| Project/Area Number |
11660081
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
応用微生物学・応用生物化学
|
|---|
| Research Institution | Shizuoka university |
|---|
Principal Investigator |
TAHARA Yasutaka Shizuoka Univ., Fac.of Agriculture, Professor, 農学部, 教授 (30022320)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
TOKUYAMA Shinji Shizuoka Univ., Fac.of Agriculture, Associate professor, 農学部, 助教授 (60283347)
|
|---|
| Project Period (FY) |
1999 – 2000
|
| Project Status |
Completed (Fiscal Year 2000)
|
| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1999: ¥2,400,000 (Direct Cost: ¥2,400,000)
|
|---|
| Keywords | Bacillus subtilis / Bacillus subtilis (natto) / γ-polyglutamic acid synthetase / gene disruption / ywsC-ywtABC genes / γ-polyglutamic acid / γ-ポリグルタミン酸合成遺伝子 / グルタミン酸合成酵素 / 相同組換え |
|---|
| Research Abstract |
The genes encoding proteins required for γ-polyglutamic acid (PGA) production were cloned from B.subtilis IFO 16449, a strain producing a high amount of PGA.Four open reading frames were found in the cloned 4.2-Kb DNA fragment and were almost identical to ywsC and ywtABC genes of B.subtlis 168. Northern blot analysis showed the four genes constitute an operon. Three genes, ywsC, ywtA and ywtB, were disrupted to investigate which genes participate in the PGA biosynthesis. No PGA was produced in ΔywsC and ΔywtA strains, however a small amount PGA was produced from ΔywsB strain. To clarify the function of YwsC protein, histidine-tag codon was introduced into the C-terminal region of ywsC gene and the histidine-tagged YwsC (YwsC-H) was expressed in ΔywsC strain. The Yws-H protein was purified from the lysozyme-treated lysate of the transformant by nickel affinity chromatography and Western blot analysis revealed YwsC-H protein consists of two subunits, 44K and 33K proteins. Northern blot analysis also indicated the two proteins was encoded by in phase overlapping in ywsC gene. The purified proteins synthesized ^<14>C-labeled PGA in the presence of ATP, MgCl_2 and ^<14>C-L-glutamate, and both 44K and 33K proteins was requisite for the in vitro PGA biosynthesis, indicating that ywsC gene codes for two subunits of PGA synthetase, a crucial enzyme in PGA production.
|
