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.     

The role of hydration in enzyme activity and stability: 1. Water adsorption by alcohol dehydrogenase in a continuous gas phase reactor

The enzymatic synthesis of the tripeptide derivative Z-Gly-Trp-Met-OEt is reported. This tripeptide is a fragment of the cholecystokinin C-terminal octapeptide CCK-8. Studies on the alpha-chymotrypsin catalyzed coupling reaction between Z-Gly-Trp-R(1) and Met-R(2) have focused on low water content media, using deposited enzyme on inert supports such as Celite and polyamide. The effect of additives (polar organic solvents), the acyl-donor ester structure, the C-alpha protecting group of the nucleophile, enzyme loading, and substrate concentration were tested. The best reaction medium found was acetonitrile containing buffer (0.5%, v/v) and triethylamine (0.5%, v/v) using the enzyme deposited on Celite as catalyst (8 mg of alpha-chymotrypsin/g of Celite). A reaction yield of 81% was obtained with Z-Gly-Trp-OCam as acyl donor, at an initial concentration of 80 mM. The tripeptide synthesis was scaled up to the production of 2 g of pure tripeptide with an overall yield of 71%, including reaction and purification steps. (c) 1996 John Wiley & Sons, Inc.     

Enzymatic condensation of cholecystokinin CCK-8 (4-6) and CCK-8 (7-8) peptide fragments in organic media

The kinetically controlled condensation reaction of Z-Gly-Trp-Met-OR(1) (R(1): Et, Al, Cam) and H-Asp-(OR(2))-Phe-NH(2) (R(2): H, Bu(t)) catalyzed by alpha-chymotrypsin deposited onto polyamide in organic media was studied. The effect of the drying process of the enzyme-support preparation, substrate concentrations, reaction medium, acyl donor, and nucleophile structure on both enzymatic activity and pentapeptide yield was investigated. The immobilized preparation directly equilibrated at a(w) = 0.113, gave higher enzymatic activities than dried with vacuum first, and then equilibrated at a(w) = 0.113. The addition of triethylamine to the reaction medium increased dramatically the enzymatic activity. However, the pentapeptide yield was affected neither by the drying procedure nor by the addition of triethylamine. The donor ester Z-Gly-Trp-Met-OAl gave initial reaction rates 2.6 times higher than the conventional ethyl ester derivative but rendered similar yields. The best results were obtained using Z-Gly-Trp-Met-OCam as acyl-donor ester; 80% yield and initial reaction rates 4 times higher than the ethyl ester derivative. In all cases, acetonitrile containing Tris-HCl 50 mM pH 9 buffer (0.5% v/v) and triethylamine (0.5% v/v) was found to be the best reaction system. Under these conditions, it was possible to use the nucleophile H-Asp-Phe-NH(2) with beta-unprotected aspartic acid residue. In this case, 50% yield was obtained, but economic considerations could lead to select it as nucleophile. Finally, the fragment condensation reaction was carried out at gram scale, obtaining a 39% yield which included the reaction, removal of protecting groups and purification steps. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 456-463, 1997.     

Kinetics and specificity of serine proteases in peptide synthesis catalyzed in organic solvents

Initial rates of peptide-bond synthesis catalyzed by poly(ethylene glycol)-modified chymotrypsin in benzene were determined using high-performance liquid chromatography. Enzymatic synthesis of N-benzoyl-L-tyrosyl-L-phenylalanine amide from N-benzoyl-L-tyrosine ethyl ester and L-phenylalanine amide was found to obey Michaelis-Menten kinetics an to be consistent with a ping-pong mechanism modified by a hydrolytic branch. The catalytic activity of modified chymotrypsin was dependent on both water concentration and type of organic solvent, the highest synthesis rate being obtained in toluene. Since the chymotrypsin specificity in the organic phase was actually altered, the enzyme's apparent kinetic parameters were determined for different substrates and compared to those obtained with other serine proteases in benzene. Both N-benzoyl-L-tyrosine ethyl ester and N-alpha-benzoyl-L-lysine methyl ester were comparable acyl donors in benzene and the (kcat/Km)app value of modified chymotrypsin was only 10-fold smaller than that obtained with poly(ethylene glycol)-modified trypsin in the synthesis of N-alpha-benzoyl-L-lysyl-L-phenylalanine amide. The change in chymotrypsin specificity was also confirmed through the binding of trypsin inhibitors in benzene. The overall results suggest that hydrophobic bonding between the enzyme and its substrate should not be taken into account during catalysis in the organic phase. In general, if hydrophobic interactions are involved in the binding of substrates to the active site in aqueous media, the replacement of water by hydrophobic solvents will induce some change in enzyme specificity. Moreover, secondary residues of enzyme-binding sites may also exert a significant influence on specificity since, as observed in this study, chymotrypsin exhibited high affinity for cationic substrates and cationic inhibitors as well in apolar solvents.     

Enzymatic reduction of a less water-soluble ketone in reverse micelles with NADH regeneration.

In enzyme catalysis there is great interest in finding suitable organic media for less water-soluble substrates in order to increase the substrate concentration and, therefore, the reaction rates. These requirements are fulfilled by using microemulsions as reaction media. In this study w/o-microemulsions were used to investigate the kinetics of the reduction of 2-Heptanone to S-2-Heptanol, catalyzed by alcohol dehydrogenase. The required cofactor NADH for this reduction is regenerated by a second enzyme, formate dehydrogenase. The influences of pH, temperature, and the kinetic parameters of the enzymes are presented. It is demonstrated that in microemulsions the reaction rate of ADH is increased up to 12 times compared to water.     

Microbial lipases: at the interface of aqueous and non-aqueous media. A review

In recent times, biotechnological applications of microbial lipases in synthesis of many organic molecules have rapidly increased in non-aqueous media. Microbial lipases are the 'working horses' in biocatalysis and have been extensively studied when their exceptionally high stability in non-aqueous media has been discovered. Stability of lipases in organic solvents makes them commercially feasibile in the enzymatic esterification reactions. Their stability is affected by temperature, reaction medium, water concentration and by the biocatalyst's preparation. An optimization process for ester synthesis from pilot scale to industrial scale in the reaction medium is discussed. The water released during the esterification process can be controlled over a wide range and has a profound effect on the activity of the lipases. Approaches to lipase catalysis like protein engineering, directed evolution and metagenome approach were studied. This review reports the recent development in the field ofnon-aqueous microbial lipase catalysis and factors controlling the esterification/transesterification processes in organic media.     

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.     

Chymotrypsin adsorbed to a polyethylene terephtalate support (SORSILEN) as a suitable model for the investigation of enzymatically catalyzed organic syntheses in nonaqueous media

Summary The kinetics of acetyl-L-tryptophan esterification by ethanol in an organic solvent immiscible with water (chloroform) and catalyzed by chymotrypsin adsorbed from aqueous solution to a polyethylene terephthalate copolymer (SORSILEN) was examined by HPLC. The esterification yield (in %) increased with the decreasing concentration of the substrate and with the increasing activity of immobilized chymotrypsin. It has been shown that there was no chymotrypsin leakage from the aqueous phase on the support surface during the catalysis.     

有机相中脂肪酶L—1754促有机硅烷醇的酯化

在有机介质中脂肪酶Ｌ－１７５４催化非天然有机硅化合物三甲基硅烷醇酯化反应的速度高于Ｌｉｐｏｚｙｍｅ。Ｌ－１７５４对脂肪酸链长有很强的特异性且不同于Ｌｉｐｏｚｙｍｅ。     

A new method for improving the enantioselectivity of lipase-catalyzed hydrolysis in organic solvent containing a small amount of water in the presence of metal ions

The hydrolysis of racemic butyl 2-(4-substituted phenoxy)propionates having various substituents catalyzed by lipase MY from Candida rugosa was achieved in di-isopropyl ether containing 0.75% (v/v) of 2.4 M LiCl or 1.2 M MgCl₂ aqueous solution. Water molecules hydrated to the metal ion in isopropyl ether acted as a nucleophile to cause the hydrolysis of these esters as with water alone. Metal ions used significantly enhanced their enantioselectivities by 100-fold or above, as compared with the ordinary reaction media.     

Effects of water activity on reaction rates and equilibrium positions in enzymatic esterifications

A technique of continuous water activity control was used to examine the effects of water activity on enzyme catalysis in organic media. Esterification catalyzed by Rhizopus arrhizus lipase was preferably carried out at a water activity of 0.33, which resulted in both maximal initial reaction rate and a high yield. When Pseudomonas lipase was used as catalyst it was beneficial to start the reaction at high water activity (giving the optimal reaction rate with this enzyme) and then shift to a lower water activity toward the end of the reaction to obtain a high yield. The apparent equilibrium constant of the reaction was influenced by the water activity of the organic solvent. (c) 1994 John Wiley & Sons, Inc.     

Mapping of the 5'-2-deoxyribose-5-phosphate lyase active site in DNA polymerase beta by mass spectrometry

The mechanism of the 5'-2-deoxyribose-5-phosphate lyase reaction catalyzed by mammalian DNA beta-polymerase (beta-pol) was investigated using a cross-linking methodology in combination with mass spectrometric analyses. The approach included proteolysis of the covalently cross-linked protein-DNA complex with trypsin, followed by isolation, peptide mapping, and mass spectrometric and tandem mass spectrometric analyses. The 8-kDa domain of beta-pol was covalently cross-linked to a 5'-2-deoxyribose-5-phosphate-containing DNA substrate by sodium borohydride reduction. Using tandem mass spectrometry, the location of the DNA adduct on the 8-kDa domain was unequivocally determined to be at the Lys(72) residue. No additional amino acid residues were found as minor cross-linked species. These data allow assignment of Lys(72) as the sole Schiff base nucleophile in the 8-kDa domain of beta-pol. These results provide the first direct evidence in support of a catalytic mechanism involving nucleophilic attack by Lys(72) at the abasic site.     

Optimization of methyl propionate production catalysed by Mucor miehei lipase

Lipase from Mucor miehei was used to catalyse the esterification reaction between propionic acid and methyl alcohol in modified organic media. Small-scale model studies were performed in order to define the optimal conditions. The specific activity of immobilized lipase, adsorbed onto hydrophilic supports, compared to free lipase, showed that enzyme activity was altered by immobilisation. Non-polar solvents were shown to be less harmful for the biocatalyst than solvents with higher polarity. Diethyl ether was used as the cosolvent of hexane to improve the solubility of substrates in the organic phase thus increasing contact with enzyme. An optimal ratio of 90/10 (v/v) was determined for a hexane/diethyl ether mixture. The mass of enzyme preparation must be high enough to display optimal diffusion of the reagents and hydration of the catalytic sites. Increased substrate concentrations were stimulatory up to a point after which inhibition and enzyme destabilisation, in repeated runs, occurred. Water saturation of the organic medium greatly lowered the biosynthetic activity of the enzyme. It was possible to reach a 96% methyl propionate biosynthesis yield after 2.30 h reaction, underlining the free-enzyme operational capacity in a quasi-anhydrous modified organic medium.     

Multi-competitive enzymatic reactions in organic media: a simple test for the determination of lipase fatty acid specificity

Multiple substrate competition was kinetically analyzed to study lipase-catalyzed reactions in organic media. For each substrate, a competitive factor (the ratio of the specificity constants kcat/Km) was measured by reference to the best substrate using a mixture of fatty acid ethyl esters submitted to a solvolysis reaction by n-propanol. A scale of competitive factors was established which quantitatively described the lipase specificity. This principle was applied to the determination of the specificity of four commercial lipase preparations towards fatty acid chain length and degree of unsaturation. The results were not affected by changes in the physicochemical conditions of the reaction (water content, substrate concentration, nature of nucleophile, etc.). The simple test will be a useful tool to characterize lipase specificity.     

Direct enantioselective HPLC monitoring of lipase‐catalyzed kinetic resolution of flurbiprofen

The solvent versatility of Chiralpak IB, a 3,5‐dimethylphenylcarbamate derivative of cellulose‐based chiral stationary phase, is demonstrated in the direct enantioselective HPLC monitoring of lipase‐catalyzed kinetic resolution of flurbiprofen in nonstandard HPLC organic solvents. Nonstandard HPLC organic solvents were used as the reaction media for the lipase‐catalysis and in mean time as diluent to dissolve the “difficult to dissolve” enzyme substrate (the acid) and as eluent for the simultaneous enantioselective HPLC baseline separation of both substrate and product in one run without any further derivatization. Chirality 2010. © 2009 Wiley‐Liss, 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.     

Revisiting the effect of water content on enzymatic peptide synthesis in non-aqueous medium

Enzymatic acyl-transfer reaction in organic medium competes with the hydrolytic side reaction depending on the water content. The effect of water content on aminolysis activity of -chymotrypsin for the synthesis of Bz-Tyr-Val-NH₂ in acetonitrile was examined under the conditions which were devoid of the hydrolytic deacylation. Excess H-Val-NH₂ (880 mM) was employed to keep the hydrolysis negligible. The aminolysis rate increased abruptly between 4 and 5% (v/v) water but a further increase in the water content did not affect the reaction rate. This suggests that water added more than 5% (v/v) does not enhance intrinsic enzyme activity but acts only as a nucleophile for the hydrolytic deacylation.     

Kinetic analysis of deactivation of immobilized alpha-chymotrypsin by water-miscible organic solvent in kyotorphin synthesis.

Two different immobilized chymotrypsin derivatives were used to synthesize kyotorphin, using N-benzoyl-L-tyrosine ethyl ester and L-arginine ethyl ester as substrates, in water-DMF media. The first was adsorbed onto Celite particles and the second was multipoint covalently attached into polyacrylamide gel. In all cases, the conversion of the carboxyl substrate was carried out in first-order reaction conditions. For the adsorbed enzyme, the reaction kinetics deviated from first-order likely due to a fast irreversible inactivation of enzyme during the reaction time even at low DMF concentration (15-20% v/v). The covalent attachment of enzyme resulted in elimination of irreversible activity loss by organic solvent up to 60% (v/v) of DMF. The catalytic activity of the covalent derivative was conserved as appropriate for performing a synthetic reaction up to 60% v/v of DMF (in comparison to 30% v/v for the adsorbed derivative), showing a clear improvement in its stability against reversible denaturation by this solvent. The selectivity of the synthetic reaction was slightly enhanced (from 40-50%) with the increase in DMF concentration to 80% v/v, but it was significantly improved (to 80%) when L-argininamide was used as nucleophile.     

Biocatalysis of lipoxygenase in selected organic solvent media

The biocatalysis of purified soybean lipoxygenase (LOX) (EC 1.13.11.12), using linoleic acid as a substrate model, was investigated in selected organic solvent media, including chloroform, dichloromethane, hexane, iso-octane, octane and toluene. The results indicated that there was a 2.6-fold increase in LOX activity in the monophasic iso-octane medium compared to that obtained in the aqueous medium. The results also showed that there was an increase of 2.2- and 1.8-fold in LOX activity in the monophasic reaction media of octane and hexane, respectively. However, an inhibitory effect on enzyme activity was observed when the monophasic reaction media of toluene, chloroform and dichloromethane were used. In addition, the results showed that the optimum concentration of octane and iso-octane in the biphasic medium containing the organic solvent and Tris–HCl buffer solution, was determined to be 3.5% and 4%, respectively, for LOX activity. Moreover, the biocatalysis of LOX in a ternary micellar system, containing either 3.5% octane or 4% iso-octane, Tris–HCl buffer solution and an emulsifier, resulted in an overall increase in enzyme activity. The K_m and V_max values, substrate specificity, optimum protein concentration, optimum reaction temperature as well as the enzymatically catalyzed end-products were investigated for LOX biocatalysis in both ternary micellar systems.     

Modification of gastric (H+ + K+)-ATPase with pyridoxal 5'-phosphate

Pig gastric membrane vesicles enriched in (H+ + K+)-ATPase were covalently modified with pyridoxal 5'-phosphate (PLP). The modification resulted in inhibition of K+-dependent ATP hydrolysis, formation of phosphoenzyme and ATP-driven H+-uptake catalyzed by (H+ + K+)-ATPase. ATP, ADP, and adenyl-5'-yl imidodiphosphate were protective ligands, whereas Mg2+ and K+ were not. Specific PLP-binding of about 4.5 nmol/mg membrane protein was necessary for complete inhibition of the enzyme activity, indicating that the stoichiometry of PLP-binding to the enzyme was about 1:1. Limited proteolysis of the enzyme modified with [3H]PLP by trypsin suggests that PLP specifically modifies the lysine residue located in the 16-kDa fragment of the enzyme cleaved by trypsin. These results suggested that PLP binds to a specific lysine residue in the nucleotide-binding site or a region in its vicinity and inhibits the substrate binding or phosphorylation step of (H+ + K+)-ATPase.