Immobilization of a Thermostable Diamine Oxidase from Vigna radiate (L.) Wilczek Seedlings

The purified diamine oxidase (DAO, E.C. 1.4.3.6.) form the germinating seedlings of Vigna radiate (L.) Wilczek cv. P-105 (Choudhary et al., 1999) and commercially available porcine kidney DAO were immobilized on zirconia coated alkylamine glass beads via gultraldehyde coupling. A coupling yield obtained was 14.2 and 19.09 mg/gm of glass beads for mungbean and porcine kidney DAO respectively and a total of 75% of mungbean enzyme was coupled to the carrier whereas the coupling of porcine kidney DAO was 19% only. The immobilized enzyme obtained from mungbean retained about 88% of the activity upto 60 days and 65% upto 180 days at 4 C in comparison to 85% of the activity of porcine kidney DAO upto 60 days and only 27% upto 180 days. The optimum incubation time, pH and temperature of the DAO from Vigna radiate on immobilization were estimated to be 18 min, 6.5 and 65�C, respectively, with putrescine (Put), with minor variation in all the parameters with porcine kidney DAO, except for temperature optima at 60�C for the later. The Km value for the immobilized enzyme, with Put as the substrate, was 0.604 and 0.484 mM for the DAO from plant and the animal sources, respectively. The Vmax value was estimated to be 0.01 and 0.0166 mole ?` pyyroline min-1 for Vigna DAO, Cu+2 binding agents and heavy metals showed almost total inhibition of the catalytic activity of the enzyme during the immobilization. However, the enzyme was less sensitive to NaCl, MgSo4 Cacl2 MnCl2 and (NH4)2 SO4. The DAO from both the sources exhibited high thermal stability and less sensitivity to the inorganic ions.