left to the right side, SEM micrographs refer to untreated enzymamples from experiment 8 of Tables 2 and 6.

ig. 4. TG thermographs of (a) untreated Novozym 435, (b) Novozym 435reated in n-butane and (c) Novozym 435 treated in carbon dioxide. In the lastwo cases, samples from experiment 8 of Tables 2 and 6.

amplification of 200 times, (c) amplification of 2000 times and (d) amplificatione, treated in n-butane and in carbon dioxide, respectively. In the last two cases,

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etween the enzymatic support and the solvent [1,15,41]. Hence,ifferences in water partitioning may also have contributed tohe higher activity values verified for the examined lipases inropane and in n-butane compared to those in carbon dioxide41].

This factor may have a crucial role when considering a rapidelease of carbon dioxide dissolved in the bound water [22].teinberger et al. [38] have pointed out that the stability ofn enzyme in supercritical carbon dioxide depends on bothts tertiary structure and on several parameters during expo-ure to high-pressure carbon dioxide. They argued that highemperatures, the water content in carbon dioxide and pressur-zation/depressurization steps can cause enzyme inactivation.

Regarding treatment in propane and in n-butane, it has beenentioned that solvents with low dielectric constant favors inter-

acial activation thus opening the lipase lid [1,41]. Furthermore,ecause the hydrophobic characteristics of propane and n-butanend the fact that they behave like a conventional liquid in theresent study, and possibly due to affinity of the enzyme parti-les for these solvents, relatively lower activity losses or evenignificant activity improvements were experimentally verified.

Considering that the number of compression/expansion stepsay be a relevant parameter for batch reactors, some tests were

erformed for Novozym 435 in carbon dioxide, propane and n-utane at the conditions of experiment 5 in Tables 2, 4 and 6.esults are presented in Table 9, from where it can be observed

hat for carbon dioxide there is a continuous decrease in enzymectivity whereas it remains nearly constant for propane and n-utane. Though the number of cycles investigated is not greatnough to get to a definitive conclusion about its influence onhe activity of the lipases tested, results for carbon dioxide werelready expected since dehydration over time is taking place.

FT-IR spectra of the Novozym 435 after treatment in theompressed fluids are presented in Fig. 3. One can see from thisgure that treatment in n-butane and in carbon dioxide (exper-

ment 8, Tables 2 and 6) has not led to significant variations inhe characteristic peaks of both materials when compared to thentreated enzyme spectra. Based on these results we can pos-ulate that the immobilized enzyme did not present chemical

odifications due to the treatment conditions used.The thermal stability of the Novozym 435 after the treat-

ent conditions are illustrated as weight loss curves, Fig. 4. Theeight loss of the enzyme samples began at 250 ◦C, and it cane observed that the weight loss temperatures of the enzymesreated in n-butane and in carbon dioxide are higher than thaterified for untreated Novozym 435. The increase in the degrada-ion temperature can be related to an increase in the crystallinityf the macroporous anionic resin induced by the high-pressurereatment in n-butane and carbon dioxide.

Fig. 5a–d depicts scanning electronic microscopy (SEM)nalysis of the morphology of Novozym 435 particles inhe untreated sample and samples after treatment in n-butanend in carbon dioxide at the conditions of experiment 8 of

ables 2 and 6. Magnification from 50 to 10,000 times in this fig-re clearly shows that treatment in supercritical carbon dioxideeads to changes of the enzyme-support structure. It seems from

icrographs (a) and (b) that carbon dioxide is capable of affect-

[

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al Fluids 38 (2006) 373–382 381

ng the original shape of the enzyme, from a nearly roundedorm with a smooth surface to a somewhat deformed shape. It ismpressive to notice from micrographs (c) and (d) that the sur-ace becomes rough and cracked, opening relatively significantoles and fissures on the enzyme-support surface with appar-nt increase of enzyme particle porosity after carbon dioxidereatment. This might be a qualitative convincing evidence ofhe damaging effect of supercritical carbon dioxide on enzymectivity.

. Conclusions

Based on the results obtained here and comparing with thoseublished in the literature, it can be concluded that the magni-ude of pressure (or reduced density), temperature, decompres-ion rate and exposure time needed to affect the lipase activitytrongly depends on the nature and the source of enzyme and,ainly, whether the enzyme is in its native or immobilized form.he results obtained in this work may be relevant with the pur-ose of selecting appropriate reaction conditions in order to usehe immobilized lipases with the lowest activity loss at typicalonditions of temperature and pressure employed in many bio-ransformation processes of raw materials.

cknowledgements

The authors thank CNPq, FAPERGS and ANP/FINEP/ETROBRAS for the financial support of this work andcholarships.

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