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国际药用蘑菇期刊
影响因子: 1.423 5年影响因子: 1.525 SJR: 0.431 SNIP: 0.661 CiteScore™: 1.38

ISSN 打印: 1521-9437
ISSN 在线: 1940-4344

国际药用蘑菇期刊

DOI: 10.1615/IntJMedMushrooms.2018025810
pages 349-358

Purification and Characterization of a Novel Protease from the Inky Cap Mushroom, Coprinopsis atramentaria (Agaricomycetes)

Weiwei Zhang
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China; State Key Laboratory for Agrobiotechnology and Department of Microbiology, China Agricultural University, Beijing, China
Xueran Geng
College of Food Science and Engineering, Shanxi Agricultural University, Taigu, Shanxi, China
Wenwen Chen
State Key Laboratory for Agrobiotechnology and Department of Microbiology, China Agricultural University, Beijing, China
Hexiang Wang
State Key Laboratory for Agrobiotechnology and Department of Microbiology, China Agricultural University, Beijing, China
Tzi Bun Ng
School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China

ABSTRACT

A novel protease was isolated from Coprinopsis atramentaria, which is, to our knowledge, the first macromolecule to be purified from this species. The protease, referred to as CAP, was purified through the use of ion exchange chromatography on sulphopropyl-sepharose, diethylaminoethyl-cellulose, Q-Sepharose, and gel filtration on a Superdex 75 column. CAP is a monomelic protein with a molecular mass of 32 kDa. The maximum activity of CAP was detected at pH 7.0 and 50°C. The preferred pH of CAP demonstrated that it was a neutral protease. Kinetic constants were determined under optimal reaction conditions, using 1% casein as the substrate. We found Km and Vmax values of 7.98 mg · mL-1 and 215 μg · mL-1 · min-1, respectively. Among the various metal ions tested, Pb2+, Zn2+, Mn2+, Hg2+, Cu2+, and Cd2+ exerted dose-dependent inhibitory actions, whereas Mg2+ exhibited a promoting action at all concentrations tested. Eight inner peptide sequences were detected by liquid chromatography on an LTQ-Orbitrap mass spectrometer and were identified using the Basic Local Alignment Search Tool, which contains proteases from other sources. Alignment results showed that 2 of them were homologous to fungal cysteine proteases. The other 5 peptide sequences also shared similarities with proteases of other origins. The isolation of a novel protease from C. atramentaria in this study not only expands the sources of proteases but also provides further information about this fungus.


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