| Project/Area Number |
18380058
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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 | 一般 |
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| Research Field |
Applied microbiology
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| Research Institution | Kyoto University |
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Principal Investigator |
YAMAMOTO Kenji Kyoto University, Graduate School of Biostudies, Professor (70109049)
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| Co-Investigator(Kenkyū-buntansha) |
ASHIDA Hisashi Kyoto University, Graduate School of Biostudies, Research Associate (40379087)
KATAYAMA Takane Ishikawa Prefectuial University, Research Institute for Bioresource and Biotechnology, Lecturer (70346104)
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| Project Period (FY) |
2006 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥16,320,000 (Direct Cost: ¥14,700,000、Indirect Cost: ¥1,620,000)
Fiscal Year 2007: ¥7,020,000 (Direct Cost: ¥5,400,000、Indirect Cost: ¥1,620,000)
Fiscal Year 2006: ¥9,300,000 (Direct Cost: ¥9,300,000)
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| Keywords | Endoglycosidase / Transgalycosylation / Glycotechnology / Bioactive Compounds / Glycosylation / Endo-β-N-acetylglucosaminidase / Endo-α-N-acetylgalactosaminidase / Glycosynthase / Endo-M / エンド-β-N-アセチルグリコサミ二ダーゼ / ムチン |
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| Research Abstract |
The purposes of this Scientific Research B is to enzymatically synthesize various bioactive glycoconjugates compounds rising transglycosylation activity of endoglycosidases and also to analyze the enzyme reaction in order to establish the preparation method. The followings are the results of this research.
1. Large preparation of the recombinant enzyme : We succeeded in the large production of recombinant Endo-β-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) by cloning the gene of Endo-M using Escherichia coil and its growing at low temperature without induction by IPTG.
2. Obtaining the mutant enzyme having higher transglycosylation activity : We obtained Y217F mutant enzyme having higher transglycosylation activity by site-directed mutagenesis of the residues in the putative catalytic region of Endo-M.
3. Obtaining mutant enzyme having glycosynthase-like activity : We obtained N175A mutant having glycosynthase-like activity by site-directed mutagenesis. This mutant shows no activities of hydrolysis and transglycosylation. However, the enzyme expressed the transglycosylation activity when the oxazoline form of sugar chain was added to the reaction mixture as donor of sugar chain in transglycosylation, and the increase of transglycosylation product continued without its hydrolysis. Using this mutant enzyme, we could synthesize the bioactive glycopeptide having anti-HIV activity with high yield.
4. Addition of mucin type sugar chain to serine or threonine residue of the peptide using the recombinant endo-α-N-acetylgalactosaminidase from Bifidobacterium: We succeeded in the addition of mucin type sugar chain (galactosylβ1,3N-acetylgalactosamine) to serine or threonine residue of Muc1a which is the peptide of a part of human mucin, in addition to free serine and threonine.
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