NEW DIAGNOSTIC ANALYSIS FOR DIABETES MELLITUS USING ENZYMES FROM FILAMENTOUS FUNGI

糖化血红蛋白的测定是糖尿病诊断分析的标准方法，也是测定血糖的标准方法。由于糖化蛋白的量反映了蛋白质半衰期（白蛋白14-20天，血红蛋白1-3个月）相应时期的血糖水平，因此糖化蛋白水平是监测糖尿病患者治疗过程中的良好指标。测定血清中糖化蛋白的常用方法是高效液相色谱法。为了克服高效液相色谱法特异性较低和操作繁琐的缺点，我们开发了一种新的酶促法，使用果糖基氨基酸氧化酶（FAOD），具有高特异性和易于进行。我们在几种丝状真菌中发现了多种FAODs，并对其酶特性进行了表征，获得了与这些酶相对应的基因。在本项目中，我们进行了FAODs的大规模制备，并开发了新的FAODs检测系统。这些酶被发现位于原始真菌的过氧化物酶体中。在了解过氧化物酶体的形成和蛋白质进入细胞器的转运机制的基础上，我们利用异质基因表达系统成功地在假丝酵母中实现了FAOD的高表达。表达的faod特异地聚集在过氧化物酶体中。此外，在一种生长在血红蛋白上作为碳和氮源的细菌分离物中发现了一种催化糖基化氨基酸裂解的蛋白酶。该检测系统包括蛋白酶（用于预处理）、FAOD和着色系统，证实对糖化蛋白的测定是有效的。

The measurement of glycated hemoglobin is a standard method for diagnostic analysis of diabetes mellitus, as well as determination of sugar in blood. Since the amount of glycated proteins reflect the level of blood glucose in periods corresponding to the half-life of the proteins (14-20 days for albumin and 1-3 months for hemoglobin), the level of the glycated proteins are good indices for monitoring diabetes mellitus patients during therapy. The prevalent methods used for the determination of glycated proteins in serum are HPLC. To overcome several demerits of lower specificity and tedious procedures for HPLC, we developed a new enzymatic method using fructosyl amino acid oxidase (FAOD), which is highly specific and easy to conduct. We have found a variety of FAODs in several filamentous fungi, characterized the enzymatic properties, and obtained the genes corresponding to the enzymes.In this project, we performed the large-scale preparations of FAODs, and developed new assay system using FAODs. The enzymes were found to locate in the peroxisome of the original fungi. On the basis of our knowledges on the formation of peroxisome, and transport mechanism of proteins into the organelle, we succeeded high expression of FAOD in Candida boidinii using the heterogeneous gene expression system. The expressed FAODs were specifically accumulated in the peroxisome. Furthermore, a protease that catalyzed cleavage of a glycated amino acid was found in a bacterial isolate which was grown on hemoglobin as carbon and nitrogen sources. The assay system involving the protease (for pretreatment), a FAOD, and coloring system was confirmed to be effective for glycated protein determination.