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

Praduman Yadav*; Taruna Yadav; Sunil Kumar; Babita Rani; Sandeep Kumar; Veena Jain; Sarla P.Malhotra

doi:10.5897/AJB2013.12193

African Journal of
Biotechnology

Abbreviation: Afr. J. Biotechnol.
Language: English
ISSN: 1684-5315
DOI: 10.5897/AJB
Start Year: 2002
Published Articles: 12504

Full Length Research Paper

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

Praduman Yadav*

Praduman Yadav*
Plant Biochemistry and Quality Control Laboratory, Directorate of Oilseeds Research, Hyderabad-500030, Andrapradesh, India.
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Taruna Yadav

Taruna Yadav
Chemistry Laboratory, Banasthali University, Banasthali, Rajasthan, India.
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Sunil Kumar

Sunil Kumar
Plant Biochemistry and Molecular Biology Laboratory, CCS HAU, Hisar, Haryana, India.
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Babita Rani

Babita Rani
Plant Biochemistry and Molecular Biology Laboratory, CCS HAU, Hisar, Haryana, India.
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Sandeep Kumar

Sandeep Kumar
Germplasm Evaluation Division, National Bureau of Plant Genetic Resources, New Delhi, India.
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Veena Jain

Veena Jain
Plant Biochemistry and Molecular Biology Laboratory, CCS HAU, Hisar, Haryana, India.
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Sarla P.Malhotra

Sarla P.Malhotra
Plant Biochemistry and Molecular Biology Laboratory, CCS HAU, Hisar, Haryana, India.
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Article Number - 050946346544
Vol.13(32), pp. 3323-3331 , August 2014
https://doi.org/10.5897/AJB2013.12193

Received: 27 February 2014
Accepted: 14 July 2014
Published: 06 August 2014

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), NaBH₄and 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.