Kinetic analysis of the mechanism for subtilisin in essentially anhydrous organic solvents

Steady-state kinetic analysis has been used to confirm the catalytic mechanism of lyophilized subtilisin suspended in a variety of organic solvents. Specifically, this article demonstrates that partial reactions can occur between subtilisin and ester substrates in organic solvents. Partitioning of common intermediates between competing acceptors at a constant ratio of products has also been described. The decomposition of a common intermediate formed from different substrates at the same rate is also further evidence of an acyl-enzyme mechanism for subtilisin suspended in anhydrous solvents. Partitioning of a common intermediate to give two products at a constant total rate, and saturation kinetics at varying substrate concentrations, complete a kinetic investigation of the enzyme mechanism. All the data generated support the formation of a stable acyl enzyme during the transesterification reaction catalzyed by subtilisin in the solvents used.     

Inhibitor-induced enzyme activation in organic solvents

The enzymatic activity of the protease subtilisin in anhydrous organic solvents can be dramatically increased by pretreating the enzyme before it is placed in the nonaqueous medium. For instance, lyophilization of subtilisin from aqueous solution containing competitive inhibitors (followed by their removal) created an enzyme which was up to 100 times more active than the enzyme lyophilized in the absence of such ligands. This phenomenon of ligand-induced "enzyme memory" also extends to the stability, affinity, and substrate specificity of subtilisin in organic solvents.     

Enzyme activation in organic solvents: co-lyophilization of subtilisin Carlsberg with methyl-beta-cyclodextrin renders an enzyme catalyst more active than the cross-linked enzyme crystals

In this study we explored the efficiency of the additive methyl-beta-cyclodextrin (M beta CD) to enhance the activity and enantioselectivity of the serine protease subtilisin Carlsberg in organic solvents. These two parameters, measured for different transesterification reactions and in several solvents, are compared with results obtained by using two additional preparations of the same enzyme: lyophilized powder and cross-linked enzyme crystals (CLEC). The results suggest that co-lyophilization of subtilisin with M beta CD preserves the enzyme's active site tertiary structure rendering a highly active and enantioselective catalyst.     

Optimizing the salt-induced activation of enzymes in organic solvents: effects of lyophilization time and water content

The addition of simple inorganic salts to aqueous enzyme solutions prior to lyophilization results in a dramatic activation of the dried powder in organic media relative to enzyme with no added salt. Activation of both the serine protease subtilisin Carlsberg and lipase from Mucor javanicus resulting from lyophilization in the presence of KCl was highly sensitive to the lyophilization time and water content of the sample. Specifically, for a preparation containing 98% (w/w) KCl, 1% (w/w) phosphate buffer, and 1% (w/w) enzyme, varying the lyophilization time showed a direct correlation between water content and activity up to an optimum, beyond which the activity decreased with increasing lyophilization time. The catalytic efficiency in hexane varied as much as 13-fold for subtilisin Carlsberg and 11-fold for lipase depending on the lyophilization time. This dependence was apparently a consequence of including the salt, as a similar result was not observed for the enzyme freeze-dried without KCl. In the case of subtilisin Carlsberg, the salt-induced optimum value of kcat/Km for transesterification in hexane was over 20,000-fold higher than that for salt-free enzyme, a substantial improvement over the previously reported enhancement of 3750-fold (Khmelnitsky, 1994). As was found previously for pure enzyme, the salt-activated enzyme exhibited greatest activity when lyophilized from a solution of pH equal to the pH for optimal activity in water. The active-site content of the lyophilized enzyme samples also depended upon lyophilization time and inclusion of salt, with opposite trends in this dependence observed for the solvents hexane and tetrahydrofuran. Finally, substrate selectivity experiments suggested that mechanism(s) other than selective partitioning of substrate into the enzyme-salt matrix are responsible for salt-induced activation of enzymes in organic solvents.     

Activation of enzymes for nonaqueous biocatalysis by denaturing concentrations of urea

Urea is one of the most commonly used denaturants of proteins. However, herein we report that enzymes lyophilized from denaturing concentrations of aqueous urea exhibited much higher activity in organic solvents than their native counterparts. Thus, instead of causing deactivation, urea effected unexpected activation of enzymes suspended in organic media. Activation of subtilisin Carlsberg (SC) in the organic solvents (hexane, tetrahydrofuran, and acetone) increased with increasing urea concentrations up to 8 M. Active-site titration results and activity assays indicated the presence of partially unfolded but catalytically active SC in 8 M urea; however, the urea-modified enzyme retained high enantioselectivity and was ca. 80 times more active than the native enzyme in anhydrous hexane. Likewise, the activity of horseradish peroxidase (HRP) lyophilized from 8 M urea was ca. 56 times and 350 times higher in 97% acetone and water-saturated hexane, respectively, than the activity of HRP lyophilized from aqueous buffer. Compared with the native enzyme, the partially unfolded enzyme may have a more pliant and less rigid conformation in organic solvents, thus enabling it to retain higher catalytic activity. However, no substantial activation was observed for alpha-chymotrypsin lyophilized from urea solutions in which the enzyme retained some activity, illustrating that the activation effect is not completely general.     

The influence of the mode of enzyme preparation on enzymatic enantioselectivity in organic solvents and its temperature dependence

The enantioselectivities of subtilisin Carlsberg and Rhizomucor miehei lipase in organic solvents are found to strongly depend on the method by which the enzymes are prepared. For the transesterification between sec-phenethyl alcohol and vinyl butyrate in dioxane at 7 degrees C, the enantioselectivity of subtilisin precipitated with isopropanol is more than twice that of the enzyme prepared by lyophilization from aqueous buffer. Furthermore, the temperature dependence of the enantioselectivity is influenced by the mode of enzyme preparation. For example, in the aforementioned process the enantioselectivities of subtilisin lyophilized from aqueous buffer and crosslinked subtilisin crystals increase when the temperature is raised from 7 to 45 degrees C. In contrast, the enantioselectivities decrease with temperature for the enzyme precipitated from aqueous solution with acetone or isopropanol and for the enzymatic hydrolysis in water. The temperature dependence of the enantioselectivity of subtilisin lyophilized from buffer is markedly affected by the solvent: In acetonitrile and nitromethane the enzyme is more enantioselective at higher temperatures, while negligible temperature effects have been found in tetrahydrofuran and pyridine. Lyophilized lipase exhibits striking temperature dependencies of its enantioselectivity in dioxane, acetonitrile, and nitromethane, while showing almost none in pyridine, triethylamine, and tetrahydrofuran. The results underscore the importance of the mode of enzyme recovery on enantioselectivity and its temperature dependence in enzymatic reactions in organic solvents (in contrast to those in water). (c) 1996 John Wiley & Sons, Inc.     

Effect of organic solvents on enantioselectivity of protease catalysis

The protease-catalyzed transesterifications between N-trifluoroacetyl-DL-phenylalanine 2,2,2-trifluoroethyl ester and 1-propanol were studied in a variety of anhydrous organic solvents at 30 degrees C. The protease preparations lyophilized from phosphate buffer solutions (pH 8.0) were used as catalysts. The organic solvent affected both rate of reaction and enantioselectivity differently. Proteases such as Aspergillus oryzae protease, subtilisin Carlsberg, and subtilisin BPN' always preferred the L-enantiomer in both hydrophilic and hydrophobic solvents, indicating no inversion of the L-specificity in hydrophobic solvents such as toluene. However, enantioselectivity was rather poor, with E (enantiomeric ratio) values not exceeding even one order of magnitude except for acetonitrile. There was a weak inverse correlation between E values of subtilisin Carlsberg and solvent hydrophobicity (logP). Acetonitrile was a preferable solvent in terms of both rate of reaction and enantioselectivity (E= 15 to 25) for processing L-amino acid derivatives in organic media. Organic solvents generally have potential advantages of processing D-amino acid derivatives. (c) 1997 John Wiley & Sons, Inc.     

Subtilisin-catalysed peptide synthesis and transesterification in organic solvents

Summary Subtilisin from Bacillus subtilis was modified with polyethylene glycol (PEG), or adsorbed either on celite or porous glass, or directly used as a suspended powder to catalyse peptide synthesis and transesterification reactions in organic solvents. The rather low yield of peptide synthesis probably resulted from the enzyme tendency to catalyse hydrolysis and transesterification side reactions. The kinetics of transesterification catalysed by PEG-subtilisin was consistent with a ping-pong mechanism modified by a hydrolytic branch. Initial rates of transesterification were found to be dependent on alcohol and organic base concentrations in the reaction mixture. The high affinity of benzyloxycarbonyl-l-serine-methyl ester for the enzyme indicated that a change in substrate specificity of subtilisin occurred in organic phase. The 50-fold increase in the rate of synthesis of benzyloxycarbonyl-l-serine-l-phenylalanine amide which was observed when PEG-subtilisin was used instead of immobilized or powdered enzyme, suggested that a higher flexibility of the polypeptide chain modified by the covalent attachment of a number of soluble PEG moieties occurred in organic solvents. This also resulted in a lower stability of PEG-subtilisin at high temperature.Offprint requests to: A. Puigserver     

Comparison of theoretical and experimental data to evaluate substrate diffusional limitations for crown ether- and methyl-beta-cyclodextrin-activated serine protease subtilisin Carlsberg in tetrahydrofuran

Simple co-lyophilization of serine protease subtilisin Carlsberg with [12]-crown ether-4 (12-crown-4) or methyl-beta-cyclodextrin (MbetaCD) drastically increases its catalytic activity in organic solvents. We investigated whether the improved activity would cause substrate diffusional limitations. To experimentally assess the issue, the enzyme was inactivated with PMSF. Different amounts of active and inactive subtilisin were codissolved in 10 mM phosphate buffer (pH 7.8) followed by lyophilization with or without 12-crown-4 or MbetaCD. Initial rates for the transesterification reaction of N-acetyl-L-phenylalanine ethyl ester and 1-propanol in anhydrous THF were plotted vs. the amount of active enzyme present in the formulations. For all three enzyme formulations a linear relationship was observed and the results clearly show that activation of subtilisin Carlsberg by crown ethers and MbetaCD did not cause diffusional limitations. This was somewhat surprising because theoretical models predicted such diffusional limitations for the activated formulations. However, investigation of the protein powder particles obtained after co-lyophilization with 12-crown-4 and MbetaCD revealed a drastically reduced particle size for these formulations when suspended in THF. The particle micronization afforded by the excipients prevented substrate diffusional limitations, a factor that should be taken into account when designing improved enzyme formulations for synthetic applications in organic solvents.     

Effect of crown ethers on structure, stability, activity, and enantioselectivity of subtilisin Carlsberg in organic solvents.

Colyophilization or codrying of subtilisin Carlsberg with the crown ethers 18-crown-6, 15-crown-5, and 12-crown-4 substantially improved enzyme activity in THF, acetonitrile, and 1,4-dioxane in the transesterification reactions of N-acetyl-L-phenylalanine ethylester and 1-propanol and that of (+/-)-1-phenylethanol and vinylbutyrate. The acceleration of the initial rate, V(0), ranged from less than 10-fold to more than 100-fold. All crown ethers activated subtilisin substantially, which excludes a specific macrocyclic effect from being responsible. The secondary structure of subtilisin was studied by Fourier-transform infrared (FTIR) spectroscopy. 18-Crown-6 and 15-crown-5 led to a more nativelike structure of subtilisin in the organic solvents employed when compared with that of the dehydrated enzyme obtained from buffer alone. However, the high level of activation with 12-crown-4 where this effect was not observed excluded overall structural preservation from being the primary cause of the observed enzyme activation. The conformational mobility of subtilisin was investigated by performing thermal denaturation experiments in 1,4-dioxane. Although only a small effect of temperature on subtilisin structure was observed for the samples prepared with or without 12-crown-4, both 18-crown-6 and 15-crown-5 caused the enzyme to denature at quite low temperatures (38 degrees C and 56 degrees C, respectively). No relationship between this property and V(0) was evident, but increased conformational mobility of the protein decreased its storage stability. The possibility of a "molecular imprinting" effect was also tested by removing 18-crown-6 from the subtilisin-18-crown-6 colyophilizate by washing. V(0) was only halved as a result of this procedure, an effect insignificant compared with the ca. 80-fold rate enhancement observed prior to washing in THF. This suggests that molecular imprinting is likely the primary cause of subtilisin activation by crown ethers, as recently suggested.     

Purification and characterization of organic solvent stable protease from Bacillus licheniformis RSP-09-37

A protease was purified from the cell-free supernatant of Bacillus licheniformis RSP-09-37, a mutant from a thermophilic bacterial strain, B. licheniformis RSP-09, using affinity chromatography with alpha-casein agarose resin. The protease was purified 85-fold to electrophoretic homogeneity. The apparent molecular mass of purified protease was 55 kDa using gel filtration in high-performance liquid chromatography, which is in agreement with the results obtained from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting a monomeric nature of the protein. The purified protease revealed temperature optima of 50 degrees C and pH optima of 10.0 and was classified as serine protease based on its complete inhibition with phenyl methyl sulfonyl fluoride. The purified protease exhibited tolerance to both detergents and organic solvent. The synthetic activity of the protease was tested using the transesterification reaction between N-acetyl-L-phenylalanine-ethyl ester and n-propanol in organic solvents varying in their log P values and the kinetic parameters of the enzyme in these organic solvents were studied. The enzyme has potential to be employed for synthetic reactions and in detergent formulations.     

Sol-gel immobilization of serine proteases for application in organic solvents

The serine proteases alpha-chymotrypsin, trypsin, and subtilisin Carlsberg were immobilized in a sol-gel matrix and the effects on the enzyme activity in organic media are evaluated. The percentage of immobilized enzyme is 90% in the case of alpha-chymotrypsin and the resulting specific enzyme activity in the transesterification of N-acetyl-L-phenylalanine ethyl ester with 1-propanol in cyclohexane is 43 times higher than that of a nonimmobilized lyophilized alpha-chymotrypsin. The activities of trypsin and subtilisin Carlsberg are enhanced with 437 and 31 times, respectively. The effect of immobilization on the enzyme activity is highest in hydrophobic solvents.     

Testing for diffusion limitations in salt-activated enzyme catalysts operating in organic solvents

The dramatic activation of serine proteases in nonaqueous media resulting from lyophilization in the presence of KCl is shown to be unrelated to relaxation of potential substrate diffusional limitations. Specifically, lyophilizing subtilisin Carlsberg in the presence of KCl and phosphate buffer in different proportions, ranging from 99% (w/w) enzyme to 1% (w/w) enzyme in the final lyophilized solids, resulted in biocatalyst preparations that were not influenced by substrate diffusion. This result was made evident through use of a classical analysis whereby initial catalytic rates, normalized per weight of total enzyme in the catalyst material, were measured as a function of active enzyme for biocatalyst preparations containing different ratios of active to inactive enzyme. The active enzyme content of a given biocatalyst preparation was controlled by mixing native subtilisin with subtilisin preinactivated with PMSF, a serine protease inhibitor, and lyophilizing the enzyme mixture in the presence of different fractions of KCl and phosphate buffer. Plots of initial reaction rates as a function of percent active subtilisin in the biocatalyst were linear for all biocatalyst preparations. Thus, enzyme activation (reported elsewhere to be as high as 3750-fold in hexane for the transesterification of N-Ac-L-Phe-OEt with n-PrOH) is a manifestation of intrinsic enzyme activation and not relaxation of diffusional limitations resulting from diluted enzyme preparations. Similar activation is reported herein for thermolysin, a nonserine protease, thereby demonstrating that enzyme activation due to lyophilization in the presence of KCl may be a general phenomenon for proteolytic enzymes.     

Inactivation and stabilization of stabilisins in neat organic solvents

The stability of the serine proteases from Bacillus amyloliquefaciens (subtillisin BPN') and Bacillus licheniformis (subtilisin Carlsberg) was investigated in various anhydrous solvents at 45 degrees C. The half-life of subtilisin BPN' in dimethyl-formamide dramatically depends on the pH of the aqueous solutions from which the enzyme was lyophilized, increasing from 48 min to 20 h when the pH is raised from 6.0 to 7.9. Both subtilisins exhibited substantial inactivation during multihour incubations in tert-amyl alcohol and acetonitrile when enzymatic activities were also measured in these solvents; however, when the enzymes were assayed in water instead, hardly any loss of activity was detected. This surprising difference appears to stem from the partitioning of the bound water essential for catalytic activity from the enzymes into the solvents. When assayed in organic solvents, this time-dependent stripping of water results in decay of enzymatic activity; however, when assayed in water, where the dehydrated subtilisins can undergo rehydration thereby recovering catalytic activity, little inactivation is observed. In agreement with this hypothesis, the addition of small quantities of water tert-amyl alcohol stabilized the subtilisins in it even when enzymatic activity was measured in the nonaqueous solvent. Ester substrates (vinyl butyrate and trichloroethyl butyrate) greatly enhanced the stability of both subtilisins in organic solvents possibly because of the formation of the acyl-enzymes.     

Determination of equilibrium and individual rate constants for subtilisin-catalyzed transesterification in anhydrous environments

We report here the first determinations of individual rate constants and equilibrium constants for enzymatic reactions in essentially anhydrous organic solvents. Using the added nucleophile method we have measured the effect of changing solvent on the binding and catalytic steps for subtilisin-catalyzed transesterification of N-protected amino acid esters. The detailed information generated indicates that once the substrate has bound to the enzyme, the catalytic machinery can work at rates equivalent to those in water. The decreased overall rates for subtilisin suspended in anhydrous solvents are merely the result of extremely high values for K(s), in most cases, coupled with low concentrations of nucleophile ( approximately 1.0M in organic solvents, and 55M in water). The method described, which is generally applicable, and straightforward experimentally, will, we believe, enable a clearer understanding of how changing solvent can predictably affect the activity and specificity of the enzyme. (c) 1992 John Wiley & Sons, Inc.     

Protease-catalyzed synthetic reactions and immobilization-activation of the enzymes in hydrophilic organic solvents.

The catalytic feature of serine proteases for synthetic reactions in hydrophilic organic solvents and effects of immobilization by complexation with polysaccharides are described. Free alpha-chymotrypsin and subtilisin Carlsberg catalyze esterification, transesterification, and peptide synthesis in hydro-organic cosolvents with less than 10% water. Subtilisin BPN' is catalytically less active. The medium effects on the reaction kinetics and product yield were investigated in terms of the nature of solvent and water content in the reaction systems. The substrate- and stereo-specificities of the enzymes suggest that the enzymes maintain their native conformations in these low-water organic solvents. The catalytic activities of the proteases markedly increase by immobilization or complexation with polysaccharides, such as chitin or chitosan. The results of the rate measurements suggest that the primary role of the support materials is the activation of the enzymes and the increase in substrate concentration at reaction sites.     

On enzymatic activity in organic solvents as a function of enzyme history

Catalytic activities of alpha-chymotrypsin and subtilisin Carlsberg in various hydrous organic solvents were measured as a function of how the enzyme suspension had been prepared. In one method, lyophilized enzyme was directly suspended in the solvent containing 1% water. In another, the enzyme was precipitated from its aqueous solution by a 100-fold dilution with an anhydrous solvent. In most cases, the reaction rate in a given nonaqueous enzymatic system strongly (up to an order of magnitude) depended on the mode of enzyme preparation. The magnitude of this dependence was markedly affected by the nature of the solvent and enzyme. A mechanistic hypothesis proposed to explain the observed dependencies was verified in additional experiments in which the water contents and enzyme history were further varied.     

Prediction of penicillin V acylase stability in water-organic co-solvent monophasic systems as a function of solvent composition

Hydrolytic activity of penicillin V acylase (EC 3.5.1.11) can be improved by using organic cosolvents in monophasic systems. However, the addition of these solvents may result in loss of stability of the enzyme. The thermal stability of penicillin V acylase from Streptomyces lavendulae in water-organic cosolvent monophasic systems depends on the nature of the organic solvent and its concentration in the media. The threshold solvent concentration (at which half enzymatic activity is displayed) is related to the denaturing capacity of the solvent. We found out linear correlations between the free energy of denaturation at 40 degrees C and the concentration of the solvent in the media. On one hand, those solvents with logP values lower than -1.8 have a protective effect that is enhanced when its concentration is increased in the medium. On the other hand, those solvents with logP values higher than -1.8 have a denaturing effect: the higher this value and concentration, the more deleterious. Deactivation constants of PVA at 40 degrees C can be predicted in any monophasic system containing a water-miscible solvent.     

High initial activity but low storage stability observed for several preparations of subtilisin Carslberg suspended in organic solvents

Colyophilization with methyl-beta-cyclodextrin activates subtilisin Carlsberg by more than 200-fold in organic solvents, though this is a short-lived effect. About 93% of the enzyme's high initial activity observed in THF (at 45 degrees C) decreases exponentially with a t(1/2) of 1.8 h, until it reaches a residual activity (of 7%) that remains constant throughout the 4 days duration of the experiment. A further study of this enzyme reveals a general trend: the initial activities of the lyophilized powder and the cross-linked enzyme crystals are also greatly reduced upon incubation in this solvent, although these preparations retain 50% of their activity after about 20 h of incubation. All of the preparations studied retained some residual activity (which persisted throughout the duration of the experiments) after the initial exponential decay. The data here presented suggest that the mode of enzyme preparation is an important issue to consider when planning lengthy reactions.     

Obtaining high transesterification activity for subtilisin in ionic liquids

It is known that subtilisin shows poor transesterification activity in ionic liquids (ILs). The present work, taking subtilisin as the system, explores approaches for biocatalyst preparations, which are capable of yielding higher/adequate transesterification activity in these solvents. Of all the approaches tried, enzyme precipitated and rinsed with n-propanol (EPRP) gave the best results (about 10,000 times increase in initial rates in 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim][PF(6)]) over what is obtained with pH tuned lyophilized powders). In case of water soluble ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim][BF(4)]), pH tuned lyophilized subtilisin did not show any transesterification activity. EPRP, however, gave an initial rate (for transesterification) of 2.78 mmol mg(-1) h(-1).