title: 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.
authors.affiliation: Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University, Seoul, South Korea.|Institute of Bioengineering, Seoul National University, Seoul, South Korea.|Institute of Molecular Biology and Genetics, Seoul National University, Seoul, South Korea.
authors.forename: Sang-Hyuk
authors.initials: SH
authors.lastname: Lee
authors.affiliation: Institute of Bioengineering, Seoul National University, Seoul, South Korea.|Institute of Molecular Biology and Genetics, Seoul National University, Seoul, South Korea.|School of Chemical and Biological Engineering, Seoul National University, 151-744, Seoul, South Korea.
authors.forename: Kiheon
authors.initials: K
authors.lastname: Baek
authors.affiliation: Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University, Seoul, South Korea.|Institute of Bioengineering, Seoul National University, Seoul, South Korea.|Institute of Molecular Biology and Genetics, Seoul National University, Seoul, South Korea.
authors.forename: Ju-Eun
authors.initials: JE
authors.lastname: Lee
authors.affiliation: Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University, Seoul, South Korea. byungkim@snu.ac.kr.|Institute of Bioengineering, Seoul National University, Seoul, South Korea. byungkim@snu.ac.kr.|Institute of Molecular Biology and Genetics, Seoul National University, Seoul, South Korea. byungkim@snu.ac.kr.|School of Chemical and Biological Engineering, Seoul National University, 151-744, Seoul, South Korea. byungkim@snu.ac.kr.
authors.forename: Byung-Gee
authors.initials: BG
authors.lastname: Kim
mesh_terms: D001205:Ascorbic Acid / Q000378:metabolism*; D001410:Bacillus megaterium / Q000201:enzymology / Q000235:genetics; D001881:Borates / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007529:Isoflavones / Q000378:metabolism; D008543:Melanins / Q000378:metabolism*; D014442:Monophenol Monooxygenase / Q000378:metabolism*
chemical_list: D001881:Borates; D007529:Isoflavones; D008543:Melanins; C004742:daidzein; D014442:Monophenol Monooxygenase; D001205:Ascorbic Acid
keywords: 7,3′,4′-trihydroxyisoflavone; Bacillus megaterium; isoflavone; monooxygenase; ortho-hydroxylation; tyrosinase