Re-Engineered β-Agarase for Efficient Agarose Oligosaccharide Preparation with Enhanced Thermostability

Area of Science:

• Biochemistry
• Enzymology
• Protein Engineering

Background:

• Agarases are key enzymes for producing bioactive agar oligosaccharides.
• Enzyme thermostability is critical for industrial applications due to temperature-dependent substrate properties.
• The agarase Aga50D was selected for protein engineering to improve its industrial utility.

Purpose of the Study:

• To re-engineer the exo-β-agarase Aga50D for enhanced thermostability and catalytic activity.
• To investigate the structural and mechanistic basis for improved enzyme performance.
• To assess the potential of the engineered enzyme for industrial agar oligosaccharide production.

Main Methods:

• Site-directed mutagenesis was used to create the A86D mutant of Aga50D.
• Enzyme activity and thermostability were assessed through catalytic performance assays and melting temperature determination.
• Structural insights were gained using Small-angle X-ray Scattering (SAXS), Differential Fluorimetry Scanning (DFS), and Size Exclusion Chromatography (SEC).
• Molecular dynamics simulations were employed to elucidate the molecular mechanisms underlying the observed improvements.

Main Results:

• The A86D mutant displayed a 23.3 °C increase in melting temperature compared to the wild-type enzyme.
• Catalytic performance for agarose hydrolysis at 50 °C improved more than seven-fold.
• Structural analyses indicated that the mutation promoted enzyme dimerization.
• Molecular dynamics simulations revealed that the mutation strengthened interchain salt-bridge interactions at the subunit interface, enhancing stability.

Conclusions:

• A single mutation near the subunit interface can significantly enhance enzyme thermostability and catalytic efficiency.
• Targeted design of intersubunit interactions is a viable strategy for developing high-stability industrial enzymes.
• The engineered A86D mutant holds considerable potential for efficient industrial production of agar oligosaccharides.