Distinct roles of carbohydrate-binding modules in multidomain β-1,3-1,4-glucanase on polysaccharide degradation

Lam16A is a novel GH16 beta-1,3-1,4-lichenase isolated from the genus Caldicellulosiruptor which can utilize untreated carbohydrate components of plant cell walls. Its catalytic module has been characterized that the six carbohydrate-binding modules (CBMs) were queued in the C-terminus, but their roles were still unclear. Here, full-length and CBM-truncated mutants of Lam16A were purified and characterized through heterologous expression in Escherichia coli. The profiles of these proteins, including the enzyme activity, degrading efficiency, substrate-binding affinity, and thermostability, were explored. Full-length Lam16A with six CBMs showed excellent thermostability and the highest activity against barley beta-glucan and laminarin with optimum pH of 6.5. The CBMs stimulated degrading ability of the catalytic module, especially against beta-1,3(4)-glucan-based polysaccharides. The released products from beta-1,3-1,4-glucan by Lam16A or its truncated mutants revealed an endo-type glycoside hydrolase. Lam16As exhibited strong binding affinities to the insoluble polysaccharides, especially Lam16A-1CBM. The degradation of yeast cell walls by Lam16A enzyme solution relative to the control reduced the absorbance values at OD800 by similar to 85% +/- 1.2, enabling the release of up to similar to 0.057 +/- 0.0039 mu g/mL of the cytoplasmic protein into the supernatant, lowering the viability of the cells by similar to 70.3% +/- 6.9, thus causing significant damage in the cell wall structure. Taken together, CBMs could influence the substrate specificity, thermal stability, and binding affinity of beta-1,3-1,4-glucanase. These results demonstrate the great potential of these enzymes to promote the bioavailability of beta-1,3-glucan oligosaccharides for health benefits.