催化学报

催化学报 ›› 2009, Vol. 30 ›› Issue (4): 291-296.

• 研究论文 • 上一篇    下一篇

定向进化提高枯草芽孢杆菌脂肪酶的活力

赵博, 陶进, 马吉胜, 廉虹, 王岩, 常琳, 高仁钧, 曹淑桂   

  1. 吉林大学分子酶学工程教育部重点实验室, 吉林长春130023
  • 收稿日期:2009-04-25 出版日期:2009-04-25 发布日期:2013-01-21

Increasing Activity of Lipase from Bacillus subtilis by Directed Evolution

ZHAO Bo, TAO Jin, MA Jisheng, LIAN Hong, WANG Yan, CHANG Lin, GAO Renjun*, CAO Shugui*   

  1. Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, Jilin University, Changchun 130023, Jilin, China
  • Received:2009-04-25 Online:2009-04-25 Published:2013-01-21

PDF

1447

被引次数

21

摘要： 通过定向进化的方法, 提高了一个新的源于枯草芽孢杆菌 (Bacillus subtilis) 的脂肪酶 (BSL2) 的活力. 经过两轮易错 PCR 以及高通量筛选, 最终获得了比活力为野生出发酶 4.5 倍的突变体 3-1B2. 基因比对结果表明, 共有两个氨基酸发生了突变. 突变酶的酶学性质研究发现, 与野生酶相比, 它的热稳定性及 pH 稳定性略有增加, 最适温度和最适 pH 值则无太大的变化. 同源模建了 BSL2 与 3-1B2 的结构, 并与底物进行了分子对接. 结果表明, 突变体 3-1B2 的结合能比野生型 BSL2 低 1.29 kcal/mol, 活性中心 Ser77 残基与底物羰基的距离也由 0.319 nm 减小为 0.278 nm, 因而加快了酶反应速度, 提高了酶活力.

关键词: 枯草芽孢杆菌, 脂肪酶, 易错聚合酶链反应, 定向进化, 分子对接

Abstract: Catalytic activity of a Bacillus subtilis lipase was enhanced by directed evolution. The distance between catalytic residue Ser77 and the substrate made an important contribution to the enzyme activity. The mutant lipase had lower energy, and its thermostability and pH stability were simultaneously increased.

Key words: Bacillus subtilis, lipase, error prone polymerase chain reaction, directed evolution, molecular docking