Production of (R)-epichlorohydrin from 1,3-dichloro-2-propanol by two-step biocatalysis using haloalcohol dehalogenase and epoxide hydrolase in two-phase system

Recombinant Escherichia coli cells harbouring haloalcohol dehalogenase and epoxide hydrolase were successfully immobilized by adsorption onto perlite and used to prepare (R)-epichlorohydrin from 1,3-dichloro-2-propanol by two-step biocatalysis in a specially designed reactor. Two-phase solution was used as the reaction system in order to improve the yield of epichlorohydrin. In the two-phase system containing 40% (v/v) cyclohexane, the yield of racemic epichlorohydrin formed in the first step was 73%, and the yield of (R)-epichlorohydrin with enantiomeric excess (ee) ≥99% increased from 19.2% to 25.1% in the second step. Ultimately, the yield of (R)-epichlorohydrin reached 26.4% by optimization of the flow rate of air and amount of immobilized cells. To our knowledge, this was the first report on production of (R)-epichlorohydrin from 1,3-dichloro-2-propanol by two-step biocatalysis using haloalcohol dehalogenase and epoxide hydrolase.     

Molecular engineering of epoxide hydrolase and its application to asymmetric and enantioconvergent hydrolysis

Safety and regulatory issues favor increasing use of enantiopure compounds in pharmaceuticals. Enantiopure epoxides and diols are valuable intermediates in organic synthesis for the production of optically active pharmaceuticals. Enantiopure epoxide can be prepared using epoxide hydrolase (EH)-catalyzed asymmetric hydrolysis of its racemate. Enantioconvergent hydrolysis of racemic epoxides by EHs possessing complementary enantioselectivity and regioselectivity can lead to the formation of enantiopure vicinal diols with high yield. EHs are cofactor-independent and easy-to-use catalysts. EHs will attract much attention as commercial biocatalysts for the preparation of enantiopure epoxides and diols. In this paper, recent progress in molecular engineering of EHs is reviewed. Some examples and prospects of asymmetric and enantioconvergent hydrolysis reactions are discussed as supplements to molecular engineering to improve EH performance.     

Biotechnological production of enantiopure epoxides by enzymatic kinetic resolution

Enantiopure epoxides are high value-added synthons for the production of pharmaceuticals, agrochemicals, as well as versatile fine chemicals and have broad scope of market demand for their applications. A major challenge in conventional organic synthesis is to generate such compounds in high enantiopurity with reasonable yield. Among possible chemical and biological technologies for enantiopure epoxide preparation, enzymatic kinetic resolution has been paid much attention with respect to its high enantioselectivity. Epoxide hydrolase (EH) has shown promising characteristics for the preparation of enantiopure epoxides and vicinal diols during enantioselective hydrolysis of racemic epoxides. EH is readily available from microbial resources thus it is being employed for biohydrolysis of a variety of epoxides. Recent technical progress in EH-catalyzed enantioselective hydrolysis is summarized in terms of exploration of novel EH, its functional improvement, high throughput assay, and preparative scale resolution process.     

Kinetic resolution of racemic alpha-methyl-beta-propiothiolactone by lipase-catalyzed hydrolysis

Kinetic resolution of racemic alpha-methyl-beta-propiothiolactone (rac-MPTL) using lipases in organic solvent was studied. The lipase from Pseudomonas cepacia (PCL) showed the highest (S)-enantioselectivity (E > 100), and cyclohexane containing 1% (v/v) buffer was identified as the best reaction medium for maintaining high enantioselectivity as well as high reaction rate. While the substrate inhibition was not observed up to 300 mM rac-MPTL, severe product inhibition was observed even at 50 mM racemic 3-mercapto-alpha-methyl propionic acid (rac-MMPA), which made the use of high substrate concentration difficult. To overcome the product inhibition, the products, (R)-MMPA, were neutralized by addition of a dilute basic solution. Although the resolution reaction proceeded further by the base titration, the enantioselectivity of the reaction decreased as a result of nonenantioselective hydrolysis of rac-MPTL in the basic solution. Under these conditions, 200 mM rac-MPTL was successfully resolved to above 95% ee(S) with 53% conversion.     

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.     

Kinetic resolution for optically active epoxides by microbial enantioselective hydrolysis

Resolution of several racemic epoxides was accomplished using an epoxide hydrolase activity of whole cells of the newly isolated Aspergillus niger. (S)-Styrene oxide, for example, was obtained from its racemates with optical purity of 100% ee and 32% yield. © Rapid Science Ltd. 1998     

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%。     

手性拆分环氧氯丙烷菌株的筛选、鉴定及产酶条件研究

从土壤中筛选到5株环氧化物水解酶生产菌,并通过ITS序列鉴定了其中的C375菌,结果为黑曲霉（Aspergillus nigerZJB-09103）。考察了培养基不同碳源、氮源、金属离子和pH等对产酶的影响,得到了较佳的培养基条件：淀粉16g／L,豆饼粉3g／L,蛋白胨3g／L,KH2PO4 0．4g／L,K2HPO4 0．8g／L,MgSO4 0．2g／L,ZnSO4 0．03g／L,pH6．5。采用优化后的培养基条件,酶活力达到156．1U／L,比优化前初始发酵培养条件下的酶活提高了252％,当环氧化物水解酶催化时间为10h时,（s）-环氧氯丙烷的对映体过量值（e．e．）可达99．0％。产率为18．6％。     

Fungal epoxide hydrolases: new landmarks in sequence-activity space

Epoxide hydrolases are useful catalysts for the hydrolytic kinetic resolution of epoxides, which are sought after intermediates for the synthesis of enantiopure fine chemicals. The epoxide hydrolases from Aspergillus niger and from the basidiomycetous yeasts Rhodotorula glutinis and Rhodosporidium toruloides have demonstrated potential as versatile, user friendly biocatalysts for organic synthesis. A recombinant A. niger epoxide hydrolase, produced by an overproducing A. niger strain, is already commercially available and recombinant yeast epoxide hydrolases expressed in Escherichia coli have shown excellent results. Within the vast body of activity information on the one hand and gene sequence information on the other hand, the epoxide hydrolases from the Rhodotorula spp. and A. niger stand out because we have sequence information as well as activity information for both the wild-type and recombinant forms of these enzymes.     

Purification and characterisation of a novel enantioselective epoxide hydrolase from Aspergillus niger M200

Purification of a novel enantioselective epoxide hydrolase from Aspergillus niger M200 has been achieved using ammonium sulphate precipitation, ionic exchange, hydrophobic interaction, and size-exclusion chromatography, in conjunction with two additional chromatographic steps employing hydroxylapatite, and Mimetic Green. The enzyme was purified 186-fold with a yield of 15%. The apparent molecular mass of the enzyme was determined to be 77 kDa under native conditions and 40 kDa under denaturing conditions, implying a dimeric structure of the native enzyme. The isoelectric point of the enzyme was estimated to be 4.0 by isoelectric focusing electrophoresis. The enzyme has a broad substrate specificity with highest specificities towards tert-butyl glycidyl ether, para-nitrostyrene oxide, benzyl glycidyl ether, and styrene oxide. Enantiomeric ratios of 30 to more than 100 were determined for the hydrolysis reactions of 4 epoxidic substrates using the purified enzyme at a reaction temperature of 10 degrees C. Product inhibition studies suggest that the enzyme is able to differentiate to a high degree between the (R)-diol and (S)-diol product of the hydrolysis reaction with tert-butyl glycidyl ether as the substrate. The highest activity of the enzyme was at 42 degrees C and a pH of 6.8. Six peptide sequences, which were obtained by cleavage of the purified enzyme with trypsin and mass spectrometry analysis of the tryptic peptides, show high similarity with corresponding sequences originated from the epoxide hydrolase from Aspergillus niger LCP 521.     

Enantioselective hydrolysis of p-nitrostyrene oxide by an epoxide hydrolase preparation from Aspergillus niger

The epoxide hydrolase activity of Aspergillus niger was synthesized during growth of the fungus and was shown to be associated with the soluble cell fraction. An enzyme preparation was worked out which could be used in place of the whole mycelium as biocatalyst for the hydrolysis of epoxides. The effect of four different cosolvents on enzyme activity was investigated. Consequently, dimethylsulfoxide (DMSO) was selected for epoxide solubilization. The effect of temperature on both reaction rate and enzyme stability was studied in the presence of DMSO (0.2 volume ratio). A temperature of 25 degrees C was selected for the reaction of bioconversion. With a substrate concentration of 4.5 mM a batch reactor showed that the enzyme preparation hydrolyzed para-nitrostyrene oxide with very high enantioselectivity. The (S) enantiomer of the epoxide remained in the reaction mixture and showed an enantiomeric excess higher than 99%. The substrate concentration could be increased to 20 mM without affecting the enantiomeric excess and degree of conversion. Therefore, the method is potentially useful for the preparative resolution of epoxides. Application are in the field of chiral synthons which are important building blocks in organic synthesis. (c) 1996 John Wiley & Sons, Inc.     

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.     

Enantioselective epoxide hydrolase activity of a newly isolated microorganism, Sphingomonas echinoides EH-983, from seawater

A marine microorganism, Sphingomonas echinoides EH-983, which possesses epoxide hydrolase (EH) activity was isolated from seawater and characterized. The EH of S. echinoides EH-983 preferentially metabolized (R)-enantiomer when the racemic styrene oxides were supplied as substrates. The optimal pH and temperature for the enantioselective hydrolysis by whole-cells ofS. echinoides EH-983 were 7.0 and 20 °C, respectively. When kinetic resolution was conducted with a racemic mixture of styrene oxides at an initial concentration of 40 mM, enantiopure (S)-styrene oxide was obtained in 180 min with a yield of 21.3%. To our best knowledge, S. echinoides EH-983 is the first marine microorganism that is reported to have EH activity.     

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).     

Preparative-scale kinetic resolution of racemic styrene oxide by immobilized epoxide hydrolase

Epoxide hydrolase from Aspergillus niger was immobilized onto the modified Eupergit C 250 L through a Schiff base formation. Eupergit C 250 L was treated with ethylenediamine to introduce primary amine groups which were subsequently activated with glutaraldehyde. The amount of introduced primary amine groups was 220 μmol/g of the support after ethylenediamine treatment, and 90% of these groups were activated with glutaraldehyde. Maximum immobilization of 80% was obtained with modified Eupergit C 250 L under the optimized conditions. The optimum pH was 7.0 for the free epoxide hydrolase and 6.5 for the immobilized epoxide hydrolase. The optimum temperature for both free and immobilized epoxide hydrolase was 40 °C. The free epoxide hydrolase retained 52 and 33% of its maximum activity at 40 and 60 °C, respectively after 24h preincubation time whereas the retained activities of immobilized epoxide hydrolase at the same conditions were 90 and 75%, respectively. Immobilized epoxide hydrolase showed about 2.5-fold higher enantioselectivity than that of free epoxide hydrolase. A preparative-scale (120 g/L) kinetic resolution of racemic styrene oxide using immobilized preparation was performed in a batch reactor and (S)-styrene oxide and (R)-1-phenyl-1,2-ethanediol were both obtained with about 50% yield and 99% enantiomeric excess. The immobilized epoxide hydrolase was retained 90% of its initial activity after 5 reuses.     

Resolution of 1,2-epoxyhexane by Rhodotorula glutinis using a two-phase membrane bioreactor

Large-scale resolution of epoxides by the yeast Rhodotorula glutinis was demonstrated in an aqueous/organic two-phase cascade membrane bioreactor. Due to the chemical instability and low solubility of epoxides in aqueous phases, an organic solvent was introduced into the reaction mixture in order to enhance the resolution of epoxide. A cascade hollow-fiber membrane bioreactor was used (1) to minimize the toxicity of organic solvents towards the epoxide hydrolase of R. glutinis, and (2) to remove inhibitory amounts of formed diol from the yeast cell containing aqueous phase. Dodecane was selected as a suitable solvent and 1,2-epoxyhexane as a model substrate. By use of this membrane bioreactor, highly concentrated (0.9 M in dodecane) enantiopure (> 98% ee) (S)-1,2-epoxyhexane (6.5 g, 30% yield) was obtained from the racemic mixture. Received: 28 June 1999 / Received revision: 26 August 1999 / Accepted: 3 September 1999     

Interfacial inactivation of epoxide hydrolase in a two-liquid-phase system

Enantioselective epoxide hydrolases are useful biocatalysts for the preparation of enantiopure epoxides and diols. The kinetic resolution of racemic epoxides can be carried out in an organic/aqueous biphasic system to allow use of high epoxide concentrations. Enzyme inactivation in such a system, however, may occur by contact with the interface. In this study, we investigated the factors which influence the interfacial inactivation of Agrobacterium radiobacter epoxide hydrolase in an octane/water biphasic system. Rates of interfacial inactivation were measured both in a stirred-cell, which has a planar interface, and in an emulsion reactor. Interfacial inactivation rates measured in the stirred-cell at a fixed interfacial area increased with mixing intensity. Interfacial inactivation rates per unit area were lower in the emulsion reactor than in the stirred-cell and increased with bulk aqueous enzyme concentration. Circular dichroism measurements showed that during biphasic incubation all unadsorbed soluble enzyme existed in the native conformation. Activity assays showed that the dissolved enzyme was also fully active, indicating that inactivated enzyme precipitated from solution. Using an inactive epoxide hydrolase mutant structurally similar to the wild-type enzyme in order to avoid the conversion of the epoxide, it was found that high concentrations of epoxide in the organic phase increased the rate of interfacial inactivation.     

Effect of carbon and nitrogen sources on the production of a highly enantioselective epoxide hydrolase from Aspergillus niger

The highly enantioselective epoxide hydrolase from Aspergillus niger is well utilized as biocatalysts for the preparation of enantiopure chiral epoxides and diols. Both growth of the fungus and EH activity production were found greatly affected by changing the carbon or the nitrogen source with fructose and corn steep liquor being the best. Their concentrations were optimized (10 g.l–1 of fructose and 15 g.l–1 of corn steep) which resulted in an increase of both the biomass produced (31%) and the epoxide hydrolase specific activity (38%). The results obtained suggested a complex regulation of the EH production. On the whole, a two times increase of the total EH activity was obtained. © Rapid Science Ltd. 1998     

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.     

Diversity and biocatalytic potential of epoxide hydrolases identified by genome analysis

Epoxide hydrolases play an important role in the biodegradation of organic compounds and are potentially useful in enantioselective biocatalysis. An analysis of various genomic databases revealed that about 20% of sequenced organisms contain one or more putative epoxide hydrolase genes. They were found in all domains of life, and many fungi and actinobacteria contain several putative epoxide hydrolase-encoding genes. Multiple sequence alignments of epoxide hydrolases with other known and putative alpha/beta-hydrolase fold enzymes that possess a nucleophilic aspartate revealed that these enzymes can be classified into eight phylogenetic groups that all contain putative epoxide hydrolases. To determine their catalytic activities, 10 putative bacterial epoxide hydrolase genes and 2 known bacterial epoxide hydrolase genes were cloned and overexpressed in Escherichia coli. The production of active enzyme was strongly improved by fusion to the maltose binding protein (MalE), which prevented inclusion body formation and facilitated protein purification. Eight of the 12 fusion proteins were active toward one or more of the 21 epoxides that were tested, and they converted both terminal and nonterminal epoxides. Four of the new epoxide hydrolases showed an uncommon enantiopreference for meso-epoxides and/or terminal aromatic epoxides, which made them suitable for the production of enantiopure (S,S)-diols and (R)-epoxides. The results show that the expression of epoxide hydrolase genes that are detected by analyses of genomic databases is a useful strategy for obtaining new biocatalysts.