Molecular Engineering Of Cytochrome P450 BM-3 For The Biosynthesis Of Indigo

To give or enhance particular function of the enzyme through modification with protein engineering methods is the pre-condition to realize the industrial application. In this paper, in order to create biocatalysts with better capability of indigo and better region-selectivivty toward 3-OH indole, we engineered P450 BM-3 by directed evolution, semi-rational design and site directed mutagenesis. In addition, the relationship between the structure and function of the mutants was also discussed.To achieve these objectives, we established a kind of high-throughput screening method with better stablility in color reaction and shorter screening cycle. With this method, we screened the library of mutants, and obtained a series of mutant enzymes with improved catalytic ability. The main work of this study was showed as follows.First, in order to introduce lactose to the screening method, expressions conditions in shake flask were optimized with lactose as the inducer. It was found that while 2.0 g/L lactose was added into the culture when the biomass reached OD600 to 0.7～1.5 and incubated at 30℃ for 10 hours, the yield of P450 BM-3 arrived at the maximum (80 mg/L), which was 2.3 times that of IPTG The solubility expression of P450 BM-3 was also improved with the inclusion bodies decreased to 9.0%(IPTG,～25%). These data indicated that lactose was an efficient inducer for the expression of P450 BM-3 in E. coli. The main reason that lactose promoted P450 BM-3 soluble expression could be that the lactose appropriately reduced the induction indensity. And this change was conducive to the synthesis of molecular chaperone and other folding related proteins, and in turn, these molecular partners ensured the correct folding of the target protein. As a result, the inclusion body proportion was effectively reduced.Based on the above facts, an efficient method for high throughput screening with lactose as inducer was developed, which could be described as follows:screening was carried out in 96-well plate with pre-adding 2.0 g/L lactose to LB liquid medium, and then the colonies were inoculated into the culture, after induction at 30℃ for 10 hours, the bacteria in any well with darkest color was selected out for further study. With this method, it took only ～12 hours to obtain the desired mutations. Compared with the colorimetric colony-based method (3～5 days), this method was more convenient and greatly saved the time consumed in screening.Then, using this method we screened the libraries of P450 BM-3 mutants,168/435 loci mutation combinatorial library and the error-prone PCR library. In 168/435 loci mutation combinatorial library, mutant D168L/E435T was identified with highest hydroxylation activity toward indole in this library. Concomitantly, its Km for indole was 16% lower than its parent enzyme. Also the turnover rate (kcat) of this mutant enzyme was increasing up to 6.6-fold that of its parent. Thus, the catalytic efficiency (kcat/Km) was correspondingly raised to 7.9 times as much as that of its parent. Moreover, the electron coupling efficiency and the proportion of the main products were improved compared with its parent. The study showed that there was synergistic effect between the mutant D168L and E435T. Take the loci 168 and 435 sited position on the protein into consideration, the 168 site was probably affected the substate binding, while the 435 site was more likely to affect the catalytic ability of the enzyme.In error-prone PCR library, mutant V445A exhibited the highest specific activity toward indole and alanine was confirmed as the best amino acid substitution by saturated mutagenesis in Val445 position. Compared with the parent enzyme, the kcat of V445A was increased by 7.5 times, while its Km value decreased by 9.2%. Consequently, the catalytic efficiency (kcat/Km) of V445A was raised to 8.2 times that of the parent enzyme. Moreover, the electron coupling efficiency was 28.2% higher than the parent and the proportion of the byproduct was futher reduced. With 3D structure analysis, it showed that alanine substitution might cause proper change of the protein structure through weakening hydrophobicity in the 445 position, but the hydrophilic or too much hydrophobic amino acid substitution in this position might cause negative effects. The alanine substitution in Val445 position might lead to some slight structural change and probably enlarged the substrate entrance thus specific activity greatly enhanced.Mutant D168L/E435T/V445A, which was from the combination of D168L/E435T and V445A, exhibited the highest hydroxylation activity of indole in this study. Though the substate affinity was reduced slightly, the kcat of the mutant enzyme is greatly increased up to 17.0-fold that of its parent enzyme. The catalytic efficiency was correspondingly raised to 12.0 times as much as that of its parent. The electronic coupling rate of the mutant is also improved by 24.7% and the proportion of the byproduct was futher reduced. All the changes of this mutant was probably due to the fine-adjusted to the structure of the enzyme caused by alanine substitution in Val445 position, V445A caused synergistic effect with Thr435 for catalytic ability and also make up the negative effect of Leu168 with the substrate binding. But on the whole, the promoting catalytic effect was given mainly, thus, the final catalytic efficiency not declined, but greatly increased.