Preparative and kinetic assessment of ß-1,4- and ß-1,3-glucan phosphorylases informs access to human milk oligosaccharide fragments and analogues thereof.

The enzymatic synthesis of oligosaccharides depends on the availability of suitable enzymes, which remains a limitation. Without recourse to enzyme engineering or evolution approaches, here we demonstrate the ability of wild-type cellodextrin phosphorylase (CDP: ß-1,4-glucan linkage-dependent) and laminaridextrin phosphorylase (Pro_7066: ß-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-mg quantities.