Optically active (R)-a-monobenzoyl glycerol (MBG) was synthesized by Candida antarctica lipase B (CHIRAZYMET L-2) catalyzed asymmetric esterification of glycerol with benzoic anhydride in organic solvents. Various conditions, such as the type and composition of the organic solvent, water content of the system, reaction temperature, and concentrations of the substrates were systematically examined and optimized in screw-capped test tubes with respect to both the reaction rate and the enzyme selectivity. 1,4-Dioxane was found to be the best solvent and no additional water was needed for the system. The optimum temperature was around 30℃, while the most suitable substrate concentrations were 100mM each for glycerol and benzoic anhydride, respectively. However, when excessive anhydride (e.g., 200mM) was used, the produced MBG could be further transformed into 1,3-dibenzoyl glycerol (DBG) by the same enzyme with a priority to (S)-MBG, resulting in a significant improvement of the product optical purity from ca. 50-70% e.e. Under optimal conditions (100mM glycerol, 100-200mM benzoic anhydride, dioxane, 25-30℃), the enzymatic synthesis of (R)-MBG was successfully operated in a packed-bed reactor for about 1 week, with an average productivity of 0.79g MBG/day/g biocatalyst in the case of continuous operation and 0.94g MBG/day/g biocatalyst in the case of semicontinuous operation. After refinement and preferential crystallization of the crude product, (R)-MBG could be obtained in an almost optically pure form (>98% e.e.).

The optimal activity of a Candida rugosa lipase (Lipase OF) for hydrolysis of 2-chloroethyl ester of Ketoprofen [2-(3-benzoyphenyl) propionic acid] was at pH 4.0, while the best enantioselectivity (E) was at pH 2.2 where the enzyme was still 60% active and stable.

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.

Lipase production and cell growth of Serratia marcescens ECU1010 were optimized in shake flasks, with lipase production being enhanced 9.5-fold (4,780 U/l) compared with the initial activity (500 U/l). Optimal carbon and nitrogen sources were Tween-80 and peptone, and the optimal ratio of Tween-80 to peptone was 1:3. The optimized cultivation conditions were 25[1]C and pH 6.5. Lipase activity, particularly specific activity, could be improved by decreasing the cultivation temperature from 35 to 25[1]C. Enzyme stability was significantly improved by simple immobilization with synthetic adsorption resin no. 8244. After five reaction cycles, enzyme activity decreased only very slightly, while enantioselectivity of the preparation remained constant, and the ees (enantiomeric excess of the remaining substrate) achieved in all cases was higher than 97%. The resin-8244-lipase preparation can be used for efficient enantioselective hydrolysis of trans-3-(4

A yeast strain, Rhodotorula sp. AS2.2241, capable of reducing acetophenone and -bromoacetophenone with high stereoselectivity, was isolated from soil samples through a novel screening procedure in which acetophenone was supplied in vapor state as the sole carbon and energy source. The biosynthesis of the ketone reductase in the yeast cells reached a maximum of 41.0 U/l at 20h of cultivation. The reductase isolated from the Rhodotorula sp. cells was partially purified by 52.6-fold through a single column chromatography of DEAE-cellulose. The catalytic performance of the partially purified reductase was examined, and the highest activitywas observed at pH 6.5 and 50 ◦C. The short-chain alkyl aldehydes such as acetaldehyde and those aldehydes or ketones with a benzoyl group were found to be good substrates for the reductase. In the preparative bioreductions of 50mM acetophenone and 2mM -bromoacetophenone using resting cells of Rhodotorula sp. AS2.2241, (S)-(−)-1-phenylethanol (>99.5% enantiomeric excess (e.e.), 34.7% yield) and (R)-(−)-2-bromo-1-phenylethanol (>99.9% e.e., 19.9% yield) were obtained, respectively.