Expression and characterization of recombinant endoglucanase from Trichoderma reesei for the production of cellulosic ethanol

Abstract

Bioethanol, unlike petroleum, is a form of renewable energy that can be produced from agricultural feedstocks. It can be used as a transport fuel, mainly as a biofuel additive for gasoline. The production of ethanol from lignocellulose has the advantage of abundant and diverse raw material compared to other sources like corn and cane sugars. However, it requires one more additional step; saccharification which the lignocellulosic biomass is hydrolyzed to glucose before converted to ethanol by fermentation. Recently, the hydrolysis of cellulose using biocatalysts, cellulase has been interestingly increased. Nevertheless, natural microbial cellulase cannot be sufficiently produced for the industrial application. Therefore, this research attempted to clone and express the genes of endoglucanase; eg1 and eg2 from Trichoderma reesei, one of the most effective producers of cellulase. From the results, the eg1 gene was composed of 713 bps, encoding 234 amino acids, molecular weight approximately 25 kD while eg2 gene was composed of 1257 bps, encoding 418 amino acids, molecular weight approximately 44 kD. The recombinant plasmid, pPICαA-eg1 and pPICαA-eg2, were transformed into Pichia pastoris X33 by electroporation. The transformants expressing the endoglucanase1 (EG1) and endoglucanase2 (EG2) were selected for optimizing expression conditions. The EG1 and EG2 were highly expressed by 4% and 5% methanol induction for 5 days, respectively. EG1 showed the highest activity at pH 5.0 and 60°C and it was stable at pH range of 4.0–5.5 and temperature below 75°C. EG2 showed the highest activity at pH 5.0 and 70°C and it was stable at pH range of 3.5–5.5 and temperature below 70°C. The steady-state kinetic study indicated that the EG2 was six folds greater catalytic efficiency toward carboxymethyl cellulose substrate than EG1. However, both of them could hydrolyze varieties of cellulosic substrate including lignocellulosic materials. From this study, it could be concluded that endoglucanase produced from recombinant Pichia pastoris can be applied for saccharification process in bioethanol production.