Production, purification and characterization of a thermostable alkaline serine protease from Bacillus lichniformis NMS-1

Abstract

Alkaline proteases are used in food industry, leather tanning and processing industry, preparation of pharmaceuticals and also in the fiber industry. An alkaline serine protease producing strain was isolated using soil sample from a natural hot water spring in Sri Lanka. It was identified based on morphological, biochemical and 16s rRNA identifications as Bacillus licheniformis NMS-1. The extacellular protease enzyme was purified by two steps procedure involving ammonium sulfate precipitation followed by DEAE-Sephadex A-25 gel chromatography. The purification gave a 56 fold increase of the specific activity with a yield of 16%. The optimal pH and optimal temperature of the protease were pH 9 and 60C, respectively. The protease was relatively stable between 20-80°C. The enzyme was stable within the pH values of 8-12. The K m and V max values calculated from Lineweaver-Burk plot were 2.7x10-3 mg/ml and 263 mU/mg. Among the protease inhibitors that were tested, PMSF completely inhibited the enzyme activity indicating that the protease is a serine protease. The enzyme retained more than 50% of its activity after 60 min incubation at 60C. The major protease types used commercially are heat stable alkaline proteases. Alkaline serine proteases are enzymes that cleave peptide bonds in protein in which serine serves as the nucleophilic amino acid at the enzyme active site. Properties of this protease have shown it's suitability for industrial applications such as detergent industry.

Production of subtilisin proteases in bacteria and yeast

“…The source of one of the promising genes encoding a serine protease is strain Bacillus licheniformis NMS-1 extracted from soil near a natural hot spring in Sri Lanka (Mathew, Gunathilaka, 2015). This protein serves for the creation of detergents.…”