Engineering of β-Agarase with Enhanced Thermostability via Multitool Consensus Prediction and Structure-Guided...

Area of Science:

• Biotechnology
• Enzyme Engineering
• Biochemistry

Background:

• β-Agarase is essential for producing bioactive agar oligosaccharides.
• Limited thermostability of wild-type β-Agarase hinders industrial applications.
• Developing thermostable β-Agarase is crucial for efficient industrial production.

Purpose of the Study:

• To enhance the thermostability of β-Agarase (AgaDcat) for industrial applications.
• To develop an efficient enzyme engineering strategy for improving thermostability.
• To investigate the structural basis for enhanced thermostability.

Main Methods:

• A stepwise design strategy combining consensus prediction, structure-based screening, and greedy optimization.
• Site-directed mutagenesis to create the M3 variant (N120S-D243N-Q246A-S287E-A335D).
• Molecular dynamics simulations to analyze structural changes and interaction networks.

Main Results:

• Mutant M3 exhibited an 11 °C higher melting temperature and a 14-fold longer half-life at 50 °C compared to wild-type.
• Molecular dynamics revealed strengthened hydrophobic interactions, salt bridges, and hydrogen bonding in M3.
• The M3 variant efficiently hydrolyzed agarose at high temperatures, producing neoagarotetraose (NA4) and neoagarohexaose (NA6).

Conclusions:

• The engineered M3 β-Agarase demonstrates significantly improved thermostability and industrial potential.
• The applied stepwise enzyme engineering framework enhances efficiency for industrial enzyme modification.
• This approach offers a general strategy for improving the thermostability of industrial enzymes.