Abstract
Hyaluronic acid (HA) in the tumor microenvironment forms a physical barrier that hinders the penetration of chemotherapeutic drugs. Hylp1 is a bacteriophage-encoded hyaluronate lyase with a unique triple-stranded β-helix structure harboring three active centers. In this study, we recombinantly expressed Hylp1 in Escherichia coli and developed a purification scheme that yielded high-purity and high-activity enzyme. Enzymatic characterization showed that recombinant Hylp1 retained substantial activity at 37 °C, degraded HA via an endolytic β‑elimination mechanism, and exhibited higher catalytic activity than recombinant human hyaluronidase PH20 (rHuPH20). In A549 lung cancer cells, Hylp1 alone showed no direct cytotoxicity. However, when combined with gemcitabine and cisplatin, Hylp1 synergistically inhibited cell proliferation, migration, and invasion. Using 3D tumor spheroids, we demonstrate that Hylp1 promotes diffusion of small-molecule drugs into the spheroid interior by degrading HA in the extracellular matrix, thereby increasing chemotherapeutic cytotoxicity. These findings establish Hylp1 as an adjuvant that improves chemotherapy efficacy in HA‑rich solid tumors.