Studies on a Fibrinolytic Enzyme Produced From Marine Serratia marcescens subsp. sakuensis

Abstract

Fibrinolytic enzymes are agents/drugs that are responsible for the breakdown of fibrin in the blood clots. They find application in treatment of myocardial infarctions, ischemic strokes, cardiac and respiratory failure. This research work was aimed at isolating a fibrinolytic enzyme producing microorganism and to assess its suitability as a potential drug candidate for therapeutic applications. Six out of the eight bacteria isolated from the sea water sample tested positive for the fibrinolytic enzyme production during the initial screening experiments. Based on the results from screening experiments, one out of the six bacteria was chosen for further work and was identified as Serratia marcescens subsp. sakuensis (KU296189.1). The medium components were optimised by one-factor-at-a-time approach and Plackett-Burman design for enhanced production of fibrinolytic enzyme. A 3.4 fold increase in fibrinolytic enzyme activity was obtained with optimised production medium. The crude enzyme solution was then purified by a three step process involving ammonium sulphate precipitation, dialysis and size exclusion chromatography. Properties of the purified enzyme such as the molecular weight, optimum pH and temperature, stability at different pH and temperature, in vitro half-life, effect of metal ions and chemical reagents, in vitro clot lysing potential and thrombolytic mechanism, proteolytic activity against several substrates and its partial amino acid sequence were determined. Chemical modification of the purified enzyme was carried out using amino acid specific modifiers and modification with 2.5 mM EDAC resulted in a 9- fold increase in the fibrinolytic activity. The circular dichroism spectrum analysis of the modified and native enzyme revealed a similar structure except a few minor changes in a- helix and ß-sheet conformation of the enzymes. The findings suggest that the fibrinolytic enzyme produced in the present work could be considered as a potential candidate, safe for human use.