Design and structural analysis of an engineered thermostable chicken lysozyme

Area of Science:

• Protein Engineering
• Biochemistry
• Structural Biology

Background:

• Wild-type chicken egg-white lysozyme serves as a model protein for stability studies.
• Understanding protein stability is crucial for protein engineering and biotechnological applications.

Purpose of the Study:

• To construct a hyperstable variant of chicken lysozyme with enhanced thermal and chemical stability.
• To investigate the combined effects of multiple stabilizing mutations on lysozyme stability and activity.

Main Methods:

• Site-directed mutagenesis to introduce specific amino acid substitutions.
• Differential scanning calorimetry (DSC) to assess thermal stability (Tm).
• Guanidine hydrochloride (GdnHCl) denaturation to evaluate chemical stability.

Main Results:

• A hyperstable (hs) lysozyme variant was created with a +10.5°C increase in Tm and a +1.3 M increase in GdnHCl denaturation midpoint.
• The hs variant exhibited a 2.5-fold greater specific activity compared to wild-type lysozyme.
• The stabilizing effects of individual mutations were found to be cumulative and nearly additive.

Conclusions:

• Combining multiple stabilizing mutations is an effective strategy for engineering hyperstable proteins.
• The engineered hyperstable lysozyme demonstrates potential for applications requiring enhanced protein robustness.
• The study highlights the additive nature of stabilizing mutations in protein engineering.