露湿漆斑菌胆红素氧化酶的分离纯化及其性质

Purification and Characterization of Bilirubin Oxidase from Myrothecim roridum

露湿漆斑菌产生的胆红素氧化酶粗酶液通过硫酸铵沉淀、DEAE-Sepharose Fast Flow和Sephadex G-100纯化,得到纯化152.54倍胆红素氧化酶,得率19.14%;用PAGE检测为单一条带,活性电泳证实该单一蛋白质组分为胆红素氧化酶,SDS-PAGE证明该酶的相对分子质量为64000。该酶最适反应温度为55℃,以胆红素为底物时最适pH值为7.5,而以ABTS为底物时最适pH值为4,该酶在pH 3-8之间都能够检测到酶活。以胆红素为底物,胆红素氧化酶的Km值和Vmax分别为552.06μmol/L和38.91μmol/（L.min）;而以ABTSA为底物,胆红素氧化酶的Km值和Vmax分别为631.84μmol/L和12.79μmol/（L.min）。

Bilirubin oxidase from Myrothecim roridum was purified to electrophoretic homogeneity by the steps of ammonium sulfate precipitation, DEAE-Sepharose Fast Flow and Sephadex G-100 column chromatography. Purification of about 152.54 fold was achieved with an overall yield of 19. 14%. It was demonstrated to be bilirubin oxidase by native PAGE, and its molecular weight was estimated to be about 64 000 by SDS-PAGE. The optimum temperature of the enzyme activity was 55℃. The optimum pH for bilirubin and ABTS were 7. 5 and 4, respectively. The Km for bilirubin and ABTS was 552.06 μmol/L and 631.84 μmol/L, respectively. In addition, The Vmax for bilirubin and ABTS was 38, 91 μmol/（L · min） and 12.79 μmol/（L · min）, respectively.