Purification and characterization of neutral invertase from chickpea nodules.
Neutral invertase from nodules of chickpea (Cicer arietinum L.) was isolated and purified by ammonium sulphate fractionation, gel filtration and DEAE-cellulose column chromatography. The purified enzyme was stable between 0 to 40 degrees C beyond which it was irreversibly denatured. Optimum temperature and pH of the enzyme were 37 degrees C and 7.0, respectively. K(m) for sucrose was 14.2 mM and Vmax was 4.8 mumole hr-1. The enzyme was inhibited by several metal ions. From the temperature effect on K(m) and Vmax values, the energy of activation (Ea), enthalpy change (delta H) and entropy change (delta S) of the enzyme were calculated to be 147 kJmol-1, -4.10 kJmol-1 and -2.33 JK-1mol-1, respectively. By employing photo-oxidation and chemical modification and by studying the effect of pH on K(m) and Vmax, the involvement of sulphydryl-, imidazole- and alpha-amino groups in the active site of the enzyme has been indicated.
Asthir
B
B
Department of Biochemistry, Punjab Agricultural University, Ludhiana, India.
Singh
R
R
D001665:Binding Sites; D002412:Cations / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D007887:Fabaceae / Q000201:enzymology*; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010946:Plants, Medicinal; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013816:Thermodynamics; D043324:beta-Fructofuranosidase
D002412:Cations; D004791:Enzyme Inhibitors; D013439:Sulfhydryl Reagents; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase