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Effect of pressure on the catalytic activity of subtilisin Carlsberg suspended in compressed gases
Biochimica et Biophysica Acta 1383 Ž1998. 165–174
Effect of pressure on the catalytic activity of subtilisin Carlsberg suspended in compressed gases Nuno Fontes, Eugenia ´ Nogueiro, A. Margarida Elvas, Teresa Correa ˆ de Sampaio, ) Susana Barreiros Instituto de Tecnologia Quımica e Biologica, UniÕersidade NoÕa de Lisboa, Quinta do Marques, ´ ´ ˆ Apt. 127, 2780 Oeiras, Portugal Received 5 September 1997; revised 14 November 1997; accepted 26 November 1997
Abstract We studied the effect of pressure up to 300 bar on the catalytic efficiency of subtilisin Carlsberg suspended in compressed propane, near-critical ethane, near-critical carbon dioxide and tert-amyl alcohol, at constant temperature and fixed enzyme hydration. Increasing pressure lowered the catalytic efficiency of the enzyme in all the solvents, resulting in positive activation volumes, DV a. The DV a values in compressed propane and in tert-amyl alcohol were similar and larger in magnitude than the value reported in the literature for the same reaction in an aqueous buffer, although within the range of typical DV a values in aqueous media. In the near-critical fluids, the DV a were much larger, e.g., an increase in pressure of only 200 bar causing a sixfold decrease in the catalytic efficiency of subtilisin in carbon dioxide. These data should reflect the proximity of ethane and carbon dioxide to the critical point, and the resulting condensation of solvent molecules about the solutes, yielding negative solute partial molar volumes. q 1998 Elsevier Science B.V. Keywords: Subtilisin; Supercritical fluid; Enzyme hydration; Activation volume
1. Introduction High pressure may be used advantageously in many areas of biotechnology. One such area where industrial applications already exist is that of food processing. For example, pressures of 4 to 9 kbar inactivate bacteria and irreversibly denature enzymes
Abbreviations: DV a, activation volume; APEE, N-acetyl-Lphenylalanine ethyl ester; APPE, N-acetyl-L-phenylalanine propyl ester; k, reaction rate constant; SAFT, Statistical Associating Fluid Theory; ´ , dielectric constant ) Corresponding author. Fax: q351-1-441-1277; E-mail: [email protected]
w1,2x, thereby increasing shelf-life while leaving unaffected vitamins and other nutrients, as opposed to thermal treatments. Pressures up to 4 or 5 kbar have been found to improve protein stability at high temperature w3x and to affect both enzyme activity w4x and selectivity w5x. The effect of pressure on the catalytic activity of an enzyme depends on the magnitude and the sign of the differences in volume between the activated and ground states for the reaction steps involved, i.e., the activation volumes for each step, DV a. A reaction step which proceeds through a reduction in volume is accelerated by an increase in pressure, and vice versa. Activation volumes for enzymatic reactions in aqueous media are typically in the range of y50 to q50
0167-4838r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 8 3 8 Ž 9 7 . 0 0 2 0 0 - 8
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cm3 moly1 w6x. For example, the rate of a reaction step with a DV a of y30 cm3 moly1 at 358C increases by about 10% as pressure is raised 100 bar and approximately triples with a pressure increase of 1000 bar. Much more moderate pressures can have a great impact on reactions rates for processes carried out in supercritical or near-critical fluids. Explored in this case is the adjustability of the properties of the solvent close to the critical point, in particular the pronounced changes in density and related compressibility and solvation ability when pressure is varied at constant temperature close to that point. For example, using solvatochromic shift data for a dye to characterize the solvent strength of supercritical ethylene, Kim and Johnston w7x were able to predict a more than two orders of magnitude increase in the rate of a chemical reaction caused by a change in pressure of only 200 bar at constant temperature in that solvent. Here we examine the effect of pressure on the catalytic activity of subtilisin Carlsberg suspended in near-critical ethane, near-critical carbon dioxide and compressed liquid propane and present activation volume data. Although pressure is a natural choice of a parameter to vary when using supercritical or nearcritical fluids and some of the authors who study biocatalysis in these media have reported on the effect of pressure on reaction rates w8–11x, activation volume data for enzymatic reactions under such conditions are rare w12x. There are also not many such data in organic solvents. Exceptions include the works of Kim and Dordick w13x and of Mozhaev et al. w14x who studied the response to pressure of subtilisin Carlsberg suspended in tert-amyl alcohol and of achymotrypsin entrapped in reverse micelles in n-octane, respectively. For the purpose of comparing data with that of Kim and Dordick, we also include some results for subtilisin in tert-amyl alcohol.
2. Materials and methods 2.1. Reagents Subtilisin Carlsberg Ž from Bacillus licheniformis, with a specific activity of 11.9 U mgy1 solid. and N-acetyl-L-phenylalanine ethyl ester ŽAPEE. were obtained from Sigma, 1-propanol from Merck, hydranal
coulomat A and C Karl Fischer reagents were from Riedel de Haen. CO 2 , ethane, propane and nitrogen were supplied by Air Liquide and guaranteed to have purities of over 99.995 mol% or 99.95 mol% Žethane and propane. . Prior to use, the enzyme was lyophilized from a 20 mM potassium phosphate buffer solution Ž5 mg cmy3 . at pH 7.8. 1-propanol was dried over molecular sieves. 2.2. High pressure bioreactor Variable volume stainless steel cells equipped with a sapphire window were used. Details of the experimental apparatus are given in Ref. w15x. 2.3. Measurement of water-sorption isotherm points and of catalytic actiÕities The cell was loaded with the enzyme Ž 1.7 mg cmy3 ., 1-propanol Ž 0.90 M. and the desired amount of water, and pressurized with the solvent to the desired volume at 100 bar and 358C. The mixture was allowed to equilibrate for 2 h. Samples were taken and released directly into a Karl Fischer apparatus to determine the water content of the solvent at equilibrium. The initial water contents of both enzyme and solvent were determined in separate experiments. Enzyme hydration at water-partitioning equilibrium with the solvent was obtained by a mass balance. To proceed with reaction, APEE Ž 10 mM. dissolved in appropriate amounts of 1-propanol and water was flushed into the cell with the solvent to the desired pressure and volume. Periodically, stirring was stopped, and samples were taken for GC analysis. More details of the experimental technique are given in Ref. w15x. At pressures higher than 100 bar and in the compressible solvents, the amounts of the two substrates were increased so as to keep the respective mole fractions constant throughout the experiments. With tert-amyl alcohol, the reaction mixture was loaded into the cell in one step and allowed to equilibrate for 1 h before any samples were taken for water quantification or to follow the course of the reaction. 2.4. GC analysis All GC analysis was performed with a 6000 Vega Series 2 Carlo-Erba gas chromatograph with a
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Chromjet Spectra-Physics integrator. GC conditions: 15-m DB-5 capillary column from J & W Scientific; oven temperature, 2008C; injection temperature, 2508C; flame ionization detection ŽFID. temperature, 2708C; carrier gas, helium Ž1 cm3 miny1 .; split ratio, 1:50.
3. Results 3.1. Dependence of enzyme hydration on pressure in compressed gases The relationship between the state of hydration of the enzyme and the water concentration in the solvent mixture, at water-partitioning equilibrium, defines a water-sorption isotherm for the enzyme in that particular solvent mixture. Fig. 1 shows the water-sorption isotherm data we obtained in compressed propane and in near-critical carbon dioxide, at various pressures. Included for comparison is an isotherm for the enzyme in CO 2 at 150 bar, in the presence of 0.2 M benzyl alcohol w15x. 3.2. Dependence of enzyme actiÕity on enzyme hydration in compressed gases With the enzyme and solvent phases at water-partitioning equilibrium, the ester substrate was added to
Fig. 2. Dependence of the catalytic activity on enzyme hydration in propane. Reaction conditions: T s 358C; P s150 bar; w1-propanolx s 0.90 M; wAPEEx s10 mM; wenzymex s1.7 mg cmy3.
start the reaction which involved no net production or consumption of water. The water-sorption data allow the representation of the catalytic activity of subtilisin as a function of enzyme hydration, as shown in Fig. 2 for compressed propane at 150 bar. Close to the maximum in the figure, small differences in the level of hydration of the enzyme have a great effect on the measured catalytic activities; towards any of the ends of the curve, the experimental error associated to the enzyme hydration level is of less consequence. A fixed enzyme hydration level for all studies in the various solvents was thus chosen as 15%. It should be noted that a value to the left of the maximum could have been similarly selected. However, the initial hydration of the enzyme powder obtained after lyophilization was usually around 15%. In contact with solvents having a low water solubility, like propane or ethane, such a powder would tend to keep most of its water, making it difficult to attain low enzyme-hydration levels in the reaction medium upon water-partitioning equilibrium. 3.3. Dependence of enzyme actiÕity on pressure
Fig. 1. Water-sorption isotherm data for subtilisin in compressed gases. Squares, propane, 0.90 M in 1-propanol: ŽB. 100 bar; ŽI. 200 bar; ŽG. 300 bar. Other symbols: carbon dioxide, approximately 0.9 M in 1-propanol: Ž%. 100 bar; Ž=. 150 bar; Ž = . 200 bar; Žq. 250 bar; Žl. 300 bar. Line: carbon dioxide, 0.2 M in benzyl alcohol, 150 bar w15x.
Fig. 3 shows the effect of pressure on the catalytic activity of subtilisin suspended in compressed propane, near-critical ethane, near-critical CO 2 and tert-amyl alcohol. Enzyme hydration was 15% in the
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molarity units at a given pressure by the molar volume of the solvent mixture at the same pressure. We took the volumes of the mixtures to be those of the solute-free solvents. The slopes of the lines in the figure multiplied by Ž RT . yield the DV a values given in Table 1. The standard deviations of these values are of the order of magnitude of that affecting the DV a reported by Mozhaev et al. w3x for a hydrolysis reaction catalyzed by a-chymotrypsin. Included for comparison are the DV a values obtained by Kim and Dordick w13x for the reaction studied in the present work, in tert-amyl alcohol and in an aqueous buffer. Fig. 3. Dependence of the logarithm of the catalytic activity, based on mole fraction units, on pressure in nonaqueous solvents at 358C. Enzyme hydration was 15% in all solvents except tert-amyl alcohol where it was approximately 2%. Žv . tert-amyl alcohol; ŽB. compressed propane; Ž'. near-critical ethane; Ž%. near-critical carbon dioxide.
compressed gases and about 2% in tert-amyl alcohol. This low hydration was reached by keeping initial conditions constant with no added water Ž the solubility of water in tert-amyl alcohol is much higher than in any of the compressed gases w16x.. As discussed in Section 1, the direct effect of pressure on the reaction rate constant for an elementary process, k, is determined by the activation volume, DV a: wŽ Eln krEP . T s yDV arRT x, where P is the pressure, T the temperature and R the gas constant, and k must be based on pressure independent units such as mole fraction. In order to have catalytic efficiencies based on mole fraction units as we do in Fig. 3, we needed to divide each value obtained in Table 1 Activation volumes for the subtilisin-catalyzed transesterification of APEE by 1-propanol Solvent
Enzyme hydration Ž%.
DV a Žcm3 moly1 .
CO 2 Ethane Propane Tert-amyl alcohol Tert-amyl alcohola Tert-amyl alcohola Tert-amyl alcohola Aqueous buffer a
15 15 15 2 3 9 25
233"24 139"19 44"6 30"5 18.8 31.2 44.0 y3.5
a
Ref. w13x.
4. Discussion 4.1. Water-sorption isotherm data Condensed phases are not very sensitive to pressure. At 358C and pressures up to 300 bar, propane behaves like a conventional liquid, e.g., its density increases by about 7% as pressure goes from 100 to 300 bar w17x. In the same range, and in spite of a density increase of about 19% w18x, the solubility of water in ethane remains practically constant w19x, as with propane w20x. This means that the water-sorption isotherms for subtilisin in each of these two solvents at pressures to 300 bar should be superimposable. The presence of 1-propanol does not significantly change this situation, as can be seen in Fig. 1 for propane. On the other hand, the density of CO 2 increases by about 27% as pressure goes from 100 to 300 bar at 358C w21x and this density increase is accompanied by an increase in water solubility from 1.21 to 1.84 g dmy3 w22,23x. Such a large variation in water-stripping ability, observed also in the presence of 1-propanol, is reflected in a marked dependence on pressure of the water-sorption isotherm for the enzyme in CO 2 , as shown also in Fig. 1. We note that it was not our objective to trace the whole isotherms but rather to define the parts of the isotherms corresponding to about 15% enzyme hydration, the selected value for activity measurements. Under the same experimental conditions, the relative positions of the water-sorption isotherms in CO 2 and in propane reflect the large differences in water solubility referred above, i.e., higher water concentrations in CO 2
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than in propane for a fixed enzyme hydration. The points taken in CO 2 –0.9 M 1-propanol at 150 bar are to the right of those taken in CO 2 –0.2 M benzyl alcohol at equal pressure, as the higher polarity and concentration of 1-propanol would lead us to expect. The data for CO 2 in Fig. 1 allowed the selection of the amounts of added water required to reach approximately 15% enzyme hydration in this solvent at varying pressure. 4.2. Catalytic actiÕity Õs. enzyme hydration The catalytic activity of subtilisin Carlsberg depends very strongly on its state of hydration. As the latter increases, so does catalytic activity until a maximum is reached, further hydration of the enzyme bringing about a decline in activity, as seen in Fig. 2. Previous evidence for such bell-shaped activity vs. enzyme hydration curves was presented by Affleck et al. w24x, Borges de Carvalho et al. w15x and Correa ˆ de w x Sampaio et al. 25 . These authors obtained a maximum in the activity of the enzyme at 10–12% enzyme hydration, for different transesterification reactions and in solvents ranging from organic to supercritical. This is again the case here. The activity maximum in the figure is of the same order of magnitude as that obtained by Affleck et al. w24x for the transesterification of N-acetyl-L-phenylalanine 2chloroethyl ester by 1 M n-propanol in tetrahydrofuran. 4.3. SolÕent effects on catalytic actiÕity at constant enzyme hydration in compressed gases As Fig. 3 shows, the enzyme is clearly more active in propane and in ethane than in CO 2 , at otherwise identical experimental conditions. Previous evidence to such differences in catalytic activity was presented by Borges de Carvalho et al. w15x for a different transesterification reaction. The authors reported activity maxima at the top of the bell-shaped activityrenzyme hydration curves in compressed propane and in supercritical CO 2 which differed by a factor of 50. The adverse effect of CO 2 is not only observed with subtilisin but also with lipases, free w26,27x or immobilized w28,29x. The possibility of CO 2 lowering the pH of a microaqueous phase associated with the enzyme or altering the state of ionization of the enzyme in the absence of such a phase has
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been suggested by several authors w26,28,30x. Kamat et al. w27x, on the other hand, believe that this is not the main reason for comparatively lower activity in CO 2 since the buffering salts present in the lyophilization solution should provide a protective matrix against possible local pH effects. These authors suggest instead a mechanism of enzyme inactivation of CO 2 involving the formation of carbamates. We note that Fig. 3 refers to a state of hydration of the enzyme beyond that corresponding to maximum enzyme activity. Much larger activity differences than those shown in the figure would be expected at enzyme hydration levels corresponding to the top of the bellshaped curves. As we discussed in an earlier paper w25x, there is evidence to suggest that the fraction of intact active centers of subtilisin does not vary significantly with enzyme hydration up to 20% in a variety of nonaqueous solvents. The studies supporting these conclusions were performed at ambient pressure. We will assume that pressure in the range used in this work similarly does not affect the fraction of intact active centers of the enzyme. Because we did not actually measure that quantity, we used the parameter Vmax instead of k cat . 4.4. Catalytic actiÕity Õs. pressure at constant enzyme hydration Subtilisin Carlsberg suspended in nonaqueous solvents acts via the ping-pong bi–bi mechanism which involves at least two activation states w31x, as shown in Scheme 1 w32x. Thus, the data in Fig. 3 allow the
Scheme 1.
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calculation of effective DV a values representative of the rate-determining step as well as other steps in the reaction, at the pressure range under study. There is evidence suggesting that the reaction studied in the present work does not suffer from diffusional limitations w33,34x, and thus our DV a values should not include pressure effects on mass transfer. The DV a in propane, 44 cm3 moly1, is larger in magnitude than the value reported in the literature for the same reaction in an aqueous buffer, as seen in Table 1, although it is within the range of typical DV a values obtained in aqueous media w6x. Chaudhary et al. w11x also studied the present reaction in propane. The authors presented their data on the effect of pressure on catalytic activity in graph form only. The corresponding DV a should be small in magnitude since pressure did not significantly affect the activity of subtilisin up to 350 bar. The DV a we obtained in propane is also similar to the values given by Kim and Dordick w13x for the same reaction in tert-amyl alcohol, included in Table 1. For the sake of comparison, experiments with tert-amyl alcohol were also done, where a DV a value of 30 cm3 moly1 was obtained at about 2% enzyme hydration. We note that we were not able to measure very low enzyme hydration levels as accurately as higher ones. Kim and Dordick, on the other hand, did not measure enzyme hydration directly at high pressure as we did, but controlled that parameter through an adjustment of initial conditions only, which might explain their observed increase in catalytic efficiency up to too high enzyme hydration levels. Experimental difficulties could help to explain the difference between our DV a value and that given by Kim and Dordick for apparently identical enzyme hydration conditions. DV a values can be very large for homogeneous reactions in supercritical or near-critical fluids. For example, Johnston and Haynes w35x obtained DV a values as low as y6000 cm3 moly1 for the decomposition of a-chlorobenzyl methyl ester in 1,1-difluoroethane. Fluids close to their critical point are highly compressible. Under these conditions, the solvent condenses about solute molecules, yielding solute partial molar volumes that can be thousands of cm3 moly1 negative w36x. These can result in very large DV a values which are differences in partial molar volumes. A similar finding was reported by Kim and Johnston w37x for the Diels–Alder reaction
of isoprene and maleic anhydride in supercritical CO 2 . The authors obtained a linear relationship between the logarithm of the rate constant w38x and the solvent strength, as measured by the transition energy of a dye. In the region of higher compressibility close to the critical point, the predicted DV a was as large as y4000 cm3 moly1 at 358C, reaching a value of y225 cm3 moly1 at 100 bar. Although the rate constant continued to increase as the density and solvent strength of CO 2 increased, it did so in a modest way, as the compressibility of the solvent farther from the critical point decreased. For example, at 300 bar, DV a was y55 cm3 moly1, a value similar to those obtained in conventional liquids. As mentioned in Section 1, DV a data for biocatalytic reactions in supercritical or near-critical fluids are rare. For example, Erickson et al. w8x and Kamat et al. w9,10x reported on the effect of pressure on reaction rates in CO 2 and in ethane, and in fluoroform, ethane and sulfur hexafluoride, respectively, without giving any DV a values. More recently, Kamat et al. w12x calculated DV a data from the reaction rates for the lipase-catalyzed transesterification of methylmethacrylate by 2-ethylhexanol in fluoroform reported earlier w10x. As with subtilisin, so does the catalytic activity of the lipase used by those authors decrease with increasing pressure, yielding positive DV a values. These were about 1800 cm3 moly1 close to the critical point of the solvent, and decreased with increasing pressure, reaching a constant value close to zero at about 200 bar. In the present study, CO 2 and ethane Žwith 1-propanol. were never as close to the respective critical points as e.g., CO 2 and fluoroform were in the studies by Kim and Johnston w37x and by Kamat et al. w10x, a condition which should be reflected on the magnitude of our DV a values: 233 cm3 moly1 in CO 2 , 139 cm3 moly1 in ethane, which could conceivably be much larger, closer to the critical point. We believe in any case that the insensitivity of these values to pressure changes, i.e., linearity of the plots in Fig. 3 for CO 2 and ethane, may not be factual but rather an artifice resulting from taking the molar volumes of the solvent mixtures to be those of the solute-free solvents, especially at the lower pressures tested and thus closer to the critical point. In fact, whereas the mole fractions of both water and the ester substrate were sufficiently low in all the solvents to be neglected in
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all calculations, the mole fraction of 1-propanol was 0.05 in CO 2 and 0.07 in ethane and in propane Žcorresponding to a concentration of 0.9 M at 110 bar. . We could not find molar-volume data for the mixtures of interest in the literature. In the case of propane and because this solvent behaves pretty much like a conventional liquid under the conditions of this study, the molar volume of the solvent mixture could be calculated by assuming a linear dependence of composition; the same is true for tert-amyl alcohol. The resulting volumes differed by less than 1% from those of the pure solvents and thus our approach was to use the latter. This approach, which was also adopted for CO 2 and ethane, is in this case an oversimplification. In fact, an extended version w39x of the SAFT ŽStatistical Associating Fluid Theory. equation of state w40,41x, which gives good pure component densities, predicts 1 for the solvent mixtures lower molar volumes than those of pure CO 2 or ethane, as shown in Table 2. This is consistent with the notion of local density enhancement of the solvent about a solute and negative solute partial molar volumes. The volume change introduced by the solute decreases as the mixtures get further away from the critical point and the solvent becomes less compressible. For example, at 200 bar, such changes are a small percentage only, as seen also in Table 2. Although the extended SAFT equation does not predict sufficiently accurate volumes for mixtures close to the critical point and thus the calculated data in the table cannot be used, we believe it correctly predicts the trend of the data. In fact, the equation similarly predicts that the volumes of mixtures of CO 2 with a few percent methanol close to the critical point are lower than the volume of the pure solvent, a trend confirmed in this case by experimental data w43x. With correct molar-volume data, our experimental points for CO 2 and ethane at the lower pressures represented in Fig. 3 should lie above the straight lines, and we would thus have a dependence of DV a on pressure. All our solvents have low dielectric constants, ´ : approximately 1.7 and 1.4 for propane w17x and ethane
1
Calculations done by Oliver Pfohl, using a van der Waals mixing rule with two parameters as in Ref. w39x, which were fit to the phase-equilibrium data published in Ref. w42x.
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Table 2 Literature w18,21x and predicted 1 values Žextended SAFT equation. w39–41x for the molar volumes of mixtures of CO 2 and ethane with 1-propanol, at given mole fractions, x, at 358C Molar volume Žcm3 moly1 . Pressure Žbar. 100
200
300
Carbon dioxideq 1-propanol x CO2 0 73.4 0.95 54.6 1 62.0 1, tabulated 61.9
72.6 49.7 50.3 50.8
71.9 47.1 46.7 47.3
Ethaneq 1-propanol x ethane 0.93 76.3 1 82.8 1, tabulated 83.4
70.6 72.7 73.4
67.4 68.3 69.0
w10x, respectively, and 1.1–1.6 for CO 2 w44x, at the conditions of our study. A correlation between DV a and ´ , obtained by Kim and Dordick w13x for the present reaction in several organic solvents, suggested that DV a values should be negative in nonpolar solvents. This is however not the case, as seen in our results. The fact that ethane and CO 2 are relatively close to the critical point could help explain a departure from the above correlation. On the other hand, propane also does not conform to that prediction, and yet it behaves like a conventional liquid. Another implication of the referred correlation is that solvents with the same ´ should yield the same DV a. This is also unlike our results. In fact, our solvents have practically the same dielectric constants and yet there are significant differences in DV a among them. Again, the fact that ethane and CO 2 are near-critical could help explain these observations. As pointed out by Mozhaev et al. w4x, the pressure dependence of the rates of enzymatic reactions may reflect changes in the rate-determining catalytic step or conformational changes of the enzyme. These changes may give rise to departures from linearity in the plots of ln k vs. pressure. Mozhaev et al. w3x, on the other hand, referred that the linear character of such a plot as they obtained for an a-chymotrypsincatalyzed hydrolysis gave an indication that the catalytic mechanism and the active conformation of the
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enzyme did not change in the pressure interval studied. As we mentioned earlier, we would have expected a departure from linearity for CO 2 and ethane in Fig. 3 at the lower pressures studied if we had been able to use the correct molar volumes of the solvent mixtures. We cannot rule out the possibility of solvent-induced mechanistic changes or conformational changes on the enzyme in these two solvents and indeed in any of them. For CO 2 in particular, Ikushima et al. w45x showed that in a very limited pressure range near its critical point the interactions between this solvent and a lipase provoked drastic conformational changes on the enzyme, causing active sites that catalyzed a stereoselective synthesis to emerge. We were never so near the critical point of CO 2 in the present study. However, we note that the positive DV a values obtained by Kim and Dordick w13x were due to high positive activation volumes for substrate binding, i.e., weaker substrate binding. As mentioned earlier, CO 2 has an adverse effect on subtilisin suggested to be due to the formation of covalent complexes between CO 2 and free amine groups at lysine residues w27x. If our positive DV a values are a result of weaker substrate binding, then the larger DV a in CO 2 could be partly due to the interactions of this solvent with the enzyme. Why should activation volumes be larger in magnitude in nonaqueous media than in aqueous solutions, as observed for the present reaction in tert-amyl alcohol and in propane? Kim and Dordick w13x explain their results in tert-amyl alcohol on the basis of pressure decreasing the affinity of bound water to the enzyme while increasing the affinity of bound water to itself w46x, a fact which would result in water stripping and diminished catalytic activity at higher pressures. As the authors referred, the transfer of water from a more polar environment to a less polar one should be a positive contribution to DV a. Mozhaev et al. w3x, on the other hand, pointed out that the application of high pressure should fortify the protein hydration shell and be directed toward preferential hydration of the protein. We note, in any case, that pressures up to 300 bar are very moderate in this context. We note also that the concept of water stripping involves the ability of the solvent to solubilize the water that is released from the enzyme. Conventional liquid solvents like propane in the present study are nearly insensitive to pressure, in partic-
ular in what concerns the amount of water they are able to solubilize and, hence, their water-stripping ability Ž and in CO 2 , where it is not so, we measured points on the various water-sorption isotherms for the enzyme so as to keep enzyme hydration constant. . An explanation based on increased water stripping at higher pressure also presents the problem that it could only hold true for the left side of the bell-shaped activity vs. enzyme hydration curve. In fact, reducing enzyme hydration from a point at the end of the bell —e.g., our fixed 15% enzyme hydration in the compressed gases—results in an increase in catalytic activity. The above explanation would also not elucidate a negative DV a. Kim and Dordick w13x also showed that the effect of pressure was more marked as the state of hydration of the enzyme increased. It is known that increasing enzyme hydration brings about an increase in the molecular mobility of an enzyme. This increase in dynamics may allow the enzyme to assume the conformation which is thermodynamically favorable in the nonaqueous medium, resulting in denaturation. Burke et al. w47x have indeed shown that the degree of flexibility of an enzyme and its degree of unfolding are highly correlated. Kim and Dordick explain the increase in DV a with pressure on the basis of increased enzyme flexibility causing increased exposure of enzyme-bound water to the solvent and, consequently, an increase in water stripping. The greater the state of hydration of the enzyme, the larger the number of water–water contacts that would form and thus the more positive the value of DV a. This, however, would seem to require the ability of the nonaqueous solvent, at constant pressure, to solubilize more or less water depending on the state of hydration of the enzyme. As explained by Mozhaev et al. w4x, pressure effects are especially pronounced on processes in which the hydration state of the system components changes significantly. The reaction mechanism for subtilisin catalysis includes several steps, as shown in Scheme 1. The enzyme–substrate and enzyme–product complexes and the activation complexes involved will likely be hydrated differently; e.g., the binding of a substrate will likely involve the release of water molecules from the enzyme active site w9x. When an enzymatic reaction takes place in an aqueous solution, changes in the hydration of the intervening
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entities can easily be accommodated by the solvent. It should not be so when the solvent is nonaqueous. This and the known sensitivity of catalytic activity to enzyme hydration in nonaqueous media should potentially lead to greater Žin magnitude. pressure effects in these systems. The way in which water redistributes itself during catalysis at constant enzyme hydration and at varying pressure needs to be better understood.
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strongly dependent on enzyme hydration, as is the case with subtilisin, is still not very clear.
Acknowledgements This work was supported by Junta Nacional de Ž Portugal. Investigac¸ao e Tecnologica ˜ Cientıfica ´ ´ through the contract PRAXIS 2r2.1rBIOr34r94. We thank Dr. Oliver Pfohl for doing the calculations with the extended SAFT equation.
5. Conclusions Increasing pressure decreased the catalytic efficiency of subtilisin Carlsberg suspended in compressed propane, in near-critical ethane and in nearcritical CO 2 . This effect was more pronounced in the near-critical solvents, and especially in CO 2 , where the enzyme became completely inactive slightly above 300 bar. This is reflected in the corresponding activation volumes: the DV a obtained in compressed propane is similar to that in tert-amyl alcohol and of the order of magnitude of the DV a measured in aqueous solutions, whereas those in the near-critical solvents are much larger. This can be explained by the higher compressibility of a solvent close to the critical point and the compression of the solvent about the solutes. The extent to which this effect contributes to the DV a measured in CO 2 and in ethane is impossible to determine here, and so is the intrinsic DV a resulting from volume changes arising from changes in bond lengths and angles only w48x. As referred to by Kamat et al. w10x, the determination of microscopic rate constants would help to evaluate the effect of the solvent physical properties on each step of the reaction mechanism. Insight into the role of the solvent is provided by Ikushima et al. w45x who used a spectroscopic technique to show how the interactions of CO 2 with a lipase triggered the activation of the enzyme. A review of the spectroscopic techniques available for the elucidation of enzyme structure is given by Mozhaev et al. w4x. There is evidence to the fact that in the absence of covalent bond formation or breaking, the largest contributions to DV a are expected to result from hydration changes accompanying noncovalent interactions w4x. How these hydration changes are accommodated by nonaqueous solvents where enzymatic activity can be
References w1x U.R. Pothakamury, G.V. Barbosa-Canovas, B.G. Swanson, ´ R.S. Meyer, Chem. Eng. Prog., March Ž1995. 45–53. w2x V.V. Mozhaev, K. Heremans, J. Frank, P. Masson, C. Balny, TIBTECH 12 Ž1994. 493–501. w3x V.V. Mozhaev, R. Lange, E.V. Kudryashova, C. Balny, Biotechnol. Bioeng. 52 Ž1996. 320–331. w4x V.V. Mozhaev, K. Heremans, J. Frank, P. Masson, C. Balny, Proteins 24 Ž1996. 81–91. w5x O. Guthmann, R. Schwerdtfeger, A. Rieks, G. Antranikian, V. Kasche, G. Brunner, in: P. von Rohr, C. Trepp ŽEds.., High Pressure Chemical Engineering, Process Technology Proceedings 12, Elsevier Ž1996. 127–131. w6x E. Morild, Adv. Protein Chem. 34 Ž1981. 93–166. w7x S. Kim, K.P. Johnston, Ind. Eng. Chem. Res. 26 Ž1987. 1206–1213. w8x J.C. Erickson, P. Schyns, C.L. Cooney, AIChE J. 36 Ž1990. 299–301. w9x S.V. Kamat, E.J. Beckman, A.J. Russell, J. Am. Chem. Soc. 115 Ž1993. 8845–8846. w10x S.V. Kamat, B. Iwaskewycz, E.J. Beckman, A.J. Russell, Proc. Natl. Acad. Sci. U.S.A. 90 Ž1993. 2940–2944. w11x A.K. Chaudhary, S.V. Kamat, E.J. Beckman, D. Nurok, R.M. Kleyle, P. Hadju, A.J. Russell, J. Am. Chem. Soc. 118 Ž1996. 12891–12901. w12x S.V. Kamat, E.J. Beckman, A.J. Russell, Crit. Rev. Biotechnol. 15 Ž1995. 41–71. w13x J. Kim, J.S. Dordick, Biotechnol. Bioeng. 42 Ž1993. 772– 776. w14x V.V. Mozhaev, N. Bec, C. Balny, Biochem. Mol. Biol. Int. 34 Ž1994. 191–199. w15x I. Borges de Carvalho, T. Correa ˆ de Sampaio, S. Barreiros, Biotechnol. Bioeng. 49 Ž1996. 399–404. w16x J.A. Riddick, W.B. Bunger, T.K. Sakano, Organic solvents, Physical Properties and Methods of Purification, 4th edn., Wiley-Interscience, New York, 1986. w17x R.D. Goodwin, W.M. Haynes, NBS Monograph 170, 1982. w18x R.D. Goodwin, H.M. Roder, G.C. Straty, NBS Technical Note 684, 1976. w19x G. Morrison, J. Phys. Chem. 85 Ž1981. 759–761.
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w20x R. Kobayashi, D.L. Katz, Ind. Eng. Chem. 45 Ž1953. 440– 451. w21x U. Sievers, Fortcshritt-Berichte der VDI Zeits Verei Deutscher Ingenieure Nr.155, Dusseldorf, 1984. ¨ w22x J. Chrastil, J. Phys. Chem. 86 Ž1982. 3016–3021. w23x R. Wiebe, V.L. Gaddy, J. Am. Chem. Soc. 63 Ž1941. 475–477. w24x R. Affleck, Z.F. Xu, V. Suzawa, K. Focht, D.S. Clark, J.S. Dordick, Proc. Natl. Acad. Sci. U.S.A. 89 Ž1992. 1100– 1104. w25x T. Correa ˆ de Sampaio, R.B. Melo, T.F. Moura, S. Michel, S. Barreiros, Biotechnol. Bioeng. 50 Ž1996. 257–264. w26x S. Kamat, J. Barrera, E.J. Beckman, A.J. Russell, Biotechnol. Bioeng. 40 Ž1992. 158–166. w27x S. Kamat, G. Critchley, E.J. Beckman, A.J. Russell, Biotechnol. Bioeng. 46 Ž1995. 610–620. w28x A. Marty, W. Chulalaksananukul, R.M. Willemot, J.S. Condoret, Biotechnol. Bioeng. 39 Ž1992. 273–280. w29x M.C. Almeida, R. Ruivo, C. Maia, L. Freire, T. Correa ˆ de Sampaio, S. Barreiros, Enzyme Microb. Technol. Ž1998. in press. w30x M. Perrut, in: C. Balny, R. Hayashi, K. Heremans, P. Masson ŽEds.., High Pressure and Biotechnology, Colloque INSERM 224, John Libbey, 1992, pp. 401–410. w31x T. Ke, R. Wescott, A.M. Klibanov, J. Am. Chem. Soc. 118 Ž1996. 3366–3374. w32x M.J. Sowa, PhD thesis, University of Delaware, Delaware, 1993. w33x Y.L. Khmelnitsky, S.H. Welch, D.S. Clark, J.S. Dordick, J. Am. Chem. Soc. 116 Ž1994. 2647–2648.
w34x S. Kamat, E.J. Beckman, A.J. Russell, Enzyme Microb. Technol. 14 Ž1992. 265–271. w35x K. Johnston, C. Haynes, AIChE. J. 33 Ž1987. 2017. w36x C.A. Eckert, D.H. Ziger, K.P. Johnston, S. Kim, J. Phys. Chem. 90 Ž1986. 2738–2746. w37x S. Kim, K.P. Johnston, Am. Chem. Soc. Symp. Ser., 329, 1987, pp. 42–55. w38x C.A. Eckert, C.R. Hsieh, J.R. McCabe, AIChE J. 20 Ž1. Ž1974. 20. w39x O. Pfohl, G. Brunner, paper submitted to Ind. Eng. Chem. Res., 1997. w40x S.H. Huang, M. Radosz, Ind. Eng. Chem. Res. 29 Ž1990. 2284–2294. w41x S.H. Huang, M. Radosz, Ind. Eng. Chem. Res. 30 Ž1991. 1994–2005. w42x K. Suzuki, H. Sue, M. Itou, R.L. Smith, H. Inomata, K. Arai, S. Saito, J. Chem. Eng. Data 35 Ž1990. 63–66. w43x E. Brunner, W. Hultenschmidt, G. Schlichtharle, J. Chem. ¨ ¨ Thermodynamics 19 Ž1987. 273–291. w44x A. Michels, L. Kleerkoper, Physica 6 Ž1939. 586–590. w45x Y. Ikushima, N. Saito, M. Arai, H.W. Blanch, J. Phys. Chem. 99 Ž1995. 8941–8944. w46x C.E. Kundrot, F.M. Richards, J. Mol. Biol. 200 Ž1988. 401–410. w47x P.A. Burke, R.G. Griffin, A.M. Klibanov, J. Biol. Chem. 267 Ž1992. 20057–20064. w48x P.E. Savage, S. Gopalan, T.I. Mizan, C.J. Martino, E.E. Brock, AIChE J. 41 Ž1995. 1723.
Effect of pressure on the catalytic activity of subtilisin Carlsberg suspended in compressed gases Nuno Fontes, Eugenia ´ Nogueiro, A. Margarida Elvas, Teresa Correa ˆ de Sampaio, ) Susana Barreiros Instituto de Tecnologia Quımica e Biologica, UniÕersidade NoÕa de Lisboa, Quinta do Marques, ´ ´ ˆ Apt. 127, 2780 Oeiras, Portugal Received 5 September 1997; revised 14 November 1997; accepted 26 November 1997
Abstract We studied the effect of pressure up to 300 bar on the catalytic efficiency of subtilisin Carlsberg suspended in compressed propane, near-critical ethane, near-critical carbon dioxide and tert-amyl alcohol, at constant temperature and fixed enzyme hydration. Increasing pressure lowered the catalytic efficiency of the enzyme in all the solvents, resulting in positive activation volumes, DV a. The DV a values in compressed propane and in tert-amyl alcohol were similar and larger in magnitude than the value reported in the literature for the same reaction in an aqueous buffer, although within the range of typical DV a values in aqueous media. In the near-critical fluids, the DV a were much larger, e.g., an increase in pressure of only 200 bar causing a sixfold decrease in the catalytic efficiency of subtilisin in carbon dioxide. These data should reflect the proximity of ethane and carbon dioxide to the critical point, and the resulting condensation of solvent molecules about the solutes, yielding negative solute partial molar volumes. q 1998 Elsevier Science B.V. Keywords: Subtilisin; Supercritical fluid; Enzyme hydration; Activation volume
1. Introduction High pressure may be used advantageously in many areas of biotechnology. One such area where industrial applications already exist is that of food processing. For example, pressures of 4 to 9 kbar inactivate bacteria and irreversibly denature enzymes
Abbreviations: DV a, activation volume; APEE, N-acetyl-Lphenylalanine ethyl ester; APPE, N-acetyl-L-phenylalanine propyl ester; k, reaction rate constant; SAFT, Statistical Associating Fluid Theory; ´ , dielectric constant ) Corresponding author. Fax: q351-1-441-1277; E-mail: [email protected]
w1,2x, thereby increasing shelf-life while leaving unaffected vitamins and other nutrients, as opposed to thermal treatments. Pressures up to 4 or 5 kbar have been found to improve protein stability at high temperature w3x and to affect both enzyme activity w4x and selectivity w5x. The effect of pressure on the catalytic activity of an enzyme depends on the magnitude and the sign of the differences in volume between the activated and ground states for the reaction steps involved, i.e., the activation volumes for each step, DV a. A reaction step which proceeds through a reduction in volume is accelerated by an increase in pressure, and vice versa. Activation volumes for enzymatic reactions in aqueous media are typically in the range of y50 to q50
0167-4838r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 8 3 8 Ž 9 7 . 0 0 2 0 0 - 8
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cm3 moly1 w6x. For example, the rate of a reaction step with a DV a of y30 cm3 moly1 at 358C increases by about 10% as pressure is raised 100 bar and approximately triples with a pressure increase of 1000 bar. Much more moderate pressures can have a great impact on reactions rates for processes carried out in supercritical or near-critical fluids. Explored in this case is the adjustability of the properties of the solvent close to the critical point, in particular the pronounced changes in density and related compressibility and solvation ability when pressure is varied at constant temperature close to that point. For example, using solvatochromic shift data for a dye to characterize the solvent strength of supercritical ethylene, Kim and Johnston w7x were able to predict a more than two orders of magnitude increase in the rate of a chemical reaction caused by a change in pressure of only 200 bar at constant temperature in that solvent. Here we examine the effect of pressure on the catalytic activity of subtilisin Carlsberg suspended in near-critical ethane, near-critical carbon dioxide and compressed liquid propane and present activation volume data. Although pressure is a natural choice of a parameter to vary when using supercritical or nearcritical fluids and some of the authors who study biocatalysis in these media have reported on the effect of pressure on reaction rates w8–11x, activation volume data for enzymatic reactions under such conditions are rare w12x. There are also not many such data in organic solvents. Exceptions include the works of Kim and Dordick w13x and of Mozhaev et al. w14x who studied the response to pressure of subtilisin Carlsberg suspended in tert-amyl alcohol and of achymotrypsin entrapped in reverse micelles in n-octane, respectively. For the purpose of comparing data with that of Kim and Dordick, we also include some results for subtilisin in tert-amyl alcohol.
2. Materials and methods 2.1. Reagents Subtilisin Carlsberg Ž from Bacillus licheniformis, with a specific activity of 11.9 U mgy1 solid. and N-acetyl-L-phenylalanine ethyl ester ŽAPEE. were obtained from Sigma, 1-propanol from Merck, hydranal
coulomat A and C Karl Fischer reagents were from Riedel de Haen. CO 2 , ethane, propane and nitrogen were supplied by Air Liquide and guaranteed to have purities of over 99.995 mol% or 99.95 mol% Žethane and propane. . Prior to use, the enzyme was lyophilized from a 20 mM potassium phosphate buffer solution Ž5 mg cmy3 . at pH 7.8. 1-propanol was dried over molecular sieves. 2.2. High pressure bioreactor Variable volume stainless steel cells equipped with a sapphire window were used. Details of the experimental apparatus are given in Ref. w15x. 2.3. Measurement of water-sorption isotherm points and of catalytic actiÕities The cell was loaded with the enzyme Ž 1.7 mg cmy3 ., 1-propanol Ž 0.90 M. and the desired amount of water, and pressurized with the solvent to the desired volume at 100 bar and 358C. The mixture was allowed to equilibrate for 2 h. Samples were taken and released directly into a Karl Fischer apparatus to determine the water content of the solvent at equilibrium. The initial water contents of both enzyme and solvent were determined in separate experiments. Enzyme hydration at water-partitioning equilibrium with the solvent was obtained by a mass balance. To proceed with reaction, APEE Ž 10 mM. dissolved in appropriate amounts of 1-propanol and water was flushed into the cell with the solvent to the desired pressure and volume. Periodically, stirring was stopped, and samples were taken for GC analysis. More details of the experimental technique are given in Ref. w15x. At pressures higher than 100 bar and in the compressible solvents, the amounts of the two substrates were increased so as to keep the respective mole fractions constant throughout the experiments. With tert-amyl alcohol, the reaction mixture was loaded into the cell in one step and allowed to equilibrate for 1 h before any samples were taken for water quantification or to follow the course of the reaction. 2.4. GC analysis All GC analysis was performed with a 6000 Vega Series 2 Carlo-Erba gas chromatograph with a
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Chromjet Spectra-Physics integrator. GC conditions: 15-m DB-5 capillary column from J & W Scientific; oven temperature, 2008C; injection temperature, 2508C; flame ionization detection ŽFID. temperature, 2708C; carrier gas, helium Ž1 cm3 miny1 .; split ratio, 1:50.
3. Results 3.1. Dependence of enzyme hydration on pressure in compressed gases The relationship between the state of hydration of the enzyme and the water concentration in the solvent mixture, at water-partitioning equilibrium, defines a water-sorption isotherm for the enzyme in that particular solvent mixture. Fig. 1 shows the water-sorption isotherm data we obtained in compressed propane and in near-critical carbon dioxide, at various pressures. Included for comparison is an isotherm for the enzyme in CO 2 at 150 bar, in the presence of 0.2 M benzyl alcohol w15x. 3.2. Dependence of enzyme actiÕity on enzyme hydration in compressed gases With the enzyme and solvent phases at water-partitioning equilibrium, the ester substrate was added to
Fig. 2. Dependence of the catalytic activity on enzyme hydration in propane. Reaction conditions: T s 358C; P s150 bar; w1-propanolx s 0.90 M; wAPEEx s10 mM; wenzymex s1.7 mg cmy3.
start the reaction which involved no net production or consumption of water. The water-sorption data allow the representation of the catalytic activity of subtilisin as a function of enzyme hydration, as shown in Fig. 2 for compressed propane at 150 bar. Close to the maximum in the figure, small differences in the level of hydration of the enzyme have a great effect on the measured catalytic activities; towards any of the ends of the curve, the experimental error associated to the enzyme hydration level is of less consequence. A fixed enzyme hydration level for all studies in the various solvents was thus chosen as 15%. It should be noted that a value to the left of the maximum could have been similarly selected. However, the initial hydration of the enzyme powder obtained after lyophilization was usually around 15%. In contact with solvents having a low water solubility, like propane or ethane, such a powder would tend to keep most of its water, making it difficult to attain low enzyme-hydration levels in the reaction medium upon water-partitioning equilibrium. 3.3. Dependence of enzyme actiÕity on pressure
Fig. 1. Water-sorption isotherm data for subtilisin in compressed gases. Squares, propane, 0.90 M in 1-propanol: ŽB. 100 bar; ŽI. 200 bar; ŽG. 300 bar. Other symbols: carbon dioxide, approximately 0.9 M in 1-propanol: Ž%. 100 bar; Ž=. 150 bar; Ž = . 200 bar; Žq. 250 bar; Žl. 300 bar. Line: carbon dioxide, 0.2 M in benzyl alcohol, 150 bar w15x.
Fig. 3 shows the effect of pressure on the catalytic activity of subtilisin suspended in compressed propane, near-critical ethane, near-critical CO 2 and tert-amyl alcohol. Enzyme hydration was 15% in the
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molarity units at a given pressure by the molar volume of the solvent mixture at the same pressure. We took the volumes of the mixtures to be those of the solute-free solvents. The slopes of the lines in the figure multiplied by Ž RT . yield the DV a values given in Table 1. The standard deviations of these values are of the order of magnitude of that affecting the DV a reported by Mozhaev et al. w3x for a hydrolysis reaction catalyzed by a-chymotrypsin. Included for comparison are the DV a values obtained by Kim and Dordick w13x for the reaction studied in the present work, in tert-amyl alcohol and in an aqueous buffer. Fig. 3. Dependence of the logarithm of the catalytic activity, based on mole fraction units, on pressure in nonaqueous solvents at 358C. Enzyme hydration was 15% in all solvents except tert-amyl alcohol where it was approximately 2%. Žv . tert-amyl alcohol; ŽB. compressed propane; Ž'. near-critical ethane; Ž%. near-critical carbon dioxide.
compressed gases and about 2% in tert-amyl alcohol. This low hydration was reached by keeping initial conditions constant with no added water Ž the solubility of water in tert-amyl alcohol is much higher than in any of the compressed gases w16x.. As discussed in Section 1, the direct effect of pressure on the reaction rate constant for an elementary process, k, is determined by the activation volume, DV a: wŽ Eln krEP . T s yDV arRT x, where P is the pressure, T the temperature and R the gas constant, and k must be based on pressure independent units such as mole fraction. In order to have catalytic efficiencies based on mole fraction units as we do in Fig. 3, we needed to divide each value obtained in Table 1 Activation volumes for the subtilisin-catalyzed transesterification of APEE by 1-propanol Solvent
Enzyme hydration Ž%.
DV a Žcm3 moly1 .
CO 2 Ethane Propane Tert-amyl alcohol Tert-amyl alcohola Tert-amyl alcohola Tert-amyl alcohola Aqueous buffer a
15 15 15 2 3 9 25
233"24 139"19 44"6 30"5 18.8 31.2 44.0 y3.5
a
Ref. w13x.
4. Discussion 4.1. Water-sorption isotherm data Condensed phases are not very sensitive to pressure. At 358C and pressures up to 300 bar, propane behaves like a conventional liquid, e.g., its density increases by about 7% as pressure goes from 100 to 300 bar w17x. In the same range, and in spite of a density increase of about 19% w18x, the solubility of water in ethane remains practically constant w19x, as with propane w20x. This means that the water-sorption isotherms for subtilisin in each of these two solvents at pressures to 300 bar should be superimposable. The presence of 1-propanol does not significantly change this situation, as can be seen in Fig. 1 for propane. On the other hand, the density of CO 2 increases by about 27% as pressure goes from 100 to 300 bar at 358C w21x and this density increase is accompanied by an increase in water solubility from 1.21 to 1.84 g dmy3 w22,23x. Such a large variation in water-stripping ability, observed also in the presence of 1-propanol, is reflected in a marked dependence on pressure of the water-sorption isotherm for the enzyme in CO 2 , as shown also in Fig. 1. We note that it was not our objective to trace the whole isotherms but rather to define the parts of the isotherms corresponding to about 15% enzyme hydration, the selected value for activity measurements. Under the same experimental conditions, the relative positions of the water-sorption isotherms in CO 2 and in propane reflect the large differences in water solubility referred above, i.e., higher water concentrations in CO 2
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than in propane for a fixed enzyme hydration. The points taken in CO 2 –0.9 M 1-propanol at 150 bar are to the right of those taken in CO 2 –0.2 M benzyl alcohol at equal pressure, as the higher polarity and concentration of 1-propanol would lead us to expect. The data for CO 2 in Fig. 1 allowed the selection of the amounts of added water required to reach approximately 15% enzyme hydration in this solvent at varying pressure. 4.2. Catalytic actiÕity Õs. enzyme hydration The catalytic activity of subtilisin Carlsberg depends very strongly on its state of hydration. As the latter increases, so does catalytic activity until a maximum is reached, further hydration of the enzyme bringing about a decline in activity, as seen in Fig. 2. Previous evidence for such bell-shaped activity vs. enzyme hydration curves was presented by Affleck et al. w24x, Borges de Carvalho et al. w15x and Correa ˆ de w x Sampaio et al. 25 . These authors obtained a maximum in the activity of the enzyme at 10–12% enzyme hydration, for different transesterification reactions and in solvents ranging from organic to supercritical. This is again the case here. The activity maximum in the figure is of the same order of magnitude as that obtained by Affleck et al. w24x for the transesterification of N-acetyl-L-phenylalanine 2chloroethyl ester by 1 M n-propanol in tetrahydrofuran. 4.3. SolÕent effects on catalytic actiÕity at constant enzyme hydration in compressed gases As Fig. 3 shows, the enzyme is clearly more active in propane and in ethane than in CO 2 , at otherwise identical experimental conditions. Previous evidence to such differences in catalytic activity was presented by Borges de Carvalho et al. w15x for a different transesterification reaction. The authors reported activity maxima at the top of the bell-shaped activityrenzyme hydration curves in compressed propane and in supercritical CO 2 which differed by a factor of 50. The adverse effect of CO 2 is not only observed with subtilisin but also with lipases, free w26,27x or immobilized w28,29x. The possibility of CO 2 lowering the pH of a microaqueous phase associated with the enzyme or altering the state of ionization of the enzyme in the absence of such a phase has
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been suggested by several authors w26,28,30x. Kamat et al. w27x, on the other hand, believe that this is not the main reason for comparatively lower activity in CO 2 since the buffering salts present in the lyophilization solution should provide a protective matrix against possible local pH effects. These authors suggest instead a mechanism of enzyme inactivation of CO 2 involving the formation of carbamates. We note that Fig. 3 refers to a state of hydration of the enzyme beyond that corresponding to maximum enzyme activity. Much larger activity differences than those shown in the figure would be expected at enzyme hydration levels corresponding to the top of the bellshaped curves. As we discussed in an earlier paper w25x, there is evidence to suggest that the fraction of intact active centers of subtilisin does not vary significantly with enzyme hydration up to 20% in a variety of nonaqueous solvents. The studies supporting these conclusions were performed at ambient pressure. We will assume that pressure in the range used in this work similarly does not affect the fraction of intact active centers of the enzyme. Because we did not actually measure that quantity, we used the parameter Vmax instead of k cat . 4.4. Catalytic actiÕity Õs. pressure at constant enzyme hydration Subtilisin Carlsberg suspended in nonaqueous solvents acts via the ping-pong bi–bi mechanism which involves at least two activation states w31x, as shown in Scheme 1 w32x. Thus, the data in Fig. 3 allow the
Scheme 1.
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calculation of effective DV a values representative of the rate-determining step as well as other steps in the reaction, at the pressure range under study. There is evidence suggesting that the reaction studied in the present work does not suffer from diffusional limitations w33,34x, and thus our DV a values should not include pressure effects on mass transfer. The DV a in propane, 44 cm3 moly1, is larger in magnitude than the value reported in the literature for the same reaction in an aqueous buffer, as seen in Table 1, although it is within the range of typical DV a values obtained in aqueous media w6x. Chaudhary et al. w11x also studied the present reaction in propane. The authors presented their data on the effect of pressure on catalytic activity in graph form only. The corresponding DV a should be small in magnitude since pressure did not significantly affect the activity of subtilisin up to 350 bar. The DV a we obtained in propane is also similar to the values given by Kim and Dordick w13x for the same reaction in tert-amyl alcohol, included in Table 1. For the sake of comparison, experiments with tert-amyl alcohol were also done, where a DV a value of 30 cm3 moly1 was obtained at about 2% enzyme hydration. We note that we were not able to measure very low enzyme hydration levels as accurately as higher ones. Kim and Dordick, on the other hand, did not measure enzyme hydration directly at high pressure as we did, but controlled that parameter through an adjustment of initial conditions only, which might explain their observed increase in catalytic efficiency up to too high enzyme hydration levels. Experimental difficulties could help to explain the difference between our DV a value and that given by Kim and Dordick for apparently identical enzyme hydration conditions. DV a values can be very large for homogeneous reactions in supercritical or near-critical fluids. For example, Johnston and Haynes w35x obtained DV a values as low as y6000 cm3 moly1 for the decomposition of a-chlorobenzyl methyl ester in 1,1-difluoroethane. Fluids close to their critical point are highly compressible. Under these conditions, the solvent condenses about solute molecules, yielding solute partial molar volumes that can be thousands of cm3 moly1 negative w36x. These can result in very large DV a values which are differences in partial molar volumes. A similar finding was reported by Kim and Johnston w37x for the Diels–Alder reaction
of isoprene and maleic anhydride in supercritical CO 2 . The authors obtained a linear relationship between the logarithm of the rate constant w38x and the solvent strength, as measured by the transition energy of a dye. In the region of higher compressibility close to the critical point, the predicted DV a was as large as y4000 cm3 moly1 at 358C, reaching a value of y225 cm3 moly1 at 100 bar. Although the rate constant continued to increase as the density and solvent strength of CO 2 increased, it did so in a modest way, as the compressibility of the solvent farther from the critical point decreased. For example, at 300 bar, DV a was y55 cm3 moly1, a value similar to those obtained in conventional liquids. As mentioned in Section 1, DV a data for biocatalytic reactions in supercritical or near-critical fluids are rare. For example, Erickson et al. w8x and Kamat et al. w9,10x reported on the effect of pressure on reaction rates in CO 2 and in ethane, and in fluoroform, ethane and sulfur hexafluoride, respectively, without giving any DV a values. More recently, Kamat et al. w12x calculated DV a data from the reaction rates for the lipase-catalyzed transesterification of methylmethacrylate by 2-ethylhexanol in fluoroform reported earlier w10x. As with subtilisin, so does the catalytic activity of the lipase used by those authors decrease with increasing pressure, yielding positive DV a values. These were about 1800 cm3 moly1 close to the critical point of the solvent, and decreased with increasing pressure, reaching a constant value close to zero at about 200 bar. In the present study, CO 2 and ethane Žwith 1-propanol. were never as close to the respective critical points as e.g., CO 2 and fluoroform were in the studies by Kim and Johnston w37x and by Kamat et al. w10x, a condition which should be reflected on the magnitude of our DV a values: 233 cm3 moly1 in CO 2 , 139 cm3 moly1 in ethane, which could conceivably be much larger, closer to the critical point. We believe in any case that the insensitivity of these values to pressure changes, i.e., linearity of the plots in Fig. 3 for CO 2 and ethane, may not be factual but rather an artifice resulting from taking the molar volumes of the solvent mixtures to be those of the solute-free solvents, especially at the lower pressures tested and thus closer to the critical point. In fact, whereas the mole fractions of both water and the ester substrate were sufficiently low in all the solvents to be neglected in
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all calculations, the mole fraction of 1-propanol was 0.05 in CO 2 and 0.07 in ethane and in propane Žcorresponding to a concentration of 0.9 M at 110 bar. . We could not find molar-volume data for the mixtures of interest in the literature. In the case of propane and because this solvent behaves pretty much like a conventional liquid under the conditions of this study, the molar volume of the solvent mixture could be calculated by assuming a linear dependence of composition; the same is true for tert-amyl alcohol. The resulting volumes differed by less than 1% from those of the pure solvents and thus our approach was to use the latter. This approach, which was also adopted for CO 2 and ethane, is in this case an oversimplification. In fact, an extended version w39x of the SAFT ŽStatistical Associating Fluid Theory. equation of state w40,41x, which gives good pure component densities, predicts 1 for the solvent mixtures lower molar volumes than those of pure CO 2 or ethane, as shown in Table 2. This is consistent with the notion of local density enhancement of the solvent about a solute and negative solute partial molar volumes. The volume change introduced by the solute decreases as the mixtures get further away from the critical point and the solvent becomes less compressible. For example, at 200 bar, such changes are a small percentage only, as seen also in Table 2. Although the extended SAFT equation does not predict sufficiently accurate volumes for mixtures close to the critical point and thus the calculated data in the table cannot be used, we believe it correctly predicts the trend of the data. In fact, the equation similarly predicts that the volumes of mixtures of CO 2 with a few percent methanol close to the critical point are lower than the volume of the pure solvent, a trend confirmed in this case by experimental data w43x. With correct molar-volume data, our experimental points for CO 2 and ethane at the lower pressures represented in Fig. 3 should lie above the straight lines, and we would thus have a dependence of DV a on pressure. All our solvents have low dielectric constants, ´ : approximately 1.7 and 1.4 for propane w17x and ethane
1
Calculations done by Oliver Pfohl, using a van der Waals mixing rule with two parameters as in Ref. w39x, which were fit to the phase-equilibrium data published in Ref. w42x.
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Table 2 Literature w18,21x and predicted 1 values Žextended SAFT equation. w39–41x for the molar volumes of mixtures of CO 2 and ethane with 1-propanol, at given mole fractions, x, at 358C Molar volume Žcm3 moly1 . Pressure Žbar. 100
200
300
Carbon dioxideq 1-propanol x CO2 0 73.4 0.95 54.6 1 62.0 1, tabulated 61.9
72.6 49.7 50.3 50.8
71.9 47.1 46.7 47.3
Ethaneq 1-propanol x ethane 0.93 76.3 1 82.8 1, tabulated 83.4
70.6 72.7 73.4
67.4 68.3 69.0
w10x, respectively, and 1.1–1.6 for CO 2 w44x, at the conditions of our study. A correlation between DV a and ´ , obtained by Kim and Dordick w13x for the present reaction in several organic solvents, suggested that DV a values should be negative in nonpolar solvents. This is however not the case, as seen in our results. The fact that ethane and CO 2 are relatively close to the critical point could help explain a departure from the above correlation. On the other hand, propane also does not conform to that prediction, and yet it behaves like a conventional liquid. Another implication of the referred correlation is that solvents with the same ´ should yield the same DV a. This is also unlike our results. In fact, our solvents have practically the same dielectric constants and yet there are significant differences in DV a among them. Again, the fact that ethane and CO 2 are near-critical could help explain these observations. As pointed out by Mozhaev et al. w4x, the pressure dependence of the rates of enzymatic reactions may reflect changes in the rate-determining catalytic step or conformational changes of the enzyme. These changes may give rise to departures from linearity in the plots of ln k vs. pressure. Mozhaev et al. w3x, on the other hand, referred that the linear character of such a plot as they obtained for an a-chymotrypsincatalyzed hydrolysis gave an indication that the catalytic mechanism and the active conformation of the
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enzyme did not change in the pressure interval studied. As we mentioned earlier, we would have expected a departure from linearity for CO 2 and ethane in Fig. 3 at the lower pressures studied if we had been able to use the correct molar volumes of the solvent mixtures. We cannot rule out the possibility of solvent-induced mechanistic changes or conformational changes on the enzyme in these two solvents and indeed in any of them. For CO 2 in particular, Ikushima et al. w45x showed that in a very limited pressure range near its critical point the interactions between this solvent and a lipase provoked drastic conformational changes on the enzyme, causing active sites that catalyzed a stereoselective synthesis to emerge. We were never so near the critical point of CO 2 in the present study. However, we note that the positive DV a values obtained by Kim and Dordick w13x were due to high positive activation volumes for substrate binding, i.e., weaker substrate binding. As mentioned earlier, CO 2 has an adverse effect on subtilisin suggested to be due to the formation of covalent complexes between CO 2 and free amine groups at lysine residues w27x. If our positive DV a values are a result of weaker substrate binding, then the larger DV a in CO 2 could be partly due to the interactions of this solvent with the enzyme. Why should activation volumes be larger in magnitude in nonaqueous media than in aqueous solutions, as observed for the present reaction in tert-amyl alcohol and in propane? Kim and Dordick w13x explain their results in tert-amyl alcohol on the basis of pressure decreasing the affinity of bound water to the enzyme while increasing the affinity of bound water to itself w46x, a fact which would result in water stripping and diminished catalytic activity at higher pressures. As the authors referred, the transfer of water from a more polar environment to a less polar one should be a positive contribution to DV a. Mozhaev et al. w3x, on the other hand, pointed out that the application of high pressure should fortify the protein hydration shell and be directed toward preferential hydration of the protein. We note, in any case, that pressures up to 300 bar are very moderate in this context. We note also that the concept of water stripping involves the ability of the solvent to solubilize the water that is released from the enzyme. Conventional liquid solvents like propane in the present study are nearly insensitive to pressure, in partic-
ular in what concerns the amount of water they are able to solubilize and, hence, their water-stripping ability Ž and in CO 2 , where it is not so, we measured points on the various water-sorption isotherms for the enzyme so as to keep enzyme hydration constant. . An explanation based on increased water stripping at higher pressure also presents the problem that it could only hold true for the left side of the bell-shaped activity vs. enzyme hydration curve. In fact, reducing enzyme hydration from a point at the end of the bell —e.g., our fixed 15% enzyme hydration in the compressed gases—results in an increase in catalytic activity. The above explanation would also not elucidate a negative DV a. Kim and Dordick w13x also showed that the effect of pressure was more marked as the state of hydration of the enzyme increased. It is known that increasing enzyme hydration brings about an increase in the molecular mobility of an enzyme. This increase in dynamics may allow the enzyme to assume the conformation which is thermodynamically favorable in the nonaqueous medium, resulting in denaturation. Burke et al. w47x have indeed shown that the degree of flexibility of an enzyme and its degree of unfolding are highly correlated. Kim and Dordick explain the increase in DV a with pressure on the basis of increased enzyme flexibility causing increased exposure of enzyme-bound water to the solvent and, consequently, an increase in water stripping. The greater the state of hydration of the enzyme, the larger the number of water–water contacts that would form and thus the more positive the value of DV a. This, however, would seem to require the ability of the nonaqueous solvent, at constant pressure, to solubilize more or less water depending on the state of hydration of the enzyme. As explained by Mozhaev et al. w4x, pressure effects are especially pronounced on processes in which the hydration state of the system components changes significantly. The reaction mechanism for subtilisin catalysis includes several steps, as shown in Scheme 1. The enzyme–substrate and enzyme–product complexes and the activation complexes involved will likely be hydrated differently; e.g., the binding of a substrate will likely involve the release of water molecules from the enzyme active site w9x. When an enzymatic reaction takes place in an aqueous solution, changes in the hydration of the intervening
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entities can easily be accommodated by the solvent. It should not be so when the solvent is nonaqueous. This and the known sensitivity of catalytic activity to enzyme hydration in nonaqueous media should potentially lead to greater Žin magnitude. pressure effects in these systems. The way in which water redistributes itself during catalysis at constant enzyme hydration and at varying pressure needs to be better understood.
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strongly dependent on enzyme hydration, as is the case with subtilisin, is still not very clear.
Acknowledgements This work was supported by Junta Nacional de Ž Portugal. Investigac¸ao e Tecnologica ˜ Cientıfica ´ ´ through the contract PRAXIS 2r2.1rBIOr34r94. We thank Dr. Oliver Pfohl for doing the calculations with the extended SAFT equation.
5. Conclusions Increasing pressure decreased the catalytic efficiency of subtilisin Carlsberg suspended in compressed propane, in near-critical ethane and in nearcritical CO 2 . This effect was more pronounced in the near-critical solvents, and especially in CO 2 , where the enzyme became completely inactive slightly above 300 bar. This is reflected in the corresponding activation volumes: the DV a obtained in compressed propane is similar to that in tert-amyl alcohol and of the order of magnitude of the DV a measured in aqueous solutions, whereas those in the near-critical solvents are much larger. This can be explained by the higher compressibility of a solvent close to the critical point and the compression of the solvent about the solutes. The extent to which this effect contributes to the DV a measured in CO 2 and in ethane is impossible to determine here, and so is the intrinsic DV a resulting from volume changes arising from changes in bond lengths and angles only w48x. As referred to by Kamat et al. w10x, the determination of microscopic rate constants would help to evaluate the effect of the solvent physical properties on each step of the reaction mechanism. Insight into the role of the solvent is provided by Ikushima et al. w45x who used a spectroscopic technique to show how the interactions of CO 2 with a lipase triggered the activation of the enzyme. A review of the spectroscopic techniques available for the elucidation of enzyme structure is given by Mozhaev et al. w4x. There is evidence to the fact that in the absence of covalent bond formation or breaking, the largest contributions to DV a are expected to result from hydration changes accompanying noncovalent interactions w4x. How these hydration changes are accommodated by nonaqueous solvents where enzymatic activity can be
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