| Project/Area Number |
63860015
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| Research Category |
Grant-in-Aid for Developmental Scientific Research
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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 | KYOTO UNIVERSITY |
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Principal Investigator |
KIMURA Akira Research Institute for Food Science, Kyoto University, Professor, 食糧科学研究所, 教授 (80026541)
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| Co-Investigator(Kenkyū-buntansha) |
TAGUCHI Jun-ichi Kyoto Prefectural Medical College, Assistant Professor, 薬理学, 助手 (50188132)
MURATA Kousaku Research Institute for Food Science, Kyoto University, Associate Professor, 食糧科学研究所, 助教授 (90142299)
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| Project Period (FY) |
1988 – 1989
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| Project Status |
Completed (Fiscal Year 1989)
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| Budget Amount *help |
¥5,600,000 (Direct Cost: ¥5,600,000)
Fiscal Year 1989: ¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1988: ¥3,700,000 (Direct Cost: ¥3,700,000)
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| Keywords | S-Lactoylglutathione / Glyoxalase I / Methylglyoxal / Glutathione / Saccharomyces cerevisiae / Escherichia ocli / HPLC / Recombinant DNA / 抗炎症作用 / グルタチオンチオ-ルエステル / グリオキサラ-ゼI / Sーラクトイルグルタチオン / グリオキサラーゼI / 遺伝子工学 / 陽イオン交換体 |
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| Research Abstract |
S-LactoyLgluththione(S-LG) is synthesized from methylglyoxal(MG) and glutathione(GSH) by an action of glyoxalase I. The enzymatic production of this compound was studied by applying glyoxalase I to glycerol-grown cells of Saccharomyces cerevisiae and Escherichia coli dosed with Pseudomonas putida glyoxalase I gene. The glyoxalase I in S. cerevistae cells was remarkably induced when the cells were grown on glycerol. The activity of the enzyme in glycerol-grown cells was more than 20-flod higher compared with that of the glucose-grown cells. By using extracts of glycerol-grown yeast cells, about 5mmol/1(2g/1) of S-LG was produced from 10 mM MG and 50 mM GSH within 1 h. The extracts of E coli cells carrying a hybrid plasmid pGI423, which contains P. putida glyoxalase I gene, showed approximately 170-fold higher glyoxalase I activity than that of E. coli cells without pGI423. The extracts were used for the production of S-LG and, under the optimal conditions, about 80 mmol/1(30 g/1) of S-L
… More
G was produced from 50 mM MG and 100 mM GSH within 1 h.
In order to purify S-LG from GSH, the reaction mixture was subjected to the high performance liquid chromatography(HPLC). After removal of protein from the reaction mixture by addition of TCA(1 %) and centrifugation, the supernatant was applied onto a Dowex lx8(HCOOH form) column. S-LG was eluted by an increasing gradient of HCOOH concentration. The eluates were collected anticoncentrated by evaporation, and then the concentrate was applied onto a reverse-phase HPLC(column ;muBondsphare 5muC-18 100A^^ﾟ). Elution was performed by 5 % methanol/9.5 % HCOOH/85.5 % water(v/v/v) with a flow rate of 1 ml/min. S-LG was eluated as a single peak and the purified compound was identical with an authentic S-LG in melting point, Rf value on thin laver chromatography and elementary analysis.
Physical properties of S-LG purified were analyzed. S-LG inhibited concanavalin A and/or compound 48/80 induced release of histamin from the mast cell of rat peritonea. Anti-inflammational activity was equivalent to those of indomethacin and 2-aminopurine. Less
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