Development of stable cholesterol oxidase used for diagnosis

目的是为了开发稳定的可用于测定样品中胆固醇的胆固醇氧化酶。这种酶必须长期稳定，因为这种酶正在出口到欧洲。我们从链霉菌中设计了胆固醇氧化酶。在大肠杆菌中高效表达，改变酶的活性部位和预测的热稳定性部位，并用计算机图形学方法推断其活性部位。三年来，我们致力于以下研究工作，取得了丰硕的成果：1.通过比较链霉菌和类固醇短杆菌胆固醇氧化酶的结构和功能，对其进行了建模和构建。2.通过定点突变，将预测的与酶活性部位和FAD结合部位相关的氨基酸替换为其他氨基酸。确定了活性部位。构建了两种新的催化胆固醇氧化或异构化的功能酶。3.通过与链霉菌和短杆菌产生的两种酶的比较，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突变，构建了一种高度稳定的酶。4.克隆并分析了胆固醇降解酶的编码基因。发现了编码酮类固醇-Delta^1-脱氢酶、酮类固醇-Delta^5-异构酶及其调控因子的基因。

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.