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 mu mol/min/ml and 0.11 mg/ml, respectively. The optimum pH and temperature values for hyaluronate lyase activity were pH 6.0 and 42 A degrees C, respectively. This enzyme was stable at pH 4-10, 5-7, and 5-7 at 4, 37, and 42 A degrees C, respectively. Investigation about temperature effects on hyaluronate lyase displayed that it was stable at 30-37 A degrees C and also showed high activity at 37 A degrees 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.