Modification of the fatty acid specificity of cytochrome P450BM-3 from Bacillus megaterium by directed evolution:: A validated assay

Cytochrome P450 BM-3 (CYP102) catalyzes the subterminal hydroxylation of fatty acids with a chain length of 12-22 carbons. The paper focuses on the regioselectivity and substrate specificity of the purified wild-type enzyme and five mutated variants towards caprylic, capric, and lauric acid. The enzymes were obtained by random mutagenic fine-tuning of the mutant F87A(LARV). F87A(LARV) was selected as the best enzyme variant in a previous study in which the single mutant F87A was subjected to rational evolution to achieve hydroxylation activity for short chain length substrates using ap-nitrophenolate-based spectrophotometric assay. The best mutants, F87V(LAR) and F87V(LARV), show a higher catalytic activity towards omega-(p-nitrophenoxy)decanoic acid (W-p-NCA) than F87A(LARV). In addition, they proved capable of hydroxylating omega-(p-nitrophenoxy)octanoic acid (8-p-NCA) which the wild-type enzyme is unable to do. Both variants catalyzed hydroxylation of capric acid, which is not a substrate for the wild-type, with a conversion rate of up to 57%. The chain length specificity of the mutants in fatty acid hydroxylation processes shows a good correlation with their activity towards p-NCA pseudosubstrates. The p-NCA assay therefore, allows high-throughput screening of large mutant libraries for the identification of enzyme variants with the desired catalytic activity towards fatty acids as the natural substrates. (C) 2001 Elsevier Science B.V. All rights reserved.