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NEW DIAGNOSTIC ANALYSIS FOR DIABETES MELLITUS USING ENZYMES FROM FILAMENTOUS FUNGI

使用丝状真菌酶对糖尿病进行新的诊断分析

基本信息

批准号：
09556017
负责人：
KATO Nobuo
金额：
$ 7.42万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

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.

糖化血红蛋白的测量是糖尿病诊断分析以及血糖测定的标准方法。由于糖化蛋白质的量反映了与蛋白质半衰期相对应的时期(白蛋白为14-20天，血红蛋白为1-3个月)的血糖水平，因此糖化蛋白质的水平是监测糖尿病患者在治疗期间的良好指标。用于测定血清中糖化蛋白的常用方法是 HPLC。为了克服高效液相色谱法特异性较低和操作繁琐的缺点，我们开发了一种使用果糖基氨基酸氧化酶（FAOD）的新酶法，该方法特异性高且易于操作。我们在多种丝状真菌中发现了多种FAODs，对其酶学性质进行了表征，并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，并开发了利用FAODs的新检测系统。发现这些酶位于原始真菌的过氧化物酶体中。基于我们对过氧化物酶体形成和蛋白质进入细胞器的转运机制的了解，我们利用异质基因表达系统成功地在博伊丁假丝酵母中高表达FAOD。表达的FAODs专门积累在过氧化物酶体中。此外，在以血红蛋白作为碳源和氮源生长的细菌分离株中发现了催化糖化氨基酸裂解的蛋白酶。经证实，涉及蛋白酶（用于预处理）、FAOD 和着色系统的测定系统对于糖化蛋白测定是有效的。