Development of mass-production of S-lactoylglutathione and its application to medical and cosmetic field.

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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