Double-Glycosylated Lysozyme at Positions 19 and 49 Constructed by Genetic Modification and Its Surface Functional Properties

Abstract

Complementary DNA encoding hen egg white lysozyme (HEWL) was subjected to site-directed mutagenesis to introduce two N-linked glycosylation sites (Asn19-Try20-Thr21 and Asn49-Ser50-Thr51) into both positions 19 and 49 by substituting Arg-21 with Thr and Gly-49 with Asn, respectively. The double-glycosylated lysozyme R21T/G49N was expressed in Saccharomyces cerevisiae carrying the yeast expression plasmid inserted the double-mutant HEWL cDNA. The mutant lysozyme mainly secreted a polymannosyl form with a small amount of two oligomannosyl forms. The polymannosyl lysozyme R21T/G49N was glycosylated at two positions, 19 and 49, with a polymannosyl and an oligomannosyl chain. The lengths of the polymannosyl and oligomannosyl chains attached to R21T/G49N were approximately 272 and 18 mannose residues, respectively. The R21T/G49N showed better emulsifying properties than two types of single-polymannosyl lysozymes R21T and G49N. With regard to single-polymannosyl lysozyme, G49N showed somewhat better emulsifying properties than R21T. In addition, the cleavage of polymannosyl chain from lysoyme with endo-β-N-acetylglucosaminidase resulted in a dramatic decrease in the emulsifying properties of polymannosyl lysozymes. Keywords: Lysozyme; yeast expression system; genetic modification; polymannosylation; double glycosylation; emulsifying properties

Production of recombinant plant gum with tobacco cell culture in bioreactor and gum characterization

“…In contrast, the fusion glycoprotein, (GA) 8 GFP, exhibited an even higher emulsifying ability than native GAGP. This is consistent with other proteinpolysaccharide conjugates, which are often excellent emulsifiers (Khan et al, 1999;Nakamura and Kato, 2000;Shu et al, 1998), presumably because the protein component adsorbs to the oil droplet while the H-bond forming polysaccharides interact with aqueous phase and stabilize the droplets through steric hindrance. In the case of (GA) 8 GFP, however, we speculate that GFP probably interacted with the oil phase while the (GA) 8 glycoprotein domain interacted with the aqueous phase.…”

Industrial Applications of Maillard-Type Protein-Polysaccharide Conjugates.

“…To further elucidate the molecular mechanism of the improvement of functional properties of proteins by attachment with polysaccharide, genetic glycosylation of lysozyme was successfully attempted using the yeast expression system (Nakamura et al, 1993a(Nakamura et al, , 1993bKato et al, 1994;Shu et al, 1998;Kato et al, 1996). In yeast cells, the proteins having an Asn-X-Thr/Ser sequence are N-glycosylated in the endoplasmic reticulum and the attached oligosaccharide chain can be elongated with further extension of a large polymannose chain in the Golgi apparatus.…”

“…The advantage of genetic modification is to be able to design the binding site as wanted. The single polyannosyl lysozyme at positions 19 and 49, and the double polymannosyl lysozyme at both positions were constructed and their emulsifying properties were compared; the latter showed much higher emulsifying properties than the former lysozymes (Shu et al , 1998). This suggests that the formation of the thick steric stabilizing adsorbed layer around the emulsion is further enhanced and the coalescence of oil droplets was more effectively inhibited by increasing the number of glycosylation sites.…”

Incorporation of N-heterocyclic cations into proteins with a highly directed chemical modification

“…13 Importantly, only 4 of the 6 total number of lysine residues present in lysozyme have been modified previously and this required several chemical steps, with double glycosylation most probable. 9,14,15 However, in this work we have been able to access and modify all 6 residues in one step, depending on the overall concentration of the reagents, see Fig. 1.…”