The fusion of a carbohydrate binding module 6 (CBM6) to GH11 xylanase improves the catalytic efficiency

Xylanases catalyze the hydrolysis of β-1,4-linked xylosyl moieties from xylan, the major hemicellulosic polysaccharide found in plants. These enzymes can exist either as a single catalytic domain or as a modular protein composed of one or more carbohydrate-binding modules appended to the catalytic core. However, the molecular mechanisms governing the synergistic effects among catalytic domains and their CBMs are not fully understood. Thus, the goal of this study was to evaluate the functional effect of the fusion of a CBM belonging to family 6 that exhibits high affinity to xylan into the GH11 xylanase from Bacillus subtilis, which does not have a CBM in its wild-type form. The recombinant native enzyme (BsXyl11) and the chimeric protein (BsXyl11-CBM6) were heterologously produced in E.coli and purified to homogeneity for biochemical characterization. Activity measurements indicated that pH and temperature dependence were not affected by the presence of CBM6. However, kinetics data revealed an increase of 65% in the catalytic efficiency of the chimeric enzyme. Furthermore, the BsXyl11-CBM6 chimera was able to supplement the activity of the commercial cocktail Accellerase® 1500, releasing 17% more reducing sugars from sugarcane bagasse. These results show that CBM6 can be used as molecular tool for improving the catalytic performance of GH11 enzymes and provide a new strategy for the development of new biocatalysts for biotechnological applications