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, H[1];Rao, ZM[1]

机构：[1]Jiangnan Univ, Sch Biotechnol, Key Lab Ind Biotechnol, Minist Educ, Wuxi, Jiangsu, Peoples R China;[2]Beijing Union Univ, Beijing Key Lab Biomass Waste Resource Utilizat, Coll Biochem Engn, Beijing, Peoples R China;[3]Ohio State Univ, Dept Chem & Biomol Engn, Columbus, OH 43210 USA

第一机构：Jiangnan Univ, Sch Biotechnol, Key Lab Ind Biotechnol, Minist Educ, Wuxi, Jiangsu, Peoples R China

通讯机构：[1]corresponding author), Jiangnan Univ, Sch Biotechnol, 1800 Lihu Rd, Wuxi 214122, Jiangsu, Peoples R 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：;WOS:【SCI-EXPANDED(收录号:WOS:000505291500003)】；

基金：This work was supported by the National Natural Science Foundation of China (31770058, 31870066), the National Key Research and Development Program of China (2018YFA090039), the Natural Science Foundation of Jiangsu Province (BK20181205), the Fundamental Research Funds for the Central Universities (JUSRP51708A), the national first-class discipline program of Light Industry Technology and Engineering (LITE2018-06) and the 111 Project (111-2-06).

语种：英文

外文关键词：4-hydroxyisoleucine; Bacillus subtilis 168; disulfide bond; l-isoleucine hydroxylase; molecular dynamics simulation

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 degrees 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-ido(T181C) 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.