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Cellodextrins are non-digestible oligosaccharides that have attracted interest from the food industry as potential prebiotics. They are typically produced through the partial hydrolysis of cellulose, resulting in a complex mixture of oligosaccharides with a varying degree of polymerisation (DP). Here, we explore the defined synthesis of cellotriose as product since this oligosaccharide is believed to be the most potent prebiotic in the mixture. To that end, the cellobiose phosphorylase (CBP) from  Cellulomonas uda  and the cellodextrin phosphorylase (CDP) from  Clostridium cellulosi  were evaluated as biocatalysts, starting from cellobiose and α- d -glucose 1-phosphate as acceptor and donor substrate, respectively. The CDP enzyme was shown to rapidly elongate the chains towards higher DPs, even after extensive mutagenesis. In contrast, an optimised variant of CBP was found to convert cellobiose to cellotriose with a molar yield of 73%. The share of cellotriose within the final soluble cellodextrin mixture (DP2-5) was 82%, resulting in a cellotriose product with the highest purity reported to date. Interestingly, the reaction could even be initiated from glucose as acceptor substrate, which should further decrease the production costs.  Key points  •  Cellobiose phosphorylase is engineered for the production of cellotriose.   •  Cellotriose is synthesised with the highest purity and yield to date.   •  Both cellobiose and glucose can be used as acceptor for cellotriose production.    Electronic supplementary material  The online version of this article (10.1007/s00253-020-10820-8) contains supplementary material, which is available to authorized users.

Engineering of cellobiose phosphorylase for the defined synthesis of cellotriose