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: Guangyan Cheng, Liying He, Zhibin Sun, Zhongli Cui, Yingxiang Du, Yi Kong
• Journal: Journal of microbiology and biotechnology (J Microbiol Biotechnol) Vol. 25 Issue 9 Pg. 1449-59 (Sep 2015) ISSN: 1738-8872 [Electronic] Korea (South)
• PMID: 26017226 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• DNA, Bacterial
• Enzyme Inhibitors
• Fibrin
• Plasminogen
• Serine Proteases
• Fibrinolysin

Topics

• Chemical Precipitation
• Chromatography, Gel
• Chromatography, Ion Exchange
• Chromatography, Liquid
• DNA, Bacterial (chemistry, genetics)
• Enzyme Inhibitors (analysis)
• Enzyme Stability
• Fibrin (metabolism)
• Fibrinolysin (chemistry, isolation & purification, metabolism)
• Hydrogen-Ion Concentration
• Molecular Weight
• Open Reading Frames
• Plasminogen (antagonists & inhibitors)
• Sequence Analysis, DNA
• Serine Proteases (chemistry, isolation & purification, metabolism)
• Soil Microbiology
• Streptomyces (enzymology, isolation & purification)
• Tandem Mass Spectrometry
• Temperature