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KMID : 0545120110210030256
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Journal of Microbiology and Biotechnology
2011 Volume.21 No. 3 p.256 ~ p.262
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A Novel Metalloprotease from the Wild Basidiomycete Mushroom Lepista nuda
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Ying Ying Wu
H. X. Wang
T. B. Ng
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Abstract
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A 20.9-kDa metalloprotease was isolated from dried fruiting bodies of the wild basidiomycete mushroom Lepista nuda. The N-terminal amino acid sequence of the protease was seen to be ATFVLTAATNTLFTA, thus displaying no similarity with the sequences of previously reported metalloproteases. The protease was purified using a procedure that entailed ion-exchange chromatography on CM-Cellulose, Q-Sepharose, and Mono S, and FPLC-gel filtration on Superdex 75. The protease functioned at an optimum pH of 7.0 and an optimum temperature of 50oC. It was also noted that the protease demonstrated a proteolytic activity of 1,756 U/mg toward casein. The Km of the purified protease toward casein was 6.36 mg/ml at a pH of 7.0 and with a temperature of 37oC, whereas the Vmax was 9.11 μg ml-1 min-1. The activity of the protease was adversely affected by EDTA-2Na, suggesting that it is a metalloprotease. PMSF, EGTA, aprotinin, and leupeptin exerted no striking inhibitory effect. The activity of the protease was enhanced by Fe2+, but was curtailed by Cd2+, Cu2+, Hg2+, Pb2+, Zn2+, and Fe3+ ions. The protease also exhibited inhibitory activity against HIV-1 reverse transcriptase with an IC50 value of 4.00 μM. The IC50 values toward hepatoma Hep G2 and leukemia L1210 cells in vitro were 4.99 μM and 3.67 μM, respectively.
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KEYWORD
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mushroom, metalloprotease, Lepista nuda, purification
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