Characterization of hyaluronate lyase from Streptococcus pyogenes bacteriophage H4489A

Hyaluronate (HA) lyase of Streptococcus pyogenes bacteriophage H4489A, was expressed in Escherichia coli, purified, and characterized. The purified homogeneous preparation of HA lyase had a molecular mass of 40 kDa. The optimum enzymatic activity was achieved at pH ∼ 5.5 and 37 °C, and the enzyme was stable at pH profile from 4 to 7 and temperature range from 25 to 45 °C. The enzymatic activity was vaguely enhanced by Mg²⁺, slightly inhibited by Ca²⁺, triton X-100, and Tween 80, strongly inhibited by Zn²⁺, and completely inhibited by Cu²⁺, Ni²⁺, Co²⁺ and sodium dodecyl sulfate. Kinetic measurements give Michaelis constant of 0.44 mg/ml, maximal velocity of 0.20 μmol ml^-1 min^-1, and showed that bacteriophage HA lyase degraded the HA efficiently. Light scattering dynamic measurements determined the denaturation temperate of HA lyase of about 46 °C. Circular dichromism and UV-visible absorption spectroscopy estimated the changes in secondary structure of native and denatureated HA lyase.