Screening, Optimization of Production and Partial Characterization of Alkaline Protease from Haloalkaliphilic Bacillus Sp

Bacillus strains isolated from the salteren pond (Kakinada) were screened and identified for high alkaline protease activity. The isolates which were positive on skim milk agar (1%) were selected as protease producing strains. Of the ten bacterial isolates screened, isolate S-8 was observed as a potential haloalkaline protease producer and it was identified as Bacillus cereus strain S8 (MTCC NO: 11901) by 16S rRNA gene sequencing, phylogenetic tree analysis and by different biochemical tests. Protease production was enhanced by optimizing the culture conditions. The nutritional factors such as carbon and nitrogen sources, NaCl and also physical parameters like temperature, incubation time, pH, inoculum size were optimized for the maximum yield of protease. Studies on the effect of different carbon and nitrogen sources revealed that maximum protease production was obtained in the medium supplemented with Molasses,1%(w/v); Potassium nitrate, 0.75%(w/v); salt solution5%(v/v) {MgSo4.7H2O, 0.5%(w/v); KH2PO4, 0.5%(w/v)}; FeSO4.7H2O, 0.01%(w/v) and CaCO3, 0.5% respectively. Thus, with selected carbon and nitrogen sources along with 1 % NaCl and 2% inoculum the maximum protease production (205.0 U/ml) was obtained in the period of 72 h incubation at pH-12.0 under 160 rpm when compared to the initial enzyme production (165.0 U/ml). The crude enzyme extract of this strain was also characterized with respect to temperature, pH, incubation period and different concentrations of casein which was used as enzyme substrate. This study shows that the enzyme has wide range of pH stability from 8 to 11 with optimum activity at pH-10.0. It is thermostable with optimum activity at 70°C (392U/ml) with 1h incubation of enzyme with 1% casein as its substrate. From the above investigations it was concluded that the protease production by these microorganisms at wide temperatures and pH ranges could be explored for varied industrial applications.