Enantioselective hydrolysis of epichlorohydrin using whole Aspergillus niger ZJB-09173 cells in organic solvents

The enantioselective hydrolysis of racemic epichlorohydrin for the production of enantiopure (S)-epichlorohydrin using whole cells of Aspergillus niger ZJB-09173 in organic solvents was investigated. Cyclohexane was used as the reaction medium based on the excellent enantioselectivity of epoxide hydrolase from A. niger ZJB- 09173 in cyclohexane. However, cyclohexane had a negative effect on the stability of epoxide hydrolase from A. niger ZJB-09173. In the cyclohexane medium, substrate inhibition, rather than product inhibition of catalysis, was observed in the hydrolysis of racemic epichlorohydrin using A. niger ZJB-09173. The racemic epichlorohydrin concentration was markedly increased by continuous feeding of substrate without significant decline of the yield. Ultimately, 18.5% of (S)-epichlorohydrin with 98 percent enantiomeric excess from 153.6 mM of racemic epichlorohydrin was obtained by the dry cells of A. niger ZJB-09173, which was the highest substrate concentration in the production of enantiopure (S)-epichlorohydrin by epoxide hydrolases using an organic solvent medium among the known reports.     

Enantioselective lipase-catalyzed kinetic resolution of N-(2-ethyl-6-methylphenyl)alanine

The enzyme (BSL2), a highly active lipase expressed from newly constructed strain of Bacillus subtilis BSL2, is used in the kinetic resolution of N-(2-ethyl-6-methylphenyl)alanine from the corresponding racemic methyl ester. Reaction conditions are optimized to enhance the enantioselectivity. The effects of various racemic alkyl esters, substrate concentration, operating temperature, pH of the aqueous medium and organic solvents on activity and enantioselectivity of BSL2 for kinetic resolution are also studied. A high enantiomeric ratio (E = 60.7) is reached in diisopropyl ether/water (10%, v/v) and the enantioselectivity is about 22-fold higher than that in pure buffered aqueous solution. The results show that the reaction medium greatly influences BSL2 reaction and its enantioselectivity in the hydrolysis of racemic methyl ester.     

Comparison of the omega-transaminases from different microorganisms and application to production of chiral amines

Microorganisms that are capable of (S)-enantioselective transamination of chiral amines were isolated from soil samples by selective enrichment using (S)-alpha-methyl-benzylamine ((S)-alpha-MBA) as a sole nitrogen source. Among them, Klebsiella pneumoniae JS2F, Bacillus thuringiensis JS64, and Vibrio fluvialis JS17 showed good omega-transaminase (omega-TA) activities and the properties of the omega-TAs were investigated. The induction level of the enzyme was strongly dependent on the nitrogen source for the strains, except for V. fluvialis JS17. All the omega-TAs showed high enantioselectivity (E>50) toward (S)-alpha-MBA and broad amino donor specificities for arylic and aliphatic chiral amines. Besides pyruvate, aldehydes such as propionaldehyde and butyraldehyde showed good amino acceptor reactivities. All the omega-TAs showed substrate inhibition by (S)-alpha-MBA above 200 mm. Moreover, substrate inhibition by pyruvate above 10 mm was observed for omega-TA from V. fluvialis JS17. In the case of product inhibition, acetophenone showed much greater inhibitions than L-alanine for all omega-TAs. Comparison of the enzyme properties indicates that omega-transaminase from V. fluvialis JS17 is the best one for both kinetic resolution and asymmetric synthesis to produce enantiomerically pure chiral amines. Kinetic resolution of sec-butylamine (20 mM) was done under reduced pressure (150 Torr) to selectively remove an inhibitory product (2-butanone) using the enzyme from V. fluvialis JS17. Enantiomeric excess of (R)-sec-butylamine reached 94.7% after 12 h of reaction.     

Dimethyl sulfoxide-induced high enantioselectivity of subtilisin Carlsberg for hydrolysis of ethyl 2-(4-substituted phenoxy)propionates

The enantioselectivity for subtilisin-catalyzed hydrolysis of ethyl 2-(4-substituted phenoxy)propionates in an aqueous buffer solution was improved by addition of DMSO (54–56% v/v). On the basis of the conformational change of subtilisin Carlsberg observed for FT-IR and CD spectra, the high enantioselectivity for subtilisin-catalyzed hydrolysis of racemic ethyl 2-(4-ethylphenoxy)propionate could be related to a partial decrease of the tertiary structure of the enzyme protein arising from an increase of the ratio of DMSO in the reaction medium. This mechanistic model for the enantiorecognition can also be supported by the discussion based on the value of the Michaelis constant (K _m) obtained for each enantiomer of the substrate.     

Enzymatic esterification of lavandulol – a partial kinetic resolution of (S)-lavandulol and preparation of optically enriched (R)-lavandulyl acetate

The asymmetric esterification of the racemic primary alcohol lavandulol was achieved using lipase B from Candida antarctica and acetic acid as acyl donor in 80% yield. The enantioselectivity of the process was characterised, and a preparative resolution of 25 mM racemic lavandulol, stopped at approx. 55% conversion, yielded (S)-lavandulol in 42% yield and 52% e.e. and (R)-lavandulyl acetate in 51% yield and 48% e.e.     

Kinetic resolution of chiral amines with omega-transaminase using an enzyme-membrane reactor

A kinetic resolution process for the production of chiral amines was developed using an enzyme-membrane reactor (EMR) and a hollow-fiber membrane contactor with (S)-specific omega-transaminases (omega-TA) from Vibrio fluvialis JS17 and Bacillus thuringiensis JS64. The substrate solution containing racemic amine and pyruvate was recirculated through the EMR and inhibitory ketone product was selectively extracted by the membrane contactor until enantiomeric excess of (R)-amine exceeded 95%. Using the reactor set-up with flat membrane reactor (10-mL working volume), kinetic resolutions of alpha-methylbenzylamine (alpha-MBA) and 1-aminotetralin (200 mM, 50 mL) were carried out. During the operation, concentration of ketone product, i.e., acetophenone or alpha-tetralone, in a substrate reservoir was maintained below 0.1 mM, suggesting efficient removal of the inhibitory ketone by the membrane contactor. After 47 and 32.5 h of operation using 5 U/mL of enzyme, 98.0 and 95.5% ee of (R)-alpha-MBA and (R)-1-aminotetralin were obtained at 49.5 and 48.8% of conversion, respectively. A hollow-fiber membrane reactor (39-mL working volume) was used for a preparative-scale kinetic resolution of 1-aminotetralin (200 mM, 1 L). After 133 h of operation, enantiomeric excess reached 95.6% and 14.3 g of (R)-1-aminotetralin was recovered (97.4% of yield). Mathematical modeling of the EMR process including the membrane contactor was performed to evaluate the effect of residence time. The simulation results suggest that residence time should be short to maintain the concentration of the ketone product in EMR sufficiently low so as to decrease conversion per cycle and, in turn, reduce the inhibition of the omega-TA activity.     

Use of stable emulsion to improve stability, activity, and enantioselectivity of lipase immobilized in a membrane reactor

The enantiocatalytic performance of immobilized lipase in an emulsion membrane reactor using stable emulsion prepared by membrane emulsification technology was studied. The production of optical pure (S)-naproxen from racemic naproxen methyl ester was used as a model reaction system. The O/W emulsion, containing the substrate in the organic phase, was fed to the enzyme membrane reactor from shell-to-lumen. The enzyme was immobilized in the sponge layer (shell side) of capillary polyamide membrane with 50 kDa cut-off. The aqueous phase was able to permeate through the membrane while the microemulsion was retained by the thin selective layer. Therefore, the substrate was kept in the enzyme-loaded membrane while the water-soluble product was continuously removed from the reaction site. The results show that lipase maintained stable activity during the entire operation time (more than 250 h), showing an enantiomeric excess (96 +/- 2%) comparable to the free enzyme (98 +/- 1%) and much higher compared to similar lipase-loaded membrane reactors used in two-separate phase systems (90%). The results demonstrate that immobilized enzymes can achieve high stability as well as high catalytic activity and enantioselectivity.     

Efficient kinetic resolution of (R,S)-1-trimethylsilylethanol via lipase-mediated enantioselective acylation in ionic liquids

Efficient enantioselective acylation of (R,S)-1-trimethylsilylethanol {(R,S)-1-TMSE} with vinyl acetate catalyzed by immobilized lipase from Candida antarctica B (i.e., Novozym 435) was successfully conducted in ionic liquids (ILs). A remarkable enhancement in the initial rate and the enantioselectivity of the acylation was observed by using ILs as the reaction media when compared to the organic solvents tested. Also, the activity, enantioselectivity, and thermostability of Novozym 435 increased with increasing hydrophobicity of ILs. Of the six ILs examined, the IL C4MIm.PF6 gave the fastest initial rate and the highest enantioselectivity, and was consequently chosen as the favorable medium for the reaction. The optimal molar ratio of vinyl acetate to (R,S)-1-TMSE, water activity, and reaction temperature range were 4:1, 0.75, and 40 -50 degrees C, respectively, under which the initial rate and the enantioselectivity (E value) were 27.6 mM/h and 149, respectively. After a reaction time of 6 h, the ee of the remaining (S)-1-TMSE reached 97.1% at the substrate conversion of 50.7%. Additionally, Novozym 435 was effectively recycled and reused in C4MIm.PF6 for five consecutive runs without substantial lose in activity and enantioselectivity. The preparative scale kinetic resolution of (R,S)-1-TMSE in C4MIm.PF6 is shown to be very promising and useful for the industrial production of enantiopure (S)-1-TMSE.     

Efficient enantioselective hydrolysis of D,L-phenylglycine methyl ester catalyzed by immobilized Candida antarctica lipase B in ionic liquid containing systems

Immobilized Candida antarctica lipase B (Novozym 435)-catalyzed enantioselective hydrolysis of D,L-phenylglycine methyl ester to enatiopure D-phenylglycine was successfully conducted in the systems with ionic liquids (ILs). Novozym 435 exhibited excellent activity and enantioselectivity in the system containing the IL BMIMxBF(4) compared to several typical organic solvents tested. It has been found that the cations and, particularly, the anions of ILs have a significant effect on the reaction, and the IL BMIMxBF(4), which shows to be the most suitable for the reaction, gave the highest initial rate and enantioselectivity among various ILs examined. The reaction became much less active and enantioselective in the systems with BMIMxHSO(4). Also, it was noticed that the enzymatic hydrolysis was strongly dependent on BMIMxBF(4) content in the co-solvent systems and the favorable content of the IL was 20% (v/v). Of the assayed four co-solvents and phosphate buffer, the lowest apparent K(m) and activation energy, and the highest V(max) of the reaction were achieved using 20% (v/v) BMIMxBF(4) co-solvent with phosphate buffer. Additionally, various influential variables were investigated. The optimum pH, substrate concentration, reaction temperature and shaking rate were 8.0, 80mM, 25-30 degrees Celsius and 150rpm, respectively, under which the initial rate, the residual substrate e.e. and the enantioselectivity were 2.46mM/min, 93.8% (at substrate conversion of 53.0%) and 38, respectively. When the hydrolysis was performed under reduced pressure, the initial rate (2.64mM/min) and the enantioselectivity (E=43) were boosted.     

酮基布洛芬拆分用酯酶产生菌的筛选及其催化特性

从土壤中筛选获得一株可以高对映选择性水解酮基布洛芬乙酯的酵母KET4,经鉴定为芸苔丝孢酵母（Trichosporon brassicae）。研究了该菌的生长和产酶过程,考察了其静息细胞对酮基布洛芬乙酯水解的催化特性。用该菌催化酯水解时,转化率为41%时,产物的对映体过量值为91%,对映选择率达到45。     

Improved production of ethyl-(R)-2-hydroxy-4-phenylbutyrate with pretreated Saccharomyces cerevisiae in water/organic solvent two-liquid phase systems

Baker's yeast pretreated with α-phenacyl chloride was employed to improve the enantioselectivity of the asymmetric reduction of ethyl-2-oxo-4-phenylbutyrate (EOPB) to ethyl-(R)-2-hydroxy-4-phenylbutyrate ((R)-EHPB) and maintain a high activity of the yeast. A water/organic solvent two-liquid phase system was also introduced to overcome the strong substrate and product inhibition of the enzyme; the highest catalytic activity and enantioselectivity were obtained in a water/benzene two-liquid phase system. When the reduction was catalyzed with pretreated yeast (300 mg mL^?1 buffer) in the water/benzene two-liquid phase system (V_aq/V_ben=20:40), 41.9% molar conversion of EOPB and 87.5% e.e. of (R)-EHPB were obtained in 48 h, using pH 8.0 phosphate buffer with 1.5% (v/v) of ethanol added as a co-substrate at 30°C, even with an initial EOPB concentration of 400 mM and a final EHPB concentration as high as 167.7 mM.     

Enantioconvergent production of (R)-1-phenyl-1,2-ethanediol from styrene oxide by combining the Solanum tuberosum and an evolved Agrobacterium radiobacter AD1 epoxide hydrolases

Soluble epoxide hydrolase (EH) from the potato Solanum tuberosum and an evolved EH of the bacterium Agrobacterium radiobacter AD1, EchA-I219F, were purified for the enantioconvergent hydrolysis of racemic styrene oxide into the single product (R)-1-phenyl-1,2-ethanediol, which is an important intermediate for pharmaceuticals. EchA-I219F has enhanced enantioselectivity (enantiomeric ratio of 91 based on products) for converting (R)-styrene oxide to (R)-1-phenyl-1,2-ethanediol (2.0 +/- 0.2 micromol/min/mg), and the potato EH converts (S)-styrene oxide primarily to the same enantiomer, (R)-1-phenyl-1,2-ethanediol (22 +/- 1 micromol/min/mg), with an enantiomeric ratio of 40 +/- 17 (based on substrates). By mixing these two purified enzymes, inexpensive racemic styrene oxide (5 mM) was converted at 100% yield to 98% enantiomeric excess (R)-1-phenyl-1,2-ethanediol at 4.7 +/- 0.7 micromol/min/mg. Hence, at least 99% of substrate is converted into a single stereospecific product at a rapid rate.     

Enantioselective resolution of racemic styrene oxide at high concentration using recombinant Pichia pastoris expressing epoxide hydrolase of Rhodotorula glutinis in the presence of surfactant and glycerol

The reaction medium was optimized to accomplish epoxide hydrolase-catalyzed, batch enantioselective hydrolysis of racemic styrene oxide at high initial substrate concentrations. The recombinant Pichia pastoris containing the epoxide hydrolase gene of Rhodotorula glutinis was used as the biocatalyst. Enantiopure (S)-styrene oxide with 98% ee was obtained with 41% yield (maximum yield = 50%) from 1.8 M racemic styrene oxide at pH 8.0, 4 degrees C in the presence of 40% (v/v) Tween 20 and 5% (v/v) glycerol.     

Purification and characterization of a recombinantCaulobacter crescentus epoxide hydrolase

ACaulobacter crescentus epoxide hydrolase (CCEH) from a recombinantEscherichia coli was purified to homogeneity using a three-step procedure. The CCEH protein was purified 7.3-fold with a 22.9% yield in overall activity. The optimal reaction temperature and pH were determined to be 37°C and pH 8.0, respectively. The addition of 10% (v/v) dimethylsulfoxide as a cosolvent improved the enantioselectivity of CCEH for a batch kinetic resolution of racemic indene oxide.     

Immobilized cells biocatalyst for the production of S-acetylthio-2-methyl propionic acid

S-acetylthio-2-methylpropionic acid (S-AMPA) is an important chiral intermediary for numerous hypertension drugs such as captopril. S-AMPA can be produced by hydrolyzing the corresponding racemic methyl MAMP (S,R-methyl-β-acetylthioisobutyrate) by lipases or esterases that have the appropriate stereo specificity. Psudomonas fluorescens IFO 12055 possessing a highly specific lipase was used to process this reaction in the form of immobilized cells. Reaction kinetic and immobilization methods were also studied. Strong product inhibition was observed, that is, at 3% S-AMPA (namely 183 mM), activity was reduced by 50%. Spontaneous hydrolysis of the ester and thioester bonds was also observed, and was independent of the cells. Thus, reaction selectivity and yield must be optimized through adjusting the substrate concentration and total biocatalyst activity. Conventional calcium alginate (3% w/w) encapsulation was modified by adding 3% w/w polyethyleneimine (PEI) and cross-linked by a biologically derived agent, genipin (5.6 mM). This method was found to be satisfactory to produce stable and functioning biocatalyst and can maintain high reactivity for repeated 25 batches with e.e. values above 90%.     

Effect of solvent on enantioselective esterification of naproxen by lipase with trimethylsilyl methanol

Improvement of stereoselective resolution of racemic Naproxen, 2-(6-methoxy-2-naphthyl)propionic acid, was attempted with esterifcation reaction by Candida cylindracea lipase. By carefully selecting the organic medium, a 72-time enhancement of yield of the desired S-ester was achieved. The optimal reaction temperature was approximately 53 degrees C, and an alcohol concentration between 20 mM and 40 mM in an 80% (v/v) isooctane and 20% (v/v) toluene mixture was found. (c) 1994 John Wiley & Sons, Inc.     

Asymmetric synthesis of (<Emphasis Type="Italic">R</Emphasis>)-3-fluoroalanine from 3-fluoropyruvate using omega-transaminase

In this study, (R)-3-fluoroalanine was asymmetrically synthesized from 3-fluoropyruvate (F-pyruvate) and (S)-α-methylbenzylamine (MBA) using recombinant ω-transaminase (TA) from Vibrio fluvialis JS17. The reaction was severely inhibited by acetophenone (deaminated product of α-MBA). In the presence of 5 mM acetophenone, the reactivity of the enzyme towards F-pyruvate decreased by 78%. To overcome the product inhibition by acetophenone, a biphasic reaction was successfully used. The conversion of F-pyruvate into (R)-3-fluoroalanine (enatiomeric exess (e.e.) > 99%) was about 95% in the biphasic system (75 mM F-pyruvate, 100 mM (S)-α-MBA, and 3.0 U/mL), whereas 31% was obtained without product extraction. The use of racemic α-MBA as an amino donor instead of (S)-α-MBA can reduce the reaction cost and also produce chiral amines through kinetic resolution. When the kinetic resolution of racemic α-MBA (40 mM) was carried out with F-pyruvate (30 mM) and ω-TA (3.0 U/mL) in 100 mM phosphate buffer (pH 7.0), the e.e. of (R)-α-MBA reached 98.4% with 52.2% conversion for 10 h and 21 mM (R)-3-fluoroalanine was produced with 70% conversion and an e.e. > 99%.     

Evaluating the impact of substrate and product concentration on a whole-cell biocatalyst during a Baeyer-Villiger reaction

The presence of high concentrations of substrate or product may impede the optimal functioning of a biocatalyst, more so in the case of whole cell biocatalysts where the metabolic status of the cells may be compromised. In this article we investigate these effects using as an example the Baeyer–Villiger oxidation of racemic bicyclo[3.2.0]hept-2-en-6-one to yield (?)-1(S),5(R)-2-oxabicyclo[3.3.0]oct-6-en-3-one and (?)-1(R),5(S)-3-oxabicyclo[3.3.0]oct-6-en-2-one by CHMO expressed in Escherichia coli TOP10. Multi parameter flow cytometry was used to illustrate that substrate (racemic bicyclo[3.2.0]hept-2-en-6-one) associated cell damage was concentration dependent. One of the two regio-isomeric products [(-)-1(S),5(R)-2-oxabicyclo[3.3.0]oct-6-en-3-one] was also used to identify that product associated cell damage was time dependent. In addition, both substrate and product concentrations affected the observed reaction rate.     

Resolution of N-(2-ethyl-6-methylphenyl) alanine catalyzed by Lipase B from Candida antarctica

A biotransformation process has been developed for the production of (S)-N-(2-ethyl-6-methylphenyl) alanine by enantioselective hydrolysis of racemic methyl ester in the presence of Candida antarctica lipase B (CAL-B). However, the enantioselectivity of CAL-B towards the resolution is not high enough to obtain enantiomerically pure product. In order to improve the enantioselectivity of the enzyme, the effects of surfactants on CAL-B-catalyzed hydrolysis were tested. The results suggest that surfactants can influence the microenvironment of the enzyme, and the addition of Tween-80, in particular, to the reaction medium markedly enhanced the selectivity of CAL-B towards the substrate used, with the enantiomeric ratio (E-value) increasing from 11.3 to 60.1.     

Modulation of lipase properties in macro-aqueous systems by controlled enzyme immobilization: enantioselective hydrolysis of a chiral ester by immobilized Pseudomonas lipase

Lipase from Pseudomonas fluorescens (PFL) has been immobilized by using different immobilization protocols. The catalytic behavior of the different PFL derivatives in the hydrolytic resolution of fully soluble (R,S) 2-hydroxy 4-phenyl butanoic acid ethyl ester (HPBE) in aqueous medium was analyzed. The soluble enzyme showed a significant but low enantioselectivity, hydrolyzing the S isomer more rapidly than the R-isomer (E = 7). The enzyme, immobilized via a limited attachment to a long and flexible spacer arm, showed almost identical activity and specificity to the soluble enzyme. However, other derivatives, e.g. PFL adsorbed on supports covered by hydrophobic moieties (octyl, decaoctyl), exhibited significant hyperactivation on immobilization (approximately 7-fold). Simultaneously, the enantioselectivity of the PFL-immobilized enzyme was significantly improved (from E = 7 to E = 80). By using such derivatives, almost pure R ester isomer (e.e. > 99%) has been obtained after 55% hydrolysis of the racemic mixture of a solution of 10% (w/v) (R,S) HPBE. The derivatives could be used for 10 cycles without any significant decrease in the activity of the biocatalyst.