High-Throughput Screening Assay for Laccase Engineering toward Lignosulfonate Valorization

Area of Science:

• Biotechnology
• Biochemistry
• Materials Science

Background:

• Lignin valorization is crucial for sustainable lignocellulose conversion.
• Lignosulfonates (LS) are widely commercialized technical lignins.
• Increasing LS molecular weight is necessary for applications like additives and dispersing agents.

Purpose of the Study:

• To develop a high-throughput screening (HTS) assay for directed evolution of laccases.
• To enable precise engineering of laccases for lignosulfonate polymerization.
• To facilitate the valorization of lignosulfonates.

Main Methods:

• Utilized fungal oxidoreductases (laccases) for lignosulfonate oxidation and cross-linking.
• Developed a colorimetric assay using Folin-Ciocalteau reagent (FCR) to detect decreased phenolic content.
• Adapted the assay to a 96-well-plate format for screening yeast-expressed laccase variants.

Main Results:

• The HTS assay demonstrated a colorimetric response (absorbance at 760 nm) correlating with laccase activity.
• The assay proved reproducible (CV = 15%) and sensitive for detecting activity differences in laccase mutant libraries.
• Validated the assay's feasibility for screening thousands of clones for enhanced laccase activity.

Conclusions:

• The developed HTS assay is effective for screening and directed evolution of laccases.
• This method supports the engineering of laccases for efficient lignosulfonate valorization.
• The approach advances the use of lignosulfonates in consumer goods and industrial applications.