A Single Mutation in the Carbohydrate-Binding Module Enhances Cellulase Activity in Bacillus Amyloliquefaciens Mutant

Abstract

From our earlier work, we modified the carbohydrate-binding module (CBM) of Bacillus amyloliquefaciens to increase cellulase activity using cold plasma technology. The cellulase gene (BglCM) from the mutant was expressed in Escherichia coli BL21(DE3) under the T7 promoter. The hydrolysis activity of the cellulase mutant (BglC-M) was approximately 2.5-fold higher than the control (BglC-W) over a wide range of pH and temperature conditions. The amino acid sequence of the mutant BglC-M contained 471 residues that were almost identical to the control BglC-W. Only a single amino acid, lysine, was replaced by glutamic acid at position 370 (K370E) within the carbohydrate-binding module (CBM). Structure prediction and substrate docking of BglC-M indicated that the single mutation (K370E) might involve cellulose-binding of the β-sandwich facilitated by hydrogen bonding. The docking study of cellopentaose with the model structure of BglC-M indicated that the replacement of lysine-370 led to the formation of a hydrogen bond with 436Y, which has a shorter distance (2.6 Å) compared with the control (5.4 Å). As a result, the structure becomes more compact and stable, resulting in increased catalytic efficiency. Finally, the biomass hydrolysis ability of cellulase was investigated on lignocellulosic wastes such as pineapple peel, corncob, and durian peel. The BglC-M enzyme showed a more significant amount of reducing sugar released from all lignocellulosic wastes than the control. This was the first evidence that altering the base composition of the cellulose-binding module enhanced the catalytic activity