Rational Design Strategy to Improve the Thermal Stability of Alginate Lyase Pedsa0632

Area of Science:

• Biochemistry and Enzymology
• Protein Engineering
• Industrial Biotechnology

Background:

• Alginate lyase enzymes are essential for producing alginate oligosaccharides (AOs), which have diverse applications in food, agriculture, and pharmaceuticals.
• The alginate lyase Pedsa0632 exhibits poor thermal stability, limiting its industrial utility.
• Improving enzyme thermal stability is critical for efficient industrial bioprocessing.

Purpose of the Study:

• To enhance the thermal stability of the alginate lyase Pedsa0632.
• To investigate the structural basis for improved thermostability.
• To demonstrate a rational design strategy for stabilizing oligomeric enzymes.

Main Methods:

• Characterization of wild-type (WT) alginate lyase Pedsa0632.
• Combinatorial engineering using disulfide bonds, salt bridges, and PROSS for mutant library generation.
• Construction and thermal stability testing of mutant L324 V-D353 V-M363T-T385 V (M3).

Main Results:

• WT Pedsa0632 showed poor thermal stability with a half-life of 6.54 min at 55 °C.
• Mutant M3 exhibited extraordinary thermal stability, retaining 60% activity after 11,000 min at 55 °C.
• Structural analysis indicated M3's enhanced stability is due to a more stable, compact interface and increased oligomer content.

Conclusions:

• Rational design effectively improved the thermal stability of alginate lyase Pedsa0632.
• The stabilization strategy, focusing on interface modification and oligomerization, is particularly effective for oligomeric enzymes.
• The thermostable M3 mutant holds significant potential for industrial applications requiring alginate oligosaccharide production.