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

TL;DR

Scientists designed a more stable enzyme that works well at high temperatures and acidic conditions, improving juice extraction efficiency.

Contribution

A novel computational strategy combining MD simulations and free-energy analysis to engineer a thermo-acidostable endo-polygalacturonase.

Findings

The mutant D249K showed a 5 °C higher optimal temperature and 68.8% longer half-life at 55 °C.

D249K retained over 76.8% activity at 75 °C and maintained pH stability from 3.0 to 8.0.

D249K increased juice extraction yields by up to 98.5% compared to the wild-type.

Abstract

The development of thermostable and pH-robust endo-polygalacturonases (endo-PGases) is crucial for industrial applications such as food processing. This study aimed to engineer the thermostability of an acidic, thermophilic endo-PGase (PoxaEnPG28B) by rigidifying its flexible regions. We employed an integrated computational strategy combining molecular dynamics (MD) simulations at elevated temperatures with in silico analyses of unfolding free-energy changes to identify and design stabilizing mutations. This approach successfully yielded the mutant D249K, which exhibited a 5 °C higher optimal temperature (70 °C) and a 68.8% longer half-life at 55 °C, and it retained over 76.8% activity at 75 °C. Notably, D249K maintained the wild-type’s optimal pH (5.0) and broad pH stability (3.0–8.0). Although it is not the absolute top performer in every single metric, D249K achieves the best overall balance between thermostability and pH robustness among all reported thermophilic endo-PGases. MD simulations revealed that its enhanced stability sems from reduced global and local flexibility and a more compact structure. In juice extraction applications, D249K increased yields by up to 98.5%, significantly surpassing the wild-type. This study demonstrates the efficacy of MD-guided flexible region engineering for the GH28 family and presents D249K as a highly promising industrial biocatalyst.

Figures