Optimization of Xylanase Enzyme Production Using Recombinant Escherichia Coli By Response Surface Methodology (RSM)

Escherichia coli BL21 (DE3) with a plasmid vector pET-22b(+) carrying xylanase coding gene isolated from an extreme thermophilic bacteria, Thermotoga sp. is used in this study for the enhanced production of recombinant xylanase. The study was focused on optimization of various physiochemical factors effecting cell growth and xylanase expression.  The statistical approaches based on Response Surface Methodology (RSM) were used to optimize the fermentation media and induction strategy in the shake flask experiments. A General Linear full factorial model was used to screen various nutrient sources in the media. A maximum xylanase activity of 48 IU mL^-1 was observed in maltose and (NH₄)₂ HPO₄ based defined media at pH 7 and temperature 37°C. The concentration of each screened nutrients in the media were then optimized by a Box-Behnken Design and an enhanced cell growth was observed at 16.2 gL^-1 of maltose and 12.7 gL^-1 of (NH₄)₂ HPO₄. Xylanase gene expression was further optimized using RSM based experimental design. Factors such as inducer type, inducer concentration, induction initiation time, post induction incubation temperature were optimized at this phase. Induction at mid log phase with 3.4 gL^-1 lactose with post induction incubation temperature at 32°C was found the optimum for enhanced xylanase gene expression. The Xylanase activity was found improved up to 65 IU mL^-1 with statistically optimized lactose based induction strategy and was found relatively higher yield and economic way of production than the IPTG based induction (38 IU mL^-1) strategies used in previous studies with unoptimized media.