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