Biocatalytic resolution of nitro-substituted phenoxypropylene oxides with Trichosporon loubierii epoxide hydrolase and prediction of their enantiopurity variation with reaction time

Biocatalytic resolution of 3-(2′-nitrophenoxy)propylene oxide (1a), 3-(3′-nitrophenoxy)propylene oxide (1b) and 3-(4′-nitrophenoxy)propylene oxide (1c) were exploited by using lyophilized cells of yeast Trichosporon loubierii ECU1040 with epoxide hydrolase (EH) activity, which preferentially hydrolyzes (S)-enantiomers of the epoxides (1a–c), yielding (S)-diols and (R)-epoxides. The activity increased as the nitro group in the phenyl ring was shifted from 4′-position (1c) to 2′-position (1a). When the substrate concentration of 1a was increased from 10 to 80 mM, the E-value increased at first, until reaching a peak at 40 mM, and then decreased at higher concentrations (>40 mM). The optically active epoxide (R)-1a was prepared at gram-scale (97% ee, 41% yield). Furthermore, a simple method was developed to predict the enantiomeric excess of substrate (ee_s) at any time of the whole reaction course based on the ee_s value determined at a certain reaction time at a relatively lower substrate concentration. This will be helpful for terminating the reaction at a proper time to get both higher optical purity and higher yield of the remaining epoxides.     

Biocatalytic preparation of (S)-phenyl glycidyl ether using newly isolated Bacillus megaterium ECU1001

A bacterial strain (ECU1001) capable of utilizing phenyl glycidyl ether as sole carbon source and energy source was isolated from soil samples through two steps of screening and was identified as a Bacillus megaterium. The epoxide hydrolase from Bacillus megaterium ECU1001 was biosynthesized in parallel with cell growth and a maximum activity of 31.0 U/l was reached after 30 h of culture when the biomass (DCW) was 9.1 g/l. A temperature of 35°C and pH 8.0 were optimal for the bioconversion. The lyophilized whole cells of Bacillus megaterium ECU1001 could preferentially hydrolyze the (R)-enantiomer of phenyl glycidyl ether, yeilding (S)-epoxide and (R)-diol with high enantioselectivity (E=47.8). The (S)-enantiomer of the epoxide remained in the reaction mixture with >99.5% ee (enantiomeric excess) at a conversion of 55.9%. The substrate concentration could be increased up to 60 mM without affecting the ee and (S)-phenyl glycidyl ether could be obtained with an optical purity of 100% ee and 25.6% yield. Therefore, the method is potentially useful for the preparative resolution of epoxides.     

A spectrophotometric method to assay epoxide hydrolase activity.

The Aspergillus niger epoxide hydrolase activity was assayed by spectrophotometric using (rac) p-nitrostryrene oxide (pNSO) as substrate. Both the substrate (pNSO) and the reaction product, p-nitrostryrene diol (pNSD), had a strong absorbance in UV at 280 nm. The assay was based on the measure of the pNSD absorbance of the water phase after extraction of the non-reacted pNSO with a solvent. Among the five solvents tested, chloroform was selected since it extracted more than 99% of the epoxide and only 32% of the produced diol. This extraction yield was independent of the diol and epoxide concentrations and it was fairly reproducible. Using different enzyme amounts, the reaction kinetics were linear for the first 10 min corresponding to degrees of conversion less than 5% for the epoxide. Two controls were run simultaneously, one with the substrate alone (epoxide hydrolysis and non-complete extraction) and one with the enzyme alone (enzyme absorbance at 280 nm). The resulting DeltaOD/min was linear with the amount of enzyme added within a large range from 2 to 80 microg of the EH preparation. The new spectrophotometric assay correlates well with the previous HPLC assay and could be used routinely for an easy and fast evaluation of EH activity. The kinetic parameters of (rac) pNSO hydrolysis by A. niger epoxide hydrolase could be easily determined and K(M) (1.1 mM) compared well with that previously reported (1.0 mM).     

Enantioselective hydrolysis of racemic styrene oxide by epoxide hydrolase of<Emphasis Type="Italic">Rhodosporidium kratochvilovae</Emphasis> SYU-08

Enantioselective hydrolysis for the production of chiral styrene oxide was investigated using the epoxide hydrolase activity of a newly isolatedRhodosporidium kratochvilovae SYU-08. The effects of reaction prameters—buffer type, pH, temperature, initial substrate concentrations, phenyl-1,2-ethanediol concentrations on hydrolysis rate, and enantioselectivity—were analyzed. Optically active (S)-styrene oxide with an enantiomeric excess higher than 99 % was obtained from its racemate with a yield of 38 % (theoretically 50% maximum yield) from an initial concentration of 80 mM.     

Effect of mass transfer limitations on the enzymatic kinetic resolution of epoxides in a two-liquid-phase system

Optically active epoxides can be obtained by kinetic resolution of racemic mixtures using enantioselective epoxide hydrolases. To increase the productivity of the conversion of sparingly aqueous soluble epoxides, we investigated the use of a two-phase aqueous/organic system. A kinetic model which takes into account interphase mass transfer, enzymatic reaction, and enzyme inactivation was developed to describe epoxide conversion in the system by the epoxide hydrolase from Agrobacterium radiobacter. A Lewis cell was used to determine model parameters and results from resolutions carried out in the Lewis cell were compared to model predictions to validate the model. It was found that n-octane is a biocompatible immiscible solvent suitable for use as the organic phase. Good agreement between the model predictions and experimental data was found when the enzyme inactivation rate was fitted. Simulations showed that mass transfer limitations have to be avoided in order to maximize the yield of enantiomerically pure epoxide. Resolution of a 39 g/L solution of racemic styrene oxide in octane was successfully carried out in an emulsion batch reactor to obtain (S)-styrene oxide in high enantiomeric excess (>95% e.e.) with a yield of 30%.     

Cloning, expression, purification, and characterization of a novel epoxide hydrolase from Aspergillus niger SQ-6

A novel epoxide hydrolase from Aspergillus niger SQ-6 has now been cloned by inverse PCR. Its gene shows eight exons including a non-coding exon at its 5'-terminal (GenBank Accession No. AY966486). Phylogenetic analysis using deduced amino acid sequence (395 aa) confirms it as an epoxide hydrolase and shares 58.3% identity with that of A. niger LCP521 (GenBank Accession No. AF238460). The predicted catalytic triad is composed of Asp(191), His(369) and Glu(343). Active recombinant epoxide hydrolase has been successfully expressed in Escherichia coli as protein fusions with a poly-His tail. Scale-up fermentation can yield 2.5g/L of recombinant protein. The electrophoretic pure recombinant protein, which shows similar characterization as natural enzyme purified from A. niger SQ-6, can be easily purified by Ni(2+)-chelated affinity and gel-filtration chromatography. Optimal pH and temperature for purified enzyme are pH 7.5 and 37 degrees C, respectively. The K(m), k(cat) and maximal velocity (V(max)) for p-nitrostyrene oxide are determined to be 1.02mM, 172s(-1) and 231micromol min(-1)mg(-1), respectively. The enzyme can be inhibited by oxidant (H(2)O(2)), solvent (Tetrahydrofuran) and several metal ions including Hg(2+), Fe(2+) and Co(2+). This (R)-stereospecific epoxide hydrolase exhibits high enantioselectivity (enantiomeric excess value, 99%) for the less hindered carbon atom of epoxide. It may be an industrial biocatalyst for the preparation of enantiopure epoxides or vicinal diols.     

Improved catalytic performance of Bacillus megaterium epoxide hydrolase in a medium containing Tween-80

A new epoxide hydrolase with high enantioselectivity toward (R)-glycidyl phenyl ether (R-GPE) was partially purified from Bacillus megaterium strain ECU1001. The maximum activity of the isolated enzyme was observed at 30 degrees C and pH 6.5 in a buffer system with 5% (v/v) of DMSO as a cosolvent. The enzyme was quite stable at pH 7.5 and retained full activity after incubation at 40 degrees C for 6 h. Interestingly, when the cosolvent DMSO was replaced by an emulsifier (Tween-80, 0.5% w/v) as an alternative additive to help disperse the water-insoluble substrate, the apparent activity of the epoxide hydrolase significantly increased by about 1.8-fold, while the temperature optimum shifted from 30 to 40 degrees C and the half-life of the enzyme at 50 degrees C increased by 2.5 times. The enzymatic hydrolysis of rac-GPE was highly enantioselective, with an E-value (enantiomeric ratio) of 69.3 in the Tween-80 emulsion system, which is obviously higher than that (41.2) observed in the DMSO-containing system.     

Epoxide hydrolases from yeasts and other sources: versatile tools in biocatalysis

Major characteristics, substrate specificities and enantioselectivities of epoxide hydrolases from various sources are described. Epoxide hydrolase activity in yeasts is discussed in more detail and is compared with activities in other microorganisms. Constitutively produced bacterial epoxide hydrolases are highly enantioselective in the hydrolysis of 2,2- and 2,3-disubstituted epoxides. A novel bacterial limonene-1,2-epoxide hydrolase, induced by growth on monoterpenes, showed high activities and selectivities in the hydrolysis of several substituted alicyclic epoxides. Constitutively produced epoxide hydrolases are found in eukaryotic microorganisms. Enzymes from filamentous fungi are useful biocatalysts in the resolution of aryl- and substituted alicyclic epoxides. Yeast epoxide hydrolase activity has been demonstrated for the enantioselective hydrolysis of various aryl-, alicyclic- and aliphatic epoxides by a strain of Rhodotorula glutinis. The yeast enzyme, moreover, is capable of asymmetric hydrolysis of meso epoxides and performs highly enantioselective resolution of unbranched aliphatic 1,2-epoxides. Screening for other yeast epoxide hydrolases shows that high enantioselectivity is restricted to a few basidiomycetes genera only. Resolution of very high substrate concentrations is possible by using selected basidiomycetes yeast strains.     

Intracellular activity of lysosomal glucosylceramidase measured with 4-nonylumbelliferyl beta-glucoside

Enzymatic activity of lysosomal glucosyl-ceramidase was determined in intact murine hybridoma and macrophage cells with the synthetic substrate nonylumbeliferyl-beta-glucoside (NUG). The substrate was applied as complex with bovine serum albumin (two binding sites, Kd 2.2 +/- 0.3 microM). The transport of the artificial substrate from medium to the enzyme was explored by measurements of substrate concentrations in cellular membranes and of endocytosis rate relative to substrate hydrolysis. The results indicated that, after enrichment in the plasma membrane, the substrate is mainly transported by simple diffusion. Release of nonylumberlliferone monitored fluorimetrically after disintegration of the cells in borate buffer containing Triton X-100 at pH 9.5 showed that 10(8) cells of both cell lines hydrolysed 1-1.5 nmol substrate/min at a total concentration of 0.1 mM NUG in the medium. Substrate hydrolysis was prevented by preincubating the cells with conduritol B epoxide (CBE), a specific active site-directed inhibitor of lysosomal glucosylceramidase. The substrate concentration at the site of the enzyme and maximal activity were evaluated by the inhibiting effect of the substrate on the inactivation rate by conduritol B epoxide. The rate of inhibitor uptake measured with bromo-[3H]conduritol B epoxide was shown to be not rate-limiting for the inactivation reaction. The molar concentration of the enzyme was determined by labeling with bromo-[3H]conduritol B epoxide. Comparison of the maximal intracellular activity with that of the enzyme after disintegration and activation by taurocholate showed a 20-fold lower activity in the native environment.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Resolution of 1,3-dioxolane derivatives using the lipase from an Acinetobacter junii SY-01

Acinetobacter junii SY-01 producing a lipase enantioselectively hydrolyzing 1,3-dioxolane derivatives was isolated from water sludge sample and the effect of solvent, acyl donor, vinyl acetate concentration, substrate concentration, operating temperature and immobilization on activity and enantioselectivity was studied for the resolution of 1,3-dioxolane derivatives through transesterification reaction using a lipase from the isolated strain. Best selectivity was obtained at lower substrate concentration (3–5 mM), higher vinyl acetate concentration (500–1000 mM) and lower temperature (30–40 °C) in the reaction mixture. Lipase immobilized onto Accurel MP-1000 (micro-porous polypropylene) gave the best results and the reactivity was about 29-fold higher than the free enzyme without the decrease of enantioselectivity. Resolution of 1,3-dioxolane derivatives was carried out in flask scale containing 100 ml solvents using the lipase immobilized onto Accurel MP-1000. In this reaction, the yield and enantiomeric excess of the remaining (2R, 4S)-alcohol were 31.2% and 98.2%, respectively. This result suggests that it can be used as an alternative method, compared to the present synthetic method, for the production of optically pure (2R, 4S)-itraconazole.     

高对映选择性环氧化物水解酶产生菌的筛选及特性研究

从土壤中分离的芽孢杆菌Bacillus megaterium ECU1001所产五氧化物水解酶能高对映选择性水解缩水甘油苯基醚（对映选择率E值可达47.8）,当转化率为55.9%时,剩余的（S）－缩水甘油苯基醚的光学纯度（对映体过量值,ee）可达99.5%;当底物浓度提高到60mmol/L时,光学纯（S）－缩水基油苯基醚的收率达到25.6%。     

Continuous production of enantiopure 1,2-epoxyhexane by yeast epoxide hydrolase in a two-phase membrane bioreactor

A two-phase membrane bioreactor was developed to continuously produce enantiopure epoxides using the epoxide hydrolase activity of Rhodotorula glutinis. An aqueous/organic cascade, hydrophilic, hollow-fiber membrane bioreactor was used: (1) to carry out large-scale resolution of epoxides, (2) to continuously extract residual enantiopure epoxides from the aqueous phase, and (3) to separate inhibitory formed diol from the yeast cells contained in the aqueous phase. Dodecane was employed to dissolve-feed epoxide as well as to extract residual epoxide. 1,2-Epoxyhexane was used as a model substrate. By use of this membrane bioreactor, enantiopure (S)-1,2-epoxyhexane (>98% enantiomeric excess) was obtained with a volumetric productivity of 3.8 g l⁻¹ h⁻¹. The continuous-production system was operated for 12 days and resulted in 38 g enantiopure (S)-1,2-epoxyhexane. Received: 14 February 2000 / Received revision: 15 June 2000 / Accepted: 18 June 2000     

Enantioselective production of 2,2-dimethylcyclopropane carboxylic acid from 2,2-dimethylcyclopropane carbonitrile using the nitrile hydratase and amidase of Rhodococcus erythropolis ATCC 25544

The enantioselective production of (S)-2,2-dimethylcyclopropane carboxylic acid was investigated in 53 Rhodococcus and Pseudomonas related strains. Rhodococcus erythropolis ATCC 25544 was selected as it showed the highest enantioselectivity. The enantioselectivity was due to the amidase activity in a two-step reaction involving nitrile hydratase. The enantiomeric excess of the amidase was highest at pH 7.0 and decreased significantly above 20 °C. For the enantioselective production of (S)-2,2-dimethylcyclopropane carboxylic acid, the optimum reaction conditions of the cells were determined to be pH 7.0, 20 °C, and 10% (v/v) methanol and were the same as the optimum pH and temperature for the enantioselective conversion by the amidase. Under these conditions, the R. erythropolis ATCC 25544 cells, which harbored nitrile hydratase and amidase enzymes, produced 45 mM (S)-2,2-dimethylcyclopropane carboxylic acid from racemic 100 mM 2,2-dimethylcyclopropane carbonitrile with an 81.8% enantiomeric excess after 64 h.     

Enantioselective epoxidation and carbon-carbon bond cleavage catalyzed by Coprinus cinereus peroxidase and myeloperoxidase

We demonstrate that myeloperoxidase (MPO) and Coprinus cinereus peroxidase (CiP) catalyze the enantioselective epoxidation of styrene and a number of substituted derivatives with a reasonable enantiomeric excess (up to 80%) and in a moderate yield. Three major differences with respect to the chloroperoxidase from Caldariomyces fumago (CPO) are observed in the reactivity of MPO and CiP toward styrene derivatives. First, in contrast to CPO, MPO and CiP produced the (S)-isomers of the epoxides in enantiomeric excess. Second, for MPO and CiP the H(2)O(2) had to be added very slowly (10 eq in 16 h) to prevent accumulation of catalytically inactive enzyme intermediates. Under these conditions, CPO hardly showed any epoxidizing activity; only with a high influx of H(2)O(2) (300 eq in 1.6 h) was epoxidation observed. Third, both MPO and CiP formed significant amounts of (substituted) benzaldehydes as side products as a consequence of C-alpha-C-beta bond cleavage of the styrene derivatives, whereas for CPO and cytochrome c peroxidase this activity is not observed. C-alpha-C-beta cleavage was the most prominent reaction catalyzed by CiP, whereas with MPO the relative amount of epoxide formed was higher. This is the first report of peroxidases catalyzing both epoxidation reactions and carbon-carbon bond cleavage. The results are discussed in terms of mechanisms involving ferryl oxygen transfer and electron transfer, respectively.     

Cloning and characterization of an epoxide hydrolase from Cupriavidus metallidurans-CH34

A putative epoxide hydrolase-encoding gene was identified from the genome sequence of Cupriavidus metallidurans CH34. The gene was cloned and overexpressed in Escherichia coli with His(6)-tag at its N-terminus. The epoxide hydrolase (CMEH) was purified to near homogeneity and was found to be a homodimer, with subunit molecular weight of 36 kDa. The CMEH had broad substrate specificity as it could hydrolyze 13 epoxides, out of 15 substrates tested. CMEH had high specific activity with 1,2-epoxyoctane, 1,2-epoxyhexane, styrene oxide (SO) and was also found to be active with meso-epoxides. The enzyme had optimum pH and temperature of 7.5 and 37°C respectively, with racemic SO. Biotransformation of 80 mM SO with recombinant whole E. coli cells expressing CMEH led to 56% ee(P) of (R)-diol with 77.23% conversion in 30 min. The enzyme could hydrolyze (R)-SO, ～2-fold faster than (S)-SO, though it accepted both (R)- and (S)-SO with similar affinity as K(m)(R) and K(m)(S) of CMEH were 2.05±0.42 and 2.11±0.16 mM, respectively. However, the k(cat)(R) and k(cat)(S) for the two enantiomers of SO were 4.80 and 3.34 s(-1), respectively. The wide substrate spectrum exhibited by CMEH combined with the fast conversion rate makes it a robust biocatalyst for industrial use. Regioselectivity studies with enantiopure (R)- and (S)-SO revealed that with slightly altered regioselectivity, CMEH has a high potential to synthesize an enantiopure (R)-PED, through an enantioconvergent hydrolytic process.     

Characterization of the epoxide hydrolase from an epichlorohydrin-degrading Pseudomonas sp.

An epoxide hydrolase was purified to homogeneity from the epichlorohydrin-utilizing bacterium Pseudomonas sp. strain AD1. The enzyme was found to be a monomeric protein with a molecular mass of 35 kDa. With epichlorohydrin as the substrate, the enzyme followed Michaelis-Menten kinetics with a Km value of 0.3 mM and a Vmax of 34 mumol.min-1.mg protein-1. The epoxide hydrolase catalyzed the hydrolysis of several epoxides, including epichlorohydrin, epibromohydrin, epoxyoctane and styrene epoxide. With all chiral compounds tested, both stereoisomers were converted. Amino acid sequencing of cyanogen bromide-generated peptides did not yield sequences with similarities to other known proteins.     

Enhancing the biocatalytic potential of carbonyl reductase of Candida viswanathii using aqueous-organic solvent system

In the present work, the toxic effect of various solvents with different Log P values was studied on the whole cells of Candida viswanathii. Experiments showed that the lower concentrations of some solvent increased both the activity retention and enzyme activity as compared to the control while this was not the case with higher concentrations of the same solvents. The model compound taken in the present study was 1-acetophenone. The percentage conversion improved from 76 to 94%. Addition of 2-propanol increased the substrate tolerance, giving the conversion of 90% compared to 9% in control at a substrate concentration of 70 mM in 1h. The operational stability increased at higher temperatures with the addition of 2-propanol in the reaction mixture with good conversion (90%) and enantiomeric excess (>99%) at 45 degrees C and 50 degrees C. The effect was also found to be prominent in other tested substrates. In order to further stabilize the cells for long term use in higher concentration of organic solvents, the cells were further immobilized, and were found to have higher activity retention than that of free cells.     

A novel enantioselective epoxide hydrolase from Agromyces mediolanus ZJB120203: Cloning,characterization and application

A new strain Agromyces mediolanus ZJB120203, capable of enantioselective epoxide hydrolase (EH) activity was isolated employing a newly established colorimetric screening and chiral GC analysis method. The partial nucleotide sequence of an epoxide hydrolase (AmEH) gene from A. mediolanus ZJB120203 was obtained by PCR using degenerate primers designed based on the conserved domains of EHs. Subsequently, an open reading frame containing 1167 bp and encoding 388 amino acids polypeptide were identified. Expression of AmEH was carried out in Escherichia coli and purification was performed by Nickel-affinity chromatography. The purified AmEH had a molecular weight of 43 kDa and showed its optimum pH and temperature at 8.0 and 35 °C, respectively. Moreover, this AmEH showed broad substrates specificity toward epoxides. In this study, it is demonstrated that the AmEH could unusually catalyze the hydrolysis of (R)-ECH to produce enantiopure (S)-ECH. Enantiopure (S)-ECH could be obtained with enantiomeric excess (ee) of >99% and yield of 21.5% from 64 mM (R,S)-ECH. It is indicated that AmEH from A. mediolanus is an attractive biocatalyst for the efficient preparation of optically active ECH.     

Epoxide hydrolase activity of Chryseomonas luteola for the asymmetric hydrolysis of aliphatic mono-substituted epoxides

Asymmetric hydrolysis of a homologous range of straight chain 1,2-epoxyalkanes was achieved using whole cells of Chryseomonas luteola. Depending on the chain length, hydrolyses of the racemic epoxides afforded optically active epoxides and diols with varying degrees of optical purity. In the case of 1,2-epoxyoctane, the enantiomeric excess of the remaining (S)-epoxide and formed (R)-diol was excellent (ees > 98% and eep = 86%). This is the first report of a bacterial epoxide hydrolase with such unusual enantioselectivity for terminal mono-substituted epoxides bearing no directing group on the chiral C-2 carbon. Benzyl glycidyl ether and the 2,2-disubstituted epoxide, 2-methyl-1,2-epoxyheptane, were hydrolysed, but no enantioselectivity was observed. © Rapid Science Ltd. 1998