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KMID : 0545120070170081271
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Journal of Microbiology and Biotechnology
2007 Volume.17 No. 8 p.1271 ~ p.1283
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Purification, Characterization, and Cloning of Fibrinolytic Metalloprotease from Pleurotus ostreatus Mycelia
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Shen Ming-Hua
Kim Jae-Sung
SAPKOTA KUMAR
Park Se-Eun
Choi Bong-Suk
Kim Se-Ung
Lee Hyun-Hwa
Kim Shun-Sung
Chun Hong-Sung
Ryoo Cheong-In
Kim Sung-Jun
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Abstract
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A fibrinolytic protease (PoFE) was purified from the cultured mycelia of the edible oyster mushroom Pleurotus ostreatus, using a combination of various chromatographies. The purification protocol resulted in an 876-fold purification of the enzyme, with a final yield of 6.5%. The apparent molecular mass of the purified enzyme was estimated to be 32 kDa by SDS-PAGE, fibrin-zymography, and size exclusion using FPLC. The optimal reaction pH value and temperature were pH 6.5 and 35oC, respectively. PoFE effectively hydrolyzed fibrinogen, preferentially digesting the Aα-chain and the Bβ-chain over the γ-chain. Enzyme activity was enhanced by the addition of Ca2+, Zn2+, and Mg2+ ions. Furthermore, PoFE activity was potently inhibited by EDTA, and it was found to exhibit a higher specificity for the chromogenic substrate S-2586 for chymotrypsin, indicating that the enzyme is a chymotrypsinlike metalloprotease. The first 19 amino acid residues of the N-terminal sequence were ALRKGGAAALNIYSVGFTS, which is extremely similar to the metalloprotease purified from the fruiting body of P. ostreatus. In addition, we cloned the PoFE protein, encoding gene, and its nucleotide sequence was determined. The cDNA of cloned PoFE is 867 nucleotides long and consists of an open reading frame encoding 288 amino acid residues. Its cDNA showed a high degree of homology with PoMEP from P. ostreatus fruiting body. The mycelia of P. ostreatus may thus represent a potential source of new therapeutic agents to treat thrombosis
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KEYWORD
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Pleurotus ostreatus, mushroom, mycelia, fibrinolysis, metalloprotease
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