Novel PNGase H<sup>+</sup> from <i>Amycolatopsis mediterranei</i>: biochemical properties and food analysis potential

Peptide-N4-(N-acetyl-<italic>β</italic>-D-glucosaminyl)asparagine amidases (PNGases) are essential biocatalysts for the deglycosylation of complex carbohydrates from glycoproteins, protein analysis and food quality assessment. In this study, the cloning, heterologous expression in <italic>Escherichia coli</italic>, and biochemical characterization of previously undescribed PNGase H⁺ enzyme (AmePNG) from <italic>Amycolatopsis mediterranei—</italic>a species known for its role in antibiotic production and fermentation processes in food biotechnology—are reported. Recombinant AmePNG showed a robust deglycosylation activity toward horseradish peroxidase (HRP), as demonstrated by HPLC analysis of released N-glycans. The enzyme's activity profile spanned a broad pH range, with maximum activity observed under acidic conditions. Structural insights were obtained through AlphaFold modeling, highlighting conserved motifs associated with the PNGase H⁺ family. These findings establish AmePNG as a promising tool for glycoprotein analysis in food science, facilitating the structural elucidation of plant glycoproteins relevant to food quality and processing.