Control of the monomer sequence in polymers is extraordinarily difficult by chemical synthesis, though Nature routinely exerts such control, including in the biosynthesis of polysaccharides. This inability has prevented us from being able to match the exquisite structure-activity control exhibited in biosynthesis of bioactive natural polysaccharides. We here address a powerful approach, whereby enzyme-catalyzed polymerization of properly modified building blocks is introduced as a simple route affording polysaccharides with controlled sequence and functionalization pattern. Targeting cellulose as a versatile scaffold for novel biomaterials, we describe the preparation of a perfectly alternating polysaccharide with repeat unit 6'-azido-6'-deoxycellobiose by a glycosynthase-catalyzed polymerization using the Humicola insolens cellulase Cel7B E197A mutant, and its further functionalization to give novel modified cellulose derivatives with a regular substitution pattern.

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 E(134)S 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 E(134)S, 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.

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