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.

The yeast strain CGMCC 0573 was identified as Citeromyces matriensis and shown to be capable of enantioselectively hydrolyzing ethyl ester of (R)-Ketoprofen (2-(3-benzoylphenyl)propionic acid). The strain was isolated for the first time from soil samples through a new and efficient screening procedure in which the probability of obtaining active strains was greatly increased by using ethanol and Tween-80 alternatively as additives during the enrichment culture. Studies of the culture conditions and catalytic performance of Citeromyces matriensis CGMCC 0573 showed that the enzyme occurs constitutively in the cells and its production is enhanced by feeding with Tween-80 during the early period of cultivation. Yeast extract was found to be beneficial both for growth and for esterase production. The optimal temperature and pH for the bioconversion were 40℃ and pH 8.0, respectively. Biotransformation using resting cells cultured in a flask with baffles and magnetic stirring and in the presence of 50mM substrate resulted in the production of (R)-ketoprofen at 93% ee (enantiomeric excess) and at 42.6% conversion.

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 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 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%).