Consensus Design of an Evolved High-Redox Potential Laccase.

Area of Science:

• Biochemistry
• Protein Engineering
• Enzyme Stabilization

Background:

• Consensus design is a powerful protein engineering strategy for enhancing enzyme stability without sacrificing catalytic activity.
• Laboratory-evolved enzymes, such as high-redox potential laccase, can benefit from further stabilization for industrial applications.

Purpose of the Study:

• To apply an in-house consensus design method to stabilize a laboratory-evolved high-redox potential laccase.
• To identify and characterize consensus mutations that improve enzyme stability, kinetics, and secretion.

Main Methods:

• Multiple sequence alignments were performed and computationally refined using relative entropy and mutual information thresholds.
• 20 consensus mutations were identified, with 18 classified as consensus/ancestral mutations.
• Consensus variants were expressed in Saccharomyces cerevisiae and analyzed individually; site-directed recombination was explored.

Main Results:

• The best single consensus variant featured the A240G mutation near the T2/T3 copper cluster.
• This A240G mutation significantly enhanced the laccase's thermostability and kinetic parameters.
• Improved secretion of the engineered laccase was also observed.

Conclusions:

• The in-house consensus design method is effective for stabilizing enzymes like high-redox potential laccase.
• The A240G mutation represents a key improvement for laccase performance, particularly in terms of stability and kinetics.
• Further optimization through recombination of mutations did not yield synergistic benefits (epistasis).