Partial purification and biochemical characterization of acid phosphatase from germinated mung bean (Vigna radiata) seeds
Mung bean (Vigna radiata) is one of the important crops of the North Eastern Region of India. In the present study, acid phosphatase enzyme was isolated and partially purified from germinated local mung bean seeds. The sequential partial purification process was performed using ammonium sulphate precipitation method. The crude enzyme having a specific activity of 0.50 U/mg was purified using 30 to 70% ammonium sulphate precipitation method. The acid phosphatase was purified by 2.6 fold with a yield of 58.9% and specific activity of 1.3 U/mg. One prominent band was obtained on sodium dodecyl sulphate-polyacrylamide gel electrophresis (SDS-PAGE) which confirmed the purity of the enzyme. Molecular weight of the enzyme was estimated as 34.5 kDa. The enzyme activity was measured at different incubation time, pH, temperature and substrate concentration. The activity increased slowly from 10 to 70 min of incubation. The maximum activity was obtained at 70 min, thereafter the activity decreased gradually. The enzyme was found to be active over a wide range of temperature (30 to 80°C) and maximum activity was observed at 70°C. The optimal pH value of the enzyme activity was found to be 5.2. There was a corresponding increase in the rate of reaction with the increase in the substrate concentration from 0.1 to 0.8 mM and a linear relationship was obtained at 2 to 8 mM. Both Km and Vmax value were calculated as 0.416 mM and 1.33 µmoles/min, respectively.
Key words: Acid phosphatase, mung bean, Vigna radiata, enzyme purification, enzyme characterization.