Partial purification and characterization of ascorbate peroxidase from ripening ber (Ziziphus mauritiana L) fruits

Abstract

Ascorbate peroxidase (EC 1.11.1.11; APX) was purified from ripe ber (Ziziphus mauritiana L.) fruits var. Illaichi using conventional techniques of ammonium sulphate fractionation, gel filtration through Sephadex G-100 and ion-exchange chromatography on DEAE-cellulose. The enzyme was purified about 47.4 fold with 34.6% recovery. The molecular weight as determined by gel filtration was found to be 58.08 kDa. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) yielded a single major protein band with molecular weight of 29.79 kDa indicating that the enzyme was a homodimer. Native PAGE revealed a single prominent band suggesting that enzyme was purified to near homogeneity. The optimum pH for APX was found to be 7.8. It exhibited the Michaelis-Menten kinetics with Km values for ascorbate and H₂O₂ of 1.82 and 2.85 mM, respectively. Mn²⁺, NO₃^-, SO₄^2- and Co²⁺ were found to be potent inhibitors of APX while K⁺, Na⁺, Ca²⁺ and Cl^- stimulated the enzyme activity. Diethylpyrocarbonate (DEPC), dithiothreitol (DTT), NaBH₄ and mercaptoethanol inhibited the enzyme activity while iodoacetate and 5, 5’-dithiobis-2-nitrobenzene (DTNB) had no inhibitory effect. Based on the inhibition studies, histidine and tryptophan have been suggested to be present at the active site.

Keywords. Fruit ripening, Ziziphus mauritiana, ascorbate peroxidase, purification

African Journal of Biotechnology, Vol 13(31) 3323-3331