title: Functional properties of glycosylated lysozyme secreted in Pichia pastoris.
abstract: Various mutant lysozymes having the N-glycosylation signal sequence, R21T (Asn(19)-Tyr(20)-Thr(21)), G49N (Asn(49)- Ser(50)-Thr(51)), R21T/G49N (Asn(19)-Tyr(20)-Thr(21)/Asn(49)-Ser(50)-Thr(51)), were secreted in the Pichia pastoris expression system. The secreted amounts of these mutant glycosylated lysozymes were almost the same as those of wild-type lysozyme (about 30 mg/liter). Glycosylation of the mutant lysozymes was confirmed by SDS-PAGE patterns, Endo-H treatment, TOF-MS analysis and chemical analysis. The composition of the carbohydrate chain attached to the single glycosylated lysozymes, R21T and G49N, was GlcNAc(2)Man(9-11), while that of the double glycosylated lysozyme, R21T/G49N, was GlcNAc(4)Man(27-32). The results of a CD analysis and lytic activity suggested that the conformation of the single glycosylated lysozymes had been conserved, while that of the double glycosylated lysozyme was less stable. The emulsifying properties of the lysozyme when glycosylated were greatly improved, being especially noteworthy in the double glycosylated lysozyme.
authors.affiliation: Department of Biological Chemistry, Yamaguchi University, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Saito
authors.forename: Yukikazu
authors.initials: Y
authors.lastname: Sako
authors.forename: Masakatsu
authors.initials: M
authors.lastname: Usui
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Azakami
authors.forename: Akio
authors.initials: A
authors.lastname: Kato
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D006031:Glycosylation; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009113:Muramidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000201:enzymology*; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013816:Thermodynamics
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D009113:Muramidase