Characterization of Detergent-compatible Alkaline Protease from Bacillus agaradhaerens MTCC 9416

Abstract

Aim: The present work deals with the purification and characterization of an alkaline protease produced by an alkalophilic bacterium, Bacillus agaradhaerens and establishment of its suitability as detergent additive. Methodology: Bacterial isolates producing alkaline protease were screened from diverse samples viz. soil, sewage and industrial effluents by enrichment culture technique. The taxonomic status and molecular characterization of the bacterium showing maximum alkaline protease activity was determined. The alkaline protease produced by the organism was purified its molecular size was determined by gel permeation chromatography. The purified enzyme was studied for its feasibility as detergent additive. Results: The bacterium under study was identified as Bacillus agaradhaerens by CSIR Institute of Microbial Technology (IMTECH), Chandigarh, India and deposited with an accession number MTCC 9416. The genotypic characterization of the 16S ribosomal DNA gene was performed and the sequence was submitted to NCBI under the name Bacillus agaradhaerens strain nandiniphanse5 (NCBI Accession No: JN703504). The alkaline protease with a molecular weight of approximately 25 kDa, demonstrated optimum activity at 55°C and pH 10.5, stability in pH range 7.0 to 12.0. The enzyme exhibited increased thermostability in presence of 25 mM CaCl2, enhanced activity in presence of chlorides of Ca2+, Mg2+,  K+, Co2+ and Mn2+. The protease exhibited highest degradation of casein followed by gelatin as compared to other protein substrates. The kinetic parameters were estimated to be 77.82 U/ml (Vmax) and 6.66 mg/ml (Km) using casein as substrate. The alkaline protease was also checked for its blood stain removal ability. The thermostable alkaline protease retained its activity in presence of detergent components with desired level stability and compatibility and therefore has a potential to be used commercially in the detergent industry. This is the first report on characterization of detergent-compatible alkaline protease from Bacillus agaradhaerens.