Addition of a carbohydrate-binding module enhances cellulase penetration into cellulose substrates

Abstract

IntroductionCellulases are of great interest for application in biomass degradation, yet the molecular details of the mode of action of glycoside hydrolases during degradation of insoluble cellulose remain elusive. To further improve these enzymes for application at industrial conditions, it is critical to gain a better understanding of not only the details of the degradation process, but also the function of accessory modules.MethodWe fused a carbohydrate-binding module (CBM) from family 2a to two thermophilic endoglucanases. We then applied neutron reflectometry to determine the mechanism of the resulting enhancements.ResultsCatalytic activity of the chimeric enzymes was enhanced up to three fold on insoluble cellulose substrates as compared to wild type. Importantly, we demonstrate that the wild type enzymes affect primarily the surface properties of an amorphous cellulose film, while the chimeras containing a CBM alter the bulk properties of the amorphous film.ConclusionOur findings suggest that the CBM improves the efficiency of these cellulases by enabling digestion within the bulk of the film.

Truncation of the processive Cel5A of Thermotoga maritima results in soluble expression and several fold increase in activity

“…All the Cel5A variants showed a broad pH range of 4-8.0 for the enzyme activity (Figure 2b), with the maximum activity at pH 6.0. These results are in agreement with the previously reported optimum pH (4.8-6.0) and temperature (80°C) for wild-type Cel5A of T. maritima (Arumugam et al, 2008;Reyes-Ortiz et al, 2013).…”

Advances in molecular engineering of carbohydrate-binding modules

“…Similarly, CBMs have biotechnological potential in the treatment of agricultural wastes for back‐end use in diverse applications. For biomass degradation, chimeric proteins have been designed by fusing CBMs to catalytic domains from multiple enzymes, which could diversify the range of carbohydrate targets and increase the enzymatic activity . For example, the fusion of CBM2a to two thermophilic endoglucanases enhances their catalytic activity on insoluble substrates up to threefold with respect to that of the WT …”

Extra carbohydrate binding module contributes to the processivity and catalytic activity of a non-modular hydrolase family 5 endoglucanase from Fomitiporia mediterranea MF3/22

“…Cellulose hydrolysis is a heterogeneous reaction in which cellulases initially bind to the surface of the insoluble cellulose substrate, followed by subsequent hydrolysis of glycosidic bonds. The processivity of cellulases is defined as the average number of consecutive cleavages that a cellulase will toward cellulose and improving the penetration of cellulases into substrates [11,12]. In addition, CBMs also play a role in cell attachment and confer enzymatic substrate specificity in other glycoside hydrolases [13][14][15].…”