CHARACTERIZATION OF PROTEASE FROM PURE BACILLUS SUBTILIS STRAIN ISOLATED FROM KINEMA

Abstract

Proteases, a varied set of enzymes, perform critical roles in a numerous biological process and have grown in relevance in a variety of industrial applications. Microbial proteases, in particular, have emerged as significant in enzyme technology due to their flexibility, cost effectiveness, and eco-friendliness. This study aims to characterize the protease from the strain isolated from kinema. The strain 24 protease was characterized for optimum temperature and pH, temperature and pH stability, substrate specificity, and enzyme kinetics. The effect of various solvents, surfactants, and metal ions were also assessed. The strain 24 protease was purified with a purification fold of 2.02, specific activity of 10.27 U/mg and yield of 15.56% by ammonium sulphate precipitation (80%) followed by dialysis. The molecular weight of protease was determined to be 32.58 KDa, by Sodium dodecyl sulphate polyacrylamide gel electrophoresis. Gelatin zymography confirmed the proteolytic activity. The molecular characterization revealed that the strain 24 belongs to the genus Bacillus subtilis. The strain 24 protease was optimally active at pH 7 and 50℃) and was stable throughout a wide temperature (0℃- 60℃) and pH (5-10) range. The protease was active over different natural substrates, with the highest on casein. The strain 24 protease was very stable in the presence of nonionic surfactants, solvents, denaturants, and metal ions (Na+, K+, Ba++, Zn++, Al+++, Ca++, and Ba++), but was inhibited by Sodium dodecyl sulphate, Hg++, Fe+++, and Cu++. The Km and Vmax values were determined to be 1.06 ±0.107 mg/ml and 2.115±0.0050 µmole/ml/min, respectively. These findings suggest tremendous prospects for this protease due to its flexibility, substrate selectivity, stability, and genetic profile. Its properties make it an attractive option for a variety of applications in the food industry and biology.