Abstract: Polygalacturonases (PGA) have important application prospects in the field of food processing. This study aims to heterologously express and characterize the thermophilic polygalacturonase AfPGA from Aspergillus fumigatus HBFH5 and explore its application for the preparation of pectin oligosaccharides (POS) form pineapple peel. The results showed that the polygalacturonase AfPGA belongs to the GH28 family, with a molecular mass of 39.2 kDa and an optimal temperature of 70 ℃. After treatment at 55 ℃ for 1 h, the enzyme’s activity remained stable. The optimal pH value was 5.0, and over 80% of the initial activity remained after treatment for 1 hour within the pH range of 2.0–12.0. It had strong resistance to various metal ions and chemical reagents. When using polygalacturonic acid as the substrate, the Km and Vmax of the recombinant enzyme were 1.05 mg/mL and 52.6 μmol/(min·mg), respectively. Molecular dynamics (MD) simulation analysis found that the root mean square deviation (RMSD) and root mean square fluctuation (RMSF) of the mesophilic polygalacturonase ADPG2 were higher than those of AfPGA. In addition, POS prepared with AfPGA had strong antibacterial activity, which could significantly inhibit the growth of Salmonella, Staphylococcus aureus, Escherichia coli, and Shigella. POS also had good antioxidant properties. In summary, this study enriches the resources of thermophilic PGA, providing theoretical support for the application of this enzyme in POS preparation.

Key words: polygalacturonase; thermophilic enzyme; molecular dynamics simulation; pectin oligosaccharides; antibacterial activity; antioxidant activity