Enhanced production of extracellular proteolytic enzyme excreted by a newly isolated Bacillus subtilis FBL-1 through combined utilization of statistical designs and response surface methodology

Abstract

An extracellular protease producing strain FBL-1 was newly isolated from soil, and it was identified as Bacillus subtilis through 16S rDNA sequence analysis. B. subtilis FBL-1 was used for extracellular protease production, and culture conditions were optimized by statistical methods. Three statistical approaches such as Plackett–Burman design, steepest ascent path analysis, and Box–Behnken design were successfully combined to optimize protease production, which resulted in significant enhancement of production. Through Plackett–Burman experimental design, fructose and yeast extract were screened as the significant components for the production of extracellular protease. The center points of each parameter were predetermined by the steepest ascent method. Based on the results obtained by Box–Behnken experimental design, the optimized level of significant parameters for protease production was determined by multiple regression analysis. The optimized levels of parameters were fructose 32.42 g L⁻¹, yeast extract 15.02 g L⁻¹, and incubation time 35.78 h. Protease activity predicted by the quadratic model developed in this work was 578.55 U mL⁻¹, which was only 2.67% different from the experimental protease activity obtained by verification studies. The protease activity was significantly enhanced by 7.0-fold compared with the activity obtained from basal medium.