Precise Immobilization Strategy Combined with Rational Design to Improve β-Agarase Stability

Area of Science:

• Biotechnology
• Enzyme Engineering
• Materials Science

Background:

• Enzyme immobilization is crucial for industrial applications.
• Precise control over enzyme orientation and activity is challenging.
• Developing stable and active immobilized enzymes is essential for green manufacturing.

Purpose of the Study:

• To develop a rational design strategy for site-specific covalent immobilization of β-agarase.
• To enhance the stability and activity of immobilized β-agarase.
• To explore the potential of immobilized enzymes in green manufacturing.

Main Methods:

• Rational screening of surface sites for cysteine mutation using molecular dynamics and energy calculations.
• Site-specific covalent immobilization via Michael addition reaction with maleimide-modified magnetic nanoparticles (MAL-MNPs).
• Enzyme activity retention, thermal deactivation kinetics, and tolerance assays (metal ions, organic reagents).

Main Results:

• Successfully achieved site-specific covalent immobilization of β-agarase onto MAL-MNPs.
• Immobilized enzymes (R66C-MAL-MNPs, K588C-MAL-MNPs) showed >96% activity retention.
• Significant improvement in thermal stability (21.25-fold increase in t1/2 at 40 °C) and enhanced tolerance to metal ions and 50% acetone/water solutions (>90% activity maintained).

Conclusions:

• The developed rational design and site-specific immobilization strategy effectively enhances β-agarase stability and functionality.
• This approach offers a robust method for creating highly stable and active immobilized enzymes.
• The findings support the application of precisely immobilized enzymes in sustainable and green manufacturing processes.