A novel chemoenzymatic glycosylation strategy: application to lysozyme modification

Area of Science:

• Biochemistry
• Protein Modification
• Enzymology

Background:

• Hen egg lysozyme is a well-studied enzyme with known antibacterial properties.
• Protein glycosylation is a common post-translational modification that can alter protein function and stability.
• Non-specific glycosylation offers a route to create novel protein derivatives with potentially enhanced characteristics.

Purpose of the Study:

• To develop a novel two-step strategy for the non-specific glycosylation of hen egg lysozyme.
• To investigate the effectiveness of different glycosyltransferases (fructosyltransferase and levansucrase) in chain elongation.
• To evaluate the impact of glycosylation on key properties of lysozyme, including hydrophobicity, enzymatic activity, substrate affinity, and thermostability.

Main Methods:

• Chemical conjugation of sucrose molecules to protein surface lysines.
• Enzymatic elongation of glycosidic chains using levansucrase (identified as most effective).
• Optimization of reaction conditions to achieve varying degrees of modification.
• Assessment of changes in protein hydrophobicity, hydrolytic activity, substrate affinity, and thermostability.

Main Results:

• A novel two-step method for hen egg lysozyme glycosylation was successfully established.
• Levansucrase proved more effective than fructosyltransferase for enzymatic chain elongation.
• The degree of protein modification was controllable.
• Significant alterations in lysozyme's hydrophobicity, activity, affinity, and thermostability were observed post-glycosylation.

Conclusions:

• The developed glycosylation strategy provides a versatile tool for modifying hen egg lysozyme.
• Glycosylation significantly impacts the functional and physical properties of lysozyme.
• These modified lysozymes represent potential candidates for applications requiring altered enzymatic or stability profiles.