Box–Behnken experimental design for the optimization of enzymatic saccharification of wheat bran

Abstract

Lignocellulosic raw materials have been widely studied for obtaining bioproducts. Pretreatments for delignification, uncertainties in feedstock composition, long hydrolysis times, and large quantities of expensive enzymes are factors that limit fermentable sugar production. Statistical optimization can help adjust these limiting factors and variables of enzymatic saccharification. This study aimed to optimize conditions for fermentable sugar production using wheat bran, Botrytis ricini URM 5627 endoglucanase, and Box–Behnken experimental design, a response surface methodology. A zymographic assay in polyacrylamide gel was stained with Congo red after hydrolysis. Enzymatic activity measurements indicated enzyme stability at different temperatures (− 20 °C and 6 °C) and periods (up to 120 days). Wheat bran was characterized using Fourier transform infrared spectroscopy (FTIR) before and after enzymatic saccharification. Saccharification time, substrate load, and enzyme load were optimized using the Box–Behnken design. The zymogram demonstrated a single activity band around 39 kDa, the molecular weight of B. ricini URM5627 endoglucanase. Enzyme activity was preserved at 78.78% and 86.08% after storage for 120 days at − 20 °C and 6 °C, respectively. FTIR analyses showed cellulose delignification and loosening after enzymatic action. Maximum production of reducing sugars (116.93 mg g−1) occurred when conditions were maintained at the central point of planning (8 h, 2% substrate load, and 4% enzyme load). The enzyme isolated from B. ricini was stable in solid medium and in stored form, and could saccharify wheat bran without pretreatments. High yield of reducing sugars and proximity between predicted and real values demonstrated effectiveness of statistical optimization in saccharification of lignocellulosic biomass.