44Noda Institute for Scientific Research GRANT
2012 Young Investigator Research Grant

Production of Novel Functional Oligosaccharides by Novel
Bacterial Phosphorylases
Hiroyuki Nakai
Food Glycoscience Laboratory, Life and Food Sciences, Graduate School of Science & Technology, Niigata University

Research aims
Recently, a variety of oligosaccharides has been reported
to have multiple functional bioactive properties such as prebiotic effects, antibacterial effects, and immunopotentiativeaction. These oligosaccharides are widely used as food additives and pharmaceuticals, and their functionality depends on their carbohydrate components and linkage types.
Therefore, it would be worthwhile producing various oligosaccharides efficiently and specifically by suitable methods.
Disaccharide phosphorylase catalyzes phosphorolysis ofparticular disaccharides to produce monosaccharide
1-phosphate. The phosphorolysis is reversible, making thephosphorylase a suitable catalyst for the synthesis of a particular disaccharide. So far, various oligosaccharides havebeen synthesized by reverse phosphorolysis using the corresponding sugar 1-phosphate as the donor and suitable acceptors. In addition, the reversibility makes it possible toproduce the oligosaccharides from abundantly available
commercial and/or natural sugar by using a single phosphorylase or the combined reaction of two phosphorylasesthat share the same monosaccharide 1-phosphate. However,utilization of disaccharide phosphorylase for the practicalproduction of oligosaccharides is limited by the fact that
there is little variation for substrate specificities, with only
21 phosphorylase activities known. Therefore, it would be
beneficial to discover phosphorylases with previously unreported substrate specificities.
Results
In this study, we focused on two proteins homologous toglycoside hydrolase family 65 (Bsel_1207 and Bsel_2816)
that are encoded in the genomic DNA of Bacillus selenitireducens MLS10. Bsel_1207 exhibits high sequence similarity with the known trehalose phosphorylase. In contrast,
Bsel_2816 could not be categorized into any of the known
phosphorylases based on sequence similarity and phylogenetic tree analysis. Therefore, we expressed the two glyco-side hydrolase family 65 homologs as His6-tag fusion proteins in Escherichia coli BL21 (DE3) to investigate their
enzymatic properties.
1. Potassium ion-dependent trehalose phosphorylase(Bsel_1207)
When tested in the presence of inorganic phosphate withseveral α-linked glucobioses such as trehalose (α1,α1),kojibiose (α-1,2), nigerose (α-1,3), maltose (α-1,4), andisomaltose (α-1,6) and including the typical substrates forglycoside hydrolase family 65 enzymes with inversion of
the anomeric configuration releasing β-Glc1P and glucose,
Bsel_1207 phosphorolyzed only trehalose, indicating that
Bsel_1207 is a trehalose phosphorylase, which is in agreement with the results of its sequence analysis. Interestingly,
Bsel_1207 strictly used potassium to regulate its enzymaticactivity. Under high potassium ion concentrations,
Bsel_1207 exists as an active dimeric form. Decreasing
potassium ion concentrations significantly reduced thethermal and pH stability of Bsel_1207, causing it to form
an inactive monomer. This is the first report of a potassium
ion-dependent phosphorylase.
2-O-α-glycosylglycerol phosphorylase (Bsel_2816)
In the presence of inorganic phosphate, Bsel_1207 did
not show obvious phosphorolytic activity with α-linkedglucobioses such as trehalose (α1,α1), kojibiose (α-1,2), nigerose (α-1,3), maltose (α-1,4), and isomaltose (α-1,6), including the typical substrates for glycoside hydrolase family 65 enzymes. Therefore, the detail acceptor specificity inthe reverse phosphorolysis was investigated. It was foundthat only a synthetic reaction with glycerol as the acceptor
gave a single product efficiently. The NMR spectra indicated that the product was a 2-O-α-glycosylglycerol. In addition, we confirmed that Bsel_2816 catalyzed phosphorolysis of 2-O-α-glycosylglycerol in the presence of inorganic
phosphate. Therefore, we propose 2-O-α-d-gluco­pyrano­
sylglycerol: phosphate β-d-glucosyltransferase as the systematic name and 2-O-α-glucosylglycerol phosphorylase as2.

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the short name for Bsel_2816. This is the first report of aphosphorylase utilizing a polyol as the real acceptor molecule.

2-O-α-glucosylglycerol phosphorylase.

Conclusion1)

We discovered two kinds of novel phosphorylases belonging to glycoside hydrolase family 65 from the halophilic strain B. selenitireducens MLS10. One is a potassium
ion-dependent trehalose phosphorylase. The other is aReferences

2)

Nihira T., Saito Y., Chiku K., Kitaoka M., Ohtsubo K., Nakai H.
(2013) Potassium ion-dependent trehalose phosphorylase from halophilic Bacillus selenitireducens MLS10. FEBS Lett. (2013) 587:
3382–3386.
Nihira T., Saito Y., Ohtsubo K., Nakai H., Kitaoka M. (2013) 2-O-αd-glucosylglycerol phosphorylase from Bacillus selenitireducens
MLS10 possessing hydrolytic activity on β-d-glucose 1-phosphate.
PLOS ONE (2013) accepted.