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

以乙二醛(MG)和谷胱甘肽(GSH)为原料，在乙二醇酶I的作用下合成了S-乳酸-谷胱甘肽(S-LG)，并将乙醛酶I应用于甘油培养的酿酒酵母细胞和转入恶臭假单胞菌乙二醛酶I基因的大肠埃希菌，研究了该化合物的酶促合成。甘油对酿酒酵母细胞的乙草酸酶I有明显的诱导作用。甘油培养细胞的酶活性比葡萄糖培养细胞高20-Flod以上。用甘油培养的酵母细胞提取液，10 mM MG和50 mM GSH在1h内可产生约5 mmol/l(2g/l)的S-LG，含有恶臭假单胞菌乙二醛酶I基因的杂合表达载体pGI423的大肠杆菌提取液的乙二醛酶I活性约为未含pGI423的大肠杆菌的170倍。提取液用于生产S-LG，在最佳条件下，S-L…的产量约为80mmoL/l(30g/l)由50 mM MG和100 mM GSH在1h内可得到较多的G。为了从GSH中分离纯化S-LG，对反应混合物进行了高效液相色谱分离。在加入1%的TCA和离心法从反应混合物中去除蛋白质后，上清液被加到Dowex lx8(HCOOH形式)柱上。S-LG用递增的甲酸浓度梯度洗脱。洗脱液通过蒸发反浓缩收集，然后将浓缩物应用于反相高效液相色谱法(柱：muBondsphare 5muC-18 100A^ﾟ)。流动相为5%甲醇/9.5%甲酸/85.5%水(v/v/v)，流速为1ml/min。S-LG为单峰洗脱，纯化后的化合物在熔点、薄层析Rf值和元素分析等方面与正品S-LG完全一致，并对纯化后的S-LG进行了物性分析。S-LG可抑制刀豆蛋白A和/或化合物48/80诱导的大鼠腹膜肥大细胞释放组胺。抗炎活性与消炎痛和2-氨基嘌呤相当。较少

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