Using tyrosinase as a monophenol monooxygenase: A combined strategy for effective inhibition of melanin formation

Abstract

Tyrosinase is a binuclear copper-containing metalloprotein that leads the fast and regio-selective o-hydroxylation of monophenols to o-diphenols. However, the subsequent second oxidation to produce o-quinones, i.e., melanin precursors, from the o-diphenols has restricted its use to the production of functional o-diphenol derivatives. Herein, we present a combined strategy for the effective inhibition of melanin formation in tyrosinase reaction, which allows the use of tyrosinase as a monophenol monooxygenase. The o-diphenolic products were protected from being oxidized in the tyrosinase reaction by borate ions and L-ascorbic acid (LAA). Borate-o-diphenol complexes were favorable formed at high pH and consequentially protected the o-diphenolic products from the catecholase activity of tyrosinase. LAA not only directly reduced the byproduct, o-quinones, into o-diphenols but also assisted the completion of the tyrosinase reaction cycle by removing a hydroxyl group attached to the copper metal cluster at the active site of the met-form tyrosinase. The regio-selective o-hydroxylation of 7,4'-dihydroxyisoflavone (daidzein) to produce 7,3',4'-trihydroxyisoflavone (3'-ODI) was successfully carried out by whole E. coli cell biotransformation with heterologously expressed tyrosinase from Bacillus megaterium. The yield of this o-hydroxylation of 5 mM daidzein in one-pot 400 mL reaction was ca. 100% in 90 min and the productivity was 16.3 mg 3'-ODI · L(-1) · h(-1) · DCW mg(-1), which is considerably higher than that of other monooxygenases. The method effectively abolished melanin synthesis, so that the o-diphenolic product remained stable without enzyme inactivation. Other monophenolic phytochemicals such as resveratrol and genistein could be subjected to the same strategy. After 1 h, 1 mM of genistein and resveratrol were both converted to orobol and piceatannol, respectively, with ca. 95% conversion yield. These results support the strong potential of tyrosinase as a monooxygenase for regio-selective o-hydroxylation of various monophenolic compounds.

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

“…The biocatalysis basically followed the method previously set-up which exploited borate-catechol chelation and reducing agents for preventing overoxidation of desired products (S. H. Lee et al, 2016).…”

“…Analysis condition of HPLC followed the previously mentioned (S. H. Lee et al, 2016). Samples were prepared by mixing with 1 N of HCl and extracted by ethyl acetate, which subsequently were evaporated and redissolved into methanol for high-performance liquid chromatography (HPLC) analysis.…”

“…To construct tyrosinase CP variants, the previous developed PCR method was used (Fischereder et al, 2014). Previous constructed tyrosinase (originated from BmTy; ATCC 10778) genes cloned in pET28a was used for a PCR template (S. H. Lee et al, 2016). In brief, the truncated tyrosinase (4Lys to 289Glu) gene was linearly duplicated in pRSFduet-1 vector with a linker (-ACTAGTGGC-) using the following primers, forward: 5′-GGCCACTAGTGGCAAGTACAGAGTT AGAAAAAACG-3′ and reverse: 5′-GGCCACTA GTTTCTATATCGTAT ACG-3′.…”

“…Samples were prepared by mixing with 1 N of HCl and extracted by ethyl acetate, which subsequently were evaporated and redissolved into methanol for high-performance liquid chromatography (HPLC) analysis. Analysis condition of HPLC followed the previously mentioned (S. H. Lee et al, 2016). Orobol standard curve, 1 H-NMR and gas chromatography-mass spectrometry data were also obtained from the previous study.…”

“…In conventional application, tyrosinase has been mainly exploited for melanin synthesis and detoxification of phenol in wastewater (Chávez-Béjar et al, 2013;Wada, Ichikawa, & Tastsumi, 1995). Recently, it was shown that fast enzyme kinetics of tyrosinase makes this simple oxygenase superior to other oxygenases for preparing a variety of beneficial catechol-like polyphenols (S. H. Lee, Baek, Lee, & Kim, 2016), and its melanin synthetic ability has been developed for visualizing live cell images and screening tyrosine-overproducing microbial variants (Jathoul et al, 2015;Santos & Stephanopoulos, 2008). Despite versatility of this enzyme, evolutional labors, and trials to enhance tyrosinases activity and substrate specificity were quite limited to only simple random mutagenesis (error-prone polymerase chain reaction [PCR]) or structure-based site-directed mutagenesis (N. Lee, Lee, Baek, & Kim, 2015; Molloy et al, 2013; Shuster Ben-Yosef, Sendovski, & Fishman, 2010), since differentiation of cresolase and catecholase activities is very difficult to select appropriate mutants for target reactions.…”