Discovery and characterization of a novel extremely acidic bacterial N-glycanase with combined advantages of PNGase F and A.

Peptide-N4-(N-acetyl-β-glucosaminyl) asparagine amidases [PNGases (peptide N-glycosidases)， N-glycanases， EC 3.5.1.52] are essential tools in the release of N-glycans from glycoproteins. We hereby report the discovery and characterization of a novel bacterial N-glycanase from Terriglobus roseus with an extremely low pH optimum of 2.6， and annotated it therefore as PNGase H+. The gene of PNGase H+ was cloned and the recombinant protein was successfully expressed in Escherichia coli. The recombinant PNGase H+ could liberate high mannose-， hybrid- and complex-type N-glycans including core α1，3-fucosylated oligosaccharides from both glycoproteins and glycopeptides. In addition， PNGase H+ exhibited better release efficiency over N-glycans without core α1，3-fucose compared with PNGase A. The facile expression， non-glycosylated nature， unusual pH optimum and broad substrate specificity of this novel type of N-glycanase makes recombinant PNGase H+ a versatile tool in N-glycan analysis.