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 30h of culture when the biomass (DCW) was 9.1g/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 60mM 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.

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 80mM, the E-value increased at first, until reaching a peak at 40mM, and then decreased at higher concentrations (>40mM). 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 (ees) at any time of the whole reaction course based on the ees 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.

A monophasic organic-water system for efficient enzymatic synthesis ofβ-d-glucopyranoside by reverse hydrolysis was constructed and optimized. p-Nitrobenzyl alcohol (pNBA), selected as a model substrate alcohol, was readily glucosylated with d-glucose through reverse hydrolysis using almond β-d-glucosidase in a monophasic aqueous-organic medium, producing a new glucoside, p-nitrobenzylβ-d-glucopyranoside (pNBG). The effects of different buffers, organic solvents and water contents were investigated. Buffer type and Ph affected the initial reaction rate but had little effect on the final yields. The ratio of organic solvent to water plays a crucial role in shifting the reaction equilibrium toward synthesis, but a minimum amount of water is necessary to maintain the enzyme activity. Dioxane, which was previously known as an unsuitable solvent forβ-d-glucosidase-catalyzed reactions, was found to be the most appropriate solvent for this synthetic procedure. The reaction equilibrium and enzyme stability in the reaction medium were also investigated. Under the optimal reaction conditions, i.e. 90% dioxane (v/v) + 10% buffer (Na2HPO4–KH2PO4, 70mM, pH 6.0) with alcohol-to-glucose molar ratio of 9:1, p-nitrobenzylβ-d-glucopyranoside was produced with a maximum yield (13.3%).

Kinetic resolution of a chiral alcohol, 4-hydroxy-3-methyl-2-(2[1]-propenyl)-2-cyclopentenone (HMPC), a key intermediate for the production of prallethrin insecticides, was successfully carried out by enantioselective hydrolysis of (RS)-HMPC acetate using calcium alginate gel-entrapped cells of a newly isolated esterase-producing bacterium Acinetobacter sp. CGMCC 0789. When the effect of different cosolvents was investigated, it was found that isopropanol could markedly enhance the activity and enantioselectivity of the immobilized cells. The optimum concentration of isopropanol was 10% (v/v) where immobilized cells still showed good operational stability. After 10 cycles of reaction, no significant decrease in the enzyme activity was observed. The catalytic specificity constants (Vmax/Km) for both enantiomers of the substrate were determined with partially purified enzyme, giving 0.0184 and 0.671 h−1for the (S)- and (R)-ester, respectively.

A highly enantioselective (R)-ester hydrolase was partially purified from a newly isolated bacterium, Acinetobacter sp. CGMCC 0789, whose resting cells exhibited a highly enantioselective activity toward the acetate of (4R)-hydroxy-3-methyl-2-(2-propynyl)- cyclopent-2-enone (R-HMPC). The optimum pH and temperature of the partially purified enzyme were 8.0 and 60℃, respectively. The enantioselectivity of the crude enzyme was increased by 1.2-fold from 16 to 20 when the reaction temperature was raised from 30 to 60℃. The activity of the crude enzyme was enhanced by 4.1-fold and the enantioselectivity (E-value) was markedly enhanced by 4.3-fold from 16 to 68 upon addition of a cationic detergent, benzethonium chloride [(diisobutyl phenoxyethoxyethyl) dimethyl benzylammoniom chloride]. The hydrolysis of 52mM (R,S)-HMPC acetate to (R)-HMPC was completed within 8 h, with optical purity of 91.4% eep and conversion of 49%.