Optimization of thermostable amylopullulanase production in solid state fermentation by Clostridium thermosulfurogenes SVM17 through Plackett-Burman and response surface methodological approaches

Abstract

A total of fifteen nutrients comprising five carbon sources, four complex organic sources and three each of nitrogen and trace mineral sources were screened in a total of sixteen experiments using Plackett-Burman design for production of thermostable amylopullulanase by Clostridium thermosulfurogenes SVM17 in solid state fermentation (SSF) system. The design comprises screening of ‘n-1’ variables in ‘n’ number of experiments. Yield of enzyme was statistically analyzed to obtain for regression coefficients and t-values. Among fifteen nutrients screened, based on their effect in terms of product promoting ability, maltose, bajra flour, peptone, CaCl2·H2O and MnCl2·4H2O were considered promising nutrients for enzyme production and selected for optimization of their concentration using response surface methodology based on the central composite rotatable design (CCRD). The design comprises a total of 54 experimental trials with first 32 organized in a fractional factorial design and experimental trials from 33–40 and 51–54 involving the replication of central points. The experimental trials from 41–50 are called star points or axial points. The design was applied to determine the effects of above medium components and their mutual interactions on amylopullulanase production. Within the tested range of concentrations, except CaCl2·2H2O, all other components had significant effect on enzyme production. The optimum level of medium components for maximum production of the enzyme was (% w/w): maltose, 22.0; peptone, 0.8; MnCl2·4H2O, 50 ppm; CaCl2·2H2O, 15 ppm and bajra flour, 11.0.