中文摘要：

为进一步提高P450BM-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（Kcat Km-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。

英文摘要：

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 （kcat Km-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.