Purification, characterization, and cloning of fibrinolytic metalloprotease from Pleurotus ostreatus mycelia.
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 35 degrees C, respectively. PoFE effectively hydrolyzed fibrinogen, preferentially digesting the A alpha-chain and the B beta-chain over the gamma-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 chymotrypsin-like 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.
Shen
Ming-Hua
MH
Department of Biotechnology, BK 21 Research Team for Protein Activity Control Gwangju 501-759, Korea.
Kim
Jae-Sung
JS
Sapkota
Kumar
K
Park
Se-Eun
SE
Choi
Bong-Suk
BS
Kim
Seung
S
Lee
Hyun-Hwa
HH
Kim
Chun-Sung
CS
Chun
Hong-Sung
HS
Ryoo
Cheon-In
CI
Kim
Sung-Jun
SJ
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003067:Coenzymes / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005337:Fibrin / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D045726:Metalloproteases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D025282:Mycelium / Q000201:enzymology*; D009842:Oligopeptides / Q000378:metabolism; D016366:Open Reading Frames; D020076:Pleurotus / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D001426:Bacterial Proteins; D002413:Cations, Divalent; D003067:Coenzymes; D004791:Enzyme Inhibitors; D008670:Metals; D009842:Oligopeptides; C414985:S 2586; D005337:Fibrin; D004492:Edetic Acid; D045726:Metalloproteases