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Purification and characterization of fibrinolytic enzyme from cultured mycelia of Armillaria mellea.

Abstract：

:A fibrinolytic enzyme was purified from the cultured mycelia of Armillaria mellea by ion-exchange chromatography followed by gel filtration, and was designated A. mellea metalloprotease (AMMP). The purification protocol resulted in a 627-fold purification of the enzyme, with a final yield of 6.05%. The apparent molecular mass of the purified enzyme was estimated to be 21kDa by SDS-PAGE, fibrin-zymography and gel filtration chromatography, which revealed a monomeric form of the enzyme. The optimal reaction pH value and temperature were, pH 6.0, and 33 degrees C, respectively. This protease effectively hydrolyzed fibrinogen, preferentially digesting the Aalpha-chain over the Bbeta- and r-chains. Enzyme activity was inhibited by Cu(2+) and Co(2+), but enhanced by the addition of Ca(2+) and Mg(2+) ions. Furthermore, AMMP activity was potently inhibited by EDTA, and was found to exhibit a higher specificity for the substrate S-2586 for chymotrypsin, indicating that the enzyme is a chymotrypsin-like metalloprotease. The first 24 amino acid residues of the N-terminal sequence were MFSLSSRFFLYTLCL SAVAVSAAP, which is extremely similar to the 24 amino acid residues of the N-terminal sequence of the fruiting body of A. mellea. These data suggest that the fibrinolytic enzyme AMMP, obtained from the A. mellea exhibits a profound fibrinolytic activity. The mycelia of A. mellea may thus represent a potential source of new therapeutic agents to treat thrombosis.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Lee SY,Kim JS,Kim JE,Sapkota K,Shen MH,Kim S,Chun HS,Yoo JC,Choi HS,Kim MK,Kim SJ

doi

10.1016/j.pep.2005.05.004

keywords:

subject

Has Abstract

pub_date

2005-09-01 00:00:00

pages

10-7

issue

1

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(05)00175-0

journal_volume

43

pub_type

杂志文章

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