| Project/Area Number |
16360418
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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 |
Biofunction/Bioprocess
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| Research Institution | TOYOTA CENTRAL R&D LABS., INC. |
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Principal Investigator |
TAKAHASHI Haruo TOYOTA CENTRAL R&D LABS., INC., Materials Dept. Biotechnology Lab., Lab. Manager・Principal Researcher, 材料分野 バイオ研究室, 室長 主席研究員 (50374088)
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| Co-Investigator(Kenkyū-buntansha) |
IMAMURA Chie TOYOTA CENTRAL R&D LABS., INC., Materials Dept. Biotechnology Lab., Senior Researcher, 材料分野 バイオ研究室, 主任研究員 (20394950)
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| Project Period (FY) |
2004 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥11,500,000 (Direct Cost: ¥11,500,000)
Fiscal Year 2006: ¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2005: ¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2004: ¥4,300,000 (Direct Cost: ¥4,300,000)
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| Keywords | xylose / yeast / xylose isomerase / xylose reductase / molecular evolution / enzyme immobilization / cell-free protein synthesis / biomass |
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| Research Abstract |
1.Improvement of cell-free expression system
An important issue is whether proteins would be expressed in active from in the screening system. In this study, a functional expression system was developed using an E.coli in vitro coupled transcription/translation system by addition of chaperones and protein disulfide isomerase. Furthermore, S30 extract had been prepared from the E.coli in which chaperones were co-expressed. Using this system, disulfide-containing proteins and dimeric proteins were successfully synthesized in active form.
2.Improvement of enzyme activity using molecular evolution method
We cloned four xylose isomerase genes from Therm us, Streptomyces, Piromyces and E.coli. Using cell-free expression system as described above, these enzymes were successfully synthesized in active form.
We have increased the activity of xylose isomerase from Piromyces by random mutagenesis and high-throughput screening using the system, designated as SIMPLEX (single-molecule-PCR-linked in vitro expression). A mutant xylose isomerase library was constructed on 384-well plates. We screened 104 wells including independently-expressed xylose isomerases led to two positive mutants of which the activity was two times higher than that of the wild type.
Xylulokinase and xylose isomerase genes, which are required for xylose assimilation, were introduced into lactic acid-producing yeast. The recombinant strain, which has xylose isomerase mutant genes, showed 30% xylose consumption / 90hr.
3.Enzyme immobilization in mesoporous materials
Xylose isomerases were successfully stabilized in mesoporous materials (FSM and SBA), which diameter is bigger than 13nm. However, enzymatic activity was not detected.
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