高等学校化学学报 ›› 2020, Vol. 41 ›› Issue (5): 1018-1025.doi: 10.7503/cjcu20190621

• 有机化学 • 上一篇    下一篇

迭代饱和突变提高Bacillus cereus胺脱氢酶对苯乙酮还原的催化效率

吴涛1,2,穆晓清1,2,*(),聂尧1,徐岩1   

  1. 1. 江南大学工业生物技术教育部重点实验室, 无锡 214122
    2. 宿迁市江南大学产业技术研究院, 宿迁 223800
  • 收稿日期:2019-12-02 出版日期:2020-05-10 发布日期:2020-02-07
  • 通讯作者: 穆晓清 E-mail:xqmu@jiangnan.edu.cn
  • 基金资助:
    国家自然科学基金(21336009);国家自然科学基金(21176103);国家“八六三”计划项目(2015AA021004);高等学校学科创新引智计划(111计划)项目(111-2-06)

Improving Catalytic Efficiency of Bacillus Cereus Amine Dehydrogenase for Acetophenone Reduction by Iterative Saturation Mutagenesis

WU Tao1,2,MU Xiaoqing1,2,*(),NIE Yao1,XU Yan1   

  1. 1. Key Laboratory of Industrial Biotechnology of Ministry of Education,Jiangnan University, Wuxi 214122, China
    2. Suqian Jiangnan University Institute of Industrial Technology, Suqian 223800, China
  • Received:2019-12-02 Online:2020-05-10 Published:2020-02-07
  • Contact: Xiaoqing MU E-mail:xqmu@jiangnan.edu.cn
  • Supported by:
    † Supported by the National Natural Science Foundation of China(21336009);† Supported by the National Natural Science Foundation of China(21176103);the National High Technology Research and Development Program of China(2015AA021004);the Overseas Expertise Introduction Project for Discipline Innovation, China(111-2-06)

摘要:

基于同源建模建立了Bacillus cereus胺脱氢酶(BcAmDH)的三维结构, 采用半理性设计方法, 对底物结合口袋附近的8个氨基酸残基(L42, G43, M67, A115, E116, T136, V293和V296)分别进行单点饱和突变, 通过显色法筛选出3个正向突变位点(116, 136和293). 进一步采用迭代饱和突变策略对这3个正向位点进行组合突变, 获得最优突变株V293A/E116V/T136S, 其对苯乙酮还原反应的催化效率达到2.54 L·min-1·mmol-1, 比BcAmDH提高了719%; 与BcAmDH相比, 最优突变株在催化苯乙酮的不对称还原反应时, 底物浓度由100 mmol/L提高至300 mmol/L, 转化率由42.1%提高至80.2%. 分子对接结果表明, 突变株底物结合口袋的位阻减小和底物进出通道的扩大是提高催化效率的主要原因.

关键词: 迭代饱和突变, 胺脱氢酶, 催化效率, 苯乙酮, (R)-α-苯乙胺

Abstract:

Based on the 3D structure of Bacillus cereus amine dehydrogenase established by homology modeling, 8 amino acid residues(L42, G43, M67, A115, E116, T136, V293 and V296) around the substrate binding pocket were selected to site-directed saturation mutagenesis. Positive mutants were screened on 3 sites(116, 136, 293) by color-rendering method. The best triple-sites mutant(V293A/E116V/T136S) was obtained by iterative saturation mutation strategy and its catalytic efficiency for acetophenone reached 2.54 L·min -1·mmol-1, which was 719% higher than BcAmDH. Compared with BcAmDH, the substrate concentrations and conversions of asymmetric reduction reaction of acetophenone were increased from 100 mmol/L to 300 mmol/L and from 42.1% to 80.2% at the optimal reaction conditions, respectively. It was suggested that the decrease of steric hindrance in the substrate binding pocket of the mutant and the expansion of the substrate entrance channel were the main reasons for improving catalytic efficiency by molecular docking results.

Key words: Iterative saturation mutagenesis, Amine dehydrogenase, Catalytic efficiency, Acetophenone, (R)-α-Phenethylamine

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