Characterization of the oligosaccharides assembled on the Pichia pastoris-expressed recombinant aspartic protease

Aspartic protease, widely used as a milk-coagulating agent in industrial ch eese production, contains three potential N-glycosylation sites. In this st udy, we report the characterization of N-linked oligosaccharides on recombi nant aspartic protease secreted from the methylotrophic yeast Pichia pastor is using a combination of mass spectrometric, 2D chromatographic, chemical and enzymatic methods. The carbohydrates from site I (Asn(79)) were found t ol range from Man(6-17)GlcNAc(2) with 50% bearing a phospho-diester-motif, site II (Asn(113)) was not occupied and site III (Asn(188)) contained mostl y uncharged species ranging from Man(8-13)GlcNAc(2). These charged groups a re not affecting the transport through the secretion pathway of the recombi nant glycoprotein, Changes from a molasses-based medium to a minimal salts- based medium led to a clear reduction of the degree of phosphorylation of t he N-glycan population.