Development of beta-glycosidases designed to improve the efficiency of noncomplexed cellulase systems

Abstract

The rate of the enzymatic hydrolysis of cellulose decreases along the reaction time, which represents a drawback for the productivity of this process. One of the factors causing that problem is the cellobiose inhibitory action upon the “cellulases” (endoglucanases and cellobiohydrolases) that catalyze the hydrolysis. This project aims to stabilize the rate of the enzymatic hydrolysis of cellulose by developing β-glycosidases designed to reduce the cellobiose inhibitory effect on the “cellulases”. Such development will be based on the β-glycosidase (cellobiase) from the fall armyworm Spodoptera frugiperda (Sfβgly) and the “carbohydrate binding domain” of the endoglucanase EngXCA from Xhantomonas axonopodis pv citri (CBMXAC). Firstly, a chimeric protein resulting from the fusion of Sfβgly and CBMXAC will be assembled. The targeting of Sfβgly to the surface of the cellulose fibers (due the presence of a CBM) could decrease the cellobiose concentration directly in the microenvironment of action of the endoglucanases and cellobiohydrolases. Thus the action of Sfβgly-CBM could reduce the cellobiose inhibitory effect and sustain a high activity of endoglucanases and cellobiohydrolases for a longer time. This project also intends to improve the participation of the β-glycosidases in the cellulose hydrolysis by selecting mutant Sfβgly that presents high hydrolytic activity upon cellobiose. Libraries of random mutant Sfβgly will be generated and screened based on the ratio of activity upon cellobiose versus synthetic substrates. Finally, amino acid residues networks involved in the determination of Sfβgly substrate specificity and catalytic activity will be identified by using structural analysis, site-directed mutagenesis studies and enzyme kinetic experiments. (AU)