Computer-Aided Rational Design Strategy to Improve the Thermal Stability of Alginate Lyase AlyMc

Area of Science:

• Biochemistry
• Enzyme Engineering
• Protein Engineering

Background:

• Alginate lyase degrades alginate into unsaturated alginate oligosaccharides (UAOS) with enhanced bioactivities.
• Improving the thermal stability of alginate lyases is critical for industrial enzyme applications.
• Current methods for enzyme stabilization often lack efficiency and precision.

Purpose of the Study:

• To develop an efficient rational design strategy for enhancing alginate lyase thermal stability.
• To create mutant alginate lyases with improved thermostability and specific activity.
• To investigate the structural basis for enhanced enzyme stability and activity.

Main Methods:

• Computer-aided ΔΔG value calculation and B-factor analysis for rational enzyme design.
• Site-directed mutagenesis to create Q246V and K249V mutants.
• Enzyme activity assays, thermal stability measurements (t1/2, 50°C), structural analysis, and molecular dynamics simulations.

Main Results:

• Two thermal stability-enhanced mutants, Q246V and K249V, were successfully generated.
• Mutant Q246V showed a t1/2, 50°C increase from 2.36 to 3.85 h; K249V increased to 3.65 h.
• Specific activities of Q246V and K249V were enhanced 2.41- and 2.96-fold, respectively.
• Mutations improved hydrogen bond networks and molecular rigidity, confirmed by structural analysis and simulations.

Conclusions:

• The developed rational design strategy effectively enhances alginate lyase thermal stability and activity.
• Mutant Q246V demonstrates superior thermal stability within the PL-7 alginate lyase family.
• This approach holds significant potential for advancing industrial applications of alginate lyase.