Journal of Supercritical Fluids 19 (2000) 79 86 www.elsevier.

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Activation and denaturation of hydrolases in dry and humid supercritical carbon dioxide (SC-CO2)
Christoph Bauer *, Doris-J. Steinberger, Gerald Schlauer, Thomas Gamse, Rolf Marr
Uni6ersity of Technology Graz, Institute fu r Thermische Verfahrens -und Umwelttechnik, Inffeldgasse 25, A -8010 Graz, Austria

Abstract The effects of enzyme treatment with dry and humid supercritical carbon dioxide (SC-CO2) were investigated using hydrolases (EC 3.1.1.1 and EC 3.1.1.3). A crude and a puried preparation of esterase EP10 from Burkholderia gladioli was incubated in SC-CO2 for long term and repeated high pressure treatments. Concerning the crude preparation, incubation for 24 h in SC-CO2 at 150 bar and 35C had no effect on enzyme activity while incubation at 75C led to a distinct loss of residual activity. After 30 pressurization and depressurization steps at 35C and 150 bar, the crude enzyme preparation showed an activity increase. Using the puried enzyme preparation of esterase EP10 from B. gladioli, no signicant effects could be observed. Fluorescence spectra indicated no conformational change before and after treatment with SC-CO2. Treatment of a preparation of esterase from porcine liver with wet and dry SC-CO2 at 200 bar at different temperatures showed a signicant denaturing inuence of the dissolved water on residual activities of the enzyme at temperatures of more than 40C. Lipase from Candida rugosa and esterase from porcine liver were treated at 150 and 300 bar at a constant temperature of 40C and an incubation time of 22 h. During these treatments, different amounts of water were introduced into the SC-CO2. The results showed an increase of the water content in the treated enzyme preparation while the enzyme activity remained stable till the maximum amount of water soluble in this medium was injected into the SC-CO2. 2000 Elsevier Science B.V. All rights reserved.
Keywords: Supercritical carbon dioxide; Hydrolases; Stability; Activity; Water content

1. Introduction The application of enzymes as biocatalysts in supercritical carbon dioxide (SC-CO2) has been described in the scientic literature since the mid
* Corresponding author. Tel.: + 43-316-8737984; fax: + 43316-8737472. E -mail address: bauer@tvtut.tu-graz.ac.at (C. Bauer).

1980s by Randolph et al. [1], Hammond et al. [2] and Nakamura et al. [3], who demonstrated that enzymes are active and stable in SC-CO2. From these rst demonstrations, various studies have been reported on the inuence of several parameters on the catalytic activity and stability of enzymes in SC-CO2, the inuence of temperature, pressure and humidity of the SC-CO2 and pressurization and depressurization steps.

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Enzymes need a specic amount of bound water to be active. This is of vital importance for biocatalysis in SC-CO2, which is a non aqueous medium. However, if the water content of the carbon dioxide is too high or water is a product in the reaction (for example, esterications), the humidity can increase to a value where the enzyme gets inactivated. SC-CO2 may dissolve 0.3 0.35% (w/w) water, depending on pressure and temperature. Kasche et al. [4] reported that a-chymotrypsin and trypsin were partly denatured by humid (3% (w/v)) SC-CO2 caused by partial unfolding of the enzyme structure during the depressurization step. The denaturation of these enzymes can be avoided when the depressurization is performed after incubation of the enzyme in dry SC-CO2. They also reported, that penicillin amidase is rapidly denatured during the depressurization step after incubation in both humid and dry SCCO2. Chulalaksananukul et al. [5] studied the thermostability of immobilized lipase from Mucor miehei at different water contents and temperatures in SC-CO2. They noted that the water content and the temperature level must be properly balanced for optimum enzyme stability and activity. After reaching a maximum value of catalytic activity, the enzyme activity decreased with increasing water content at all temperatures. By examining lipase catalyzed reactions is SC-CO2, Dumont et al. [6], Marty et al. [7] and Yoon et al. [8] came to the conclusion, that there is always a distinct optimum amount of dissolved water depending on the enzyme. Zagrobelny et al. [9] showed by uorescence spectroscopic studies with trypsin, that pressurization steps are the reason for a change in protein conformation. The inuence of purity of the enzyme preparation was examined in only one publication by Giebauf et al. [10] using two preparations of lipase from Aspergillus niger (3.4 and 1 U mg 1; triolein as substrate). No inuence on temperature stability in SC-CO2 and on stability against pressurization and depressurization steps was detected.

Only few studies deal with the fact that SCCO2 treatment leads to a signicant increase of enzyme activity. However, SC-CO2 is known to be a nonpolar solvent, which exhibits excellent solubility for many impurities of crude enzyme preparations like carbohydrates, fatty acids and triglycerides, while the protein itself is generally insoluble in SC-CO2. Kamihira et al. [11] reported an activity increase (21 and 35%) of a crude a-amylase mixed with Escherichia coli or bakers yeast at a weight ratio of 9:1 during a sterilization step at 200 atm and 35C for 2 h. Giebauf et al. [10] showed, that treatment of lipase from Pseudomonas species with SC-CO2 at 150 bar and 45C for 1 h led to an activity increase of 20%, and incubation of lipase from porcine pancreas at 150 bar and 75C for 24 h caused an activity increase of several hundred percent [12]. Up to the present, these purication effects have not been found at enzyme preparations from other sources. The aim of this paper is to investigate the inuence of SC-CO2 treatment on stability and the catalytic activity of hydrolases (esterases (EC 3.1.1.1) and lipases (EC 3.1.1.3)), which are used for esterications and transesterications in non aqueous media. The inuence of purity on long term stability at various temperatures and on stability against pressurization and depressurization steps was examined using a crude and a puried preparation of esterase EP10 from Burkholderia gladioli. Michor et al. [13] reported that esterase EP10 from B. gladioli proved to be a suitable catalyst for transesterication of D,Lmenthol for the production of enantiomerically pure L-menthyl acetat with high enantioselectivity. To examine the possibility that the enzyme becomes modied during processing, the samples were characterized by uorescence spectroscopy. The inuence of water on enzyme stability was studied by two experiments, treatment of esterase from porcine liver with wet and dry SCO2 at different temperatures. Treatment of esterase from porcine liver and lipase from C. rugosa with SC-CO2 at different pressures with different amounts of water at a temperature commonly used in biocatalysis with hydrolases. Esterase from porcine liver is applied for resolution of

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racemic esters by esterication and lipase from C. rugosa is the lipase of choice for resolution of esters of secondary alcohols. Esterase fromporcine liver was used instead from B. glad ioli because Michor et al. [14] have already demonstrated, that any amount of additional water in the SC-CO2 inactivates this hydrolase. Both esterase from porcine liver and lipase from C. rugosa proved to be stable and active in dry SC-CO2 as reported by Giebauf et al. [10] and Steinberger et al. [15].

2.2. Determination of enzyme acti6ity

Photometric assays were used for enzyme activity measurements before and after treatment with SC-CO2 (all absorbance measurements were performed with a Shimadzu UV 160A spectrophotometer equipped with a Lauda RM6 temperature control). The activity was measured after storage of the preparations in plastic tubes (closed with a screw cap) in a freezer at 18C for at least 24 h.

2. Materials and methods

2.1. Materials
All chemicals used, if not otherwise stated, were purchased from Merck (Darmstadt) and were of p.a. (pro analysi) quality. The carbon dioxide with a purity \ 99.94% (v/v) and a dewing point lower than 60C was purchased from Linde (Graz) and stored in a tank with a capacity of 3200 l. Esterase EP10 from B. gladi oli was a gift from the Department of Biotechnology, University of Technology, Graz. Two preparations were used, crude (0.2 U mg 1 lyophilisate, 2-nitrophenyl butyrate as substrate) and chromatographically (HIC, hydrophobic interaction chromatography) puried (0.4 U mg 1 lyophilisate, 2-nitrophenyl butyrate as substrate). Specication of esterase EP10, recombinant enzyme overpressed in E. coli BL21 [DE3]. A preparation of esterase from porcine liver (19 U mg 1 preparation, ethyl butyrate as substrate) was purchased from Sigma Aldrich (Vienna). Lipase AY 30 from C. rugosa (30 U mg 1 preparation, olive oil as substrate) was supplied by Amano (Nagoya). 2-nitrophenyl butyrate (o NPB) was supplied by Sigma Aldrich (Vienna). Thesit and 1,2-O -dilauryl-rac-glycero-3-glutaric acid-resorun ester (DGGR) were purchased from Boehringer Mannheim Biochemica (Mannheim). Coomassie Brilliant Blue G-250 was provided by Serva (Heidelberg). Celluloseacetate lters (not sterile, 0.22 mm) were supplied by Roth (Karlsruhe). Karl Fischer solutions were purchased from Merck (Darmstadt).

2.2.1. Esterase assay Ten microliter of substrate solution (84 ml o NPB dissolved in 916 ml ethanol) were added to 990 ml of an enzyme solution (100 mg esterase ml 1 in 0.1-M tris(hydroxymethyl)aminomethan/HCl, pH 7.0). The linear increase of absorbance at 420 nm after 7 min of incubation at 25C was used to determine the enzyme activity. Three independent activity measurements for each sample were performed and the standard deviation (S.D.) was calculated. 2.2.2. Lipase assay A hundred microliter of substrate solution (6 mg 1,2-O -dilauryl-rac-glycero-3-glutaric acid-resorun ester were dissolved in 12 ml of a 1:1 mixture of dioxan/Thesit) were added to 900 ml of the enzyme solution (2.2 mg lipase ml 1 dissolved in 0.1-M KH2PO4, pH 6.8). The linear increase of absorbance at 572 nm after 2 min of incubation was used to measure the activities. Three independent activity measurements for each sample were performed and the S.D. was calculated. 2.3. Measurement of water content
Water contents of the untreated and treated enzymes were determined by the Karl Fischer titration method at 55C. The device consisted of a ABU93 triburette automatically controlled by the Radiometer Copenhagen VIT 90 video titrator. Three independent measurements were performed and the S.D. was calculated.

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2.4. Fluorescence spectroscopy 2.4.1. Tryptophan uorescence Fluorescence spectra of the intrinsic uorescence of the clear solutions of the proteins (0.1 mg ml 1) after ltering through the cellulose acetate lters were recorded with a Perkin Elmer LS50B spectrouorimeter (instrument settings, excitation wavelength, 280 nm; emission wavelength, 300 420 nm; excitation/emission slits, 3 nm; scan speed, 400 nm min 1). The emission spectra recorded are the average of ten scans. The proteins were dissolved in the same buffer solution as used for activity measurements and the emission spectra were recorded at 30C. 2.5. Long term stability of crude and puried enzyme preparations of esterase EP10 from B. gladioli
Thirty milligram of enzyme preparation was balanced on a lter paper and the folded lter was put into a high-pressure reactor (140 ml) placed in a water bath (Fig. 1). The temperature inside the reactor was measured by means of a platinum sensor. CO2 was compressed by a piston pump up to the required pressure and the enzyme was incubated under these conditions for 24 h at 150 bar and 35 and 75C, respectively. After treatment, the enzyme was stored at 18C and the activity was measured. Fluorescence spectra were recorded of the preparation with treatment at 75C.

2.6. Pressurization and depressurization experiments with crude and puried enzyme preparations of esterase EP10 from B. gladioli
The same device, as described above, was used. The enzyme preparation (30 mg) was incubated for 1 h in SC-CO2 at 150 bar and 35C. Then the reactor was depressurized to atmospheric pressure (duration about 6 min) and again pressurized to 150 bar. This procedure was repeated 30 times. Afterwards, the enzyme was stored at 18C in a freezer, the activity was measured and uorescence spectra were recorded.

2.7. Inuence of temperature on acti6ity of esterase from porcine li6er in wet and dry SC -CO2
To check the inuence of dissolved water in SC-CO2 during enzyme incubation, two high pressure reactors (140 ml) as shown in Fig. 1 were used, one lled with dry SC-CO2, the other with dry SC-CO2 and 30 ml of water placed at the bottom of the reactor. The enzyme preparation (30 mg) was placed at the top of both reactors (to avoid a direct contact to the liquid, if water was inside). During the experiment at 200 bar and 1-h incubation time, the inuence of different temperatures (20100C) on enzyme stability in wet and dry CO2 was investigated. Before measuring enzyme activity, the enzyme was stored at 18C in a freezer after treatment.

2.8. Inuence of water on lipase from C. rugosa and esterase from porcine li6er
These experiments were carried out with enzyme preparations (400 mg) incubated in a high pressure reactor placed in a water bath at 150 and 300 bar at a constant temperature of 40C and an incubation time of 22 h (Fig. 2). During these treatments, different amounts of water (100, 200 and 300 ml) were injected through a HPLC valve to the system (total volume, 170 ml), while SCCO2 was pumped in a cycle by a gear pump. Water was given to the system in 50 ml portions, waiting about 30 min between the injections.

Fig. 1. Schematic illustration of the device, used for the long term experiments and pressurization/depressurization experiments, a, cryostat; b, piston pump; c, high pressure reactor; d, heater; e, wash bottle; PI, pressure indicator; TI, temperature indicator; V1, inlet valve; V2, outlet valve.

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3. Results and discussions

3.1. Long term stability of crude and puried enzyme preparations of esterase EP10 from B. gladioli
Table 1 shows the residual activities of the crude and puried esterase preparations after treatment with SC-CO2 at 75 and 35C at a pressure of 150 bar for 24 h. While there is no signicant change in enzyme activity of the puried enzyme preparation, the activity of the crude esterase preparation did not change after high pressure treatment at 35C (103.8%) but decreased to 62.3%, of the untreated preparation after incubation at 75C. The treatment at 75C had the effect that the appearance of the crude enzyme preparation changed from yellow color and a fatty consistence to a caramel brown color and to rm pieces while the color and consistency of the puried enzyme preparation did not change remaining a white power. In contrast to lipase from A. niger examined by Giebauf et al. [10], the purity of the enzyme preparation of esterase EP10 from B. gladioli is an important factor for thermal stability in SC-CO2.

Fig. 2. Schematic illustration of the device with SC-CO2 cycle, a, cryostat; b, piston pump; c, high pressure reactor; d, heater; e, gear pump; f, wash bottle; g, HPLC valve; V1, inlet valve; V2, valve for the cycle; V3, outlet valve; PI, pressure indicator; TI, temperature indicator.

Table 1 Comparison between the residual activity of crude and puried esterase from B. gladioli after treatment with SC-CO2 at 150 bar and different temperatures for 24 ha Temperature (C) Residual activity (%), crude (mean 9 S.D.) 103.8 9 4.10 62.3 9 5.82 Residual activity (%), puried (mean 9 S.D.) 98.7 9 5.46 93.7 9 8.11

35 75
a

Value of the untreated enzyme was set to 100%.

3.2. Pressurization and depressurization experiments with crude and puried enzyme preparations of esterase EP10 from B. gladioli
Table 2 shows the enzyme activities after 30 pressurization and depressurization cycles with SC-CO2 at 35C and 150 bar. Each incubation step lasted 1 h. A temperature of 35C was chosen for this experiment to avoid thermal inactivation. The experiment had no effect on the puried enzyme preparation and the appearance of the white powder did not change at all. The 30 pressurization and depressurization cycles did not generate any activity loss. The enzyme activity of crude esterase, however, increased to 120.7% of the untreated preparation and the crude enzyme preparation became a bright yellow dry power. Repeated extraction had a greater purifying effect than the long-term extraction, because the preparation was contacted with fresh CO2 at the beginning of each pressurization cycle. Therefore, the

Table 2 Comparison between enzyme activity of crude and puried esterase from B. gladioli before and after 30 pressurization and depressurization cyclesa Residual activity (%), crude (mean 9 S.D.) 120.7 9 3.58
a

Residual activity (%), puried (mean 9 S.D.) 101.9 9 2.93

Value of the untreated enzyme was set to 100%.

After depressurization, the water content of the treated enzyme was measured immediately by Karl Fischer titration and the activity was measured after storage in a freezer.

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mass transfer of the impurities into the supercritical phase was enhanced.

3.3. Fluorescence spectroscopy

In the study of the intrinsic uorescence of esterase EP10 from B. gladioli treated with SCCO2, it was found out that no change of emission maxima occurred. The exposure to SC-CO2 (longTable 3 Fluorescence emission maxima of esterase from B. gladioli in Tris-buffer at pH 7.0a Treatment Untreated 24 h, 75C, 150 bar 30 pressurization/depressurization steps, 35C, 150 bar
a

Fig. 5. Residual activity of esterase from porcine liver after treatment with various amounts of water dissolved in the SC-CO2.

A (nm) 340.0 341.0 339.5

B (nm) 331.5 331.0 330.0

A, crude preparation; B, puried preparation.

term treatment of pressurization and depressurization steps, see Table 3) causes, therefore, no larger protein conformational change. So, the activity decrease of the crude preparation at 75C might be caused by chemical modications on protein reactive groups by impurities in the preparation.

3.4. Inuence of temperature on acti6ity of esterase from porcine li6er in wet and dry SC -CO2
Incubation of the enzyme at different temperatures showed clear differences between wet (30 ml water inside the reactor) and dry SC-CO2 at temperatures of more than 40C (see Fig. 3). Working with dry carbon dioxide, only a slight loss of enzyme activity up to a temperature of 80C was observed. At 100C, the activity of the esterase sharply decreased to 76.6%. The results with wet carbon dioxide show this decrease to a residual activity of 71.3% already at 65C, remaining at this level at higher temperatures.

Fig. 3. Residual activity of esterase from porcine liver after treatment with dry and wet SC-CO2 at 200 bar at different temperatures.

3.5. Inuence of water on lipase from C. rugosa and esterase from porcine li6er
Figs. 4 and 5 show the residual enzyme activities after SC-CO2 treatment at 150 and 300 bar at 40C with increasing amounts of water for lipase from C. rugosa and esterase from porcine liver. The added water led to a signicant change in the water content of the preparation of both enzymes

Fig. 4. Residual activity of lipase from C. rugosa after treatment with various amounts of water dissolved in the SC-CO2.

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(see Figs. 6 and 7) but had no effect on the activity over a wide range. Even at a water content of more than 170% of the untreated preparation at 300 bar and 200 ml added water, the enzyme preparation of lipase from C. rugosa remained stable and active (residual activity, 91.6%). In contrast to previous studies about enzyme stability in SC-CO2, no optimum amount of water could be detected. Both preparations remained stable and active (more than 85% residual activity at 150 bar and more than 90% at 300 bar) at all amounts of water added, that did not exceed the maximum amount of water soluble in the SC-CO2. For the used system volume (170 ml), this is 270 ml at 150 bar and 40C and 350 ml for 300 bar. Injection of 300 ml water at 150 bar led to a signicant loss of enzyme activity (residual activity, around 40%), because of denaturing effects of the undissolved water during the depressuriza-

tion step. Both enzymes are well suited for biocatalysis with production of high amounts of water. High pressure should be used to maximize the solubility of water in SC-CO2 at a given temperature.

4. Conclusion The results show that SC-CO2 treatment leads in certain cases to an increase of enzymatic activity when crude preparations are used. Purity was proved to be a factor, that inuences temperature stability, because impurities decreased the stability of the examined esterase during incubation in SC-CO2 at high temperatures. The water content of the SC-CO2 had a signicant inuence on activity and stability only at temperatures of more than 40C, the temperature level normally used for biocatalysis with hydrolases. So both enzymes are t for use for biocatalysis in SC-CO2 with high accumulation of water like esterication reactions. The purication effect, shown in this paper, might be another benet of the use of supercritical carbon dioxide in biocatalysis, which is still restricted to research on laboratory scale.

Acknowledgements This work was supported by a grant of the Austrian Science Fund FWF (project no. 13269CHE). Fluorescence spectra were recorded at the Department of Biochemistry at the Karl-Franzens University, Graz. Special thanks to Professor Schwab (Department of Biotechnology, University of Technology, Graz) and his co-workers for donating us esterase from B. gladioli.

Fig. 6. Water content of lipase from C. rugosa after treatment with various amounts of water dissolved in the SC-CO2.

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Fig. 7. Water content of esterase from porcine liver after treatment with various amounts of water dissolved in the SC-CO2.

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C. Bauer et al. / J. of Supercritical Fluids 19 (2000) 7986 [10] A. Giebauf, W. Magor, D.-J. Steinberger, R. Marr, A study of hydrolases stability in supercritical carbon dioxide (SC-CO2), Enzyme Microb. Technol. 24 (1999) 577 583. [11] M. Kamihira, M. Taniguchi, T. Kobayashi, Sterilization of micro-organisms with supercritical carbon dioxide, Agric. Biol. Chem. 51 (2) (1987) 407 412. [12] A. Gieauf, T. Gamse, A simple process for increasing the specic activity of porcine pancreatic lipase by supercritical carbon dioxide treatment, J. Mol. Catal. B: Enzymatic 9 (2000) 57 64. [13] H. Michor, R. Marr, T. Gamse, T. Schilling, E. Klingsbichel, H. Schwab, Enzymatic catalysis in supercritical carbon dioxide: Comparison of different lipases and a novel esterase, Biotechnol. Lett. 18 (1996) 79 84. [14] H. Michor, R. Marr, T. Gamse, Enzymatic catalysis in supercritical carbon dioxide: effect of water activity, Proceedings of the Third International Symposium in High Pressure Chemical Engineering, In: R. Rohr, C. Trepp (Eds.), High Pressure Chemical Engineering, Netherlands, 1996, pp. 115 120. [15] D.-J. Steinberger, T. Gamse, R. Marr, Enzyme inactivation and prepurication effects of supercritical carbon dioxide (SC-CO2), Proceedings of the Fifth Conference on Supercritical Fluids and their Applications, Garda, 1999, pp. 339 346.

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