Characterization of a Hyaluronidase from Paenibacillus residui BSSK58 and Evaluation of Its Industrial Applicability

Hyaluronidase is a hydrolytic enzyme that cleaves β-1,4-glycosidic linkages in high-molecular-weight hyaluronic acid, generating low-molecular-weight oligosaccharides with enhanced biological functions. These products exhibit immunomodulatory, antioxidant, and tissue-regenerative properties, making them valuable in pharmaceutical, cosmetic, and functional food applications. However, most commercial hyaluronidases originate from pathogenic bacteria or recombinant hosts, raising concerns over their biosafety and regulatory acceptance, particularly in food-grade applications. In this study, we report the isolation and characterization of a novel non-pathogenic soil bacterium, Paenibacillus residui BSSK58, which produces an extracellular hyaluronidase. Whole-genome sequencing revealed the absence of known virulence factors and antibiotic resistance genes. Phenotypic safety evaluations confirmed that there was no hemolytic activity, biogenic amine production, or cytotoxicity against human intestinal epithelial cell lines (Caco-2 and HT-29). The purified BSSK58 hyaluronidase exhibited a molecular weight of approximately 170 kDa, with optimal activity at pH 8.0–9.0 and 50 °C. The enzyme showed broad substrate specificity toward hyaluronic acid, chondroitin sulfate, and alginate, and its depolymerizing activity was confirmed using gel permeation chromatography. Furthermore, a 13-week oral repeated-dose toxicity study under Good Laboratory Practice conditions demonstrated no adverse effects. These findings support the use of BSSK58 hyaluronidase as a safe, non-recombinant biocatalyst suitable for industrial applications under regulatory-compliant frameworks.