Characterisation of an exo-(α-1,3)-3,6-anhydro-D-galactosidase produced by the marine bacterium Zobellia galactanivorans Dsij^T: Insight into enzyme preference for natural carrageenan oligosaccharides and kinetic characterisation on a novel chromogenic substrate

Flavobacteriia are important degraders in the marine carbon cycle, due to their ability to efficiently degrade complex algal polysaccharides. A novel exo-(α-1,3)-3,6-anhydro-D-galactosidase activity was recently discovered from a marine Flavobacteriia (Zobellia galactanivorans Dsij^T) on red algal carrageenan oligosaccharides. The enzyme activity is encoded by a gene found in the first described carrageenan-specific polysaccharide utilization locus (CarPUL) that codes for a family 129 glycoside hydrolase (GH129). The GH129 family is a CAZy family that is strictly partitioned into two niche-based clades: clade 1 contains human host bacterial enzymes and clade 2 contains marine bacterial enzymes. Clade 2 includes the GH129 exo-(α-1,3)-3,6-anhydro-D-galactosidase from Z. galactanivorans (ZgGH129). Despite the discovery of the unique activity for ZgGH129, finer details on the natural substrate specificity for this enzyme are lacking. Examination of enzyme activity on natural carrageenan oligomers using mass spectrometry demonstrated that ZgGH129 hydrolyses terminal 3,6-anhydro-D-galactose from unsulfated non-reducing end neo-β-carrabiose motifs. Due to the lack of chromogenic substrates to examine exo-(α-1,3)-3,6-anhydro-D-galactosidase activity, a novel substrate was synthesised to facilitate the first kinetic characterisation of an exo-(α-1,3)-3,6-anhydro-D-galactosidase, allowing determination of pH and temperature optimums and Michaelis-Menten steady state kinetic data.