Optimization of protease production using Bacillus velezensis through response surface methodology and investigating its applications as stain remover

The aim of this research work was to evaluate the potential of Bacillus velezensis for proteases production by employing eggshells and egg membrane powder as substrate followed by statistical optimization via RSM. While, optimizing the physical parameters maximum protease production was observed at incubation period of 72-h, inoculum size of 2% and substrate concentration of 1.5%. Optimization of nutritional parameters was done through PBD by running 12 experiments, among 7 screened factors (Glucose, peptone, KH₂PO₄, MgSO₄, NaCl, CaCl₂ and yeast extract), 2 factors (Glucose and CaCl₂) was found significant. Significant factors were further optimized through CCD and statistical analysis of results were also performed by running 2-Factor ANOVA. Maximum protease activity of 301.0010 U/mlU/ml was observed. Then, enzyme characterization revealed maximum protease activity at 45 ˚C, pH-8, with highest thermal stability at 60 ˚C. Furthermore, effects of metal ions (Ca^+ 2, Cu^+ 2, Mn^+ 2, Mg^+ 2, K⁺, Na⁺) on protease activity was also evaluated and results have shown Cu^+ 2 ions increase protease activity up-to 100-folds while Na⁺, Mg^+ 2 and Mn^+ 2 ions enhanced protease activity by 103.40, 115.42 and 106.01% respectively. The produced protease could be investigated further for potential application as excellent stain remover.