Purification, Characterization and Applications of Proteases Produced by Bacillus amyloliquefaciens 35s Isolated from Soil of the Nile Delta of Egypt

Aims: The current study aimed at purifying and determining general characteristics of proteases produced by Bacillus amyloliquefaciens 35s isolated from the soil of Nile Delta of Egypt and to study its applications in environmental and industrial purposes.
Study Design: The produced proteases were purified by ammonium sulfate precipitation method followed by dialysis. The effects of some physical and chemical factors on the activity and stability of the partially purified proteases were determined. The produced proteases were tested in various environmental and industrial applications.
Place and Duration of Study: Department of Agricultural Microbiology, Faculty of Agriculture, Ain Shams University, between July 2014 and September 2014.
Methodology: Proteases produced in optimized medium and conditions were extracted by centrifugation, purified by ammonium sulfate precipitation and followed by dialysis. The effects of pH, temperature, metal ions, inhibitors, organic solvents, H2O2 and surfactants on enzyme’s activity and stability were determined. Protease’s ability to hydrolyze gelatin was employed to recover silver from used X-ray films, and used a detergent additive for stain removal from cloths. Both proteases and the producing isolate were used to digest chicken feathers to produce amino acids, peptides and soluble proteins. All generated data were analyzed using one-way ANOVA with post hoc multiple comparison analysis using Tukey’s HSD.
Results: Ammonium sulfate at 70% had the best effect on precipitating the produced protease increasing specific activity to 5143 (u/mg), a 2.6 fold of that of the crude enzyme (1408.5 u/ml). Characterization experiments showed optimum conditions for activity to be pH 9 and 60ºC. Ca2+, Mn2+, Pb2+, Mg2+, Co2+ , Zn2+ and Ba2+ negatively affected the enzyme activity at 5mM. The enzyme was stable at low concentrations of oxidizing agent and surfactants and retained nearly 100% and 65% activity in presence of 0.1% and 0.5% of SDS, respectively. Purified proteases was 23 fold active than the commercial Savinase® under the standard assay conditions. As a detergent additive, it was effective in removing blood and egg stains from cotton fabric when combined with detergent. In digesting chicken feather, the isolate showed maximum caseinase activity of 645 u/ml in the initial screening process and an ability to degrade raw feather to free protein hydrolysates under optimal conditions (after 96 h of fermentation in 30 g/l chicken feather and pH 8.0).
Conclusion: Optimum pH indicated alkalinity class and optimum indicated thermal classification. Purified proteases was 23 fold more active than the commercial Savinase® under the standard assay conditions. Applications of the partially purified proteases proved it to be a good profitable industrial tool.