PB design considered insignificant dummy variables, which were used to estimate the experimental error and check the adequacy of the first-order model. In the present work, 10 variables were evaluated in 20 experimental trials (Table 2). All the trials were carried out in duplicate, and the average protease yield for each trial was used as the response variable. Regression analysis determined the variables that had a significant (95% level (P< 0.05)) effect on protease yield, and these variables were evaluated in further optimization experiments