| Project/Area Number |
04556010
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
応用微生物学・発酵学
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| Research Institution | Nagoya University |
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Principal Investigator |
UDAKA Shigezo Nagoya Univ., Agriculture Professor, 農学部, 教授 (70023463)
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| Co-Investigator(Kenkyū-buntansha) |
YAMANE Tsuneo Nagoya Univ., Agriculture Professor, 農学部, 教授 (70026102)
山形 秀夫 名古屋大学, 農学部, 助教授 (20023468)
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| Project Period (FY) |
1992 – 1993
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| Project Status |
Completed (Fiscal Year 1993)
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| Budget Amount *help |
¥10,200,000 (Direct Cost: ¥10,200,000)
Fiscal Year 1993: ¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 1992: ¥6,600,000 (Direct Cost: ¥6,600,000)
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| Keywords | Xylose isomerase / Thermus thermophilus / Protein engineering |
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| Research Abstract |
D-xylose (glucose) isomerase is used industrially to catalyse the isomerization of glucose to fructose. Since the isomerization is reversible, and the final fructose content dependes on the reaction temperature, a higher temperature gives a higher fructose content, which is the important component of the syrup. We cloned the gene encoding an extremely thermostable xylose isomerase from Thermus thermophilus and determined the amino acid sequence of the gene. In order to prevent the heat inactivation of lysine residue which has a vital role for enzyme catalysis, effect of alterations of amino acids surrounding the lysine residue on the enzyme properties was tested by means of site specific mutageneasis and PCR reactions. As the results, it became clearn that alterration of Val (no. 135) to alanine, proline (137) to glycine etc. gave no gross change on enzyme, but heat stability with Mg^<2+> of the mutated enzyme in the vicinity of Trp (no. 136) lowered greatly. Aspartic and (247) was alterd to glycine, a big charge in the metal requiremet was observed. For the preparation of a large amount of xylose isomerase in B.brevis, an antomatic nutrient feed system was devised. Furthermore, a method was developed for immobilization of B.brevis cells containing the enzyme and glucose was isomerized to fructose.
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