Optimization of culture media for extracellular expression of Trichoderma reesei endoglucanase II in Escherichia coli using response surface methodology (RSM)

Cellulose is considered to be the most abundant carbohydrate polymer on the earth. Bioconversion of cellulosic biomass has recently garnered a lot of attention for sustainable production of eco-friendly biofuels and chemicals. Enzymatic total hydrolysis of cellulosic biomass could break it down into its constituent monomeric sugars which could be further fermented into commercial products such as alternative fuels. Endoglucanase (EC 3.2.1.4) capable of hydrolyzing the internal -1,4-glycosidic bonds of cellulose and disrupting its crystalline structure which exposes the individual cellulose polysaccharide chains. Enzyme production costs remain to be a limiting factor in the industrial synthesis of bioethanol. A great deal of research has been geared to decrease costs of enzyme production (down to 20–30 cents per gallon of ethanol), yet less-sustainable corn-derived fuel remains to be a cheaper alternative (3–4 cents per gallon). An appealing strategy for further reducing of enzyme production costs involves the optimization of the culture medium composition and the expression conditions for recombinant production. In this study, the expression of synthetic Trichoderma reesei endoglucanase II gene (EGII) in E. coli was greatly improved by adjusting the expression condition. In this regard, a recombinant gene was designed and codon optimized for periplasmic expression this protein. Then, gene subcloning employed to insert the synthesized gene into the pET-26b expression vector. Thereafter, the response surface methodology method was employed to design 20 experiments to find out the optimum points for isopropyl β-D-1-thiogalactopyranoside concentration, post-induction period and cell density of induction (OD600). The expression fluctuations were assessed using standard activity assay. Our results indicated that the synthetic EGII gene was successfully codon optimized and subcloned into the expression vector. The enzyme activity results revealed that the optimum levels of the selected parameters are0.331 mM for isopropyl β-D-1-thiogalactopyranoside concentration, 10.98 H for the post-induction period and 3.41 for cell density (OD600). These optimized conditions led to a 1.7-fold increase in the EGII activity which is highly promising for large-scale EGII overexpression for industrial applications

Keywords:

Endoglucanase , Trichoderma reesei , Recombinant protein , RSM