化学
转氨酶
酶
基质(水族馆)
脱氢酶
辅因子
生物化学
生物转化
立体化学
赖氨酸
产量(工程)
突变体
酶动力学
酶分析
活动站点
氨基酸
生物
基因
冶金
材料科学
生态学
作者
Liqun Jin,Jun-Kang Shentu,Jianlin Liu,Tian-Chen Shao,Zhi‐Qiang Liu,Ya‐Ping Xue,Yu‐Guo Zheng
标识
DOI:10.1016/j.jbiotec.2021.11.002
摘要
Transaminases catalyze the transfer of an amino group from a donor to a keto group of an acceptor substrate and are applicable to the asymmetric synthesis of herbicide L-phosphinothricin (L-PPT). Here, the important residue sites (C390, I22, V52, R141, Y138 and D239) of transaminase from Salmonella enterica (SeTA) were modified at the adjacency of the substrate-binding pocket to improve the enzyme activity. Among the constructed mutant library, the SeTA-Y138F mutant displayed higher activity than the wild-type enzyme. Compared to the wild-type, SeTA-Y138F showed improved catalytic efficiency with a 4.36-fold increase. The Km and kcat of SeTA -Y138F toward 4-(hydroxy(methyl) phosphoryl)-2-oxobutanoic acid (PPO) were 26.39 mM and 34.28 s-1, respectively. Subsequently, the three-enzyme co-expression system of E. coli BL21 (DE3)/pACYCDuet-SeTA-Y138F/pETDuet-AlaDH-BsGDH was developed by combining a alanine dehydrogenase (AlaDH) to recycle the byproduct of amino donor, a glucose dehydrogenase (BsGDH) for cofactor recycling. Under the optimized conditions, an excellent L-PPT yield of 90.8% was achieved by the whole-cell biotransformation with 500 mM PPO. It exhibited the tri-enzymatic coupling system was potential for effective production of target L-PPT.
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