Computational Design of a Thermo-Acidostable Endo-Polygalacturonase for Efficient Juice Extraction

Area of Science:

• Enzyme engineering
• Biocatalysis
• Protein engineering

Background:

• Thermostable and pH-robust endo-polygalacturonases (endo-PGases) are vital for industrial applications, particularly in food processing.
• Existing enzymes often lack the required stability under harsh processing conditions.

Purpose of the Study:

• To engineer the thermostability of an acidic, thermophilic endo-PGase (PoxaEnPG28B) by rigidifying its flexible regions.
• To identify and design stabilizing mutations using computational methods.

Main Methods:

• Integrated computational strategy combining molecular dynamics (MD) simulations at elevated temperatures.
• In silico analyses of unfolding free-energy changes to identify stabilizing mutations.
• Characterization of mutant enzyme properties, including optimal temperature, half-life, activity at high temperatures, and pH stability.

Main Results:

• The D249K mutant exhibited a 5 °C higher optimal temperature (70 °C) and a 68.8% longer half-life at 55 °C.
• D249K retained over 76.8% activity at 75 °C and maintained the wild-type's optimal pH (5.0) and broad pH stability (3.0-8.0).
• In juice extraction, D249K increased yields by up to 98.5%, significantly surpassing the wild-type enzyme.

Conclusions:

• MD-guided flexible region engineering is effective for enhancing enzyme stability in the GH28 family.
• The D249K mutant offers an excellent balance of thermostability and pH robustness, making it a promising industrial biocatalyst.
• The engineered enzyme significantly improves efficiency in juice extraction applications.