© 2005 The Japanese Biochemical Society
Regular Paper |
Catalytic Properties of an Organic Solvent–Resistant Tyrosinase from Streptomyces sp. REN-21 and Its High-Level Production in E. coli
1 Central Laboratory, Rengo Co., Ltd., Fukushima-ku, Osaka 553-0007; and 2 Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502
* To whom correspondence should be addressed at the present address: Life Science Laboratory, Shimadzu Corporation, Nakagyo-ku, Kyoto 604-8511. Tel: +81-75-823-1351, Fax: +81-75-823-1364, E-mail: ma-itou{at}shimadzu.co.jp
An organic solvent-resistant tyrosinase (OSRT) from Streptomyces sp. REN-21 is a unique enzyme showing high activity in the presence of organic solvents. The OSRT-catalyzed oxidation of monophenols such as tyrosine-containing peptides and proteins was examined. The catalytic properties of OSRT were compared with those of mushroom tyrosinase. OSRT was shown to oxidize Gly-L-Tyr most effectively among four peptide substrates tested. On the other hand, mushroom tyrosinase showed the highest activity toward L-Tyr-Gly under the condition of 1 mM substrate. OSRT oxidized several proteins, including casein and hemoglobin, with relatively higher activity compared with mushroom tyrosinase under the condition of 1% (w/v) substrate. Thus, it was clarified that the catalytic properties of OSRT toward tyrosine-containing peptides and proteins are different from those of mushroom tyrosinase under these conditions. The OSRT-encoding gene operon was cloned, and found to consist of two genes, designated ORF-OSRT and ORF-393. The former encodes apo-OSRT, and the latter encodes the putative activator protein of apo-OSRT. A binuclear copper-binding site (type-3 copper site) characteristic of tyrosinases is contained in the deduced amino acid sequence for apo-OSRT. A high-level production system for the OSRT was constructed using pET20b(+) and Escherichia coli BL21(DE3)pLysS. Approximately 54 mg of active OSRT was synthesized in a 1-liter broth culture by this system. The properties of the recombinant OSRT were similar to those of the wild-type enzyme. In conclusion, we succeeded in constructing a high-level production system for OSRT.