Production of Novel Bioactive Compounds Using Microbial Endoglycosidases and Its Enzymatic Analyses
| 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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Report
(3 results)
Research Products
(25 results)
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[Journal Article] Mutants of Mucor hiemalis Endo-β-N-acetylglucosaminidase Show Enhanced Transglycosylation and Glycosynthase-like Activities2008
Author(s)
Umekawa, M., Huang, W., Li, B., Fujita, K., Ashida, H., Wang, L. -X. and Yamamoto, K.
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Journal Title
J. Biol. Chem. 283
Pages: 4469-4479
Description
「研究成果報告書概要(和文)」より
Related Report
Peer Reviewed
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[Journal Article] Mutants of Mucor hiemalis Endo- 8 -N-acetylglucosaminidase Show Enhanced Transglycosylation and Glycosynthase-like Activities2008
Author(s)
Umekawa, M., Huang, W., Li, B., Fujita, K., Ashida, H., Wang, L.-X., Yamamoto, K
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Journal Title
J. Biol. Chem 283
Pages: 4469-4479
Description
「研究成果報告書概要(欧文)」より
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[Presentation] Alteration of Transglycosylation Function of Endo-β-N-acetyl-glucosaminidase from Mucor hiemalis (Endo-M) and Analyses of Its Reaction2007
Author(s)
Umekawa, M., Fujita, K., Huang, W., Wang, L-X., Ashida, H., Yamamoto, K
Organizer
Annual Meeting of The Japanese Society of Applied Glycoscience in 2007
Place of Presentation
Fujisawa
Year and Date
2007-08-29
Description
「研究成果報告書概要(欧文)」より
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[Presentation] Alteration and Analyses of Transglycosylation Activity which is Specific Function of Mucor hiemalis Endo-β-N-acetylglucosaminidase (Endo-M)2007
Author(s)
Umekawa, M., Fujita, IC, Huang, W., Kato, M., Ashida, H., Wang, L-X, Yamamoto, K
Organizer
Meeting of Kansai Branch of Japan Society for Bioscience(Biotechnology and Agrochemistry)
Place of Presentation
Kyoto
Year and Date
2007-05-26
Description
「研究成果報告書概要(欧文)」より
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[Book] Endoglycosidases2006
Author(s)
Endo, M., Hase, S., Yamamoto, K. and Takagaki, K.
Total Pages
255
Publisher
Kodansha & Springer
Description
「研究成果報告書概要(和文)」より
Related Report
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[Book] Endoglycosidases2006
Author(s)
Endo, M., Hase, S., Yamamoto, K., Takagaki, K
Publisher
Kodansha & Springer
Description
「研究成果報告書概要(欧文)」より
Related Report
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[Book] Endoglycosidases2006
Author(s)
Endo, M., Hase, S., Yamamoto, K., Takagaki, K.
Total Pages
255
Publisher
Kodansha ＆ Springer
Related Report
