Engineering of cellobiose phosphorylase for the defined synthesis of cellotriose

Abstract

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.

β-Glucan phosphorylases in carbohydrate synthesis

“…The final product consisted of DP3, DP4, DP5, and DP6 with a distribution of 33, 34, 24, and 9 wt%, respectively, a purity of over 95%, and a yield of 88% (Zhong et al 2020b ). We previously described the synthesis of predominantly cellotriose from both glucose and cellobiose by using a cellobiose phosphorylase variant (Ubiparip et al 2020 ). Facilitated synthesis of cellodextrins from glucose will certainly unlock further application studies, especially related to the use of these carbohydrates as food and feed additives.…”

“…A double mutant (T508I/N667A) that no longer required a glucosyl acceptor with a free anomeric hydroxyl group and, hence showed some activity on cellobiose as acceptor was slightly improved (0.113 U/mg) by three additional mutations (N156D, N163D, and E649G) (De Groeve et al 2010b ). Most recently, it was determined that the mutant enzyme synthesizes mostly cellotriose with both glucose and cellobiose as acceptors (Ubiparip et al 2020 ). The finding became particularly interesting since cellotriose was shown to be the most potent prebiotic among soluble cellodextrins (DP2-6) when tested against bifidobacteria of the human gut (Pokusaeva et al 2011 ).…”

“…The finding became particularly interesting since cellotriose was shown to be the most potent prebiotic among soluble cellodextrins (DP2-6) when tested against bifidobacteria of the human gut (Pokusaeva et al 2011 ). The addition of M52R substitution improved the variant’s kinetic properties for the acceptor cellobiose and slightly increased the cellotriose yield (Ubiparip et al 2020 ). This study was the first to demonstrate the possibility of controlled, bottom-up, enzymatic synthesis of cellodextrins with a specific degree of polymerization.…”

“…Contrary to the engineering of Cu CBP, single-point mutagenesis of CDP from Clostridium cellulosi did not result in a change of the enzyme’s preference to synthesize specific cellodextrins, most probably due to its broader active site (Ubiparip et al 2020 ). The creation of the C485A, Y648F, and Y648V mutants of the CDP from Ruminococcus albus resulted in the higher preference for d -glucosamine, more rapid synthesis of 4- O -β- d -glucopyranosyl- d -mannose, and synthetic activity on 4- O -β- d -glucopyranosyl-N-acetyl- d -glucosamine, respectively (Hamura et al 2013 ).…”

“…1 Example of the coupled reaction by β-glucobiose (cellobiose) phosphorylase, β-glucan (cellodextrin) phosphorylase, and sucrose phosphorylase for cellodextrin synthesis. Adapted from Ubiparip et al ( 2020 ) …”