| The production of fuel ethanol from cellulosic material is a process that occurs in several steps, one of which is the enzymatic hydrolysis of the cellulose chains. In order to keep costs down, it is imperative that this step be as efficient as possible. The understanding of the interactions between different cellulases allows researchers to design optimal mixtures of enzymes for a given application.;In this study, cel48A was cloned into pET30(a)+, effectively adding a C-terminal His6-tag to expressed protein. Purification of Cel48A-His6 was optimized using this construct, and enzymatic activity was determined on AviceleRTM PH-101 using the phenol sulfuric acid assay. Synergistic activity between Cel48A-His6 and a recombinant endoglucanase from C. fimi, Cel6A-His 6, was demonstrated. The calculated degree of synergy was determined to be ∼1.37 when the ratio of Cel48A-His6 to Cel6A-His 6 is 2:1.;Cellulomonas fimi produces four endoglucanases, two cellobiohydrolases, and one mixed function xylanase/beta-glucosidase for the degradation of cellulose. One of the cellobiohydrolases is Cel48A, a family 48 enzyme with few known substrates and particularly low activity. The family 48 glycoside hydrolases are unique in that they are produced mainly by bacteria. Many of the family 48 glycoside hydrolases are produced as part of complexes called cellulosomes but several, including Cel48A, are secreted as a component of a non-complexed system. Whereas studies have provided insight into the function and substrate specificity of Cel48A, it is still unclear why C. fimi produces an enzyme with such low activity. This study tests the hypothesis that Cel48A contributes to the synergistic activity of the cellulolytic system of C. fimi, and in particular acts with cellulase A (Cel6A). |
