Three-Enzyme Phosphorylase Cascade for Integrated Production of Short-chain Cellodextrins

Cellodextrins are linear β-1,4-gluco-oligosaccharides that are well soluble in water up to a degree of polymerization (DP) of ~6. Soluble cellodextrins have promising applications as nutritional ingredients. A DP-controlled, bottom-up synthesis from expedient substrates is desired for their bulk production. Here, we developed a three-enzyme glycoside phosphorylase cascade for the conversion of sucrose and glucose into the short-chain cellodextrins (DP range 3 – 6). The cascade reaction involves iterative β-1,4-glucosylation of glucose from α-glucose 1-phosphate (αGlc1-P) donor that is formed in situ from sucrose and phosphate. With final concentration and yield of soluble cellodextrins set as targets for the biocatalytic synthesis, we identified, and partly optimized, three major factors of reaction efficiency: the ratio of enzyme activities; the ratio of sucrose and glucose; and the phosphate concentration used. We demonstrate efficient use of the phosphate/αGlc1-P shuttle for cellodextrins production and obtain the soluble product at 40 g/L under near complete utilization of the donor substrate offered (88 mol.% from 200 mM sucrose). The productivity was 16 g/(L h). Through a simple two-step route, the soluble cellodextrins were recovered from the reaction mixtures in ≥ 95% purity and ~92% yield. Overall, this study provides the basis for their integrated production.