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 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℃ 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℃ for 6h. 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℃ and the half-life of the enzyme at 50℃ 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.

A racemic glycidyl butyrate resolving strain, preliminarily identified as a Rhizopus sp., had been isolated from soil. Its extracellular lipase was found to enantioselectively hydrolyze the (S)-enantiomer of the chiral ester, with optimal activities at pH 5.3 and 42℃. Higher enantioselectivity of the enzyme was observed at lower temperatures, while the best enantioselectivity was obtained at pH 5.5-6.0, with an E value (enantiomeric ratio) of 57.

A yeast strain CGMCC 0574, identified as Trichosporon brassicae, was selected from 92 strains for its high (S) selectivity in the hydrolysis of ketoprofen ethyl ester. The effective strains of the microorganisms were isolated from soil samples with the ester as the sole carbon source. The ethyl ester proved to be the best substrate for resolution of ketoprofen among several ketoprofen esters examined. The resting cells of CGMCC 0574 could catalyze the hydrolysis of ketoprofen ethyl ester with an enantiomeric ratio of 44.9, giving (S)-ketoprofen an enantiomeric excess of 91.5% at 42% conversion. Une souche de levure désignée CGMCC 0574, identifiée comme étant Trichosporon brassicae, a été sélectionnée parmi 92 souches sur la base de sa (S)-sélectivité élevée lors de l'hydrolyse de l’ester éthylique de ketoprofen. La sélection s'est effectuée sur des souches de micro-organismes ayant été isolées à partir d'échantillons de sol avec l'ester comme seule source de carbone. L'ester éthylique s'est avéré être le meilleur substrat pour la resolution du ketoprofen parmi plusieurs esters de ketoprofen analysés. Les cellules de CGMCC 0574 au repos ont pu catalyser l'hydrolyse de l'ester éthylique de ketoprofen avec un rapport énantiomérique de 44,9, produisant du (S)-ketoprofen à 91,5% excès énantiomérique, à un taux de conversion de 42%.

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%.