caa a22 4500 605498636 CHVBK 20210128100547.0 cr unu---uuuuu 210128e20151001xx s 000 0 eng 10.1007/s00253-015-6691-1 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6691-1 Heterologous expression of tyrosinase (MelC2) from Streptomyces avermitilis MA4680 in E [Elektronische Daten] coli and its application for ortho -hydroxylation of resveratrol to produce piceatannol [Nahum Lee, Sang-Hyuk Lee, Kiheon Baek, Byung-Gee Kim] Recombinant tyrosinase from Streptomyces avermitilis MA4680, MelC2 (gi:499291317), was heterologously expressed in Escherichia coli BL21 (DE3). The expression level of active MelC2 was increased by the codon-optimized MelC1 caddie protein (KP198295.1). By performing saturation mutagenesis of the Y91 residue of MelC1, it was found that aromatic residues such as Y, F, and W at the 91st position help produce a correctly folded conformation of MelC2. The recombinant MelC2 was utilized as a biocatalyst to convert trans-resveratrol into piceatannol. In order to improve the product yield through suppression of the formation of melanin, a by-product, an increase in the ratio of monooxygenation (k 1) to dioxygenation (k 2) of MelC2 is desirable. This was achieved by a combination of protein engineering and regeneration of NADH with glucose dehydrogenase (GDH). Saturation mutagenesis was performed at 15 residues within 8-Å radius from copper ions of MelC2. A total of 2760 mutants were examined (99.7% probability for NNK codon) and I41Y, a mutant, was screened. The ratio of k 1 to k 2 of the mutant increased sevenfold on tyrosine and fivefold on resveratrol, when compared to wild-type MelC2. As a result, the overall product yield from 500μM resveratrol in 50-mL reaction was 15.4% (77.4μM piceatannol), 1.7 times higher than wild type. When I41Y was incorporated with the NADH regeneration system, the total product yield was 58.0%, an eightfold increase (290.2μM of piceatannol). Springer-Verlag Berlin Heidelberg, 2015 Tyrosinase nationallicence Streptomyces avermitilis nationallicence Resveratrol nationallicence Piceatannol nationallicence Lee Nahum School of Chemical and Biological Engineering, Seoul National University, 151-744, Seoul, South Korea aut Lee Sang-Hyuk Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University, 151-744, Seoul, South Korea aut Baek Kiheon School of Chemical and Biological Engineering, Seoul National University, 151-744, Seoul, South Korea aut Kim Byung-Gee School of Chemical and Biological Engineering, Seoul National University, 151-744, Seoul, South Korea aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/19(2015-10-01), 7915-7924 0175-7598 99:19<7915 2015 99 253 https://doi.org/10.1007/s00253-015-6691-1 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6691-1 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Lee Nahum School of Chemical and Biological Engineering, Seoul National University, 151-744, Seoul, South Korea aut NATIONALLICENCE 700 1- Lee Sang-Hyuk Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University, 151-744, Seoul, South Korea aut NATIONALLICENCE 700 1- Baek Kiheon School of Chemical and Biological Engineering, Seoul National University, 151-744, Seoul, South Korea aut NATIONALLICENCE 700 1- Kim Byung-Gee School of Chemical and Biological Engineering, Seoul National University, 151-744, Seoul, South Korea aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/19(2015-10-01), 7915-7924 0175-7598 99:19<7915 2015 99 253