A low molecular weight chymotrypsin-like novel fibrinolytic enzyme from Streptomyces sp. CS624

Abstract

A proteolytic enzyme with fibrinolytic activity (FES624) was purified from recently isolated Streptomyces sp. CS624. SDS-PAGE of the enzyme showed a single polypeptide chain with molecular weight of 18 kDa, which is the lowest among the so far reported Streptomyces fibrinolytic enzymes. The enzyme activity was optimum and highly stable at pH 7.0, suggesting that it is a neutral enzyme. Furthermore, the activity was maximum at 60 °C and stable at or below 50 °C. In fibrin plate assay, FES624 showed stronger fibrinolytic activity than that of plasmin. It hydrolyzed Aα-, Bβ- and γ-chains of fibrinogen within 5, 10 and 150 min, respectively. FES624 showed higher specificity towards N-succinyl-Ala-Ala-Pro-Phe-pNA, a substrate for chymotrypsin. The activity was inhibited by serine protease inhibitor pefabloc as well as metalloprotease inhibitors EDTA and EGTA. In addition, metal ions showed varied effects on the activity. Altogether, these results suggest that FES624 is a chymotrypsin-like serine metalloprotease. K m and V max for the substrate N-succinyl-Ala-Ala-Pro-Phe-pNA were 0.218 mM and 84.03 mM min −1 mg −1, respectively. The first fifteen amino acid residues of the N-terminal sequence were APNVDAIYLPQYRLS, which are significantly dissimilar from the sequences of previously reported fibrinolytic enzymes; therefore, FES624 may be a new enzyme of its kind.