摘要
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.
出处
《微生物学报》
CAS
CSCD
北大核心
2005年第5期711-715,共5页
Acta Microbiologica Sinica
基金
国家"863计划"(2001AA24161)~~
关键词
变色栓菌
锰过氧化物酶
纯化
性质
Trametes versicolor, Manganese peroxidase, Purification, Properties
