Purification and Characterization of Hyaluronate Lyase from Arthrobacter globiformis A152

A hyaluronate lyase was obtained by cultivating Arthrobacter globiformis strain A152. The enzyme was purified to homogeneity from the supernatant by ammonium sulfate fractionation, Q Sepharose Fast Flow, and Sephadex G-100 chromatography. The purification resulted in a 32.78-fold increase in hyaluronate lyase activity with specific activity of 297.2 U/mg. The molecular weight of the enzyme determined by SDS-PAGE was approximately 73.7 kDa. Using hyaluronic acid (HA) as a substrate, the maximal reaction rate (V_max) and the Michaelis–Menten constant (K_m) of hyaluronate lyase were found to be 4.76 μmol/min/ml and 0.11 mg/ml, respectively. The optimum pH and temperature values for hyaluronate lyase activity were pH 6.0 and 42 °C, respectively. This enzyme was stable at pH 4–10, 5–7, and 5–7 at 4, 37, and 42 °C, respectively. Investigation about temperature effects on hyaluronate lyase displayed that it was stable at 30–37 °C and also showed high activity at 37 °C. The enzymatic activity was enhanced by Ca²⁺ and was strongly inhibited by Cu²⁺ and SDS. These properties suggested that the hyaluronate lyase in this study could bring promising prospects in medical and industry applications.