文献类型：期刊文献

英文题名：Engineering Isopropanol Dehydrogenase for Efficient Regeneration of Nicotinamide Cofactors

作者：Jia, Qiao[1];Zheng, Yu-Cong[1];Li, Hai-Peng[1];Qian, Xiao-Long[1,2];Zhang, Zhi-Jun[1,3];Xu, Jian-He[1,3]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai, Peoples R China;[2]Suzhou Bioforany EnzyTech Co Ltd, Changshu, Jiangsu, Peoples R China;[3]East China Univ Sci & Technol, Shanghai Collaborat Innovat Ctr Biomfg, Sch Biotechnol, Shanghai, Peoples R China

年份：2022

卷号：88

期号：9

外文期刊名：APPLIED AND ENVIRONMENTAL MICROBIOLOGY

收录：;EI(收录号：20222012117118);WOS:【SCI-EXPANDED(收录号:WOS:000784538900001)】；

基金：This work was financially supported by the National Key Research and Development Program of China (no. 2019YFA0905000), the National Natural Science Foundation of China (no. 21536004, 21871085, and 32071475), the Natural Science Foundation of Shanghai (no. 18ZR1408400), and the Fundamental Research Funds for the Central Universities (no. 22221818014).

语种：英文

外文关键词：isopropanol dehydrogenase; cofactor regeneration; thermostability evolution; cofactor specificity reversal; protein engineering

摘要：Isopropanol dehydrogenase (IPADH) is one of the most attractive options for nicotinamide cofactor regeneration due to its low cost and simple downstream processing. However, poor thermostability and strict cofactor dependency hinder its practical application for bioconversions. In this study, we simultaneously improved the thermostability (433-fold) and catalytic activity (3.3-fold) of IPADH from Brucella suis via a flexible segment engineering strategy. Meanwhile, the cofactor preference of IPADH was successfully switched from NAD(H) to NADP(H) by 1.23 x 10(6)-fold. When these variants were employed in three typical bioredox reactions to drive the synthesis of important chiral pharmaceutical building blocks, they outperformed the commonly used cofactor regeneration systems (glucose dehydrogenase [GDH], formate dehydrogenase [FDH], and lactate dehydrogenase [LDH]) with respect to efficiency of cofactor regeneration. Overall, our study provides two promising IPADH variants with complementary cofactor specificities that have great potential for wide applications. IMPORTANCE Oxidoreductases represent one group of the most important biocatalysts for synthesis of various chiral synthons. However, their practical application was hindered by the expensive nicotinamide cofactors used. Isopropanol dehydrogenase (IPADH) is one of the most attractive biocatalysts for nicotinamide cofactor regeneration. However, poor thermostability and strict cofactor dependency hinder its practical application. In this work, the thermostability and catalytic activity of an IPADH were simultaneously improved via a flexible segment engineering strategy. Meanwhile, the cofactor preference of IPADH was successfully switched from NAD(H) to NADP(H). The resultant variants show great potential for regeneration of nicotinamide cofactors, and the engineering strategy might serve as a useful approach for future engineering of other oxidoreductases. Oxidoreductases represent one group of the most important biocatalysts for synthesis of various chiral synthons. However, their practical application was hindered by the expensive nicotinamide cofactors used.