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 H 2 O 2 of 1.82 and 2.85 mM, respectively. Mn 2+ , NO 3 - , SO 4 2- and Co 2+ were found to be potent inhibitors of APX while K + , Na + , Ca 2+ and Cl - stimulated the enzyme activity. Diethylpyrocarbonate (DEPC), dithiothreitol (DTT), NaBH 4 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
Highlights
MATERIALS AND METHODSBer fruits (varieties Umran and Illaichi) were harvested at different stages of maturity viz. immature green (IG), mature green (MG), colour turning (CT), ripe (R) and over-ripe (OR) from the orchards of CCS Haryana Agricultural University, Hisar, India for the purpose of studying the APX profile since Illaichi fruits harvested at ripe stage were used for the purpose of enzyme purification
Partia al purification n and charac c cterizattion off ascorrbate peroxidase from f ripening ber ((Ziziph hus ma auritian na L)
We report here the extraction, partial purification and characterization of APX
Summary
Ber fruits (varieties Umran and Illaichi) were harvested at different stages of maturity viz. immature green (IG), mature green (MG), colour turning (CT), ripe (R) and over-ripe (OR) from the orchards of CCS Haryana Agricultural University, Hisar, India for the purpose of studying the APX profile since Illaichi fruits harvested at ripe stage were used for the purpose of enzyme purification. Immature green (IG), mature green (MG), colour turning (CT), ripe (R) and over-ripe (OR) from the orchards of CCS Haryana Agricultural University, Hisar, India for the purpose of studying the APX profile since Illaichi fruits harvested at ripe stage were used for the purpose of enzyme purification. 95 mM potassium phosphate buffer (pH=7.0), 0.5 mM L-ascorbate, and 0.5 mM H2O2. Two hundred grams of ripe fruits was ground with 500 ml of potassium phosphate extraction buffer (pH=7.5, 0.1 M) containing 3% PVP, 1 mM EDTA and 1 mM. The dialyzed (NH4)2SO4 fraction was concentrated against solid sucrose and used for gel filtration chromatography. The e concentrated fraction obtaine ed after gel filtration was load ded over the top of the e DEAE cellulos se column (60 × 3 cm) previou usly.
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