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.