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化工学报  2013 

定点突变提高细胞色素P450BM-3吲哚羟基化能力

DOI: 10.3969/j.issn.0438-1157.2013.09.035, PP. 3331-3337

Keywords: 细胞色素P450,BM-3,定点突变,羟基化,吲哚,靛蓝

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Abstract:

为进一步提高细胞色素P450BM-3(A74G/F87V/L188Q)对吲哚的羟基化能力,根据酶结构与功能的关系,以突变酶E435T为基础,在168位点引入D168L突变,获得了吲哚羟基化能力得到显著提高的突变酶D168L/E435T。突变酶对吲哚的Km为1.72mmol·L-1(父本2.09mmol·L-1),转化数(kcat)为28.15min-1(父本4.04min-1),表明D168L定点突变可以略微提高酶对底物的亲和力,但主要的效应是促进了底物的转化速率,这两个效应的综合表现是使酶的催化效率(kcatKm-1)比父本酶提高了8.48倍。此外,产物中副产物靛玉红的比例也降低为1.2%(父本7.3%),这说明该突变酶催化吲哚的区域选择性上也更有利于靛蓝的生成。

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