Preparative and Kinetic Analysis of β‐1,4‐ and β‐1,3‐Glucan Phosphorylases Informs Access to Human Milk Oligosaccharide Fragments and Analogues Thereof

Abstract

The enzymatic synthesis of oligosaccharides depends on the availability of suitable enzymes, which remains a limitation. Without recourse to enzyme engineering or evolution approaches, herein we demonstrate the ability of wild‐type cellodextrin phosphorylase (CDP: β‐1,4‐glucan linkage‐dependent) and laminaridextrin phosphorylase (Pro_7066: β‐1,3‐glucan linkage‐dependent) to tolerate a number of sugar‐1‐ phosphate substrates, albeit with reduced kinetic efficiency. In spite of catalytic efficiencies of <1 % of the natural reactions, we demonstrate the utility of given phosphorylase–sugar phosphate pairs to access new‐to‐nature fragments of human milk oligosaccharides, or analogues thereof, in multi‐milligram quantities.

Recent advances in enzymatic synthesis of β-glucan and cellulose

“…This study pioneered the molecular detailed recognition of an unnatural donor substrate by GPs as a means to provide background knowledge to harness their prospective as biocatalyst for synthetic applications. More recently, wild-type Pro_7066 and CDP showed reasonable tolerance to a variety of sugar 1- phosphates in a multi-milligram-scale synthesis of fragments of human milk oligosaccharides (HMOs) [ 131 ], which are currently a hot topic in enzymatic syntheses [ 132 ]. Kinetic data of these enzymes indicated general low efficiency (<1%) for the unnatural donors α-D-Gal-1P, α-D-GlcN-1P and α-D-Man-1P compared to α-D-Glc-1P in the presence of both d -laminaribiose and d -cellobiose, though the transference of glucose onto both acceptors was effective.…”

β-Glucan phosphorylases in carbohydrate synthesis

“…Most (β-glucan) phosphorylases show promiscuity towards various alternative donors and/or acceptors (Table 2 ) (Singh et al 2020 ). For example, the cellodextrin phosphorylase from Clostridium thermocellum was shown to successfully utilize anomeric phosphates of xylose (Shintate et al 2003 ), galactose (Tran et al 2012 ), and glucosamine (O’Neill et al 2017 ), although only a single monomer could be added to the acceptor substrate in these cases (Singh et al 2020 ).…”

“…Most (β-glucan) phosphorylases show promiscuity towards various alternative donors and/or acceptors (Table 2 ) (Singh et al 2020 ). For example, the cellodextrin phosphorylase from Clostridium thermocellum was shown to successfully utilize anomeric phosphates of xylose (Shintate et al 2003 ), galactose (Tran et al 2012 ), and glucosamine (O’Neill et al 2017 ), although only a single monomer could be added to the acceptor substrate in these cases (Singh et al 2020 ). In turn, a variety of sugar phosphates (of glucose, galactose, glucosamine, and mannose) could be offered to the β-1,3-oligoglucan phosphorylase Pro_7066 for the synthesis of new-to-nature analogs of human milk oligosaccharides (HMO) (Singh et al 2020 ).…”

“…In turn, a variety of sugar phosphates (of glucose, galactose, glucosamine, and mannose) could be offered to the β-1,3-oligoglucan phosphorylase Pro_7066 for the synthesis of new-to-nature analogs of human milk oligosaccharides (HMO) (Singh et al 2020 ). These studies highlighted the innate ability of β-glucan phosphorylases to synthesize smaller glycans (Singh et al 2020 ). Although the kinetic efficiencies of these processes are frequently over 100-fold lower than those of their natural reaction, they enable single turnover that is not typical for the enzymes naturally prone to build carbohydrate polymers ( Singh et al 2020 ) .…”

“…These studies highlighted the innate ability of β-glucan phosphorylases to synthesize smaller glycans (Singh et al 2020 ). Although the kinetic efficiencies of these processes are frequently over 100-fold lower than those of their natural reaction, they enable single turnover that is not typical for the enzymes naturally prone to build carbohydrate polymers ( Singh et al 2020 ) .…”