Development of stable cholesterol oxidase used for diagnosis

目的研制稳定的胆固醇氧化酶，用于样品中胆固醇的测定。这种酶必须长期稳定，因为这种酶要出口到欧洲。我们设计了链霉菌胆固醇氧化酶，使其在大肠杆菌中过量产生，并改变了酶的活性位点和热稳定性预测位点，并通过计算机图形化推导出酶的活性位点。三年来，我们致力于以下研究工作，并取得了丰硕的成果。通过比较链霉菌和甾醇短杆菌中胆固醇氧化酶的结构-功能，建立了模型。通过位点定向诱变，将与酶活性位点和FAD结合位点有关的预测位点的氨基酸替换为其他氨基酸。确定了活性部位。构建了两种新的功能酶，它们可以催化每种胆固醇的氧化或异构化。通过对链霉菌和短杆菌两种酶的比较，推导出与热稳定性有关的预测位点。通过对预测位点的定点诱变，构建了一个高度稳定的酶。克隆并分析了编码胆固醇降解酶的基因。发现了酮类固醇- delta ^1脱氢酶、酮类固醇- delta ^5异构酶和调节因子的编码基因。

For the purpose of development of stable cholesterol oxidase which can be used for determination of cholesterol in specimen. The enzyme has to be stable for long term since the enzyme are exporting to Europe. We design the cholesterol oxidase from Streptomyces sp. to overproduce in Escherichia coli and change the active site and predicted site of thermal stability of the enzyme in which the sites were deduced by computer graphic. For three years, we have devoted the following research work and obtained fruitful results.1.We made modeling and constructed the structure-function by comparison of cholesterol oxidases between the enzymes from Streptomyces and Brevibacterium sterolicum.2.Amino acids of the predicted sites concerned with enzymatic active site and FAD binding site were substituted with other amino acids by site-directed mutagenesis. The active site was determined. Two new functional enzymes that can catalyze each oxidation or isomerization of cholesterol were constructed.3.By comparison with two enzymes from Streptomyces and Brevibcterium, we deduced the predicted sites concerned with thermal stability. By site-directed mutagenesis of the predicted site, a highly stable enzyme was constructed.4.The genes encoded cholesterol degradating enzymes were cloned and analyzed. The genes encoded ketosteroid-DELTA^1-dehydrogenase, ketosteroid-DELTA^5-isomerase and the regulator were found.