A highly stable laccase obtained by swapping the second cupredoxin domain

Area of Science:

• Biochemistry
• Protein Engineering
• Enzyme Technology

Background:

• Laccases are multi-copper oxidases with diverse industrial applications.
• Enhancing laccase stability is crucial for expanding their utility.
• Domain swapping offers a strategy for protein engineering.

Purpose of the Study:

• To improve the robustness and stability of a high-redox potential laccase.
• To investigate the impact of domain swapping on enzyme characteristics.
• To explore the application potential of the engineered laccase.

Main Methods:

• Domain swapping between fungal laccase genes.
• Engineering of signal peptides for enhanced secretion in Saccharomyces cerevisiae.
• Site-directed mutagenesis to identify stability determinants.
• Enzymatic treatment of kraft lignin.

Main Results:

• The domain-swapped laccase exhibited enhanced stability across a broad pH range (2-9), temperature (50-70°C), and in organic solvents.
• Improved activity in the presence of ethanol and methanol was observed.
• A newly formed salt bridge contributed significantly to pH stability.
• The engineered laccase demonstrated effective kraft lignin treatment under challenging conditions.

Conclusions:

• Domain swapping is an effective strategy for creating robust and stable laccases.
• The engineered laccase shows significant potential for industrial applications, particularly in biomass conversion.
• Further research into the structural determinants of laccase stability can guide future protein engineering efforts.