Biochemical characterization and homology modelling of cold-active alkophilic protease from Northwestern Himalayas and its application in detergent industry

Proteolytic enzymes are one of the largest classes of commercial enzymes used in industrial applications especially alkophilic protease is required by detergent industry. Cold-active alkophilic protease producing psychrotrophic bacterium was isolated from soil sediment of Wular lake Kashmir and identified as Bacillus subtilis WLCP1. The molecular weight of purified protease was 38 kDa, determined by SDS-PAGE. The optimal pH of the purified protease was 10.0 and was stable at pH 7.0–11.0. The optimum temperature of the enzyme was 15 °C, lowest to be reported for protease producing Bacillus sp. and can work up to 40 °C at pH 10. The protease activity was inhibited by phenylmethylsulfonyl fluoride (PMSF) while improved by Ca2+ and Cu2+ indicating the enzyme is serine alkaline protease. Purified genomic DNA from Bacillus subtilis WLCP1 was cloned and sequenced that encodes a protein of 378 amino acids. Two 3D structure models were built using the structure of Subtilisin E and SUBTILISN BPNꞌ as templates and concluded by Ramachandran Plot. The purified protease from Bacillus subtilis WLCP1 with detergent enhanced the removal of blood stains under cold conditions, making it extremely useful as detergent additive for cold washing due to its ability to work in alkophilic pH and low temperature ranges, and first ever to be reported from the North Western Himalayas, exploring the range of habitat for industrial enzymes.