Isolation characterization and applications of an alkaliphilic thermo tolerant protease from Bacillus species

Abstract

ABSTRACT newlineAlkaline protease of microbial origin posses considerable industrial potential due to their biochemical diversity and wide application in food industry, medicinal formulation, detergents, waste treatment and silver recovery. The protease producing bacteria were screened in glucose yeast casein agar medium using casein as the substrate. The bacterial isolate ATP-P5 isolated from Tatapani area of Mandi district of H.P (India) showed maximum zone of hydrolysis on casein agar plate was selected for further optimization studies. The isolate was identified as Bacillus subtilis based on morphological, biochemical and molecular characterizations 16S rRNA. The isolate was able to grow under alkaline conditions at pH 9.0 and a temperature of 50°C. 1.9 fold purification of enzyme following ammonium sulphate precipitation and DEAE-cellulose chromatography (Ion Exchange chromatography) was achieved. 29KDa molecular weight alkaline protease was obtained by SDS-Polyacryamide gel electrophoresis. Interestingly Ca2+ (5mM) activated enzyme activity, while Mg2+, Mn2+ Co2+ moderately activated enzyme activity, where as Fe2+ and Cu2+ inhibited the activity of enzyme. The purified protease was highly compatible with the detergents and help in removing protein based stain from clothes. Purified alkaline protease was also used for recovery of silver from waste X-Ray films and about 0.054g silver was recovered from X-Ray film. This indicated that alkaline protease from Bacillus subtilis has a potential of being applied for recovery of silver from waste X-Ray film as well as for biofilm degradation. In the present study 0.2 ml of purified protease has been used for degradation of the biofilm from Pseudomonas mendocina and E.coli in 10 minutes. Purified alkaline protease from Bacillus subtilis was efficiently immobilized by entrapment method using 0.2M CaCl2 and 3% sodium alginate. The immobilized enzyme retained its optimum activity upto 24 days of incubation and was stable at 9.0 pH and temperature 50°C. newline