文献类型：期刊文献

英文题名：Engineered disulfide bonds improve thermostability and activity of L-isoleucine hydroxylase for efficient 4-HIL production in Bacillus subtilis 168

作者：Qiao, Zhina[1]; Xu, Meijuan[1]; Shao, Minglong[1]; Zhao, Youxi[2]; Long, Mengfei[1]; Yang, Taowei[1]; Zhang, Xian[1]; Yang, Shangtian[3]; Nakanishi, Hideki[1]; Rao, Zhiming[1]

第一作者：Qiao, Zhina

通讯作者：Nakanishi, Hideki

机构：[1] The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu Province, China; [2] Beijing Key Laboratory of Biomass Waste Resource Utilization, College of Biochemical Engineering, Beijing Union University, Beijing, China; [3] Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH, United States

第一机构：The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu Province, China

年份：2020

卷号：20

期号：1-2

起止页码：7-16

外文期刊名：Engineering in Life Sciences

收录：EI(收录号：20194407598052);Scopus(收录号：2-s2.0-85074040960)

语种：英文

外文关键词：Covalent bonds - Stability - Sulfur compounds - Biochemical engineering - Enzyme activity - Amino acids - Bacteriology

摘要：4-Hydroxyisoleucine, a promising drug, has mainly been applied in the clinical treatment of type 2 diabetes in the pharmaceutical industry. l-Isoleucine hydroxylase specifically converts l-Ile to 4-hydroxyisoleucine. However, due to its poor thermostability, the industrial production of 4-hydroxyisoleucine has been largely restricted. In the present study, the disulfide bond in l-isoleucine hydroxylase protein was rationally designed to improve its thermostability to facilitate industrial application. The half-life of variant T181C was 4.03?h at 50°C, 10.27-fold the half-life of wild type (0.39?h). The specific enzyme activity of mutant T181C was 2.42?±?0.08?U/mg, which was 3.56-fold the specific enzyme activity of wild type 0.68?±?0.06?U/mg. In addition, molecular dynamics simulation was performed to determine the reason for the improvement of thermostability. Based on five repeated batches of whole-cell biotransformation, Bacillus subtilis 168/pMA5-idoT181C recombinant strain produced a cumulative yield of 856.91?mM (126.11?g/L) 4-hydroxyisoleucine, which is the highest level of productivity reported based on a microbial process. The results could facilitate industrial scale production of 4-hydroxyisoleucine. Rational design of disulfide bond improved l-isoleucine hydroxylase thermostability and may be suitable for protein engineering of other hydroxylases. ? 2019 The Authors. Engineering in Life Sciences published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.