Abstract
Cytochrome P450 BM-3 from Bacillus megaterium is a fatty acid hydroxylase exhibiting selectivity for long-chain substrates (12–20 carbons). Replacement of Phe87 in P450 BM-3 by Val (F87V) greatly increased its activity towards a variety of aromatic and phenolic compounds. The apparent initial reaction rates of F87V as to benzothiophene, indan, 2,6-dichlorophenol, and 2-(benzyloxy)phenol were 227, 204, 129, and 385 nmol min−1 nmol−1 P450, which are 220-, 66-, 99-, and 963-fold those of the wild type, respectively. These results indicate that Phe87 plays a critical role in the control of the substrate specificity of P450 BM-3. Furthermore, F87V catalyzed regioselective hydroxylation at the para position of various phenolic compounds. In particular, F87V showed high activity as to the hydroxylation of 2-(benzyloxy)phenol to 2-(benzyloxy)hydroquinone. With F87V as the catalyst, 0.71 mg ml−1 2-(benzyloxy)hydroquinone was produced from 1.0 mg ml−1 2-(benzyloxy)phenol in 4 h, with a molar yield of 66%.
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Acknowledgements
The authors wish to thank Prof. T. Sakaki, (Toyama Prefectural University) for helpful discussions. Q.S.L. received a post-doctoral fellowship (no. P99115) from the Japan Society for Promotion of Science. This work was supported in part (S.S.) by the Project for the Development of a Technological Infrastructure for Industrial Bioprocesses (R&D of New Industrial Science and Technology Frontiers, New Energy and Industrial Technology Development Organization, Japan) and by the COE for Microbial-Process Development Pioneering Future Production Systems (COE Program, Ministry of Education, Culture, Sports, Science and Technology, Japan).
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Sulistyaningdyah, W.T., Ogawa, J., Li, QS. et al. Hydroxylation activity of P450 BM-3 mutant F87V towards aromatic compounds and its application to the synthesis of hydroquinone derivatives from phenolic compounds. Appl Microbiol Biotechnol 67, 556–562 (2005). https://doi.org/10.1007/s00253-004-1761-9
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DOI: https://doi.org/10.1007/s00253-004-1761-9