Enantioselective reduction of prochiral ketones by engineered bifunctional fusion proteins
Enantioselective reduction of prochiral ketones by engineered bifunctional fusion proteins
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DOI：
10.1042/ba20100143
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发表时间：
2010-08-01
影响因子：
2.8
通讯作者：
Weuster-Botz, Dirk
中科院分区：
文献类型：
--
作者：
Hoelsch, Kathrin;Weuster-Botz, Dirk
nadph依赖的氧化还原酶是产生手性合成子的有用催化剂。然而，氧化还原酶的制备应用需要有效的方法来原位再生昂贵的烟酰胺辅助因子。辅助因子再生的有利方法是构建由两种酶组成的双功能融合蛋白，一种酶催化还原反应，另一种酶介导辅助因子的再循环。本文描述了母牛分枝杆菌NI0的NADP（+）接受突变体FDH（甲酸脱氢酶）与聚球菌菌株PCC 7942的3-酮酰基（酰基载体蛋白）还原酶KR （3-ketoacyl（酰基载体蛋白）还原酶）的框架内融合。连接体插入突变体的产生导致融合蛋白的酶活性分别为天然KR和FDH的100%和80%。表达融合蛋白的大肠杆菌细胞表现出近似。产生手性醇的初始反应速率比单独表达酶的细胞高2倍。工程融合蛋白应用于全细胞生物还原五氟苯酮，底物转化率为99.97%，对映体过量99.9% (S)- 1 -（五氟苯基）乙醇。
NADPH-dependent oxidoreductases are useful catalysts for the production of chiral synthons. However, preparative applications of oxidoreductases require efficient methods for in situ regeneration of the expensive nicotinamide cofactors. An advantageous method for cofactor regeneration is the construction of bifunctional fusion proteins composed of two enzymes, one catalysing the reduction reaction and the other one mediating the recycling of cofactors. Herein, we describe the in-frame fusion between an NADP(+)-accepting mutant of FDH (formate dehydrogenase) from Mycobacterium vaccae NI0 and KR [3-ketoacyl(acyl-carrier-protein) reductase] from Synechococcus sp. strain PCC 7942. The generation of linker insertion mutants led to a fusion protein exhibiting 100 and 80% of the enzymatic activities of native KR and FDH respectively. Escherichia coli cells expressing the fusion protein showed an approx. 2-fold higher initial reaction rate in the production of chiral alcohols than cells expressing the enzymes separately. The application of the engineered fusion protein in whole-cell bioreduction of pentafluoroacetophenone resulted in a substrate conversion of 99.97% with an excellent enantiomeric excess of 99.9% (S)-I-(pentafluorophenyl)ethanol.