Rational evolution of a medium chain-specific cytochrome P-450 BM-3 variant.

摘要: The single mutant F87A of cytochrome P-450 BM-3 from Bacillus megaterium was engineered by rational evolution to achieve improved hydroxylation activity for medium chain length substrates (C8-C10). Rational combines design and directed overcome the drawbacks these methods when applied individually. Based on X-ray structure enzyme, eight mutation sites (P25, V26, R47, Y51, S72, A74, L188, M354) were identified modeling. Sublibraries created site-specific randomization mutagenesis each site screened using a spectroscopic assay based omega-p-nitrophenoxycarboxylic acids (pNCA). mutants showing shorter combined, combi-libraries again with even better catalytic properties. Using this approach, variant five mutations (V26T, R47F, A74G, L188K, F87A) that efficiently hydrolyzes 8-pNCA obtained. efficiency towards omega-p-nitrophenoxydecanoic acid (10-pNCA) omega-p-nitrophenoxydodecanoic (12-pNCA) is comparable wild-type BM-3.