Arabinogalactan-proteins (AGPs) are plant proteoglycans, which are widely encountered in the plant kingdom, usually localized on the cell surface. The carbohydrate moieties of AGPs consist of β-1,3-galactan main chains and β-1,6-galactan side chains, to which other auxiliary sugars are attached. To date, FvEn3GAL isolated from Flammulina velutipes is the sole β-1,3-galactanase acting on β-1,3-galactan in an endo-manner. Here we cloned two homologous genes, designated Af3G and NcEn3GAL, possibly encoding endo-β-1,3-galactanase from Aspergillus flavus and Neurospora crassa, respectively. The recombinant Af3G (rAf3G) and rNcEn3GAL expressed in Pichia pastoris specifically hydrolyzed β-1,3-galactan in an endo-manner, as did the rFvEn3GAL. Among galactooligosaccharides, β-1,3-galactotriose was identified as the smallest substrate for these enzymes. These results suggest that enzymatic characteristics are conserved in many endo-β-1,3-galactanases belonging to the glycoside hydrolase 16 family. On the other hand, rAf3G and rNcEn3GAL generated more β-1,3-galactobiose from β-1,3-galactotetraose than did rFvEn3GAL, suggesting that rAf3G and rNcEn3GAL prefer hydrolyzing the central β-1,3-glycosidic linkage of three in β-1,3-galactotetraose. Although rAf3G and rNcEn3GAL alone hardly hydrolyze native AGP, they acted synergistically with a fungal exo-β-1,3-galactanase on the AGP. These endo-β-1,3-galactanases presumably aid hydrolysis by internally breaking up AGPs, which creates more sites of attack for exo-β-1,3-galactanase.
Keywords: Arabinogalactan-protein; Aspergillus flavus; Endo-β-1,3-galactanase; Glycoside hydrolase family 16; Neurospora crassa.
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