NEW DIAGNOSTIC ANALYSIS FOR DIABETES MELLITUS USING ENZYMES FROM FILAMENTOUS FUNGI

糖化血红蛋白的测量是糖尿病诊断分析的标准方法，也是血糖测定的标准方法。由于糖化蛋白质的量反映了对应于蛋白质半衰期（白蛋白为14-20天，血红蛋白为1-3个月）的时期内的血糖水平，因此糖化蛋白质的水平是在治疗期间监测糖尿病患者的良好指标。用于测定血清中糖化蛋白的常用方法是HPLC。针对高效液相色谱法特异性差、操作繁琐等缺点，建立了一种特异性强、操作简便的果糖基氨基酸氧化酶（FAOD）酶法。我们在几种丝状真菌中发现了多种FAODs，并对其酶学性质进行了表征，获得了相应的酶基因，本项目进行了FAODs的大规模制备，并建立了新的FAODs检测体系。发现这些酶位于原始真菌的过氧化物酶体中。在对过氧化物酶体的形成和蛋白质向细胞器的转运机制了解的基础上，我们利用异源基因表达系统成功地在博伊丁假丝酵母中高效表达了FAOD。表达的FAODs在过氧化物酶体中特异性积累。此外，在以血红蛋白为碳源和氮源生长的细菌分离物中发现了催化糖化氨基酸裂解的蛋白酶。涉及蛋白酶（用于预处理）、FAOD和显色系统的测定系统被证实可有效用于糖化蛋白测定。

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.