胆红素氧化酶的分离纯化及其性质的研究

【作者】 龚艳秋；

【导师】 李大力； 陆路德；

【作者基本信息】 南京理工大学 ， 生物医学工程， 2004， 硕士

【摘要】 露湿漆斑菌发酵4天后，从发酵液中通过盐析、透析浓缩、SephadexG—100凝胶柱层析，分离纯化出胆红素氧化酶，通过SDS-聚丙烯酰胺电泳鉴定，显示为一条带，分子量为68kDa左右。通过紫外诱变选育到两株产酶能力稍高的菌株，其产酶能力是原先的1.5～2倍。该酶在37℃以胆红素为底物的Km值为150μmol／L，最适反应温度为50℃，从60℃开始酶的稳定性变差。该酶在pH7.5时酶促反应最快，酸碱稳定性较高，金属离子对酶促反应有很大影响，EDTA、尿素等能提高酶促反应。利用胆红素氧化酶可用来测定胆红素含量，进行总胆红素测定实验时表明该酶用于诊断具有线性范围较宽、准确性和精密度都较好，可为黄疸等肝胆疾病的诊断提供可靠的依据。更多还原

【Abstract】 Having been cultured 4 days in potato culture medium, Bilirubin oxidase(BOX) is purified from the culture filtrate of Myrothecium roridum after 4 days. The purified enzyme is obtained by the SephadexG-100 column chromatography. It shows a homogenous stain on the gel of SDS-PAGE. After treated by UV, two mutant strains with high yield of bilirubin oxidase are obtained. The molecular weight of the enzyme is about 68kDa. its optimum temperature is 50℃ and its optimum pH is 7.5. Metal ions have effect on its activity, EDTA and Urea can improve its activity and improve the stability of substrate. This enzyme can oxidaze bilirubin to biliverdin and further to an unknown substance, so it can be used to determine the bilirubin in serum in the clinical field. As an analytical tool, it has a wide line range and is accurate and exact.更多还原