Artificial N-Glycosylation Motifs Engineering for Heterologous Glycoside Hydrolases Production in Aspergillus niger

    Aspergillus niger is an excellent protein production host as evidenced by its wide use in industry to produce a variety of enzymes for hydrolysis of polysaccharides to sugars. In native fungal enzymes, glycosylation is associated with secreted enzyme stability and function, but the effects on normally a glycosylated prokaryotic enzymes is not understood. Therefore, we introduced several artificial N-glycosylation sites into the sequence of the bacterial β-glucosidase A5IL97. An interesting revelation was that many variants appear to have greater activity than the unmodified strain. We are performing top-down proteomic analyses to determine which variants are glycosylated and correlating those results with our enzyme kinetic and thermodynamic characterizations to deepen our understanding of protein glycosylation.