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

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.

Orobol from enzyme biotransformation attenuatesDermatophagoides farinae-induced atopic dermatitis-like symptoms in NC/Nga mice

“…To solve this issue, our co-operating group recently developed an orobol bio-engineering system with low-cost, fast, and large-scale production. 8,9 Therefore, orobol might become applicable in numerous industries.…”

“…Recently, our group developed a bio-engineering system with low-cost, fast, and large-scale production. 8,9 Due to this enzyme technology, orobol could be applicable in the cosmeceutical or pharmaceutical industry for the prevention and treatment of AD. We also revealed the multiple effects of orobol through recent studies.…”

Polyphenol-Hydroxylating Tyrosinase Activity under Acidic pH Enables Efficient Synthesis of Plant Catechols and Gallols

“…Enzyme expression and purification were conducted with slight modifications of previous work [ 27 ]. Six bacterial tyrosinases had been transformed in E. coli BL21 (DE3) and their cell stocks were used to express each tyrosinase ( Table S1 ).…”

Regioselective One-Pot Synthesis of Hydroxy-(S)-Equols Using Isoflavonoid Reductases and Monooxygenases and Evaluation of the Hydroxyequol Derivatives as Selective Estrogen Receptor Modulators and Antioxidants

“…In order to synthesize such hydroxylated polyphenols efficiently, scientists isolated several microbial enzymes and constructed the respective biocatalytic reaction systems. Those studies harnessed microbial cytochrome P450s, flavin-dependent monooxygenases (FMO), or tyrosinases in their natural or mutated forms for an efficient and regioselective production of plant polyphenols ( Pandey et al, 2010 ; Lee et al, 2012 ; Lee et al, 2014 ; Lee S.-H. et al, 2016 ; Lee et al, 2019 ). In recent studies, a two-component FMO called HpaBC derived from E. coli or P. aeruginosa has been introduced as another potent candidate to hydroxylate aromatic chemicals.…”