Laboratory evolution of high-redox potential laccases

Area of Science:

• Biotechnology
• Enzyme Engineering
• Protein Engineering

Background:

• Laccases are multi-copper oxidases with broad industrial applications.
• Enhancing laccase activity, stability, and secretion is crucial for biotechnological use.
• Previous efforts focused on improving specific properties, but combined enhancements remain challenging.

Purpose of the Study:

• To engineer a highly active and thermostable laccase with improved secretion in Saccharomyces cerevisiae.
• To combine directed evolution with rational design for synergistic improvements in enzyme kinetics and stability.
• To identify key mutations responsible for enhanced enzyme function and expression.

Main Methods:

• A strategy combining directed evolution with rational approaches was employed.
• The laccase signal sequence was replaced with the α-factor prepro-leader.
• Molecular evolution was performed over eight rounds using Saccharomyces cerevisiae for improved kinetics and secretion.

Main Results:

• Total laccase activity was enhanced 34,000-fold, yielding the OB-1 mutant.
• The OB-1 mutant demonstrated high activity and stability across a wide range of temperatures, pH, and organic cosolvents.
• Mutations in the hydrophobic core of the leader sequence enhanced functional expression.
• Mutations in the mature protein improved catalytic capacities through altered residue interactions.

Conclusions:

• The engineered OB-1 laccase represents a significant advancement in enzyme engineering for industrial applications.
• The combined strategy of directed evolution and rational design is effective for creating superior biocatalysts.
• Further studies can explore the detailed structural basis of the OB-1 mutant's enhanced properties.