خالص سازی و بررسی خواص سینتیکی آنزیم هیستامیناز از گونه خلر ( Lathyrus sativus ) با استفاده از کروماتوگرافی تعویض آنیون

Background : The histamine poisoning directly correlates to improper preservation of foods. Diamine oxidase catalyses the oxidative deamination of histamine and other biogenic amines such as cadaverine and putrescine. Plant diamine oxidases are of widespread occurrence in Leguminaceae such as Cicer arietinum, Lathyrus sativus and Vigna radiata. For this reason, this study was carried out to examine purification and enzyme kinetic properties of histaminase from the Lathyrus sativus seedling. Materials and Methods: The purification of histaminase was according to the method given by Federico et al., with some modifications. In short, using ammonium sulfate precipitation of proteins, dialysis against potassium phosphate buffer 50mM, pH=7 over night, concentrating via dialysis bag and PEG, loading on DEAE52-sephadex column previously equilibrated with 15 mM potassium phosphate buffer pH=5.5 containing 1 µmol CuCl2 and finally enzyme eluted by increasing the NaCl concentration to 0.2 M. The homogenicity of enzyme was confirmed by SDS-PAGE in each step and diamine oxidase activity was determined by a spectrophotometer. Results: The specific activity of the final purified enzyme was recorded at 50 U/mg protein for histamine substrate and enzyme kinetic properties of this enzyme (Km=2.5 mM, Vmax=3.05), and the homogenicity of purified enzyme by SDS-PAGE at 72 kDa region were indicated. Conclusion: Overall, the herbal histaminase as a new enzyme in prevention of histamine poisoning is promising.