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 Km 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 kcat=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(kcatKm-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.