Water activity and substrate concentration effects on lipase activity

Catalytic activity of lipases (from Rhizopus arrhizus, Canadida rugosa, and Pseudomonas sp. was studied in organic media, mainly diisopropyl ether. The effect of water activity (a(w)) on V(max) showed that the enzyme activity in general increased with increasing amounts of water for the three enzymes. This was shown both for esterification and hydrolysis reactions catalyzed by R. arrhizus lipase. In the esterification reaction the K(m) for the acid substrate showed a slight increase with increasing water activities. On the other hand, the K(m) for the alcohol substrate increased 10-20-fold with increasing water activity. The relative changes in K(m) were shown to be independent of the enzyme studied and solvent used. The effect was attributed to the increasing competition of water as a nucleophile for the acyl-enzyme at higher water activities. In a hydrolysis reaction the K(m) for the ester was also shown to increase as the water activity increased. The effect of water in this case was due to the fact that increased concentration of one substrate (water), and thereby increased saturation of the enzyme, will increase the apparent K(m) of the substrate (ester) to be determined. This explained why the hydrolysis rate decreased with increasing water activity at a fixed, low ester concentration. The apparent V(max) for R. arrhizus lipase was similar in four of six different solvents that were tested; exceptions were toulene and trichloroethylene, which showed lower values. The apparent K(m) for the alcohol in the solvents correlated with the hydrophobicity of the solvent, hydrophobic solvents giving lower apparent K(m). (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 798-806, 1997.     

Papain in organic solvents: determination of conditions suitable for biocatalysis and the effect on substrate specificity and inhibition

Synthesis of N-CBZ-(N-Carbobenzoxy)-1-amino-acid methyl esters from N-CBZ-amino acids and methanol has been used as an assay to examine the properties of papain in organic solvents containing small amounts of water. Papain is active in solvents ranging in polarity from acetonitrile to tetrachloromethane. The optimal activity in each solvent varied only about three to four fold, but the amount of added water required to achieve it varied from 4% (v/v) in acetonitrile to 0.05% (v/v) in tetrachloromethane. The enzyme was generally more stable in hydrophobic solvents and at lower water contents. The apparent K(m) value of CBZ-glycine was 26 times higher in acetonitrile than in toluene due to differential partitioning of the substrate between aqueous and organic phases. The substrate specificity of the enzyme was qualitatively little different from that in aqueous solution, with amino acid derivatives still the best substrates. Nitrile analogs of substrates inhibited the enzyme, as they do in aqueous solution, and inhibition by a variety of substituted aromatic hydrocarbons showed that the main specificity of papain for hydrophobic side chains at its S(2) subsite, was little affected. The results show that papain can catalyze reactions under a variety of conditions in organic solvents but its substrate specificity is little changed from that in aqueous media.     

Peptide synthesis catalyzed by polyethylene glycol-modified chymotrypsin in organic solvents

Chymotrypsin modified with polyethylene glycol was successfully used for peptide synthesis in organic solvents. The benzene-soluble modified enzyme readily catalyzed both aminolysis of N-benzoyl-L-tyrosine p-nitroanilide and synthesis of N-benzoyl-L-tyrosine butylamide in the presence of trace amounts of water. A quantitative reaction was obtained when either hydrophobic or bulky amides of L- as well as D-amino acids were used as acceptor nucleophiles, while almost no reaction occurred with free amino acids or ester derivatives. The acceptor nucleophile specificity of modified chymotrypsin as a catalyst in the formation of both amide and peptide bonds in organic solvents was quite comparable to that in aqueous solution as well as to that of the leaving group in hydrolysis reactions. By contrast, the substrate specificity of modified chymotrypsin in organic solvents was different from that in water since arginine and lysine esters were found to be as effective as aromatic amino acids to form the acyl-enzyme with subsequent synthesis of a peptide bond.     

Kinetics of lipase-catalyzed esterification in supercritical CO(2)

This study compares two solvents for enzymatic reactions: supercritical carbon dioxide (SCCO(2)) and organic solvent (n-hexane). The model reaction that was chosen was the esterification of oleic acid by ethanol catalyzed by an immobilized lipase from Mucor miehei (Lypozyme). The stability of the enzyme appeared to be quite good and similar in both media but was affected by the water content. Partition of water between solvents and immobilized enzyme has been calculated from experimental adsorption isotherms. The water content of the solid phase has a dramatic influence on the activity of the enzyme and its optimum value for activity was about 10% (w/w) in both media. A kinetic study enabled a Ping-Pong Bi-Bi reaction mechanism with inhibition by ethanol to be suggested. Despite some differences in kinetic constants, activity was in the same range in both media. Hypotheses for explaining the kinetic constant variations have been proposed and particular attention has been paid to the pH effects.     

Stabilization of Candida antarctica lipase B in hydrophilic organic solvent by rational design of hydrogen bond

Enzymatic reactions conducted in organic solvents have many advantages. However, organic solvent molecules may replace water molecules at the protein surface and penetrate into the enzyme, which could lead to the denaturation of the enzyme or changes in its reaction kinetics and substrate specificity. Thus, it is important to enhance the stability of enzymes in organic solvents. To date, there has been no efficient rational approach developed to enhance enzyme stability in hydrophilic solvents. We developed a rational approach to enzyme design. The design rules were established by investigating stable mutants from previous studies of directed evolution. Candida antarctica lipase B (CalB) was used as a target enzyme due to its versatile applications in organic solvents. The N97Q, N264Q, and D265E mutants of CalB showed higher organic solvent stability than the wild type.     

Reaction Kinetics of Immobilized α-Chymotrypsin in Organic Media 1. Influence at Solvent Polarity

Esterification of N-acetyl phenylalanine with ethanol catalyzed by immobilized α-chymotrypsin (EC 3.4.21.1) was studied in various reaction media. The effect of reaction medium polarity on enzymatic activity as well as equilibrium yield was measured. The reaction rate increased with increasing amounts of added water, reaching an optimum corresponding to water saturation of the reaction medium. Further additions of water resulted in decreased activity. Bell-shaped activity profiles were obtained for all reaction media tested. Reaction media consisting of pure solvents and of mixtures of solvents were used. The enzymatic activity and the equilibrium yield increased with decreased polarity of the medium. Maximum activity was found in a reaction medium consisting of 80% diisopropyl ether and 20% heptane. The enzymatic activity obtained at optimal water additions in the different solvents and solvents mixtures could be correlated to the solubility of water and the log P of the medium. The equilibrium yield of the reaction was much more closely correlated to the solubility of water than the log P. Much lower enzymatic activity was obtained when solvent mixtures producing water-miscible media were created, than in mixtures producing water-immiscible media, such as mixtures of acetonitrile and diisopropyl ether. The equilibrium yield could be increased by decreasing the water content in the reaction medium, which reduced the water activity.     

Influence of Water Activity and Support Material on the Enzymatic Synthesis of a Cck-8 Tripeptide Fragment

The kinetically controlled condensation of Z-Gly-Trp-OMe and H-Met-OEt catalyzed by α-chymo-trypsin in organic media is reported. The influence of thermodynamic water activity and the support material used to adsorb α-chymotrypsin, on both the product yield and enzymatic activity was investigated. Polyamide based materials were the best support at low water activity rendering the highest reaction rates and yields. The activity of the adsorbed enzyme at low water activities depends on both the accessible surface area and the hydrophobicity of the support. Polyamide had both adequate hydrophilicity and high surface area yielding the best results. Polypropylene based supports were strongly hydrophobic and, although they presented a high surface area, the enzymatic activity was much lower. The solvents used to carry out the synthesis were acetonitrile and ethyl acetate. No significant differences were observed on the performance of the reaction in either solvent. The tripeptide selected is a fragment of the cholecystokinin C-terminal octapeptide (CCK-8), a biological active peptide involved in the control of gastrointestinal function.     

Substrate desolvation as a governing factor in enzymatic transformations of PAHs in aqueous-acetonitrile mixtures

Conversion of hydrophobic substrates such as polyaromatic hydrocarbons (PAHs) was studied in aqueous-organic media using transformation of pyrene by cytochrome c. The experiments were conducted in pure solvents and aqueous-acetonitrile mixtures. The reaction rates dropped gradually as the solvent concentration was increased and were negligible in pure solvents. Thermodynamic calculations show that substrate desolvation was the governing factor in this reaction. The decrease in free energy of activation upon increasing the solvent concentration was found to be proportional to the substrate transfer free energy. This suggests that in order to make such reactions feasible in organic media, it will be necessary to improve the binding between the proteins and the substrates.     

Protein-protein interaction in low water organic media

Trypsin either modified with polyethylene glycol or as a suspended powder was used to catalyze digestion of protein substrates in benzene in order to get insight into protein-protein interactions in water-immiscible organic media. Depending on whether suspended or soluble trypsin was used, catalysis was found to proceed differently. In the first case, the amount of water in the reaction mixture (up to 1% v/v) appeared to be critical, and adsorption of water from the reaction medium by the protein substrate allowed it to behave as a hydrophilic support material comparable to that involved in immobilized enzymes. In the latter case, the presence of an additional nucleophile was a prerequisite for catalysis to proceed, and thus both water and nucleophile concentrations had some influence on trypsin activity. Phe-NH(2) was the most potent nucleophile for proteolysis catalyzed by polyethylene glycol-modified trypsin in organic media containing 1-2% water (v/v). The organic solvent-soluble enzyme was found to bind reversibly to the protein substrate as a function of both extent of hydration of the reaction medium and time of incubation. The overall results strongly suggested that modified trypsin catalyzed peptide bond hydrolysis at the protein substrate-organic solvent interface. Peptide mapping of bovine insulin digest by reversed-phase high-performance liquid chromatography definitely showed that enzyme-catalyzed proteolysis did occur in organic solvents with a concomitant and significant transpeptidation reaction.     

A comparison of lipase-catalyzed ester hydrolysis in reverse micelles, organic solvents, and biphasic systems

The performance of lipases from Candida rugosa and wheat germ have been investigated in three reaction media using three acetate hydrolyses as model reactions (ethyl acetate, allyl acetate, and prenyl acetate). The effect of substrate properties and water content were studied for each system (organic solvent, biphasic system, and reverse micelles). Not unexpectedly, the effect of water content is distinct for each system, and the optimal water content for enzyme activity is not always the same as that for productivity. A theoretical model has been used to simulate and predict enzyme performance in reverse micelles, and a proposed partitioning model for biphasic systems agrees well with experimental results. While the highest activities observed were in the micellar system, productivity in microemulsions is limited by low enzyme concentrations. Biphasic systems, however, support relatively good activity and productivity. The addition of water to dry organic solvents, combined with the dispersion of lyophilized enzyme powders in the solvent, resulted in significant enzyme aggregation, which not surprisingly limits the applicability of the "anhydrous" enzyme suspension approach. (c) 1995 John Wiley & Sons, Inc.     

Activity and flexibility of alcohol dehydrogenase in organic solvents

The oxidation of cinnamyl alcohol to cinnamaldehyde by horse liver alcohol dehydrogenase (LADH) was carried out in nearly anhydrous organic solvents and in solvents containing from 0.1 to 10% added water. In nearly anhydrous solvents containing less than 0.02% water, the oxidation rate increased as the water solubility in the solvent decreased, but the reaction did not require active LADH. Moreover, the highest activity in nearly anhydrous heptane was obtained by lyophilizing the enzyme from a solution of pH 2.0, even though LADH exhibits virtually no enzymatic activity in water at this pH. The catalytic activity of LADH was restored and increased dramatically as small amounts of water were added to each solvent. In conjunction with the activity measurements, electron paramagnetic resonance (EPR) spectroscopy and two active-site directed spin labels were used to examine solvent-dependent structural features of LADH. The EPR spectra indicated that LADH became more rigid as the dielectric constant of the solvent decreased. The degree of rigidity also depended on the pH from which the enzyme was lyophilized, indicating that the ionization state of the enzyme can have an important influence on its dynamics in organic solvents. Finally, adding 1% water to organic solvents had no apparent effect on the enzyme's conformation or flexibility near the spin label, even though enzyme activity was an order of magnitude higher when 1% water was present.     

Effect of water activity on thermolysin-catalyzed peptide synthesis in organic solvents

Summary The effect of water activity on the rate of thermolysin-catalyzed synthesis of an aspartame precursor has been investigated in water-miscible and water-immiscible solvents. In both cases, the enzyme reaction rate at a given water activity was found to be significantly different depending on the nature of the solvent. The reaction rates in water-immiscible solvents, where the water activities were close to 1.0, were found to be significantly dependent on the volume ratio of water to organic media and the hydrophobicity of the solvent. These data suggest that the enzyme reaction in the solvent is influenced appreciably by other factors in addition to the water activity.     

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.     

Comparison of hydrolytic activity in water and heptane for thirty-two commercial lipase preparations

The protein content and the rates of hydrolysis of p-nitrophenyl palmitate (pNPP) in water (soluble enzyme and emulsified substrate) and in heptane (soluble substrate and insoluble enzyme) were measured for thirty-two commercial lipase preparations. The protein content of the powders varied in a wide range as well as the activity on emulsified pNPP showing the high heterogeneity of the commercial samples. Activity in heptane also varied but to a lesser extent than that in water. There was no direct correlation between activities in water and in heptane as assayed with the same hydrolytic reaction. The ratio of activity in heptane to that in water, R(O/A) ratio, was introduced to characterize activity in organic media. Six lipases showed R(O/A) values higher than 1 demonstrating a higher activity in organic solvent than in water. A linear correlation of R(O/A) with activity in water (log plot) suggested the strong influence of diffusional limitations on activity of solid enzyme suspended in organic solvents.     

Ester synthesis from trimethylammonium alcohols in dry organic media catalyzed by immobilized Candida antarctica lipase B

Twenty-one different organic solvents were assayed as possible reaction media for the synthesis of butyryl esters from trimethylammonium alcohols in dry conditions catalyzed by immobilized Candida antarctica lipase B. The reactions were carried out following a transesterification kinetic approach, using choline and L-carnitine as primary and secondary trimethylammonium alcohols, respectively, and vinyl butyrate as acyl donor. The synthetic activity of the enzyme was strictly dependent on the water content, the position of the hydroxyl group in the trimethylammonium molecule, and the Log P parameter of the assayed solvent. Anhydrous conditions and a high excess of vinyl butyrate over L-carnitine were necessary to synthesize butyryl-L-carnitine. The synthetic reaction rates of butyryl choline were practically 100-fold those of butyryl-L-carnitine with all the assayed solvents. In both cases, the synthetic activity of the enzyme was dependent on the hydrophobicity of the solvent, with the optimal reaction media showing a Log P parameter of between -0.5 and 0.5. In all cases, 2-methyl-2-propanol and 2-methyl-2-butanol were shown to be the best solvents for both their high synthetic activity and negligible loss of enzyme activity after 6 days.     

Thermodynamic and kinetic aspects on water vs. organic solvent as reaction media in the enzyme-catalysed reduction of ketones

The stereoselective reduction of ketones catalysed by alcohol dehydrogenase from Thermoanaerobium brockii was studied in different reaction media, hexane at controlled water activities, hexane with 2. 5% water (biphasic) and pure water. The reactions were studied in the temperature range from -1 to 50 degrees C. Increasing the water activity from 0.53 to 0.97 increased the reaction rate 16-fold. The rate was further enhanced in hexane when exceeding the water solubility and in pure water the rates were even higher. This was general for all ketones studied. At controlled water activity the entropy of activation (DeltaSdouble dagger) was the dominating factor. Large negative DeltaSdouble dagger values caused low reaction rates at low aw. When increasing the carbon chain length of the substrate, for reactions in hexane, the decrease of reaction rate was mainly due to a decrease in DeltaSdouble dagger. In the comparison between hexane and pure water, DeltaGdouble dagger values were higher in hexane due to higher DeltaHdouble dagger (activation enthalpy) values. The enantioselectivity (E value) increased from 2.6 at water activity 0. 53 to 4.6 at water activity 0.97. Changing media from hexane (2.5%, v/v water) to pure water was not affecting the enantioselectivity or the specificity for different ketones.     

On the importance of the support material for enzymatic synthesis in organic media. Support effects at controlled water activity

Enzymes were deposited on different porous support materials and these preparations were used to catalyze reactions in organic media. Reactions were carried out at specific water activities, achieved by equilibrating both the enzyme preparation and the substrate solution at the desired water activity before mixing them and thereby starting the reactions. The reaction rates obtained at the same water activity with different supports differed greatly, indicating a direct influence of the support on the enzyme. For horse liver alcohol dehydrogenase, Celite was the best support, and the reaction rate increased with increasing water activity. In the alpha-chymotrypsin-catalyzed alcoholysis of N-acetyl-L-phenylalanine ethyl ester with 1-butanol, high rates were again obtained with Celite, but with this support only about one third of the ethyl ester was converted to butyl ester, the rest was hydrolyzed. With the polyamide support, Accurel PA6, alcoholysis was the dominating reaction, and by using a low water activity (0.33), hydrolysis was completely suppressed while still maintaining a high alcoholysis activity. Controlled pore glass (CPG), derivatized with either hexyl or glucosyl groups, had quite different properties as enzyme supports. For horse liver alcohol dehydrogenase, glucose-CPG was a much better support than hexyl-CPG, and in the alpha-chymotrypsin-catalyzed reactions, glucose-CPG favored hydrolysis, and hexyl-CPG alcoholysis, at water activities exceeding 0.8. The results are discussed considering the absorption of water on the enzymes, on the supports and the solubility of water in the reaction media; all these parameters were measured separately.     

Enzymatic asymmetrization of prochiral 2-benzyl-1,3-propanediol through esterification in solvent media

The enzymatic esterification of the prochiral substrate, 2-benzyl-1,3-propanediol, has been studied in solvent media. Among the five tested lipases, Lipozyme and Novozym 435 led to higher reaction rates. Novozym 435 catalyzed faster reactions at low water activity and in solvents having log P above 2. However, the two positions of the diol, pro-(R) and pro-(S), led to the same reaction rate trends and no prochiral selectivity was obtained. When using Lipozyme in toluene, the reaction rates for the formation of both (R) and (S) products presented an optimum at a water activity of 0.22. In this case, the prochiral selectivity increased with the water activity, from a value of 5 at a _w < 0.01, to a value of 8 at a _w = 0.22, at which point it remained constant.     

Enzymatic properties of polyethylene glycol-modified cholesterol esterase in organic solvents.

The solvent dependency and substrate specificity of polyethylene glycol (PEG)-modified cholesterol esterase (CEH) catalyzing cholesterol ester synthesis in organic solvents were studied. When cholesterol and linoleic acid were used as the substrates, PEG-modified CEH synthesized cholesterol linoleate only in water-immiscible organic solvents. Among some solvents capable of solubilizing all of the reaction components (PEG-modified CEH, cholesterol, and linoleic acid), chloroform was most suitable for enzymatic cholesterol linoleate synthesis, and the synthetic activity for cholesterol linoleate decreased in the order chloroform, benzene, toluene, and cyclohexane. PEG-modified CEH synthesized various cholesterol esters with significant substrate specificity. The substrate specificity for cholesterol ester synthesis in benzene was analogous to that for cholesterol ester hydrolysis in aqueous solution.