Application of Rotatable Central Composite Design for Optimization of Extracellular Alkaline Protease Production from newly isolated Bacillus pseudofirmus MSB4

Introduction: Microbial extracellularalkaline proteases are one of the most important industrial enzymes with a wide range of applications in various industries. In the present study, the production of an alkaline protease by Bacillus pseudofirmus MSB4 was evaluated using response surface methodology (RSM). Materials and methods: Four significant independent variables were selected, based on the results of one-factor-at-a-time (OFAT) approach (xylose concentration, beef extract concentration, pH and temperature), for rotatable central composite design (RCCD) to analyze the response pattern and to establish a model. The design consisted of total 30 runs, at five levels for each factor, with four replications of the center points. In addition, the presence of extracellular alkaline protease genes was evaluated by using PCR. Results: Maximum protease production (185.397 U/ml), 2.2 times higher than that of OFAT method and 3.7 times higher than the unoptimized conditions, was obtained by using 3% w/v beef extract, at pH 9 and 37ºC according to the results of RCCD. Furthermore, the genes sub I, II and apr with the expected size (319, 486 and 194 bp, respectively) corresponding to the extracellular alkaline serine protease and metalloprotease were detected in MSB4 by using PCR. Discussion and conclusion: The experimental data well fitted the model (Adj R2: 0.9982) and the established quadratic model has a great ability to predict responses for new observations (Pred R2: 0.9967). The results showed that the OFAT and RSM strategies were a useful screening and optimization method for enhancing protease production of MSB4, respectively