Cloning, Expression, and Characterization of Endo-β-1,6-galactanase PoGal30 from Penicillium oxalicum

Because beta-1,6-galactans are significant components in arabinogalactans from plant cell walls, identifying selective endo-beta-1,6-galactanases is crucial to degrading these polysaccharides and to analyzing and modifying their structures. Here, we cloned and expressed in E. coli a novel endo-beta-1,6-galactanase in the glycosidic hydrolase family 30 (GH30) from Penicillium oxalicum. Our recombinant PoGa130 hydrolase (1464 bp gene) that contains an N-terminal His-tag for purification by nickel affinity chromatography has a specific activity of 3.8 U/mg on the substrate de-arabinosylated gum Arabic (dGA) polysaccharide. The enzyme has 487 residues with a molecular mass of 60 kDa, an isoelectric point of 6, and functional pH and temperature optima of pH 2.5 to pH 5.0 and 40 degrees C, respectively. While the activity of PoGa130 is activated by Mg2+ (5 or 50 mmol/L), it is completely inhibited by Cu2+ and Fe3+ (50 mmol/L) and partially inhibited by Hg2+, EDTA, and SDS (50 mmol/L). The enzyme demonstrates high specificity towards beta-1,6-galactosidic linkages in dGA, but is inactive against aryl-glycosides and galactobioses with different linkages. Using PoGa130 is, therefore, an effective approach to analyzing the fine structure of polysaccharides and preparing bioactive oligosaccharides.