| Project/Area Number |
61560113
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
発酵・醸造
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| Research Institution | The University of Tokyo |
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Principal Investigator |
UOZUMI Takeshi Faculty of Agriculture, The University of Tokyo, 農学部, 教授 (40011978)
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| Co-Investigator(Kenkyū-buntansha) |
HORINOUCHI Sueharu Faculty of Agriculture, The University of Tokyo, 農学部, 助教授 (80143410)
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| Project Period (FY) |
1986 – 1987
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| Project Status |
Completed (Fiscal Year 1988)
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| Budget Amount *help |
¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 1987: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1986: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | tryptophanase / gene / cloning / thermophile / heat-stable enzyme / symbiosis / tryptophan / 発現 / 耐熱性 |
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| Research Abstract |
An obligatorily symbiotic bacterium, strain T, associating with a specific strain S of a thermophilic Bacillus sp. produces heat-stable tryptophanase. We purified the tryptophanase and determined the NH_2-terminal amino acid sequence. Two corresponding oligonucleotide sequences were synthesized as the probes for cloning of this enzyme gene. A Bam HI fragment of about 5.4-kb hybridized with these probes was isolated and E.coli harboring the subcloned 2.0-kb fragment on expression vector pUC19 produced approximately ten-fold higher level of the tryptophanase than that in strain T after induction with IPTG. The determination of nucleotide sequence of this fragment is now in progress. Since the sequence corresponding to NH_2-terminal sequence is already found, the fragment must contain an entire gene for the tryptophanase.
A molecular weight of the purified enzyme estimated by gel filtration column chromatography was 208 kd, and the molecular weight of subunit was determined by SDS-PAGE to be 46 kd, indicating that the native enzyme is composed of four homologous subunits. The optimum temperature for the enzyme activity was 70 C and the enzyme was stable at 65 C. These temperatures are higher than those of the tryptophanase from E. coli, which are 55 C and 40 C, respectively. So this heat-stable tryptophanase will be available for industrial production of tryptophan using reverse reaction of this enzyme activity.
Growth of strain T required co-culture with Bacillus strain S which was not replaced by other strains of thermophilic Bacillus species. No independent growth of strain T was observed in any of the medium so far tested. Further studies are required for elucidating the mechanism of symbiosis of the tryptophanase-producing microorganism strain T.
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