Preparation, Characterization and Anticancer Applications of HAase from Pedobacter heparinus

Background Hyaluronidase (HAase) are a group of glycosidases that catalyze the degradation of hyaluronic acid (HA) into low-molecular-weight oligosaccharides, playing pivotal roles in physiological and pathological processes. Their applications span from drug delivery enhancers to diagnostic and therapeutic tools in oncology. Results In this work, a novel hyaluronidase (Ph-HAase) was produced by Pedobacter heparinus cultivated in a HA acid-enriched fermentation medium. Extraction was performed via ultrasonication, followed by a two-step purification process employing affinity and ion-exchange chromatography. The protocol yielded 43.08% recovery with a 47.5-fold purification and a specific activity of 32.32 IU/mg. SDS-PAGE and LC-MS analyses confirmed the homogeneity of Ph-HAase, showing a single band with a molecular mass of 79.6 kDa. The enzyme demonstrated optimal stability at pH 6.5–7.5 and temperatures up to 30°C. While HA served as the preferred substrate, Ph-HAase also exhibited degradative activity toward Chondroitin Sulfate (CS) and Dermatan Sulfate (DS). Notably, Ph-HAase displayed significant anti-melanoma activity against B16F10 cells (IC₅₀=28.63 µg/mL) with minimal cytotoxicity toward HaCaT keratinocytes (IC₅₀=86.12 µg/mL). Mechanistic evaluation revealed that Ph-HAase triggers apoptosis in B16F10 cells through the mitochondrial pathway, associated with loss of membrane potential and ROS accumulation. In vivo studies further corroborated its anti-tumor efficacy in a murine melanoma model. Conclusion This study constitutes the first report on the production, purification, and characterization of a HAase from P. heparinus . Ph-HAase shows promise as a novel biocatalyst with potential therapeutic applications in melanoma treatment.