Proteases Production by a Bacterial Isolate Bacillus amyloliquefaciens 35s Obtained from Soil of the Nile Delta of Egypt

Abstract

Aims: The present work was designed to investigate the production of proteases from an Egyptian bacterial isolate. Study Design: Samples were collected from soil, water and food, and screened for the isolation of proteases producing bacteria. The highest proteases producing isolate was selected for proteases production. Place and Duration of Study: Department of Agricultural Microbiology, Faculty of Agriculture, Ain Shams University, between March 2014 and September 2014. Methodology: Twenty samples were obtained. Primary screening of proteolytic bacteria was done by inoculating plates of skim milk agar. Secondary screening of proteolytic bacteria was done by quantitative determination of proteases concentration. One isolate from soil was chosen due its high proteolytic activity. Identification of the selected isolated was done 16S rRNA gene sequencing. Optimization of proteases production was conducted using a two-step approach. First, a quick identification of the important factors by simple screening experiments (this manuscript) including inoculum size, incubation period, carbon and nitrogen sources, temperature and pH. In a subsequent manuscript, application of complex response surface methodology for further optimization will be studied. Data generated from the above experiments were analyzed using one-way ANOVA with post hoc multiple comparison analysis performed using Tukey’s HSD. Results: In the first optimization step, the production of extracellular proteases was favored in the presence of starch, and peptone. A 2.1 fold increase in proteases production was obtained using the design space in the optimized medium as compared with the un-optimized basal medium. Enzyme production increased significantly with optimized medium (845 U/ml) compared to un-optimized medium (405 U/ml). Optimum production conditions were composed of incubation period of 24 h at 37ºC, pH 8 and agitation speeds of 120 rpm. Optimum medium for proteases production was composed of (g/l): peptone (10), starch (10), KH2PO4, MgSO4.7H2O, and CaCl2. Interaction between the variables and optimizing these variables will be studied in a later manuscript using a Plackett-Burman design and further with the Face Centered Central Composite Design (FCCCD) of Response Surface Methodology (RSM). Conclusion: This work succeeded to obtain a novel bacterial isolate capable of producing proteases and the production process was optimized by screening the physical and nutritional parameters influencing proteases production by employing one-variable-at-time approach.