Purification and characterization of fibrinolytic metalloprotease from Perenniporia fraxinea mycelia.
In this study we purified and characterized a fibrinolytic protease from the mycelia of Perenniporia fraxinea. The apparent molecular mass of the purified enzyme was estimated to be 42kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), fibrin zymography and size exclusion using fast protein liquid chromatography (FPLC). The first 20 amino acid residues of the N-terminal sequence were ASYRVLPITKELLPPEFFVA, which shows a high degree of similarity with a fungalysin metallopeptidase from Coprinopsis cinerea. The optimal reaction pH value and temperature were pH 6.0 and 35-40 degrees C, respectively. Results for the fibrinolysis pattern showed that the protease rapidly hydrolyzed the fibrin alpha-chain followed by the beta-chain. The gamma-gamma chains were also hydrolyzed, but more slowly. The purified protease effectively hydrolyzed fibrinogen, preferentially digesting the Aalpha-chains of fibrinogen, followed by Bbeta- and gamma-chains. We found that protease activity was inhibited by Cu(2+), Fe(3+), and Zn(2+), but enhanced by the additions of Mn(2+), Mg(2+) and Ca(2+) metal ions. Furthermore, the protease activity was 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 mycelia of P. fraxinea may thus represent a source of new therapeutic agents to treat thrombosis.
Kim
Jae-Sung
JS
Department of Biotechnology and BK21 Research Team for Protein Activity Control, Chosun University, Gwangju 501-759, Republic of Korea.
Kim
Ji-Eun
JE
Choi
Bong-Suk
BS
Park
Se-Eun
SE
Sapkota
Kumar
K
Kim
Seung
S
Lee
Hyun-Hwa
HH
Kim
Chun-Sung
CS
Park
Yeal
Y
Kim
Myung-Kon
MK
Kim
Yoon-Sik
YS
Kim
Sung-Jun
SJ
D000595:Amino Acid Sequence; D004795:Enzyme Stability; D005343:Fibrinolytic Agents / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D045726:Metalloproteases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D025282:Mycelium / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D020072:Polyporales / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D013379:Substrate Specificity; D014934:Wood / Q000382:microbiology
D005343:Fibrinolytic Agents; D005656:Fungal Proteins; D045726:Metalloproteases