ω-转氨酶的基因挖掘与酶学性质研究
Gene mining and enzymatic properties analysis of ω-transaminase
ω-转氨酶(EC 2.6.1.X)是一类5\'-磷酸吡哆醛依赖性酶,能介导醛酮不对称还原胺化或手性胺动力学拆分,因此在医药中间体手性胺合成领域具有重要研究价值。本研究采用基因挖掘技术,从基因组数据库中筛选获得一个来自Paracoccus pantotrophus的新型ω-转氨酶(ppTA)基因,将负载该基因的重组载体转化大肠杆菌BL21(DE3)进行表达、纯化和酶学性质分析。其最适温度和pH分别为38℃和9;与来源于杆菌的ω-转氨酶不同,ppTA对大于两个碳的长侧链胺仍然具有活性,对酮的活力大于醛,并且偏好于芳香族胺;低温贮藏稳定性好,半衰期高达1560 h;Na+对该酶活力具有一定的促进作用,低浓度Fe2+与Fe3+、Cu2+与金属离子螯合剂对酶活力抑制作用明显。酶动力学分析表明该酶对手性胺L-2-氨基丁酸的前体2-酮丁酸的Km为3.3mM, Vmax为2292 μmol min-1 L-1,偶联苏氨酸脱氨酶催化合成L-2-氨基丁酸的产率>80%,ee值>99%。ω-转氨酶基因的成功表达为其生物合成手性胺如L-2-氨基丁酸提供了可能。
ω-transaminase (EC2.6.1.X) is a kind of pyridoxal-5-phosphate-dependent enzymes, which can mediate asymmetric reductive amination of aldehydes and ketones or kinetic resolution of chiral amines, so it has important research value in field of chiral amine synthesis ofpharmaceutical intermediates. In this study, a novel ω-transaminase gene (ppTA), from Paracoccus pantotrophus was screened by gene mining technique. The recombinant vector carrying the gene was transformed into E.coli BL21 (DE3) for expression, purification and enzyme properties analysis. The optimum temperature and pH were 38℃ and 9, respectively; Different from ω-transaminases from bacilli, it was found that ppTA was still active for long side chain amines with more than two carbon, and the activity for ketones was higher than that for aldehydes, and preferred aromatic amines; the storage stability at low temperature was very good, the half-life was 1560 h; Metal ion Na+ can promote the enzyme activity to a certain extent, while iron ion, Cu2+ and metal ion chelating agent have obvious inhibitory effect on the enzyme activity. The kinetic analysis of enzyme activity showed that the Km of 2-ketobutyric acid, was 3.3mM, and the Vmax was 2292 μmol min-1 L-1. The yield of L-2-aminobutyric acid was>80%, ee value>99%. The successful expression of ω-transaminase gene provides the possibility for its biosynthesis of chiral amines such as L-2-aminobutyric acid.
罗玮、胡佳桂、王小平、刘元涛、孙钦波
生物科学研究方法、生物科学研究技术生物化学生物工程学
分子生物学ω-转氨酶苄胺基因挖掘酶学性质
molecular biologyω-transaminasebenzylaminegene miningenzymatic properties
罗玮,胡佳桂,王小平,刘元涛,孙钦波.ω-转氨酶的基因挖掘与酶学性质研究[EB/OL].(2021-03-04)[2025-08-23].http://www.paper.edu.cn/releasepaper/content/202103-59.点此复制
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