Unraveling the subtleties of ß-(1→3)-glucan phosphorylase specificity in the GH94, GH149, and GH161 glycoside hydrolase families.

Glycoside phosphorylases (GPs) catalyze the phosphorolysis of glycans into the corresponding sugar 1-phosphates and shortened glycan chains. Given the diversity of natural ß-(1→3)-glucans and their wide range of biotechnological applications, the identification of enzymatic tools that can act on ß-(1→3)-glucooligosaccharides is an attractive area of research. GP activities acting on ß-(1→3)-glucooligosaccharides have been described in bacteria, the photosynthetic excavate Euglena gracilis, and the heterokont Ochromonas spp. Previously, we characterized ß-(1→3)-glucan GPs from bacteria and E. gracilis, leading to their classification in glycoside hydrolase family GH149. Here, we characterized GPs from Gram-positive bacteria and heterokont algae acting on ß-(1→3)-glucooligosaccharides. We identified a phosphorylase sequence from Ochromonas spp. (OcP1) together with its orthologs from other species, leading us to propose the establishment of a new GH family, designated GH161. To establish the activity of GH161 members, we recombinantly expressed a bacterial GH161 gene sequence (PapP) from the Gram-positive bacterium Paenibacillus polymyxa ATCC 842 in Escherichia coli We found that PapP acts on ß-(1→3)-glucooligosaccharide acceptors with a degree of polymerization (DP) ≥ 2. This activity was distinct from that of characterized GH149 ß-(1→3)-glucan phosphorylases, which operate on acceptors with DP ≥ 1. We also found that bacterial GH161 genes co-localize with genes encoding ß-glucosidases and ATP-binding cassette transporters, highlighting a probable involvement of GH161 enzymes in carbohydrate degradation. Importantly, in some species, GH161 and GH94 genes were present in tandem, providing evidence that GPs from different CAZy families may work sequentially to degrade oligosaccharides.

Purification and characterization of 1,3-ß-D-glucan phosphorylase from Ochromonas danica.

1,3-ß-D-glucan phosphorylase (BGP) is an enzyme that catalyzes the reversible phosphorolysis of 1,3-ß-glucosidic linkages to form α-D-glucose 1-phosphate (G1P). Here we report on the purification and characterization of BGP from Ochromonas danica (OdBGP). The purified enzyme preparation showed three bands (113, 118, and 124 kDa) on SDS-polyacrylamide gel electrophoresis. The optimum pH and temperature were 5.5 and 25 °C-30 °C. OdBGP phosphorolysed laminaritriose, larger laminarioligosaccharides, and laminarin, but not laminaribiose. In the synthesis reaction, laminarin and laminarioligosaccharides served as good acceptors, but OdBGP did not act on glucose. Kinetic analysis indicated that the phosphorolysis reaction of OdBGP follows a sequential Bi Bi mechanism. The equilibrium of the enzymatic reaction indicated that OdBGP favors the reaction in the synthetic direction. Overnight incubation of OdBGP with laminaribiose and G1P resulted in the formation of precipitates, which were probably 1,3-ß-glucans.