Kinetic resolution of (R,S)-alpha-hydroxy acid esters via Klebsiella oxytoca hydrolase in biphasic media

• Main Authors: Pei-Yun Wang, 王培筠
• Other Authors: Teh-Liang Chen
• Format: Others
• Language: en_US
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/82176362570658476800

博士 === 國立成功大學 === 化學工程學系碩博士班 === 95 === For the first time, the thermally stable Klebsiella oxytoca hydrolase (SNSM-87) is explored as an enantioselective biocatalyst for the hydrolytic resolution of (R,S)-alpha-hydroxy acid esters in biphasic media. In this study, we show how medium engineering (operating parameters), substrate engineering (acyl part and leaving alcohol) and enzyme immobilization can be combined to improve the enzymatic enantioselectivity or activity. The hydrolytic resolution of (R,S)-ethyl mandelate via SNSM-87 in biphasic media is first investigated. Effects of various process parameters such as solvent type, temperature, pH, product inhibition, enzyme loading and substrate concentration on the enzyme performance were studied, leading to the high enzyme (S)-enantioselectivity of E = 56 at 45oC for the reaction media consisting of isooctane and pH 7 buffer. The optimal conditions of pH between 7 and 8, temperature 45oC and enzyme loading no less than 0.5 mg/ml were proposed for obtaining optically pure (R)-ethyl mandelate remained in the organic phase. The hydrolytic resolution of (R,S)-ethyl 2-chloromandelate in biphasic media by using SNSM-87 as a promising biocatalyst is then reported for producing the desired (R)-ethyl 2-chloromandelate as an important intermediate for the synthesis of Clopidogrel. Effects of various operating parameters on the enzyme activity and enantioselectivity were systematically studied, from which the optimal condition of using isooctane as the organic phase, enzyme concentration of 2 mg/mL, temperature of 55oC, and pH 6 of the aqueous phase was selected. The kinetic constants were further estimated from the kinetic analysis at the optimal condition. By further considering the acid product inhibition and non-enzymatic hydrolysis for the enzymatic resolution, good agreements of the time-course conversions from experiments and the theoretical prediction were also obtained. For studying the effect of leaving alcohol on the enzymatic activity and enantioselectivity using (R,S)-2-chloromandelates as model substrates, an expanded Michaelis-Menten mechanism for the rate-limiting acylation step was adopted for the kinetic analysis. The fast-reacting (S)-2-chloromandelates containing a difficult leaving alcohol moiety, as well as that the slow-reacting (R)-2-chloromandelates in the whole range of leaving alcohol moieties, indicates that the breakdown of tetrahedral intermediates to acyl-enzyme intermediates is rate-limiting. However, the rate-limiting step shifts to the formation of tetrahedral intermediates for the (S)-2-chloromandelates containing an easy leaving alcohol moiety, and leads to an optimal enantioselectivity for the methyl ester substrate. The SNSM-87 is also explored as an enantioselective biocatalyst for the hydrolytic resolution of the other (R,S)-alpha-hydroxy acid esters in biphasic media, where the best methyl esters possessing the highest enantioselectivity and reactivity are selected and elucidated in terms of the structure-enantioselectivity correlations and substrate partitioning in the aqueous phase. Two-step Br�幯sted slopes for the fast-reacting (R)-ester but only one-step for the slow-reacting (S)-ester are further obtained in the hydrolytic resolution of (R,S)-alpha-chlorophenyl acetate in biphasic media. A replacement of the alpha-hydroxy moiety to alpha-methyl or alpha-chloro group has profound effects on changing the enzymatic activity, enantioselectivity and optical-preference for SNSM-87 and lipases. The Klebsiella oxytoca hydrolase was immobilized on epoxy-activated acrylic polymers Eupergit�� C 250L via covalent attachment. The effects of temperature, pH, and organic solvent on the enzymatic activity and enantioselective for the hydrolysis of (R,S)-ethyl mandelate were investigated. The immobilized enzyme retains more than 30% catalytic activity for the (S)-ethyl mandelate and increases the E value from 43.6 to 319 at pH 6 and 55oC with isooctane as the organic phase. An analysis of the thermodynamic parameters indicates that –ΔΔH increased from 20.1 to 42.0 kJ/mol as well as –ΔΔS from 31.0 to 81.5 J/mol K after immobilization when comparing with the free enzyme. The kinetic analysis for the immobilized SNSM-87 shows the slower proton transfer for the breakdown of transition state, especial for the slow reacting (R)-ester. In addition, the combination of substrate engineering and enzyme immobilization for the hydrolytic resolution of other (R,S)-alpha-hydroxy acid esters also results in an increase of enantioselectivity. The immobilized enzyme could be used in the repetitive manner more than 8 times without loss the activity and enantioselectivity in the biphasic system. Therefore, the immobilization procedure developed may provide a promising solution for the industrial application of SNSM-87 in biphasic system.