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Circular permutation of a bacterial tyrosinase enables efficient polyphenol‐specific oxidation and quantitative preparation of orobol

Pyung-Gang Lee,Sanghyuk Lee,E. Hong,S. Lutz,Byung-gee Kim

Published 2018 in Biotechnology and Bioengineering

ABSTRACT

Tyrosinase is a type 3 copper oxygenase that catalyzes a phenol moiety into ortho‐diphenol, and subsequently to ortho‐quinone. Diverse tyrosinases have been observed across the kingdom including Animalia, Bacteria, Plantae, and Fungi. Among the tyrosinases, bacterial, and mushroom tyrosinases have been extensively exploited to prepare melanin, ortho‐hydroxy‐polyphenols, or novel plant secondary metabolites during the past decade. And their use as a biocatalyst to prepare various functional biocompounds have drawn great attention worldwide. Herein, we tailored a bacterial tyrosinase from Bacillus megaterium (BmTy) using circular permutation (CP) engineering technique which is a novel enzyme engineering technique to covalently link original N and C termini and create new termini on the middle of its polypeptide. To construct a smart rationally‐designed CP library, we introduced 18 new termini at the edge of each nine loops that link α‐helical secondary structure in BmTy. Among the small library, seven functional CP variants were successfully identified and they represented dramatic change in their enzyme characteristics including kinetic properties and substrate specificity. Especially, cp48, 102, and 245 showed dramatically decreased tyrosine hydroxylase activity, behaving like a catechol oxidase. Exploiting the dramatic increased polyphenol oxidation activity of cp48, orobol (3′‐hydroxy‐genistein) was quantitatively synthesized with 1.48 g/L, which was a 6‐fold higher yield of truncated wild‐type. We examined their kinetic characters through structural speculation, and suggest a strategy to solubilize the insoluble artificial variants effectively.

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