Abstract
Polyvinyl alcohol (PVA) is a widely used industrial polymer, and its persistence in the environment poses significant challenges, making the study of its biodegradation both a major production concern and an important scientific issue. This study aimed to elucidate the enzymatic mechanisms of PVA biodegradation by identifying and characterizing a novel oxidized polyvinyl alcohol hydrolase (OPH) from Stenotrophomonas rhizophila QL-P4, and to investigate its evolutionary divergence across microbial species. Through genomic analysis, we identified a gene, BAY15_0160 (996 bp), encoding a putative OPH. Its essential role in PVA degradation was confirmed via gene deletion, overexpression, and functional complementation assays. Site-directed mutagenesis demonstrated that the signal peptide and active site are indispensable for enzymatic function. The recombinant enzyme expressed in E. coli BL21(DE3) exhibited optimal specific activity (5.19 U/mg) at pH 7.0 and 30 °C, with a Michaelis constant (Km) of 0.1765 mM. Structural modeling and domain analysis revealed conserved catalytic features, which were further supported by simulated docking between the catalytic domain and substrate molecules. These results experimentally validate the functional importance of these domains. Furthermore, comparative sequence alignment with bacterial and fungal homologs uncovered a highly adapted fungal homolog, suggesting evolutionary divergence in OPH functionality. These findings provide a theoretical foundation for understanding PVA biodegradation mechanisms and developing microbial-based bioremediation strategies, while also demonstrating the potential of BAY15_0160 in environmental engineering applications-paving the way for designing efficient industrial microbial agents and enzyme preparations.