Kataoka M, Rohani L P, Yamamoto K, Wada M, Kawabata H, Kita K, Yanase H, Shimizu S
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Japan.
Appl Microbiol Biotechnol. 1997 Dec;48(6):699-703. doi: 10.1007/s002530051118.
The asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (R)-4-chloro-3-hydroxybutanoate (CHBE) using Escherichia coli JM109 (pKAR) cells expressing the aldehyde reductase gene from Sporobolomyces salmonicolor AKU4429 as a catalyst was studied. The reduction required NADP+, glucose and glucose dehydrogenase for NADPH regeneration. In an aqueous system, the substrate was unstable, and inhibition of the reaction by the substrate was also observed. Efficient conversion of COBE to (R)-CHBE with a satisfactory enantiomeric excess (ee) was attained on incubation with transformant cells in an n-butyl acetate/water two-phase system containing the above NADPH-regeneration system. Under the optimized conditions, with the periodical addition of COBE, glucose and glucose dehydrogenase, the (R)-CHBE yield reached 1530 mM (255 mg/ml) in the organic phase, with a molar conversion yield of 91.1% and an optical purity of 91% ee. The calculated turnover of NADP+, based on the amounts of NADP+ added and CHBE formed, was about 5100 mol/mol.
研究了使用表达来自鲑鱼色掷孢酵母AKU4429醛还原酶基因的大肠杆菌JM109(pKAR)细胞作为催化剂，将4-氯-3-氧代丁酸乙酯(COBE)不对称还原为(R)-4-氯-3-羟基丁酸乙酯(CHBE)。该还原反应需要NADP⁺、葡萄糖和葡萄糖脱氢酶来再生NADPH。在水体系中，底物不稳定，且观察到底物对反应的抑制作用。在含有上述NADPH再生体系的乙酸正丁酯/水两相体系中，与转化细胞一起孵育时，可实现COBE高效转化为(R)-CHBE，并具有令人满意的对映体过量(ee)。在优化条件下，通过定期添加COBE、葡萄糖和葡萄糖脱氢酶，有机相中(R)-CHBE的产量达到1530 mM(255 mg/ml)，摩尔转化率为91.1%，光学纯度为91% ee。根据添加的NADP⁺量和形成的CHBE量计算，NADP⁺的周转数约为5100 mol/mol。
