Thermal stabilization of an endoglucanase by cyclization

Area of Science:

• Biochemistry
• Protein Engineering
• Enzymology

Background:

• Intein-mediated protein splicing enables the creation of cyclic proteins.
• Cyclic protein structures can offer advantages over linear forms.
• Bacillus licheniformis endo-β-1,3-1,4-glucanase (LicA) is a target for stability engineering.

Purpose of the Study:

• To engineer circular variants of LicA using intein-driven protein splicing.
• To evaluate the impact of cyclization on LicA's catalytic activity and thermal stability.
• To investigate the role of calcium ions in the stability and function of circular LicA.

Main Methods:

• Intein-driven protein splicing to generate circular LicA variants (LicA-C1, LicA-C2).
• Enzyme activity assays to compare catalytic efficiency.
• Thermal stability assessments using incubation at elevated temperatures and differential scanning calorimetry.
• Fluorescence spectroscopy to monitor conformational changes.

Main Results:

• Circular LicA variants (LicA-C1, LicA-C2) displayed 2-3 fold higher catalytic activity than linear LicA (LicA-L1).
• Circular variants exhibited significantly enhanced thermal stability, with 6-fold and 16-fold longer half-lives of inactivation at 65 °C.
• Circular enzymes showed increased thermal transition temperatures, indicating greater structural stability.
• Calcium ions further enhanced the thermal stability of circular LicA.

Conclusions:

• Protein cyclization via intein-driven splicing is an effective strategy to enhance enzyme activity and stability.
• The length of connecting loops influences the stability of circular proteins.
• Circular LicA variants represent promising biocatalysts with improved robustness for industrial applications.