Directed-Evolution Analysis of Human Cytochrome P450 2A6 for Enhanced Enzymatic Catalysis
作者： Hwayoun Lee , Joo-Hwan Kim , Songhee Han , Young-Ran Lim , Hyoung-Goo Park
DOI: 10.1080/15287394.2014.951757
关键词: Enzyme 、 Biochemistry 、 Enzyme catalysis 、 Molecular biology 、 Mutant 、 Mutagenesis 、 Chemistry 、 Hydroxylation 、 Directed evolution 、 Coumarin 、 CYP2A6
摘要: Cytochrome P450 2A6 (P450 2A6) is the major enzyme responsible for oxidation of coumarin, nicotine, and tobacco-specific nitrosamines in human liver. In this study, catalytic turnover coumarin was improved by directed-evolution analysis enzyme. A random mutant library constructed using error-prone polymerase chain reaction (PCR) open reading frame gene individual clones were screened activity fluorescent 7-hydroxylation. Four consecutive rounds mutagenesis screening performed catalytically enhanced mutants selected each round screening. The showed sequentially accumulated mutations amino acid residues 2A6: B1 (F209S), C1 (F209S, S369G), D1 S369G, E277K), E1 E277K, A10V). displayed approximately 13-fold increased based on hydroxylation assays at bacterial who...
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