Isolation and Affinity Purification of Peroxidase from Daikon

Peroxidase enzyme plays an important role in pharmaceutical, Industrial and various research laboratories. This enzyme can be purified by various different method. Our research trend was to purify the enzyme in a convenient and easy way. For this purpose a new affinity column was prepare by immobilizing PABA to N-HydroxySuccinamaideagarose gel. The presence of peroxidase was investigated on various Bangladeshi vegetables, such as, daikon, cabbage, cauliflower, tomato and sweet potato. Based on a previous screening experiment, daikon contains the highest amount of peroxidase. The juice of daikon was first extracted by tincture press and subjected to ammonium sulphate precipitation. The precipitate was dissolved in 25 mM phosphate buffer, pH7.4 containing 500 mMNaCl, 1 mM MnCl2 and 1 mM CaCl2. The crude extract thus obtained was subjected to an inhibitor affinity chromatography (IAC) method. Here, we have taken advantage of the affinity of peroxidase toward p-aminobenzoic acid hydrazide (PABAH) and subsequent recovery of the peroxidase by a known destabilizing agent ascorbic acid. The partially purified sample was applied to the column containing p-aminobenzoic acid hydrazide immobilized on agarose equilibrated with the same buffer. After washing the column with this buffer, daikon peroxidase was eluted with 5 mM ascorbic acid in the initial buffer. The eluted fraction showed 60 folds purification as judged by peroxidase activity measurement INTRODUCTION: Peroxidase has a wide range of application in health science, food industry and in diagnostic purposes. It has been used in delignification of paper pulp. It is use in laundry detergents for bleaching of dyes in solution and thus preventing surplus dye from one garment to deposit on and decolorize other garments. QUICK RESPONSE CODE DOI: 10.13040/IJPSR.0975-8232.6(7).2813-19 Article can be accessed online on: www.ijpsr.com DOI link: http://dx.doi.org/10.13040/IJPSR.0975-8232.6(7).2813-19 Other potential applications are polymerization of chlorinated aromatic compounds in the treatment of waste-water, enhancement of flavors in food and polymerization of lignin in the production of various types of composite materials, where the use of artificial resins thus may be avoided. Various medical diagnostic kits contain appreciable amount of peroxidase in their compositions. In spite of having the reported demand for peroxidase, no such efforts to produce peroxidase from Bangladeshi vegetables been reported in the literature to our knowledge. We have found that some indigenous vegetables contain considerable