变色栓菌锰过氧化物酶同工酶的纯化及其性质研究

Purification and properties of manganese peroxidase from Trametes versicolor

变色栓菌(Trametes versicolor)胞外产酶培养液经硫酸铵沉淀、DEAE-cellulose DE52离子交换柱层析后,获得两个活性组分D1和D2,其中活性组分D2经Phenyl Sepharose TM 6 Fast Flow疏水层析后,所得样品MnP1经SDS-PAGE检测已达到电泳纯.活性组分D1经Phenyl SepharoseTM 6 Fast Flow疏水层析、Sephacryl S-200HR凝胶过滤层析后,所得样品MnP2经SDS-PAGE检测已达到电泳纯.两种同工酶MnP1及MnP2,各自的比活力为579.09、425.00U/mg;纯化倍数为17.51、12.85;活力回收率为6.17%、2.47%.由SDS-PAGE法测得MnP1及MnP2的表观分子量分别为46.3kD、43.0kD.两种同工酶催化DMP(2,6-二甲氧基酚)氧化反应的最适pH值及最适反应温度有所不同,最适pH值分别为pH5.8、pH6.2,最适反应温度分别为60℃、65℃.在45℃以下,pH4.0～7.0之间,MnP1及MnP2的稳定性好.DMP为最佳酶促反应底物,以DMP为底物的Km分别为13.43μmol/L、12.45μmol/L.在无Mn2+存在的条件下,酶促反应几乎不发生.EDTA在较高浓度时抑制酶的活性,DTT在所试浓度下都完全抑制酶的活性.

Two manganese peroxidase （MnP） active fractions D1 and D2 were got from the extracellular culture of Trametes versicolor by using ammonium sulfate precipitation, DEAE-cellulose DE52 chromatography. MnP1 was purified to electrophoretic homogeneity from the D2 by Phenyl Sepharose^TM 6 Fast Flow chromatography and MnP2 was purified to electrophoretic homogeneity from the D1 by Sephacryl S-200HR chromatography and Phenyl SepharoseTM 6 Fast Flow chromatography. The specific activities of two MnP isozymes are 579.1U/mg and 425.0U/mg; purification folds are 17.51 and 12.85 and the yields are 6.17% and 2.47%, respectively. MnP1 and MnP2 have approximate molecular masses of 46.3kD and 43.0kD respectively, as determined by SDS-PAGE. The isoenzymes differed in optimum temperature （60℃ and 65℃ ） and optimum pH（5.8 and 6.2） for oxidation of DMP （2,6-dimethoxyphenol） . MnP1 and MnP2 are stable below 45℃ and ranging from pH4.0 to pH7.0. DMP is the best substrate, the Km values of MnP1 and MnP2 for DMP are 13.43μmol/L and 12.45μmol/L respectively. Catalysis doesn＇t occur in the complete absence of Mn. EDTA inhibites the activities of MnP1 and MnP2 at the higher concentration and DTT inhibites the enzyme activities completely.