title: Purification and Biochemical Characterization of a Novel Fibrinolytic Enzyme from Streptomyces sp. P3.
abstract: A novel proteolytic enzyme with fibrinolytic activity, FSP3, was purified from the recently isolated Streptomyces sp. P3, which is a novel bacterial strain isolated from soil. FSP3 was purified to electrophoretic homogeneity by ammonium sulfate precipitation, anion exchange, and gel filtration. FSP3 is considered to be a single peptide chain with a molecular mass of 44 kDa. The maximum activity of the enzyme was observed at 50°C and pH 6.5, and the enzyme was stable between pH 6 and 8 and below 40°C. In a fibrin plate assay, FSP3 showed more potent fibrinolytic activity than urokinase, which is a clinical thrombolytic agent acting as a plasminogen activitor. The activity was strongly inhibited by the serine protease inhibitor PMSF, indicating that it is a serine protease. Additionally, metal ions showed different effects on the activity. It was significantly suppressed by Mg(2+) and Ca(2+) and completely inhibited by Cu(2+), but slightly enhanced by Fe(2+). According to LC-MS/MS results, its partial amino acid sequences are significantly dissimilar from those of previously reported fibrinolytic enzymes. The sequence of a DNA fragment encoding FSP3 contained an open reading frame of 1287 base pairs encoding 428 amino acids. FSP3 is a bifunctional enzyme in nature. It hydrolyzes the fibrin directly and activates plasminogen, which may reduce the occurrence of side effects. These results suggest that FSP3 is a novel serine protease with potential applications in thrombolytic therapy.
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, P.R. China.
authors.forename: Guangyan
authors.initials: G
authors.lastname: Cheng
authors.affiliation: Department of Analytical Chemistry, China Pharmaceutical University, Nanjing 210009, P.R. China.
authors.forename: Liying
authors.initials: L
authors.lastname: He
authors.affiliation: School of Life Science, Nanjing Agriculture University, Nanjing 210095, P.R. China.
authors.forename: Zhibin
authors.initials: Z
authors.lastname: Sun
authors.affiliation: School of Life Science, Nanjing Agriculture University, Nanjing 210095, P.R. China.
authors.forename: Zhongli
authors.initials: Z
authors.lastname: Cui
authors.affiliation: Department of Analytical Chemistry, China Pharmaceutical University, Nanjing 210009, P.R. China.
authors.forename: Yingxiang
authors.initials: Y
authors.lastname: Du
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, P.R. China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Kong
mesh_terms: D011232:Chemical Precipitation; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004791:Enzyme Inhibitors / Q000032:analysis; D004795:Enzyme Stability; D005337:Fibrin / Q000378:metabolism; D005341:Fibrinolysin / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D016366:Open Reading Frames; D010958:Plasminogen / Q000037:antagonists & inhibitors; D017422:Sequence Analysis, DNA; D057057:Serine Proteases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D012988:Soil Microbiology; D013302:Streptomyces / Q000201:enzymology* / Q000302:isolation & purification; D053719:Tandem Mass Spectrometry; D013696:Temperature
chemical_list: D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D005337:Fibrin; D010958:Plasminogen; D057057:Serine Proteases; D005341:Fibrinolysin
keywords: Fibrinolytic activity; Purification; Serine protease; Streptomyces