Purification, Characterization And Gene Cloning Of A Novel Fibrinolytic Enzyme From Streptomyces Sp. And Studies On Its Fibrinolytic Mechanism

A novel protease with fibrinolytic activity designated as SW-1 , was isolated and purified from the fermentation broth of Streptomyces sp. strain Y405, a soil isolate. The characterization, fibrinolytic mechanism, primary pharmacodynamics and gene cloning of SW-1 were studied.SW-1 was purified by fractional precipitation with ammonium sulphate in 40-80% saturation, decolorization on 290 resin, gel filtration on Sephadex G75, anion-exchange chromatography on DEAE Sephadex A25 (eluted with linear gradiant of 0-1.0 mol/L sodium chloride) and affinity chromatography on Lysine Sepharose 4B (eluted with linear gradiant of 0-0.5mol/L sodium chloride) from the fermentation broth of Streptomyces sp. strain Y405 which was cultured for 96 hours. About 4.2mg purified enzyme per liter of fermentation broth was obtained and the recovery yield was 12.0%. The purified enzyme showed specific activity of 2952.3 urokinase unit per milligram, which increased by 230.6 fold over the fermentation broth. The purity of the purified enzyme determined by HPLC was 83.5%. The large-scale purification of SW-1 succeeded by scale-up the above purification procedure.SW-1 is a single chain peptide with a molecular weight of 30,000 Dalton in SDS-PAGE and an isoelectric point of 8.5. The N-terminal sequence of the enzyme purified by HPLC isArg/Asn/Phe-Pro/Asp-Gly-Met-Thr-Met-Thr-Ala-Ile-Ala-Asn-Gln-Asn-Thr-Gln-Ile-AsnThere is nonhomogeneity in the first and second amino acid residue of its...