A STATISTICAL AND DOWNSTREAM APPROACH FOR THE IMPROVEMENT OF PROTEASE PRODUCTION FROM BACILLUS TOYONENSIS VKB5 ISOLATED FROM ACTINIDIA DELICIOSA

Abstract

Endophytes are endosymbiotic microbes that reside inside the plant without affecting the host. Bacillus spp., are known to produce highly active extracellular proteases. This study aims for the optimization and partial purification of fibrinolytic enzyme production from Bacillus toyonensis VKB5 isolate from the fruit Actinidia deliciosa. Optimization studies on medium components show that maximum enzyme production can be achieved in alkaline conditions of pH 8 with nutrient sources mannitol, yeast extract and ammonium chloride with 1% (w/v) concentration. The statistical optimization studies using Plackett-Burman and Response surface methodology determines that the interaction of yeast extract with the mannitol and ammonium chloride enhances enzyme production up to 2 fold. The purification of the enzyme from ammonium sulfate to gel filtration step describes that purity of protein enhance up to 7.4 with 68.9% recovery as compared to the crude enzyme. The molecular weight was determined to be 21.9 kDa using Sodium Dodecyl Sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE). The purity of the protease was analyzed using High-performance liquid chromatography (HPLC). Further studies on the effect of temperature, pH, inhibitors, detergents and metal ions confirm that the purified enzyme could be alkaline serine protease that can withstand thermal conditions up to 60 °C. The study concludes that the purified enzyme showing fibrinolytic activity having strong stability which can be further exploited for medicinal purpose.