Development of optimized enzyme cocktails for hydrolysis of sugar cane bagasse

The high cost of lignocellulose conversion by enzymatic hydrolysis is a recurring problem that limits the production of bioethanol from a renewable and highly energetic source. Many factors affect the efficiency of the hydrolysis process, such as lignocellulosic substrates, pretreatment, enzyme ratios and loadings, inhibitors and adsorption. Among the promising approaches to optimize hydrolysis reaction, may be mentioned the development of individually more efficient and stable enzymes through the use of genetic engineering tools and well-balanced cocktails involving different classes of enzymes. Thus, this project proposes the development of optimized enzyme cocktails based on the rational and minimum mixture of main and accessory enzymes for hydrolysis of pretreated sugar cane bagasse. The enzymes were provided from Trichoderma reesei and genetically modified Escherichia colli cultures, followed by purification, and from commercial source. The optimization of the enzymatic mixture composition was performed in two stages: firstly the ratio between hemicellulases (endo-xylanase, α-L-arabinofuranosidase and acetilxylan esterase) was established; afterwards the cellulases (cellobiohydrolase, endoglucanase and β-glucosidase) ratio and their synergism with hemicellulases (already fixed ratio between their) were studied. The protein loading was 10 mg/g bagasse. Experimental mixture design, set up as a simplex centroid design, was used to achieve the experiments goals. Hydrolysis micro assays, validated in this study, were used to evaluation the synergism between enzymes. We intend to contribute to the understanding of the enzymatic synergism regarding hydrolysis of pretreated bagasse and to generate a more efficient, minimum and optimized enzyme mixture for the saccharification of bagasse.