Extraction, Purification And Characteristic Of POD From Agaricus Bisporus

Peroxidase(POD), a variety of enzyme, widely found in fruit and vegetable,can promote many reactions and generally will not be inhibited by other enzymes,which plays a vital role in the browning of fruit and vegetable during storage.The crude enzyme was extracted from the Agaricus bisporus, whose pileus unopened and without mechanical damage, after homogenization, ammonium sulfate precipitation(using 20% and 80% ammonium sulfate solution one after another). whereafter, Electrophoresis-purity peroxidase, relative molecular mass74.08 KDa, was obtained sequentially after DEAE-Cellulose-52 chromatography and Sephadex G-100 gel filtration chromatography from this crude enzyme. Its specific activity was 426.69 U/mg, the purification fold was 66.77 and the recovery rate was 13.41%.The analysis of purified POD, its apparent Km of guaiacol(as substrate) was5.704mmol/L, showed that it had an an affinity for guaiacol. The enzyme activity of POD reached the maximum when the concentration of H2O2 was 0.8%. The optimum p H of POD was 5.0, the optimum temperature was 45°C; the thermostability of POD was good at the condition of 30-50°C heated for 5h, which accounted for strong heat resistance of POD. DTT had the strongest inhibition of POD, followed by ASA, L-Cys, SDS, anhydrous sodium sulfite, citric acid and Na Cl.The comparison of the change in POD activity and the whiteness of Agaricus bisporus’ centrum and pulp, during the storage by lower temperature, preservative and controlled atmosphere, showed that POD activity in the Agaricus bisporus stored in controlled atmosphere was lowest, but the highest whiteness arose in which treated with preservative. This indicated that POD activity has no direct effect on the browning of Agaricus bisporus. The enzyme activity of POD declined prominently at the condition of 2°C and 10°C in comparison with the condition of20°C, which was consistent with the characteristics of POD. Furthermore, the trend of whiteness of Agaricus bisporus tended to drop off over time during the storage.