ω-转氨酶的筛选和异源表达用于制备S-甲氧基异丙胺  

Screening and heterologous expression of ω-transaminase for the preparation of S -methoxyisopropylamine

作　　者：张涛[1] 周海胜[1] 徐佳琪[1] 杨立荣[1] ZHANG Tao;ZHOU Haisheng;XU Jiaqi;YANG Lirong(Institute of Bioengineering,College of Chemical and Biological Engineering,Zhejiang University,Hangzhou 310027,China)

机构地区：[1]浙江大学化学工程与生物工程学院生物工程研究所,杭州310027

出　　处：《生物学杂志》2024年第2期8-15,56,共9页Journal of Biology

基　　金：国家重点研发计划项目(2021YFC2102003)。

摘　　要：根据转氨酶家族进化分类和底物特异性,构建一个包含36个ω-转氨酶的酶库;将酶库中的基因克隆进大肠杆菌E.coli BL21 (DE3)中进行异源重组表达,考察酶蛋白表达水平并测定相应的酶活以及对映体选择性。通过筛选,获得最佳的ω-转氨酶为来源于巨大芽孢杆菌Bacillus megaterium的ω-转氨酶BmeTA,其重组表达粗酶活为2.0 U/mL,纯酶比活为9.5 U/mg蛋白;酶学性质表征发现其最适温度为35℃,最适pH为8.0。在此基础上进一步优化其催化反应工艺条件,在20 g/L加酶量、0.5 mmol/L辅酶添加量以及1.4的氨基供体/氨基受体比例条件下,反应18 h获得450 mmol/L的S-甲氧基异丙胺,原料转化率达到90%,为实现生物催化制备S-甲氧基异丙胺的工业化奠定基础。An enzyme library containing 36ω-transaminase was constructed according to the evolutionary classification of the aminotransferase family and substrate specificity.Genes from the library were cloned into E.coli BL21(DE3)for heterologous recombinant expression of the enzymes.The enzyme expression levels were analyzed and corresponding enzyme activity and enantiomeric selectivity were determined.Through screening,the bestω-aminotransferase was found to be the one from Bacillus megaterium(Bme TA),which exhibited a crude enzyme activity of 2.0 U/mL and a pure enzyme activity of 9.5 U/mg protein.Enzymology characterization showed that the optimal temperature of Bme TA was 35℃,and the optimal pH was 8.0.Based on these,the catalytic reaction process conditions were further optimized,and 450 mmol/L of S-methoxyisopropylamine was obtained after an 18 h catalytic reaction under the conditions of 20 g/L crude enzyme and 0.5 mmol/L coenzyme dosage,and 1.4 amino donor/amino acceptor ratio,reaching a 90%conversion rate of substrate.The investigation of this study laid the foundation for the industrialization of biocatalytic preparation of S-methoxyisopropylamine.

关 键 词：S-甲氧基异丙胺 ω-转氨酶 异源表达 氨基转移 生物催化 

分 类 号：Q555.6[生物学—生物化学]