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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. | Turkish Journal of Biology http://journals.tubitak.gov.tr/biology/ Research Article Turk J Biol (2013) 37: 661-669 © TÜBİTAK doi:10.3906/biy-1208-37 Purification and characterization of trehalase from seeds of chickpea (Cicer arietinum L.) Maimona KORD, Elhusseiny YOUSSEF, Hanaa AHMED*, Ebtesam QAID Department of Botany, Faculty of Science, University of Cairo, Giza, Egypt Received: 17.08.2012 Accepted: 03.05.2013 Published Online: 08.10.2013 Printed: 04.11.2013 Abstract: 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. Key words: Cicer arietinum, trehalase, trehalose, purification, molecular mass, validamycin A 1. Introduction Trehalose is a nonreducing disaccharide, formed of 2 α-Dglucose molecules linked by an α,α-1,1-glycosidic linkage. It is widely distributed through the biological world. This sugar plays multiple physiological roles in stabilizing and protecting proteins and membranes against environmental stresses. It also serves as a source of energy and a source of carbon, and as a sensing and .