눈꽃동충하초로부터 혈전분해효소의 정제 및 특성분석

Abstract

ABSTRACT

Purification and characterization of fibrinolytic enzyme from Paecilomyces japonica

Kim, Hoi-Chang Advisor : Prof. Kim, Sung-Jun, Ph. D. Department of Biotechnology, Graduate School of Chosun University

This study was carried out to investigate the purification and characteristics of the fibrinolytic protease from medical cordyceps, Paecilomyces japonica. The fibrinolytic enzyme was purified by using DEAE sepharose CL-6B fast flow chromatography followed by Sephadex G-75 gel filtration and POROS 20 HQ chromatography. The final specific activity of purified enzyme was 1431.81 units per milligram and increased 550.69 fold comparing homogenate and the final recovery yield was 3.8%. The apparent molecular weight of purified fibrinolytic enzyme was estimated to be about 14 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The optimal reaction pH value and temperature for the enzyme activity were pH 5.0 and 35℃, respectively. The purified enzyme had fibrigenolytic activity showing that the protease rapidly hydrolyzed the Aα-chain followed by the Bβ-chain. The purified protease also hydrolyzed fibrin, preferentially digesting the α-chains, followed by γ- γ chains. Interestingly the γ- chains of fibrinogen and the β-chains of fibrin were not hydrolyzed. The activity of the purified enzyme was totally inhibited by ZnCl2, and moderately MgCl2, CoCl2, or MnCl2, but enhanced by the additions of CaCl2 metal ions. The protease activity was potently inhibited by PMSF(serine protease inhibitor), and it was found to exhibit a higher specificity for the chromogenic substrate S-2222 and S-2765 for factor Xa, indicating that the purified enzyme is a factor Xa-like serine protease. The N-terminal amino acid sequence of purified enzyme was identified to be AQNIGAVVNLSPPKQ. Therefore, the purified enzyme from Paecilomyces japonica can be an effective thrombolytic source.