期刊
SCIENTIFIC REPORTS
卷 7, 期 -, 页码 -出版社
NATURE RESEARCH
DOI: 10.1038/s41598-017-01813-1
关键词
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资金
- Austrian Science Fund (FWF) [P25217]
- DESY [P11, I-20120633 EC]
- ESRF [ID23-2, mx1616]
- Austrian Science Fund (FWF) [P 25217] Funding Source: researchfish
Tyrosinases are an ubiquitous group of copper containing metalloenzymes that hydroxylate and oxidize phenolic molecules. In an application context the term 'tyrosinase' usually refers to 'mushroom tyrosinase' consisting of a mixture of isoenzymes and containing a number of enzymatic side-activities. We describe a protocol for the efficient heterologous production of tyrosinase 4 from Agaricus bisporus in Escherichia coli. Applying this procedure a pure preparation of a single isoform of latent tyrosinase can be achieved at a yield of 140 mg per liter of autoinducing culture medium. This recombinant protein possesses the same fold as the enzyme purified from the natural source as evidenced by single crystal X-ray diffraction. The latent enzyme can be activated by limited proteolysis with proteinase K which cleaves the polypeptide chain after K382, only one The latent enzyme can amino acid before the main in-vivo activation site. Latent tyrosinase can be used as obtained and enzymatic activity may be induced in the reaction mixture by the addition of an ionic detergent (e.g. 2 mM SDS). The proteolytically activated mushroom tyrosinase shows >50% of its maximal activity in the range of pH 5 to 10 and accepts a wide range of substrates including mono-and diphenols, flavonols and chalcones.
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