Multifunctional elastin-like polypeptide renders β-glucosidase enzyme phase transition and high stability

Area of Science:

• Biotechnology
• Enzyme Engineering
• Biomass Conversion

Background:

• Efficient degradation of cellulose into glucose is crucial for biomass conversion.
• β-Glucosidase (Bgluc) is essential for cellulose degradation, but its stability is a bottleneck.
• Traditional protein purification methods are costly and reduce enzyme activity.

Purpose of the Study:

• To develop a simplified and efficient purification method for Bgluc.
• To enhance the stability and activity of Bgluc using a novel tag system.
• To create a cost-effective biocatalyst for lignocellulosic biomass degradation.

Main Methods:

• Constructed a recombinant fusion protein (BglucLEH) with Bgluc, ELP, and His tags.
• Expressed and purified BglucLEH in E. coli using inverse transition cycling (ITC) and Ni-NTA resin.
• Analyzed enzyme activity, stability, and kinetic parameters (Km, Kcat/Km).

Main Results:

• ITC purification yielded higher specific activity (75.5 U/mg) and recovery rate (77.8%) compared to Ni-NTA.
• The ELP tag did not affect Bgluc's secondary structure or enzyme activity.
• ELP significantly improved Bgluc stability under harsh conditions (heating, denaturants).

Conclusions:

• The Bgluc-linker-ELP-His system facilitates efficient purification and enhances Bgluc stability.
• This engineered BglucLEH is a promising tool for reducing costs and improving biocatalyst efficiency.
• The system has broad applications in lignocellulosic biomass degradation.