Enhanced production of dehairing alkaline protease from Bacillus subtilis mutant E29 by consolidated bioprocessing using response surface modeling

Protease is used in various industries and the uprising demand urges for its enhanced production. Herein, strain improvement by mutagenic treatment with ethyl methanesulfonate and ultra-violet light was carried out in proteolytic Bacillus subtilis AKAL7. Mutants were screened through proteolytic clear zone ratio on the skim milk agar. The highest protease producer mutant E29 was selected for enhanced protease production in shake flask fermentation using one-factor-at-a-time (OFAT) and statistical methodology. The OFAT approach resulted in maximum level of protease (161.7 U/mL) after 24 h of fermentation at 30 °C, pH 10 in a basal medium. The statistical Plackett–Burman design predicted peptone, yeast extract, MgSO 4 , pH, and temperature as the significant factors among eight independent variables for the protease production. The response surface methodology (RSM) determined the optimal concentration of the significant factors for maximum protease production (741.284 U/mL). The experimental protease level (722.7 U/mL), which was ~5-fold of that produced with the basal media, validated the predicted values of RSM. The partial purification resulted in a final ~14-fold purified protease with specific activity of ~9508 U/mg protein and molecular weight ~40 kDa. The protease showed maximum activity at 50 °C and stability up to 45 °C for 1 h. However, 10 mM CaCl 2 increased its thermostability. Severe inhibition of protease activity by phenylmethylsulfonyl fluoride and HgCl 2 revealed the partially purified protease as serine and cysteine type, which was active at pH 7.0–12.0 with the optima at pH 10.0. The protease could substantially dehair the cow skin within 14 h of treatment and retain 98% activity after 30 days of lyophilization.