Purification and characterization of a novel fibrinolytic enzyme from Streptomyces spp.
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 purification procedure involved ammonium sulphate fractionation, decolorization on 290 resin, gel filtration on Sephadex G75, anion-exchange chromatography on DEAE Sephadex A25, and affinity chromatography on Lysine Sepharose 4B. About 4.2 mg purified enzyme was obtained from a liter of fermentation broth and the recovery yield was 12.0%. The purified enzyme showed the specific activity of 2952.3 urokinase units per milligram, which was increased by 230.6 fold over the fermentation broth. The purity determined by HPLC was 83.5%. SW-1 is a single chain polypeptide with a predicted molecular weight of 30 kDa in SDS-PAGE and an isoelectric point of 8.5. The N-terminal sequence of SW-1 is R/N/F-P/D-G-M-T-M-T-A-I-A-N-Q-N-T-Q-I-N. There may be nonhomogeneity in the first and second amino acid residue of its N-terminal sequence. The analysis of amino acid composition showed that SW-1 consisted of 262 amino acids. The fibrinolytic activity of SW-1 was entirely inhibited by 10 mmol/L PMSF, 1 mmol/L EDTA and 1 mol/L lysine, respectively, suggesting that SW-1 is a serine protease and metalloprotease, and that the lysine binding site might play a role in the activity. The fibrinolytic activity of SW-1 is stable between 4-37 degrees C and pH 4.0-9.0, and the optimum pH is 8.0. On plasminogen-free fibrin plates, SW-1 showed the same fibrinolytic activity as the mixture of SW-1 with plasminogen, indicating that SW-1 is a fibrinolytic enzyme which affects fibrin directly, but not a plasminogen activator which affects fibrin by activating plasminogen.
Wang
J
J
Institute of Medicinal Biotechnology, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China.
Wang
M
M
Wang
Y
Y
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005337:Fibrin / Q000737:chemistry; D005343:Fibrinolytic Agents / Q000737:chemistry / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases / Q000737:chemistry / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D020539:Sequence Analysis, Protein; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification*; D013302:Streptomyces / Q000737:chemistry*; D013696:Temperature
D001426:Bacterial Proteins; D005343:Fibrinolytic Agents; D005337:Fibrin; C406280:SW-1 enzyme; D012697:Serine Endopeptidases; D008666:Metalloendopeptidases