Isolation, Purification, Enzymatic Characterizations And Immobilization Of Peroxidase From Leaves Of Ipomoea Aquatica Forsk

Peroxidase(POD, EC1.11.1.7) as one of the widely hemoglobinoxidases, consists of peptide and ferriporphyrin, which can catalyze a variety of oxidation-reduction reaction involving H2O2, such as the oxidation of amines and phenols.POD plays an important role in organisms, for example, decomposition of IAA, synthesis of lignin and defense responses. Currently, it can not only be widely used in western blot, enzyme-linked immunosorbent assay and electron microscopy, but also show the significance in sewage treatment and biological sensors.Electrophoresis pure peroxidase was obtained from Ipomoea Aquaticsuccessively by means of homogenation, buffer solution extraction, ammonium sulfate precipitation,ion exchange chromatography on DEAE-Sepharose Fast Flow column and gel filtration chromatography on Superdex-200. Its specific activity was35972.96U/mg, the purification fold was168.48, and the recovery rate is12.21%. Molecular mass of this purified enzyme was around42.7KD determined by electrophoresis on SDS-PAGE gel filtration chromatography on Superdex-200.Besides, the peroxidase, whose optimal temperature and pH were60℃and6, was relatively stable in the range of20～50℃and pH5-8. And the peroxidase showed Km value of18.32mmol/L under definite conditions. In addition, the peroxidase was found to be activated by NaCl、Urea、Zn2+、Mg2+, but inhibited by SDS, KSCN, AsA, Ba2+Mn2+, Fe2-, methanol, ethanol, n-butyl alcohol and isopropanol, especially without activity when the concentration of AsA reached0.01mmol/L.After isolated and purified from Ipomoea Aquatica Forsk, peroxidase is selectively modified by using BD, DTT, pCMB, Maleic anhydride, Chloramine-T, PMSF, NAI and BrAc separately and its change on activity is also determined. The result shows:Arg, Lys, His and sulfhydryl involving in the composition of the enzyme active center should be considered as indispensable functional groups of peroxidase from Ipomoea Aquatica Forsk, while Met, Ser and Tyr residues should not be because they were not directly related to the activity of peroxidase from Ipomoea Aquatica Forsk.The enzyme was immobilized with0.2%PVA-CA as carrier, saturated boric acid solution as crosslinker and CaCl2as fixing agent. Their effects on POD immobilization were determinedby means of single factor analysis, and the optimal combination of their agentsviaorthogonal test were0.2%PVA-3%CA, the1:2ratio between PVA-VA and enzyme, saturated boric acid-6%CaCl2and45min immobilizing time. After immobilization, the optimal temperature, stability in high temperature and the reuse stability enhanced.