beta-1,3-Glucan phosphorylase: enzyme discovery

Glycoside phosphorylases are enzymes catalysing the reversible addition of phosphate across the glycosidic bond (phosphorolysis), in the following reaction:


In certain conditions the reverse reaction leading to glycoside bond formation can prevail and that is why glycoside phosphorylases are of particular interest to us. Biosynthesis of glycans under glycoside phosphorylase catalysis is very attractive because it can utilise relatively inexpensive substrates and is regio- and stereospecific by its own nature. Among several types of phosphorylases we study β-1,3-glucan phosphorylases which are represented by two enzymes with different sugar substrate preferences. The first, laminaribiose phosphorylase, acts on a disaccharide and the second, β-1,3-oligoglucan phosphorylase, has the preference for longer β-1,3-linked oligoglucans. The phosphorolytic reaction in these cases can be described by the following equations.


We propose that a combination of laminaribiose and β-1,3-oligoglucan phosphorylases could be used for a one-pot enzymatic synthesis of β-1,3-oligosaccharides starting from readily available starting materials, glucose and glucose-1-phosphate. Thus, enzymatic synthesis could provide an effective way of producing designer β-1,3-oligosaccharides, compounds which potentially have several biotechnological application. However, successful biotechnological application of phosphorylases is hampered by rather limited availability of these enzymes, in fact only a small number of organisms has proven to possess enzymes with β-1,3-glucan phosphorylase activity.  Our goal is to uncover potential candidates capable of demonstrating such activities and test them in practical applications. In order to achieve this goal we have undertaken research which is based on several approaches:

  • We have performed de novo transcriptome sequencing and preliminary analysis of Euglena gracilis, a species of single-celled Eukaryote algae which has been known to contain the phosphorylase activity (1, 2).
  • We use enzymatic assays, such as phosphate release assay, to screen protein lysate of candidate species for the phosphorylase activity. The organisms which we are interested in are Euglena gracilis and Ochromonas danica. This assay was also be adopted for high-throughput screening of a wide variety of sugar acceptor – sugar phosphate donor combinations in order to probe the promiscuity of the enzymes which can be useful tools for creating other types of glycosidic linkages.
Phosphate release assay: A high-throughput screen for phosphorylase activity.
  • We use bioinformatics tools and phylogenetic analysis to study the evolutionary relationship of these candidates with other known carbohydrate active enzymes.

We perform structural study of a well-characterised laminaribiose phosphorylase in order to understand the molecular basis of the preference of this enzyme towards disaccharide substrates.


187. Enzymatic synthesis using glycoside phosphorylases.
O'Neill, E. C.; Field, R. A.
Carbohydr. Res. 2015, 403, 23-37.

194. The transcriptome of Euglena gracilis reveals unexpected metabolic capabilities for carbohydrate and natural product biochemistry.
O'Neill, E. C.; Trick, M.; Hill, L.; Rejzek, M.; Dusi, R. G.; Hamilton, C. J.; Zimba, P. V.; Henrissat, B.; Field, R. A.
Mol. Biosyst. 2015, 11, 2808-20.