Purification and characterization of trehalase from seeds of chickpea (Cicer arietinum L.)
In the present study, trehalase was purified and characterized from the seeds of Cicer arietinum L. 'Giza 1'. Crude extract was prepared and purified for electrophoretic homogeneity using ammonium sulfate, chromatography on DEAE-cellulose, CM Sepharose, and Sephadex G-200. The final specific activity was 7 U/mg protein, with 232-fold purification. The purified enzyme exhibited its pH optimum at 5.5. The optimum temperature was 60 °C. The determined Km value was 3.64 mM trehalose. The enzyme activity was stimulated by 20 mM Mn2+, Ni2+, or Co2+, while it was inhibited by 20 mM Na+, K+, Li+, Ca2+, Zn2+, Cu2+, or Fe3+. Zn2+ proved to be a noncompetitive inhibitor, while mannitol and validamycin A proved to be competitive inhibitors. The inhibition constants (Ki) of Zn2+, mannitol, and validamycin A were 7 mM, 9 mM, and 4 nM, respectively. The molecular mass of the native enzyme was 223 kDa by gel filtration. SDS-PAGE indicated that the enzyme consisted of 6 identical subunits with a molecular mass of 38 kDa.
Purification and characterization of trehalase from seeds of chickpea (Cicer arietinum L.)
In the present study, trehalase was purified and characterized from the seeds of Cicer arietinum L. 'Giza 1'. Crude extract was prepared and purified for electrophoretic homogeneity using ammonium sulfate, chromatography on DEAE-cellulose, CM Sepharose, and Sephadex G-200. The final specific activity was 7 U/mg protein, with 232-fold purification. The purified enzyme exhibited its pH optimum at 5.5. The optimum temperature was 60 °C. The determined Km value was 3.64 mM trehalose. The enzyme activity was stimulated by 20 mM Mn2+, Ni2+, or Co2+, while it was inhibited by 20 mM Na+, K+, Li+, Ca2+, Zn2+, Cu2+, or Fe3+. Zn2+ proved to be a noncompetitive inhibitor, while mannitol and validamycin A proved to be competitive inhibitors. The inhibition constants (Ki) of Zn2+, mannitol, and validamycin A were 7 mM, 9 mM, and 4 nM, respectively. The molecular mass of the native enzyme was 223 kDa by gel filtration. SDS-PAGE indicated that the enzyme consisted of 6 identical subunits with a molecular mass of 38 kDa.
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