文献类型：期刊文献

英文题名：Identification and Rational Engineering of a High Substrate-Tolerant Leucine Dehydrogenase Effective for the Synthesis of L-tert-Leucine

作者：Meng, Xiangqi[1];Yang, Lin[1];Liu, Yan[1];Wang, Hualei[1];Shen, Yaling[1];Wei, Dongzhi[1]

机构：[1]East China Univ Sci & Technol, New World Inst Biotechnol, State Key Lab Bioreactor Engn, 130 Meilong Rd, Shanghai 200237, Peoples R China

年份：2021

卷号：13

期号：14

起止页码：3340

外文期刊名：CHEMCATCHEM

收录：;EI(收录号：20212310466707);WOS:【SCI-EXPANDED(收录号:WOS:000656611800001)】；

基金：This work was supported by the National Natural Science Foundation of China (Nos. 21776084 and 22078097).

语种：英文

外文关键词：Coenzyme; Catalysis; Directed evolution; Enzyme; L-tert-leucine; Leucine dehydrogenase; Substrate tolerance

摘要：The asymmetric synthesis of chiral amino acids by leucine dehydrogenases has great potential for industrialization; however, the inhibitory effect of high-concentration substrates limits its large-scale application. Herein, using structure-guided genome mining based on sequence-structure prediction of substrate tolerance and specificity, a novel leucine dehydrogenase (LaLeuDH) from Labrenzia aggregate was identified and characterized, which exhibited the highest substrate tolerance and excellent activity to trimethyl pyruvate, even at 1.5 M concentration. Moreover, based on coenzyme binding structural information and sequence alignment, directed evolution of LaLeuDH was performed to increase affinity for NADH. The obtained variant D153 N/H191 N resulted in 25-fold improved affinity for NADH, with 50-fold enhanced catalytic efficiency (kcat/Km) of 40464.6 mM(-1) s(-1). Finally, through a combination of the above two strategies, as high as 1.5 M substrate could be completely converted in 18 h without coenzyme addition, demonstrating that this engineered enzyme has promising prospects for industrialization.