RESUMO

Aspartic protease, widely used as a milk-coagulating agent in industrial cheese production, contains three potential N-glycosylation sites. In this study, we report the characterization of N-linked oligosaccharides on recombinant aspartic protease secreted from the methylotrophic yeast Pichia pastoris using a combination of mass spectrometric, 2D chromatographic, chemical and enzymatic methods. The carbohydrates from site I (Asn79) were found to range from Man6-17GlcNAc2 with 50% bearing a phospho-diester-motif, site II (Asn113) was not occupied and site III (Asn188) contained mostly uncharged species ranging from Man-13GlcNAc2. These charged groups are not affecting the transport through the secretion pathway of the recombinant glycoprotein. Changes from a molasses-based medium to a minimal salts-based medium led to a clear reduction of the degree of phosphorylation of the N-glycan population.

Assuntos

Ácido Aspártico Endopeptidases/química , Oligossacarídeos/química , Pichia/enzimologia , Pichia/genética , Sequência de Aminoácidos , Ácido Aspártico Endopeptidases/genética , Sítios de Ligação , Queijo , Cromatografia Líquida de Alta Pressão , Tecnologia de Alimentos , Glicosilação , Dados de Sequência Molecular , Mucor/enzimologia , Mucor/genética , Pichia/ultraestrutura , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz