| Project/Area Number |
63560102
|
|---|
| Research Category |
Grant-in-Aid for General Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
発酵・醸造
|
|---|
| Research Institution | KYOTO UNIVERSITY |
|---|
Principal Investigator |
KUMAGI Hidehiko Kyoto Univ., Fac.Agric., Assoc. Prof., 農学部, 助教授 (70027192)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
YANO Toshihiro Kyoto Univ., Fac.Agric., Official. Researcher, 農学部, 教務職員 (30135553)
YAMAMOTO Kenji Kyoto Univ., Fac.Agric., Instructor, 農学部, 助手 (70109049)
|
|---|
| Project Period (FY) |
1988 – 1989
|
| Project Status |
Completed (Fiscal Year 1989)
|
| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1989: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1988: ¥1,300,000 (Direct Cost: ¥1,300,000)
|
|---|
| Keywords | gamma-Glutamyltranspeptidase / Escherichia coli / gamma-Glutamyl compounds / Glutathione / グルタチオン / γーグルタミルトランスペプチダーゼ / γーグルタミルトランスペプチダーゼ遺伝子 / 塩基配列 / γーグルタミル化合物 |
|---|
| Research Abstract |
1. The nucleotide sequence of ggt, the gene that codes for gamma -glutamyltrans- peptidase(GGT) of Escerichia coli K-12, was determined. The sequence contains a single open reading frame encoding the signal peptide(25 amino acid residues) and large(365) and small(190) subunits, in that order. This result suggests that E. coli gamma-glutamyltranspeptidase is processed posttranslationally, as in the case of mammalian GGTs. The amino acids sequence was compared with those of mammalian GGTs and we found that E. coli GGT has 30% homology with mammalian ones and most part of the protein is conserved through conservative substitutions of amino acid.
2. By a simple two-step method, a large amount of GGT was purified from E. coli SH643 harboring ggt-cloned plassid pSH101.
3. S-Benzyl glutathione methyl ester, gamma-glutamyl-L-tyrosine methylester and gamma - glutamyl-L-histidine were synthesized by GGT in the yield of 31.2, 35.7 and 41.2 g/L, respectively.
4. Cloning of the structure gene of ggt into the high-expression vector pKK223-3 was carried out by the following two-step method. First, EcoRV fragment which encodes the large subunit was inserted into the vector plasmid. And then the HpaI-PstI fragment of ggt which encodes small subunit was tried to insert into the vector having the EcoRV fragment. Now we are successful in the first step.
5. Ser75 residue in the small subunit was replaced with Ala or Cys by the method of site directed mutation. The mutant cells exhibited almost same activity with the wild type. The properties of mutant enzymes are under investigation now.
|
