Impact of different carbon and nitrogen sources on protease production byBacillus sp. was investigated using Plackett Burman (PB) method. The inoculum concentration, incubation time, incubation temperature, medium pH, aeration and agitation were evaluated by conventional one factor at a time (OFAT) method. Fractional Factorial Central Composite Design (FFCCD) of Response Surface Methodology (RSM) was used to further optimize with respect to glucose (carbon source), soyabean meal (nitrogen source), inoculum concentration, medium volume, temperature and initial medium pH. High determination co-efficient (R2 = 0.9165) and lower value of the coefficient of variation (CV = 8.19%) indicated a better precision and reliability of experimental data. ANOVA indicated linear effect by inoculum concentration whereas; incubation temperature vs volume, pH vs volume and volume vs inoculum size were more significant for protease production at interactive level. Response surface data revealed medium pH, glucose and soyabean meal concentrations were most significant with respect to inoculum concentration. Software predicted optimized conditions were validated at shake flask and improved protease production to the tune of 37 % was observed. Incubation temperature and medium volume depicted maximum interactive influence among all selected factors. Glucose, soyabean meal and medium pH play critical regulatory role in protease production by this isolate. Considerable improvement of enzyme production 37 % was noticed at optimized environment at shake flask. Carbon and nitrogen source ratio and medium pH were the major regulatory factors in protease production by Bacillus sp. Significant improvement 37% in enzyme production was achieved at optimized environment. Among all selected fermentation parameters, medium volume was the most interactive factor and showed significant interaction with inoculum concentration and medium pH while inoculum concentration had linear effect on enzyme production.
