Abstract:

Fresh chives were homogenized, extracted with buffer and precipitated by ammonium sulfate. The precipitant
was dissolved in HAc-NaAc and then purified by CM-Sepharose ion-exchange chromatography and superdex-200 gel
filtration chromatography. A new type of electrophoretically pure acid phosphatase (ACP) from fresh Chinese chives was
then obtained. The enzyme activity of the purified ACP reached 645.83 U/mg. The purification factor was 454.81 and the
activity recovery was 10.50%. Characterization studies showed that the molecular mass of this enzyme was approximately
58.97 kD and the optimum reaction temperature and pH value were 65 ℃ and 5.4, respectively. The Km value for this
enzyme was 0.896 × 10-3 mol/L when using nitrophenyl phosphate disodium (pNPP) as the substrate. High concentrations
of Mg2+ and Mn2+ showed stronger activation of the enzyme while the higher concentration of ascorbic acid, methanol,
ethanol, chloroform, isopropanol, Cu2+ and Ag+, the stronger the inhibition of the enzyme.

Key words: Chinese chives, acid phosphatase, isolation and purification, characterization