Carbohydrate-binding module assisting glycosynthase-catalysed polymerizations

Carbohydrate-binding modules (CBMs) are found within multi-modular polysaccharide degrading enzymes [glycoside hydrolases (GHs)]. CBMs play a critical role in the recognition of plant cell-wall polysaccharides and enhance the hydrolase activity of their cognate catalytic domains by increasing enzyme substrate proximity. Mimicking their role in Nature, we, in the present study, propose that CBMs may assist in vitro glycosynthase-catalysed polymerization reactions to produce artificial polysaccharides. Glycosynthases are GHs that have been engineered to catalyse glycoside bond formation for the synthesis of oligosaccharides, glycoconjugates and glycans. The degree of polymerization (DP) of the glycans generated is limited by the solubility of the polymeric product. In the present study, we have targeted the synthesis of artificial 1,3-1,4-β-glucans with a regular sequence using the glycosynthase E¹³⁴S derived from a Bacillus licheniformis lichenase. We show that the addition of CBM11, which binds mixed-linked β-glucans, either as an isolated protein or fused to the glycosynthase E¹³⁴S, has an effect on the DP of the polysaccharide products that is dependent on the rate of polymerization. The mechanism by which CBM influences the DP of the synthesized glycans is discussed.