2004 Fiscal Year Final Research Report Summary
Studies on biosynthesis, biodegradation, and regulation mechanism γ-polyglutamic acid in Bacillus subtilis
| Project/Area Number |
15580058
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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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 | Shizuoka university |
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Principal Investigator |
TAHARA Yasutaka Shizuoka univ., Agriculture, professor, 農学部, 教授 (30022320)
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| Project Period (FY) |
2003 – 2004
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| Keywords | natto / Bacillus subtilis / γ-polyglutamic acid / PGA-degrading enzyme / gene disruption / glutamate racemase / γ-glutamyltranspeptidase / endo-L-glutamylhydrolase |
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| Research Abstract |
Among PGA operon (ywsC-ywtABC) involved in γ-polyglutamic acid biosynthesis in Bacillus subtilis, no production of PGA was observed in ΔywsC and ΔywtA strains, but ΔywtB strain producing a small amount of PGA with 100% L-Glu. However, the ΔywtB revertant produced PGA with 70 : 30 in ratio of D, L-Glu, which is same to that of the wild-type. There are two glutamate racemases, GIr and YrpC, in B. subtilis, of which the genes were disrupted in an attempt to make clear determination of D, L-glutamate in PGA. ΔywtB-ΔgIr-ΔyrpC strain produced PGA with only L-Glu as well as that of the wild type. These results, together with the fact that the purified YwsC is able to biosynthesizes ^<14>C-PGA only from ^<14>C-L-glutamate under presence of ATP and Mg^<2+>, suggest that YwtB should possess the activity of glutamate racemase for producing PGA heterogeneous with D, L-glutamate.
The structure of γ-polyglutamate-hydrolyzed product (F2) having about 2 kDa in molecular mass with D-glutamate and L-glutamate in an 80 : 20 ratio released by YwtD of B. subtilis was analyzed using γ-glutamyltranspeptidase, rat γ-glutamyl hydrolase and carboxypeptidase G. The analysis of the digested products showed the F2 product is an optically heterogeneous polymer consisting of D-γ-glutamate and L-γ-glutamate peptide units with D-glutamate in both sides, suggesting that the YwtD should be an unique endopeptidase that cleaves γ-glutamyl bond between D-and D-glutamate recognizing adjacent L-glutamate in the N-terminal side.
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