Xu Zhinan, Liu Ying, Fang Limei, Jiang Xiaoxia, Jing Keju, Cen Peilin
Institute of Bioengineering, Department of Chemical Engineering and Bioengineering, Zhejiang University, Hangzhou, Zhejiang 310027, People's Republic of China.
Appl Microbiol Biotechnol. 2006 Mar;70(1):40-6. doi: 10.1007/s00253-005-0037-3. Epub 2005 Sep 21.
Escherichia coli M15 (pQE30-car0210) was constructed to express carbonyl reductase (CAR) by cloning the car gene from Candida magnoliae and inserting it into pQE30. By cultivating E. coli M15 (pQE30-car0210) and M15 (pQE30-gdh0310), 8.2-fold and 12.3-fold enhancements in specific enzymatic activity over the corresponding original strain were achieved, respectively. After separate cultivations, these two strains were then mixed together at appropriate ratio to construct a novel two-strain system, in which M15 (pQE30-car0210) expressed CAR for ethyl 4-chloro-3-oxobutanoate (COBE) bioreduction and M15 (pQE30-gdh0310) expressed glucose dehydrogenase (GDH) for nicotinamide adenine dinucleotide phosphate (NADPH) regeneration. In this complex system, the effects of substrate concentration, the biomass ratio between two strains as well as reaction temperature were investigated for efficient bioreduction. The results showed that the bioreduction reaction could be completed effectively without any addition of GDH or NADPH/NADP(+). An optical purity of 99% (enantiometric efficiency) was obtained, and the yield of (S)-4-chloro-3-hydroxybutanoate ethyl ester reached 96.6% when initial concentration of COBE was 36.9 mM. The coupling reactions between two different strains were further explored by determining the profile of NADPH in the reaction broth.
通过从大花假丝酵母中克隆car基因并将其插入pQE30，构建了大肠杆菌M15 (pQE30-car0210)以表达羰基还原酶(CAR)。通过培养大肠杆菌M15 (pQE30-car0210)和M15 (pQE30-gdh0310)，其比酶活分别比相应的原始菌株提高了8.2倍和12.3倍。单独培养后，将这两种菌株按适当比例混合在一起，构建了一种新型的双菌株系统，其中M15 (pQE30-car0210)表达用于4-氯-3-氧代丁酸乙酯(COBE)生物还原的CAR，M15 (pQE30-gdh0310)表达用于烟酰胺腺嘌呤二核苷酸磷酸(NADPH)再生的葡萄糖脱氢酶(GDH)。在这个复杂系统中，研究了底物浓度、两种菌株的生物量比以及反应温度对高效生物还原的影响。结果表明，无需添加任何GDH或NADPH/NADP(+)即可有效完成生物还原反应。当COBE的初始浓度为36.9 mM时，获得了99%的光学纯度(对映体效率)，(S)-4-氯-3-羟基丁酸乙酯的产率达到96.6%。通过测定反应液中NADPH的含量，进一步探索了两种不同菌株之间的偶联反应。
