| Project/Area Number |
09556017
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
KATO Nobuo Kyoto University, Graduate School of Agriculture, Professor, 農学研究科, 教授 (50026556)
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| Co-Investigator(Kenkyū-buntansha) |
UENOYAMA Seizo Kyoto Dai-ichi Kagaku, Basic Technology Laboratory, Manager, 基盤研究所, 所長
YURIMOTO Hiroya Kyoto University, Graduate School of Agriculture, Instructor, 農学研究科, 助手 (00283648)
SAKAI Yasuyoshi Kyoto University, Graduate School of Agriculture, Associate Professor, 農学研究科, 助教授 (60202082)
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| Project Period (FY) |
1997 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥11,600,000 (Direct Cost: ¥11,600,000)
Fiscal Year 1999: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1998: ¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 1997: ¥7,400,000 (Direct Cost: ¥7,400,000)
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| Keywords | Albumin / Hemoglobin / heterogeneous gene expression / Fructosyl amino acid oxidase / Diagnostic analysis for diabetes mellitous / Methylotrophic yeast / 糖化タンパク質 / フルクトシルアミノ酸オキシダーゼ / メタノール質化性酵母 / ペルオキシソーム / Candida boidinii / 糖化アミノ酸 / 酵素定量法 / フルクトシルアミノ酸オキシターゼ |
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| Research Abstract |
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.
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