State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing 210009, People's Republic of China.
Appl Microbiol Biotechnol. 2010 Dec;88(6):1277-85. doi: 10.1007/s00253-010-2836-4. Epub 2010 Aug 20.
A cofactor regeneration system for enzymatic biosynthesis was constructed by coexpressing a carbonyl reductase from Pichia stipitis and a glucose dehydrogenase from Bacillus megaterium in Escherichia coli Rosetta (DE3) PlySs. Transformants containing the polycistronic plasmid pET-PII-SD2-AS1-B exhibited an activity of 13.5 U/mg protein with 4-chloro-3-oxobutanoate ethyl ester (COBE) as the substrate and an activity of 14.4 U/mg protein with glucose as the substrate; NAD(H) was the coenzyme in both cases. Asymmetric reduction of COBE to (S)-4-chloro-3-hydroxybutanoate ethyl ester [(S)-CHBE] with more than 99% enantiomeric excess was demonstrated by transformants. Furthermore, the paper made a comparison of crude enzyme catalysis and whole-cell catalysis in an aqueous monophasic system and a water/organic solvent biphasic system. In the water/n-butyl acetate system, the coexpression system produced 1,398 mM CHBE in the organic phase, which is the highest yield ever reported for CHBE production by NADH-dependent reductases from yeasts. In this case, the molar yield of CHBE was 90.7%, and the total turnover number, defined as moles (S)-CHBE formed per mole NAD+, was 13,980.
构建了一种用于酶生物合成的辅助因子再生系统，通过在大肠杆菌 Rosetta (DE3) PlySs 中共同表达来自毕赤酵母的羰基还原酶和来自巨大芽孢杆菌的葡萄糖脱氢酶来实现。含有多顺反子质粒 pET-PII-SD2-AS1-B 的转化体在以 4-氯-3-氧代丁酸乙酯 (COBE) 为底物时表现出 13.5 U/mg 蛋白的活性，在以葡萄糖为底物时表现出 14.4 U/mg 蛋白的活性；在这两种情况下，NAD(H) 都是辅酶。转化体证明了 COBE 不对称还原为（S）-4-氯-3-羟基丁酸乙酯 [(S)-CHBE]，对映体过量超过 99%。此外，本文比较了在单相水体系和水/有机溶剂两相体系中粗酶催化和全细胞催化。在水/正丁酯体系中，共表达系统在有机相中产生 1398 mM CHBE，这是迄今为止报道的酵母来源的 NADH 依赖型还原酶生产 CHBE 的最高产量。在这种情况下，CHBE 的摩尔产率为 90.7%，定义为每摩尔 NAD+形成的（S）-CHBE 摩尔数的总周转率数为 13980。
