Kinetic study of sphingomyelin hydrolysis for ceramide production

Kinetic study of sphingomyelin hydrolysis catalyzed by Clostridium perfringens phospholipase C was, at the first time, conducted for ceramide production. Ceramide has the major role in maintaining the water-retaining properties of the epidermis. Hence, it is of great commercial potential in cosmetic and pharmaceutical industries such as in hair and skin care products. The enzymatic hydrolysis of sphingomyelin has been proved to be a feasible method to produce ceramide. The kinetic performance of sphingomyelin hydrolysis in the optimal two-phase (water:organic solvent) reaction system was investigated to elucidate the possible reaction mechanism and also to further improve the hydrolysis performance. Enzyme in solution had less thermal stability than the enzyme powder and the immobilized enzyme. The thermal inactivation of phospholipase C in all the three forms did not follow the first order reaction at 65 °C. The reactions for both the soluble and immobilized enzymes followed Michaelis–Menten kinetics. K_m's for the soluble and immobilized enzymes were 1.07 ± 0.32 and 1.26 ± 0.19 mM, respectively. The value of V_max was markedly decreased by the immobilization without much change in K_m, as if the immobilization functioned as the non-competitive inhibition. Ceramide as product activated the hydrolysis reaction, however, and its addition mainly caused the increase in the affinity of the enzyme–substrate complex.     

Designing enzymatic kyotorphin synthesis in organic media with low water content

Design of enzymatic kyotorphin synthesis in low water media has been carried out as a function of enzyme nature, the immobilization support material and the reaction medium, by using N-benzoyl-L-tyrosine ethyl ester and L-argininamide as substrates. Native and chemically-glycated alpha-chymotrypsin deposited on supports with different degrees of aquaphilicity (celite, polypropylene PP, and polyamide PA6) were used as catalysts. Binary organic solvent systems of ethanol and different water-immiscible organic cosolvents (ethylacetate, tert-butanol, chloroform, toluene, n-hexane, and n-octane) were studied as reaction media at constant water content (3% v/v). The greater the water binding affinity of the support the lower the synthetic activity of deposited enzymes: the activity of the celite derivative was 4x greater than the polyamide derivative. The enzyme glycation process hardly modified the catalytic ability of the celite derivative, but resulted in a moderate increase in operational stability. The presence of hydrophobic organic cosolvents in the water/ethanol reaction medium significantly increased enzyme activity, whereas the selectivity of the reaction remained high. Hexane was shown to be the best cosolvent, the synthetic activity of the celite derivative in hexane-ethanol (77 : 20%, v/v) being 130x greater than that in 97% (v/v) ethanol.     

Enhancement of hydrolytic activity of sphingolipid ceramide N-deacylase in the aqueous-organic biphasic system

Lysoglycosphingolipids were produced from glycosphingolipids by using sphingolipid ceramide N-deacylase, which cleaves the N-acyl linkage between fatty acids and sphingosine bases in various glycosphingolipids. The enzyme reaction was done in a biphasic media prepared with water;-immiscible organic solvent and aqueous buffer solution containing the enzyme. We investigated the effects of organic solvents and detergents on lysoglycosphingolipid production in the biphasic system. Among the organic solvents tested, n-butylbenzene, cumene, cyclodecane, cyclohexane, n-decane, diisopropylether, n-heptadecane, and methylcyclohexane promoted hydrolysis of GM1, whereas benzene, chloroform, ethyl acetate, and toluene inhibited GM1 hydrolysis. Hydrolysis of asialo GM1, GD1a, GalCer, and sulfatide was also enhanced by the addition of n-decane. The hydrolytic activity of the enzyme was enhanced by the addition of 0.8% sodium taurodeoxycholate or sodium cholate to the aqueous phase. The most effective hydrolysis of various glycosphingolipids by the enzyme was thus obtained in the aqueous-n-decane biphasic system containing 0.8% sodium taurodeoxycholate. Under this condition, the fatty acids released from GM1 by the action of the enzyme were trapped and diffused into the organic phase, while lysoGM1 remained in the aqueous phase.Thus the almost complete hydrolysis of GM1 was achieved using the biphasic system, while at most 70% of hydrolysis was obtained using normal aqueous media possibly due to the inhibition of hydrolysis reaction by accumulation of fatty acids in the reaction mixture.     

Enzymatic esterification in organic media: role of water and organic solvent in kinetics and yield of butyl butyrate synthesis

Summary The respective roles of organic solvent and of water in butyl butyrate synthesis from n-butanol and n-butyric acid in n-hexane by Mucor miehei lipase have been investigated by analysis of the kinetics and the reaction balances. Esterificaton was found to take place in both low water systems containing solid enzyme in hexane and in biphasic aqueous enzyme solution/hexane systems. In the solid enzyme system, the enzyme adsorbed the water produced, thus delaying the appearance of a discrete aqueous phase. As expected, the presence of some water was indispensable for this system, as its removal or exclusion by various means (adsorption, distillation) affected enzyme activity. However, water removal had little effect on the final yield of esterification. Reaction velocities were quite similar for the solid enzyme/hexane system and for the biphasic aqueous enzyme solution/hexane system. In the latter case, the butyl butyrate formed was almost exclusively found in the organic phase. Ethyl butyrate, a more polar compound, was synthesized with a lower yield. These results allow the conclusion that the reaction took place in a phase consisting of either solid hydrated enzyme with no discrete aqueous phase or of an aqueous enzyme solution by basically similar mechanisms according to the amount of water available to the system, the esterification being driven to completion by transfer of the ester into the organic phase because of a favourable partition coefficient. Offprint requests to: F. Monot     

Whole cell yeast biotransformations in two-phase systems: Effect of solvent on product formation and cell structure

Summary Biotransformation of benzaldehyde and pyruvate to (R)-phenylacetyl carbinol bySaccharomyces cerevisiae was investigated in two-phase aqueous-organic reaction media. With hexane as organic solvent, maximum biotransformation activity was observed with a moisture content of 10%. Of the organic solvents tested, highest biotransformation activities were observed with hexane and hexadecane, and lowest activities occurred with chloroform and toluene. Biocatalyst samples from biphasic media containing hexane, decane and toluene manifested no apparent cell structural damage when examined using scanning electron microscopy. In contrast, cellular biocatalyst recovered from two-phase systems containing chloroform, butylacetate and ethylacetate exhibited damage in the form of cell puncturing after different incubation periods. Phospholipids were detected in reaction media from biocatalytic systems which exhibited cell damage in electron micrographs. Phospholipid release was much lower in the two-phase systems containing toluene or hexane or in 100% aqueous biocatalytic system.     

Towards more active biocatalysts in organic media: increasing the activity of salt-activated enzymes

The activation of freeze-dried subtilisin Carlsberg (SC) in hexane has been systematically studied and partially optimized with respect to the freezing method, the addition of inorganic salts and lyoprotectants, the initial concentration and final weight percent of additives, and the amount of water added to the organic solvent. Activity and water content were found to correlate directly with the kosmotropicity of the activating salt (kosmotropic salts bind water molecules strongly relative to the strength of water-water interactions in bulk solution). Combinations of kosmotropic salts with known lyoprotectants such as poly(ethylene glycol) (PEG) and sugars did not yield an appreciably more active catalyst. However, the combination of the kosmotropic sodium acetate with the strongly buffering sodium carbonate activated the enzyme more than the individual additives alone. Enzyme activity was enhanced further by the addition of small amounts of water to the organic solvent. Under optimal conditions, enzyme activity in hexane was improved over 27,000-fold relative to the salt-free enzyme, reaching a catalytic efficiency that was within one order of magnitude of k(cat)/K(m) for hydrolysis of the same substrate in aqueous buffer. Further activation to attain even higher catalytic efficiencies may be possible with additional optimization.     

Lipase-catalyzed acyl exchange of soybean phosphatidylcholine in n-Hexane: a critical evaluation of both acyl incorporation and product recovery

Lipase-catalyzed acidolysis was examined for the production of structured phospholipids in a hexane system. In a practical operation of the reaction system, the formation of lyso-phospholipids from hydrolysis is often a serious problem, as demonstrated from previous studies. A clear elucidation of the issue and optimization of the system are essential for the practical applications in reality. The effects of enzyme dosage, reaction temperature, solvent amount, reaction time, and substrate ratio were optimized in terms of the acyl incorporation, which led to the products, and lyso-phospholipids formed by hydrolysis, which led to the low yields. The biocatalyst used was the commercial immobilized lipase Lipozyme TL IM and substrates used were phosphatidylcholine (PC) from soybean and caprylic acid. A response surface design was used to evaluate the influence of selected parameters and their relationships on the incorporation of caprylic acid and the corresponding recovery of PC. Incorporation of fatty acids increased with increasing enzyme dosage, reaction temperature, solvent amount, reaction time, and substrate ratio. Enzyme dosage had the most significant effect on the incorporation, followed by reaction time, reaction temperature, solvent amount, and substrate ratio. However the parameters had also a negative influence on the PC recovery. Solvent amount had the most negative effect on recovery, followed by enzyme dosage, temperature, and reaction time. Individually substrate ratio had no significant effect on the PC recovery. Interactions were observed between different parameters. On the basis of the models, the reaction was optimized for the maximum incorporation and maximum PC recovery. With all of the considerations, the optimal conditions are recommended as enzyme dosage 29%, reaction time 50 h, temperature 54 degrees C, substrate ratio 15 mol/mol caprylic acid/PC, and 5 mL of hexane per 3 g substrate. No additional water is necessary. Under these conditions, an incorporation of caprylic acid up to 46% and recovery of PC up to 60% can be obtained from the prediction. The prediction was confirmed from the verification experiments.     

Preparation of fluorescence-labeled GM1 and sphingomyelin by the reverse hydrolysis reaction of sphingolipid ceramide N-deacylase as substrates for assay of sphingolipid-degrading enzymes and for detection of sphingolipid-binding proteins.

Sphingolipid ceramide N-deacylase is an enzyme capable of hydrolyzing the N-acyl linkages of ceramides of various sphingolipids. Recently, it was found that the enzyme catalyzes the reverse hydrolysis reaction in which free fatty acids are condensed to lyso-sphingolipids to produce sphingolipids. This paper describes a simple method for the synthesis of fluorescence-labeled sphingolipids utilizing the condensation reaction of the enzyme. N-TFAc-aminododecanoic acids were efficiently condensed by the enzyme to the lyso-forms of GM1 and sphingomyelin in glycine buffer (pH 10). The reaction products, N-TFAc-amino-GM1 and sphingomyelin, were obtained with overall yields of 60%. The purified products were identified to be omega-amino-GM1 and omega-amino-sphingomyelin, respectively, by TLC and FAB-MS or ESI-LC/MS analysis after removal of the N-TFAc by mild alkaline treatment. NBD-labeled GM1 and sphingomyelin were prepared from omega-amino-GM1 and omega-amino-sphingomyelin by coupling with 4-fluoro-NBD. These fluorescence-labeled substrates, C12-NBD-GM1 and C12-NBD-sphingomyelin, were hydrolyzed by endoglycoceramidase and sphingomyelinase, respectively, to produce NBD-dodecanoylsphingosines, but were resistant to hydrolysis by sphingolipid ceramide N-deacylase. C12-NBD-sphingomyelin was found to be a better substrate than the commercially available C6-NBD-sphingomyelin for the assay of sphingomyelinase from various sources. We also describe a new method to detect GM1-binding proteins using fluorescence-labeled GM1.     

Modeling hydration mechanisms of enzymes in nonpolar and polar organic solvents

A comprehensive study of the hydration mechanism of an enzyme in nonaqueous media was done using molecular dynamics simulations in five organic solvents with different polarities, namely, hexane, 3-pentanone, diisopropyl ether, ethanol, and acetonitrile. In these solvents, the serine protease cutinase from Fusarium solani pisi was increasingly hydrated with 12 different hydration levels ranging from 5% to 100% (w/w) (weight of water/weight of protein). The ability of organic solvents to 'strip off' water from the enzyme surface was clearly dependent on the nature of the organic solvent. The rmsd of the enzyme from the crystal structure was shown to be lower at specific hydration levels, depending on the organic solvent used. It was also shown that organic solvents determine the structure and dynamics of water at the enzyme surface. Nonpolar solvents enhance the formation of large clusters of water that are tightly bound to the enzyme, whereas water in polar organic solvents is fragmented in small clusters loosely bound to the enzyme surface. Ions seem to play an important role in the stabilization of exposed charged residues, mainly at low hydration levels. A common feature is found for the preferential localization of water molecules at particular regions of the enzyme surface in all organic solvents: water seems to be localized at equivalent regions of the enzyme surface independently of the organic solvent employed.     

Enzymatic hydrolysis of hydrophobic triolein by lipase in a mono-phase reaction system containing cyclodextrin; Reaction characteristics

A hydrophobic substrate triolein was hydrolyzed by lipase in a mono-phase reaction system containing cyclodextrin(CD) as emulsifier. The triolein was transformed to an emulsion-like state in the CD containing reaction system in contrast to the oil-droplet like state without CD due to the formation of an inclusion complex between the lipids and CDs. The hydrolysis reaction increased substantially in the CD containing reaction system, and the optimum reaction conditions including the amount of lipase, β-CD concentration, and mixing ratio of triolein and β-CD, were determined. The performance of the enzyme reaction in a mono-phase reaction system was compared with that of a two-phase reaction system which used water immiscible hexane as the organic solvent. The role of a CD in the mono-phase reaction system was elucidated by comparing the degree of the inclusion complex formation with triolein and oleic acid, K_m and V_max values, and product inhibition by oleic acid in aqueous and CD containing reaction systems. The resulting enhanced reaction seems to be caused by two phenomena; the increased accessibility of lipase to triolein and reduced product inhibition by oleic acid through the formation of an inclusion complex.     

Implication of Sphingomyelin/Ceramide Molar Ratio on the Biological Activity of Sphingomyelinase

Sphingolipid signaling plays an important, yet not fully understood, role in diverse aspects of cellular life. Sphingomyelinase is a major enzyme in these signaling pathways, catalyzing hydrolysis of sphingomyelin to ceramide and phosphocholine. To address the related membrane dynamical structural changes and their feedback to enzyme activity, we have studied the effect of enzymatically generated ceramide in situ on the properties of a well-defined lipid model system. We found a gel-phase formation that was about four times faster than ceramide generation due to ceramide-sphingomyelin pairing. The gel-phase formation slowed down when the ceramide molar ratios exceeded those of sphingomyelin and stopped just at the solubility limit of ceramide, due to unfavorable pairwise interactions of ceramide with itself and with monounsaturated phosphatidylcholine. A remarkable correlation to in vitro experiments suggests a regulation of sphingomyelinase activity based on the sphingomyelin/ceramide molar ratio.     

A lipid analogue that inhibits sphingomyelin hydrolysis and synthesis, increases ceramide, and leads to cell death

We report the synthesis and characterization of a novel thiourea derivative of sphingomyelin (AD2765). In vitro assays using pure enzyme and/or cell extracts revealed that this compound inhibited the hydrolysis of BODIPY-conjugated or 14C-labeled sphingomyelin by acid sphingomyelinase and Mg2+-dependent neutral sphingomyelinase. Studies in normal human skin fibroblasts further revealed that AD2765 was taken up by cells and inhibited the hydrolysis of BODIPY-conjugated sphingomyelin in situ. In situ and in vitro studies also showed that this compound inhibited the synthesis of sphingomyelin from BODIPY-conjugated ceramide. The specificity of AD2765 for enzymes involved in sphingomyelin metabolism was demonstrated by the fact that it had no effect on the hydrolysis of BODIPY-conjugated ceramide by acid ceramidase or on the synthesis of BODIPY-conjugated glucosylceramide from BODIPY-conjugated ceramide. The overall effect of AD2765 on sphingomyelin metabolism was concentration-dependent, and treatment of normal human skin fibroblasts or cancer cells with this compound at concentrations > 10 microM led to an increase in cellular ceramide and cell death. Thus, AD2765 might be used to manipulate sphingomyelin metabolism in various ways, potentially to reduce substrate accumulation in cells from types A and B Niemann-Pick disease patients, and/or to affect the growth of human cancer cells.     

有机溶剂可溶的超氧化物歧化酶的制备及其性质

本文报道用谷氨酸、十二醇、葡萄糖酸内酯合成了一种精脂（２Ｃ（１２）ＧＥ）,并制备了ＳＯＤ－糖脂复合体．所得的ＳＯＤ－糖脂复合体是脂溶性的而不是水溶性的,它在乙醇等有机溶剂中的活性比在水中高,而且存在一最适有机溶剂浓度。其对温度、ｐＨ、蛋白酶水解的稳定性比天然ＳＯＤ明显增强。     

The lipase-catalyzed hydrolysis of lutein diesters in non-aqueous media is favored at extremely low water activities

The enzymatic hydrolysis of a mixture of lutein diesters from Marigold flower (Tagetes erecta) was performed both in organic solvents and supercritical CO(2) (SC-CO(2)) using two commercial lipases: lipase B from Candida antarctica (Novozym 435) and the lipase from Mucor miehei (Lipozyme RM IM). Both lipases showed an unexpected dependence of initial reaction rate with the initial water activity (a(wi)) in hexane, with the highest rates of hydrolysis taking place at the lowest a(wi) of the biocatalyst particles. The same result was observed using isooctane, toluene, or SC-CO(2). It is proposed that an increase in a(wi) generates a hydrophilic microenvironment that prevents efficient partitioning of the highly hydrophobic lutein diesters to the enzyme. The critical role of water in this system has not been reported for other hydrolytic reactions in low water media. Calculations of water available for hydrolysis from isotherm analysis, Karl-Fischer titration, and substrate conversion at a(wi) = 0.13, indicate that the extent of reaction is not limited by the amount of available water. Accordingly, the enzyme that holds the largest amount of water after prehydration at the same a(wi) (0.13) will yield the greatest substrate conversion and concentration of the free lutein product. The highest conversion occurred in SC-CO(2), which opens up new opportunities to develop a combined extraction-reaction process for the environmentally benign synthesis of lutein, an important nutraceutical compound.     

Sphingomyelin metabolites inhibit sphingomyelin synthase and CTP:phosphocholine cytidylyltransferase

Tissue injury in inflammation involves the release of several cytokines that activate sphingomyelinases and generate ceramide. In the lung, the impaired metabolism of surfactant phosphatidylcholine (PC) accompanies this acute and chronic injury. These effects are long-lived and extend beyond the time frame over which tumor necrosis factor (TNF)-alpha and interleukin-1beta are elevated. In this paper, we demonstrate that in H441 lung cells these two processes, cytokine-induced metabolism of sphingomyelin and the inhibition of PC metabolism, are directly interrelated. First, metabolites of sphingomyelin hydrolysis themselves inhibit key enzymes necessary for restoring homeostasis between sphingomyelin and its metabolites. Ceramide stimulates sphingomyelinases as effectively as TNF-alpha, thereby amplifying the sphingomyelinase activation, and TNF-alpha, ceramide, and sphingosine all inhibit PC:ceramide phosphocholine transferase (sphingomyelin synthase), the enzyme that restores homeostasis between sphingomyelin and ceramide pools. Second, ceramide inhibits PC synthesis, probably because of its effects on CTP:phosphocholine cytidylyltransferase, the rate-limiting enzymatic step in de novo PC synthesis. The data presented here suggest that TNF-alpha may be an inhibitor of phospholipid metabolism in inflammatory tissue injury. These actions may be amplified because of the ability of metabolites of sphingomyelin to inhibit the pathways that should restore the normal ceramide-sphingomyelin homeostasis.     

Effects of Polar Organic Solvents on the Biocatalysis of Chlorophyllase in a Biphasic Organic System

The effects of polarity of various organic solvents, including acetone, ethanol, and propanol, used in a biphasic organic system, on the hydrolytic activity of a partially purified chlorophyllase from Phaeodactylum tricornutum were investigated. The different concentrations of each polar organic solvent, from 0 to 40%, were added to a mixture (45:55, v/v) of hexane and a buffer solution of Tris–HCl (20 mm, pH 7.5). The most appropriate concentrations of acetone, ethanol, and propanol for the hydrolytic activity of chlorophyllase were 12.5, 5.0, and 2.5%, respectively. The results indicated that the optimum reaction time for the chlorophyllase activity in the biphasic system decreased from 7.0 h to 3.0, 5.0, and 5.0 h, respectively, upon the addition of an appropriate amount of acetone, ethanol, or propanol. The V_max and K_m as well as the inhibitory effect of phytol on the chlorophyllase activity in the biphasic organic system containing a polar organic solvent were also investigated.     

Resolution of rac-ketoprofen esters by enzymatic reactions in organic media

Enzyme-catalyzed reactions in organic media of rac-ketoprofen esters with different nucleophiles such as alcohols, amines, and water have been studied. Among the parameters optimized are the enzyme, the activated substrate, and the solvent. With the enzymes used in this study the preferred substrate was the trifluoroethyl ester of rac-ketoprofen (rac- 2 ), whose (R)-enantiomer reacted preferentially. The enzyme of choice was the lipase M-AP-10 from Mucor miehei and best results were obtained with diisopropyl ether as solvent. Three different methods have been scaled-up for the resolution of 75–150 g of substrate: transesterification with 1-butanol (90% yield of (S)-ketoprofen, 88% ee), transesterification with 2-(2-pyridyl)ethanol (94% yield, 92% ee), and hydrolysis in wet organic solvent (93% yield, 97% ee). Despite the comparable chemical and optical yields obtained with these three methods, the use of 2-(2-pyridyl)ethanol and the hydrolysis allowed a much easier work-up and isolation of the desired (+)-(S)-ketoprofen. © 1993 Wiley-Liss, Inc.     

Kinetically controlled synthesis of ampicillin and cephalexin in highly condensed systems in the absence of a liquid aqueous phase

Advantages of performing penicillin G amidase catalysed synthesis of ampicillin and cephalexin by enzymatic acyl transfer to the β-lactam antibiotic nuclei in a highly condensed system using mainly undissolved substrates, with no apparent aqueous liquid phase, were demonstrated. It was shown that synthesis can be performed in the absence of a liquid phase formed by water or an organic co-solvent. This highly condensed system is formed by a liquid phase given by one of the reactant, the phenylglycine methyl ester (PGM), that remains liquid in these operative conditions and the partially dissolved β-lactam nucleus. Operating in such highly condensed system, the water that causes the hydrolysis of PGM is limited to the water hydrating the support on which the enzyme is covalently immobilised. In this way the reaction system is maintained at a controlled degree of hydration.

In the present work the reaction system was modulated by eliminating the solvent (aqueous or aqueous/organic), reducing the amount of water to the minimum for the biocatalytic activity and using PGM as solvent and reagent at the same time. The synthesis was conducted with equimolar amounts of PGM and the β-lactam nucleus, with a reduced hydrolysis of the activated acyl donor. We have also studied a simple and efficient method for the workup of the reaction where the unreacted reagents can be recovered after selective filtration and precipitation.     

Optimization of Pseudomonas cepacia lipase preparations for catalysis in organic solvents

The activity of different lipase (from Pseudomonas cepacia) forms, such as crude powder (crude PC), purified and lyophilized with PEG (PEG + PC), covalently linked to PEG (PEG-PC), cross-linked enzyme crystals (CLEC-PC), and immobilized in Sol-Gel-AK (Sol-Gel-AK-PC) was determined, at various water activities (aw), in carbon tetrachloride, benzene and 1,4-dioxane. The reaction of vinyl butyrate with 1-octanol was employed as a model and both transesterification (formation of 1-octyl butyrate) and hydrolysis (formation of butyric acid from vinyl butyrate) rates were determined. Both rates depended on the lipase form, solvent employed, and aw value. Hydrolysis rates always increased as a function of aw, while the optimum of aw for transesterification depended on the enzyme form and nature of the solvent. At proper aw, some lipase forms such as PEG + PC, PEG-PC, and Sol-Gel-AK-PC had a total activity in organic solvents (transesterification plus hydrolysis) which was close to (39 and 48%) or even higher than (130%) that displayed by the same amount of lipase protein in the hydrolysis of tributyrin-one of the substrates most commonly used as standard for the assay of lipase activity-in aqueous buffer. Instead, CLEC-PC and crude PC were much less active in organic solvents (2 and 12%) than in buffer. The results suggest that enzyme dispersion and/or proper enzyme conformation (favored by interaction with PEG or the hydrophobic Sol-Gel-AK matrix) are essential for the expression of high lipase activity in organic media.     

A new approach to preparative enzymatic synthesis

A new approach to preparative organic synthesis in aqueous–organic systems is suggested. It is based on the idea that the enzymatic process is carried out in a biphasic system “water–water-immiscible organic solvent.” Thereby the enzyme is localized in the aqueous phase—this eliminates the traditional problem of stabilizing the enzyme against inactivation by a nonaqueous solvent. Hence, in contrast to the commonly used combinations “water–water-miscible organic solvent,” in the suggested system the content of water may be infinitely low. This allows one to dramatically shift the equilibrium of the reactions forming water as a reaction product (synthesis of esters and amides, polymerization of amino acids, sugars and nucleotides, dehydration reactions, etc.) toward the products. The fact that the system consists of two phases provides another very important source for an equilibrium shift, i.e., free energies of the transfer of a reagent from one phase to the other. Equations are derived describing the dependence of the equilibrium constant in a biphasic system on the ratio of the volumes of the aqueous and nonaqueous phases and the partition coefficients of the reagents between the phases. The approach has been experimentally verified with the synthesis of N-acetyl-L -tryptophan ethyl ester from the respective alcohol and acid. Porous glass was impregnated with aqueous buffer solution of chymotrypsin and suspended in chloroform containing N-acetyl-L -tryptophan and ethanol. In water (no organic phase) the yield of the ester is about 0.01%, whereas in this biphasic system it is practically 100%. The idea is applicable to a great number of preparative enzymatic reactions.