Purification and Characterization of Peroxidase from Papaya (Carica papaya) Fruit
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Ripening of papaya fruit was found to be characterized with a decrease in peroxidase activity and its transcript. This peroxidase was purified to homogeneity through successive steps of ammonium sulfate fractionation, ion exchange and molecular exclusion chromatography. The peroxidase was purified 30.22-folds with overall recovery of 44.37 % and specific activity of 68.59. Purified peroxidase was found to be a heterotrimer of ~240 kDa, containing two subunits each of 85 and one of 70 kDa. Purified enzyme exhibited pH and temperature optima of 7.0 and 40 °C, respectively. K m values for substrates o-dianicidin, guaiacol and ascorbic acid were found to be 0.125, 0.8 and 5.2 mM, respectively. K m for H2O2 was found to be 0.25 mM. Salicylic acid was found to activate peroxidase up to 50 μM concentration, beyond which it acted as inhibitor. Ca2+ and Mg2+ activated peroxidase while sodium azide, SDS, and Triton X-100 were found to inhibit peroxidase.
KeywordsFruit ripening Peroxidase Real-time PCR Papaya Salicylic acid
Financial assistances from UGC, New Delhi (in the form of UGC-JRF to VPP), Department of Biotechnology, New Delhi, Council of Scientific and Industrial Research, New Delhi and Department of Higher Education, Government of U.P. under Centre of Excellence Grant are gratefully acknowledged.
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