Preparation of Bioactive and Surface Functional Oligomannosyl Neoglycoprotein Using Extracellular pH-Sensitive Glycosylation of Mutant Lysozyme Having N-Linked Signal Sequence in Yeast

Abstract

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.

Synthesis and Characterization of Cholesteryl Conjugated Lysozyme (CHLysozyme)

“…There are several ways to chemically modify proteins, for instance conjugating polyethylene glycol to protein (PEGylated protein) is of value as this enhances the chemical stability, solubility, and blood retention of the protein during clinical use [ 3 , 4 , 5 , 6 , 7 , 8 ]. The chemical stability (e.g., against thermal stress) of proteins has also been improved by modification with polysaccharides, which are hydrophilic groups [ 9 , 10 , 11 ]. The chemical modification of proteins has mostly been via conjugation with hydrophilic groups, but there are a few reports on chemical modification using hydrophobic groups [ 12 , 13 ].…”

Complex Coacervate Core Micelles with a Lysozyme-Modified Corona

“…The initial decrease in the absorbance at 450 nm of the mixture caused by lysis of M. lysodeikticus cells was measured at 25 °C for 3 min with a Cary 100 Bio UV-vis spectrophotometer as described elsewhere. 27,28 An example of an enzymatic curve is given in the inset in Figure 5. The initial slope of these curves was taken as a measurement of the enzymatic activity.…”

“…A solution containing lysozyme or lysozyme−C3M conjugates (0.2 mL of 0.1 mg/mL) was added to 2 mL of the cell suspension in 10 mM sodium phosphate buffer with 50 mM NaNO 3 (pH 7.7). The initial decrease in the absorbance at 450 nm of the mixture caused by lysis of M. lysodeikticus cells was measured at 25 °C for 3 min with a Cary 100 Bio UV−vis spectrophotometer as described elsewhere. , An example of an enzymatic curve is given in the inset in Figure . The initial slope of these curves was taken as a measurement of the enzymatic activity.…”

Cholesteryl-Conjugated Ribonuclease A Exhibits Enzyme Activity in Aqueous Solution and Resistance to Dimethyl Sulfoxide

“…The goal of PCM is to enhance the functionality of proteins by increasing their structural and functional diversity. , This technique has also been used in the development of new drugs, including antibody–drug conjugates. − Proteins can be chemically modified in several ways, including through conjugation to polyethylene glycol (PEG), which has proved useful in clinical applications. PEG conjugation not only improves the stability and solubility of proteins but also increases their retention time in the body. − Many proteins are modified with hydrophilic groups; for example, polysaccharides may be conjugated to proteins in this manner, which improves their thermal stability against stress conditions. − Although most proteins are modified with hydrophilic groups, some studies have introduced modifications with hydrophobic groups. , …”