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

Ravindra Pal Singh,Peterson De Andrade,Giulia Pergolizzi,Sergey A Nepogodiev,Sakonwan Kuhaudomlarp,Robert A Field

doi:10.1002/cbic.201900440

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.

Highlights

The synthesis of oligosaccharides and glycoconjugates remains a major challenge, it is beginning to yield to enzymatic methods.[1]
We investigated the action of b-1,4-glucan linkagedependent cellodextrin phosphorylase (CDP, GH94)[6] and b1,3-glucan linkage-dependent laminaridextrin phosphorylase (Pro_7066, GH149;[7] natural reactions shown in Figure 1) in reactions with a range of both natural and unnatural sugar-1phosphate donors and glucan acceptor substrates
In selecting sugar phosphates to assess with CDP and Pro_ 7066, we considered the synthesis of fragments of human milk oligosaccharides (HMOs), which have been the focus of recent enzymatic syntheses.[16]

Summary

Introduction

Supporting information and the ORCID identification numbers for the authors of this article can be found under https://doi.org/10.1002/ cbic.201900440. Phosphorylases are generally thought to be involved physiologically in the degradation of di- or oligosaccharides, by feeding elevated levels of sugar-1-phosphate or removing inorganic phosphate by-products, preparatively useful reactions can be accomplished This is well exemplified by the commercial, kilogram-scale production of the cosmetic humectant a-glucosyl glycerol by using sucrose phosphorylase.[8] a-1,4-Glucan phosphorylase has been used with variants of its natural donor substrate to synthesise unnatural oligo- and polysaccharides;[9] a-1,4-glucan polymermodified nanomaterials can be accessed.[10] In a similar way, CDP-catalysed reactions provide access to b-1,4-glucanlinked, cellulose-like materials.[11,12] In terms of single sugar addition, as opposed to oligo/polymerisation reactions with its natural substrate Glc1P, CDP has been shown to be capable of using the anomeric phosphates of xylose (synthesis of a library of b-(1,4) hetero-oligosaccharides),[13] galactose (biocatalytic production of novel glycolipids)[14] and glucosamine (microscale reaction; preparative utility not demonstrated).[6] We recently identified algal and bacterial b-1,3-glucan phosphorylases (e.g., Pro_7066) that are capable of producing b-1,3-glucan. We have recently reported on structural rationalisation of the promiscuity of laminaribiose phosphorylase, which produces disaccharides Glc-b-1,3-Glc and Man-b-1,3-Glc when fed glucose and Glc1P or Man1P, respectively.[19]

Results and Discussion

Conclusion

Conflict of Interest