Preparation, Characterization and Anticancer Applications of HAase from Flavobacterium heparinum

Hyaluronidase (HAase) is attracting considerable attention in the field of medical cosmetics and exhibits considerable potential. In this study, a novel HAase (FH-HAase) was obtained from ultrasonication cells of Flavobacterium heparinum cultivated in fermentation broth containing hyaluronic acid. FH-HAase was separated and purified through affinity chromatography and ion exchange chromatography. The final purification fold of enzyme obtained was 48.76, the yield was 43.08%, and the specific activity was 33.06 IU/mL. The purified enzyme was displayed as a single band on electrophoresis, and its molecular weight was determined to be 79.6 KDa by LC-MS. The enzyme was stable at pH of 6.5-7.5 and temperature below 30℃. Hyaluronic acid is the optimal substrate for enzyme degradation, while chondroitin sulfate and dermatan sulfate can also be degraded. The purified HAase showed potent anticancer activities against melanoma B16F10 cells with low toxicity against HaCaTcell. The cell viability of HAase-treated B16F10 cells was found to be in a dose dependent manner. This is the first report, to our knowledge, on preparing HAase from microorganism Flavobacterium heparinum and the application of it in inhibiting tumor cell growth.