Catalytic properties of an organic solvent-resistant tyrosinase from Streptomyces sp. REN-21 and its high-level production in E. coli.
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.
Ito
Masaaki
M
Central Laboratory, Rengo Co. Ltd., Fukushima-ku, Osaka 553-0007, Japan. ma-itou@shimadzu.co.jp
Inouye
Kuniyo
K
D000363:Agaricales / Q000201:enzymology; D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D004156:Catechol Oxidase; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D011506:Proteins / Q000378:metabolism; D012997:Solvents; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity
D011506:Proteins; D012997:Solvents; D004156:Catechol Oxidase; D014442:Monophenol Monooxygenase