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

doi:10.3969/j.issn.0438-1157.2014.04.030

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

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 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 Lehe^1,2, LEI Yinlin², JIN Zhihua², YAO Shanjing¹

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/Abstract

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（k_cat）分别提高1.61倍及1.31倍，催化效率（k_catK_m^-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 (k_cat). Correspondingly, catalytic efficiency (k_catK_m^-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

中图分类号:

TQ962.2

张彭湃, 胡升, 梅乐和, 雷引林, 金志华, 姚善泾. 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[J]. 化工学报, 2014, 65(4): 1374-1380.

ZHANG Pengpai, HU Sheng, MEI Lehe, LEI Yinlin, JIN Zhihua, YAO Shanjing. A triple mutant improving activity of cytochrome P450 BM-3 to catalyze indole hydroxylation：D168L/E435T/V445A[J]. CIESC Journal, 2014, 65(4): 1374-1380.

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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

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

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