Molecular Basis of Thermostability in a Novel Polygalacturonase MlPG28A and Its Application in Producing Bioactive Oligogalacturonides for Sustainable Aquaculture

A novel thermostable polygalacturonase, MlPG28A, was identified from Mucor lusitanicus and characterized for biochemical properties, structural determinants of thermostability, and potential in the enzymatic production of functional oligogalacturonides (OGs). MlPG28A exhibited exceptional heat tolerance, retaining over 70% of catalytic activity at 100 °C and maintaining stability across pH 3–11. Structural modeling and site-directed mutagenesis revealed that a unique disulfide bond (Cys275–Cys283) and hydrophobic interactions involving Tyr274 jointly stabilize the T1 loop structure, while Lys106 and Asn160 contribute to active-site stabilization via electrostatic interactions and hydrogen bonding. Under optimized hydrolysis conditions determined by response surface methodology, MlPG28A produced OGs with a maximal reducing-sugar yield of 3.233 mg·mL–1. The resulting OGs exhibited concentration-dependent antioxidant activity, demonstrating measurable DPPH and ABTS radical-scavenging effects within the tested concentration range, and displayed selective antibacterial activity. Application tests in simulated aquaculture systems confirmed that OGs effectively inhibited both Gram-negative and Gram-positive pathogens.