Screening and Enumeration of Rhizospheric Soil Bacteria for Production of Extracellular Protease

Plant rhizosphere is an important ecological niche having a rich array of plant released metabolites that provide carbon and nutrients for augmentation of a bioactive microbial zone. This diverse range of bacteria, including protease producers, plays a central role in nutrient cycling and biodegradation of organic matter. A comprehensive study was carried out to enumerate cultivable microflora underneath Areca catechu L., Averrhoa carambola L., Mangifera indica L. and Cycas circinalis L. and primarily screen their protease-producing ability in casein supplemented nutrient agar. It was observed that 85.2 % of 27 bacterial strains produced extracellular protease out of which six strains showed good proteolytic index (>1.7). Bacterium Bacillus CGR04 from mango rhizosphere, showed highest protease production under submerged growth in mineral salts medium. Optimal enzyme production occurred during the early stationary phase (24 h) in presence of 1% casein. Bacillus CGR04 showed maximum proteolytic activity at 1.2% (w/v) substrate (335.86 U/ml) and enzyme kinetics followed a linearized Line weaver Burk plot. The Km and Vmax values were calculated as 4.77 mg/ml and 454.55 U/ml, respectively. Enzyme production was optimal in presence of 1 % (w/v) glucose and peptone showing protease production of 279.6 U / ml and 304.84 U/ml respectively. Moreover, the study revealed that enzyme production was optimum at pH 7.5 (307.1 U / ml) and 35⁰C (297.3 U / ml) indicating production of a neutral protease. Furthermore, purification and characterization of the protease from Bacillus CGR04 could be of potential application for dairy and beverage industries.