Preparation, characterization and anticancer applications of HAase from Pedobacter heparinus

Hyaluronidase (HAase) is an enzyme capable of degrading hyaluronic acid (HA). In anti-tumor therapy, HAase can enhance the efficacy of anti-cancer drugs by regulating the tumor microenvironment and overcoming drug delivery barriers, and it can also be used directly to inhibit tumor growth. In this study, we identified a previously uncharacterized HAase (Ph-HAase) from Pedobacter heparinus. After purification, the enzyme was obtained with a 43.08% recovery, a 47.5-fold purification, and a specific activity of 32.32 IU/mg. SDS-PAGE and LC-MS analyses revealed that the molecular weight of Ph-HAase was 79.6 kDa. Ph-HAase exhibited excellent stability at temperatures below 30 °C and within the pH range of 6.5 to 7.5. The enzyme activity was found to be relatively high at pH 6.5 and 45℃. Although HA served as the preferred substrate, Ph-HAase also exhibited degradative activity toward Chondroitin Sulfate (CS) and Dermatan Sulfate (DS). Notably, we found that Ph-HAase induced apoptosis in melanoma B16F10 cells via the mitochondrial pathway, characterized by loss of membrane potential and ROS accumulation, and affected the expression of apoptosis-related genes and proteins. In vivo studies further confirmed its anti-melanoma effect. This study is the first to report HAase derived from P. heparinus and to demonstrate its potential for melanoma treatment.