化工学报 ›› 2014, Vol. 65 ›› Issue (4): 1374-1380.DOI: 10.3969/j.issn.0438-1157.2014.04.030

• 生物化学工程与技术 • 上一篇    下一篇

一个有利于细胞色素P450 BM-3催化吲哚合成靛蓝的三位点突变酶:D168L/E435T/V445A

张彭湃1, 胡升2, 梅乐和1,2, 雷引林2, 金志华2, 姚善泾1   

  1. 1 浙江大学生物工程研究所, 浙江 杭州 310027;
    2 浙江大学宁波理工学院生物与化学工程学院, 浙江 宁波 315100
  • 收稿日期:2013-08-11 修回日期:2013-09-25 出版日期:2014-04-05 发布日期:2013-10-30
  • 通讯作者: 梅乐和
  • 作者简介:张彭湃(1978—),男,博士研究生。
  • 基金资助:

    国家自然科学基金项目(21176220,31240054);浙江省自然科学基金重点项目(Z13B060008);宁波市重大科技项目(2011C11023)。

A triple mutant improving activity of cytochrome P450 BM-3 to catalyze indole hydroxylation:D168L/E435T/V445A

ZHANG Pengpai1, HU Sheng2, MEI Lehe1,2, LEI Yinlin2, JIN Zhihua2, YAO Shanjing1   

  1. 1 Institute of Bioengineering, Zhejiang University, Hangzhou 310027, Zhejiang, China;
    2 School of Biological and Chemical Engineering, Ningbo Institute of Technology, Ningbo 315100, Zhejiang, China
  • Received:2013-08-11 Revised:2013-09-25 Online:2014-04-05 Published:2013-10-30
  • Supported by:

    supported by the National Natural Science Foundation of China(21176220, 31240054), the Natural Science Foundation of Zhejiang Province (Z13B060008) and the Key Technology Research and Develop Project of Ningbo(2011C11023).

摘要: 为进一步提高P450 BM-3催化吲哚合成靛蓝的能力,在前期获得的突变酶D168L/E435T基础上引入V445A突变,获得突变酶D168L/E435T/V445A,该突变酶与D168L/E435T及V445A相比,对吲哚的亲和力分别降低41.5%及35.8%,转化数(kcat)分别提高1.61倍及1.31倍,催化效率(kcatKm-1)分别提高1.53倍及1.47倍;该突变酶对电子的耦合率也提高至24.7%,较D168L/E435T提高39.5%,较V445A提高64.7%,说明该突变酶在电子的利用率上有明显改善;此外,该突变酶催化副产物靛玉红的比例大幅降低,降低为D168L/E435T的41.7%及V445A的30.6%,说明该突变酶催化吲哚的区域选择性上也更加有利于靛蓝形成。

关键词: 生物催化, 酶, 分子生物学, P450 BM-3, 吲哚, 羟基化, 靛蓝

Abstract: To further enhance hydroxylation ability with indole of P450 BM-3, site-directed mutagenesis with V445A was introduced to the mutant D168L/E435T obtained in earlier research, and mutant D168L/E435T/V445A was obtained. Compared with D168L/E435T and V445A, mutant D168L/E435T/V445A exhibited lower affinity with indole, but higher improved turnover rate (kcat). Correspondingly, catalytic efficiency (kcatKm-1) was increased to 1.53 times and 1.47 times respectively. The electronic coupling rate of the mutant was also improved by 24.7%, 39.5% higher than that of D168L/E435T and 64.7% higher than that of V445A, showing better efficiency of electronic utilization of this new mutant enzyme. Mutant D168L/E435T/V445A also displayed improved regioselectivity with indole in indigo synthesis, with the proportion of by-product indirubin 41.7% of that of D168L/E435T and 30.6% of that of V445A.

Key words: bio-catalysis, enzyme, molecular biology, P450 BM-3, indole, hydroxylation, indigo

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