定点突变提高细胞色素P450 BM-3吲哚羟基化能力

为进一步提高细胞色素P450 BM-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K_m为1.72 mmol·L^-1(父本2.09 mmol·L^-1),转化数(k_cat)为28.15 min^-1(父本4.04 min^-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(k_cat K_m^-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。

Improving indole-hydroxylation ability of cytochrome P450 BM-3 by site-directed mutagenesis

According to the relationship between the structure and function of enzyme,it was speculated that the combination of the mutant enzyme D168L and E435T might improve the catalytic ability of indole to generate indigo.The mutant enzyme D168L/E435T was produced by site-directed mutagenesis conducted on the 168th site of the P450 BM-3(A74G/F87V/L188Q/E435T)and it was identified with higher hydroxylation activity toward indole in comparison to its parent enzyme.Concomitantly,its K_m for indole was 1.72 mmol·L^-1 and this value was lower compared to its parent enzyme(2.09 mmol·L^-1).Also with the k_cat=28.15 min^-1,the turnover rate of the mutant enzyme was increasing up to 6.97-fold that of its parent enzyme(4.04 min^-1).The catalytic efficiency(k_catK_m^-1)was correspondingly raised to 8.48 times as much as that of its parent enzyme.Moreover,the proportion of the byproducts indirubin reduced to 1.2%,much lower than that of its parent mutant(7.3%),which indicated that this regioselectivity of mutant enzyme to catalyze indole was more conducive to generating indigo.