枇杷果肉过氧化物酶的分离纯化及其性质研究

枇杷果肉制成丙酮粉,通过硫酸铵沉淀分级分离、透析、DEAE-纤维素离子交换柱层析纯化,获得两个酶活力峰,第二活力峰的酶制剂经过PAGE和SDS-PAGE电泳获得单一纯的过氧化物酶(PODⅡ),纯酶比活力为710.5U/mg。酶亚基分子量为22.6kDa,等电点pI为3.6。酶的动力学研究表明:酶的最适pH为5.0,在pH2.6～10区域较稳定;酶最适温度是35℃,对热敏感,高于45℃以上酶的稳定性差。酶对愈创木酚和H2O2的表观Km值分别为20.58和12.04mmol/L,以愈创木酚为底物时,酶比活力最大,其次是苯酚、邻苯二酚、对苯二酚和间苯二酚,酶未能催化以焦性没食子酸为底物的氧化反应。Al3+、Mn2+、Zn2+和Hg2+对酶有抑制作用,Mg2+、Ca2+、Cu2+、Fe2+、Ba2+和Pb2+对酶有激活作用;半胱氨酸等12种化合物对酶活力均有不同程度的抑制作用,在防止或减轻枇杷采后的酶促褐变中将起到重要的作用。

The Study on Purification and Properties of Peroxidase from Loquat Fruit

Peroxidase in the acetone powder of loquat(Eriobotrya japonica Lindl.cv.Zaozhong 6) fruit was extracted and purified through ammonium sulfate fractionation,dialysis and chromatography on DEAE-cellulose(DE-32).Two enzyme activity peaks were obtained,and the purified enzyme from the second activity peak determined to be homogeneous by polyacrylamide gel electrophoresis(PAGE) and SDS-PAGE.The specific activity of the purified enzyme was 710.5 U/mg.Molecular weight of enzyme subunit was 22.6 kDa and the pI value was calculated to be 3.6 by isoelectric focusing.The optimum pH and optimum temperature of the enzyme were pH 5.0 and 35 ℃,respectively.The stability of the enzyme was investigated,and the results showed that the enzyme was stable relatively in a pH range from 2.6 to 10.0 and at temperatures<45 ℃.Kinetic studies showed that the reaction catalyzed by the enzyme followed the typical behavior of ordered mechanism of two-substrate reaction.The Kmapp values of 4-methylcatechol and H2O2 were 20.58 and 12.04 mmol/L,respectively.Several compounds including phenolic compounds were used as the substrates of the enzyme for specificity study.Meanwhile,the effects of metal ions on the enzyme were studied.The result indicated Mg2+,Ca2+,Cu2+,Fe2+,Ba2+ and Pb2+ activated the enzyme,while Al3+,Mn2+,Zn2+ and Hg2+ showed various degrees of inhibitory effects on the enzyme.The effects of twelve inhibitors on this peroxidsae were also assayed,and this study may provide some enzymatic inhibitors for post-harvest treatment to delay such browning.