Preparation of bioactive and surface functional oligomannosyl neoglycoprotein using extracellular pH-sensitive glycosylation of mutant lysozyme having N-linked signal sequence in yeast.

Bioactive oligomannosyl lysozyme with improved surface functionalities was successfully prepared by using an extracellular pH-sensitive glycosylation system for heterogeneous protein in yeast cell. A recombinant Saccharomyces cerevisiae carrying a mutant lysozyme gene encoding the signal sequence of an N-linked glycosylation site at position 49 was cultivated in various pH conditions to investigate the effects of extracellular pH on the glycosylation patterns and the expression of the protein. A large polymannose (Man(310)GlcNAc(2)) chain-linked lysozyme was predominantly expressed accompanied by small amounts of a core-type oligomannose chain (Man(14)GlcNAc(2))-linked lysozyme in the yeast medium where the extracellular pH was kept at 3.5 or above, while an oligomannose chain lysozyme was preferentially expressed in the yeast medium where the pH was less than 3. The lytic activities of the oligomannosyl and the polymannosyl lysozymes were found to be 70.4 and 5.1%, respectively, of the wild-type lysozyme when Micrococcus lysodeikticus cells were used as the substrate. The enzymatic activity of the oligomannosyl lysozyme was totally conserved for the glycolysis assay with a soluble substrate, glycol chitin, whereas that of the polymannosyl lysozyme was not. After heating the sample up to 95 degrees C at pH 7.0 where no visible protein coagulation was observed, thermostability of the enzymatic activity of the oligomannosyl lysozyme was drastically improved with more than 60% of residual lytic activity. Emulsifying properties of the protein also were highly improved by the oligomannosylation, in which the emulsifying activity was 3.2 times higher than that of the wild-type protein. Corresponding to the increase of the surface functionalities, the surface tension of the oligomannosyl protein exhibited a significantly (p < 0.05) lower value compared to that of the wild-type. By using the lower pH medium at 3.0, it was revealed that a substantial amount (0.31 mg/L) of the oligomannosyl lysozyme was successfully obtained in the culture medium. Therefore, the extracellular pH-sensitive glycosylation system can be used to obtain bioactive and surface functional neoglycoproteins.