Purification and Biochemical Characterization of a Novel Fibrinolytic Enzyme from Streptomyces sp. P3

J Microbiol Biotechnol. 2015 Sep;25(9):1449-59. doi: 10.4014/jmb.1503.03015.

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.

Keywords: Fibrinolytic activity; Purification; Serine protease; Streptomyces.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

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

Substances

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