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

S-乳酰谷胱甘肽的量产开发及其在医疗美容领域的应用。

基本信息

批准号：
63860015
负责人：
KIMURA Akira
金额：
$ 3.58万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

项目摘要

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

S-乳酰谷胱甘肽（S-LG）是由甲基乙二醛（MG）和谷胱甘肽（GSH）在谷胱甘肽转移酶I的作用下合成的。该化合物的酶促生产进行了研究，通过应用glycoprotein酶I的甘油生长的细胞的酿酒酵母和大肠杆菌给药的恶臭假单胞菌glycoprotein酶I基因。S.当细胞在甘油上生长时，cerevistae细胞被显著诱导。甘油培养的细胞酶活力比葡萄糖培养的细胞高20倍以上。通过使用甘油培养的酵母细胞的提取物，从10 mM MG和50 mM GSH在1小时内产生约5 mmol/l（2g/l）的S-LG。携带含有恶臭假单胞菌glycoprotein酶I基因的杂合质粒pGI 423的大肠杆菌细胞提取物的glycoprotein酶I活性比大肠杆菌高约170倍。coli细胞中表达。提取物用于生产S-LG，在最佳条件下，约80 mmol/l（30 g/l）的S-L ...更多信息在1小时内由50 mM MG和100 mM GSH产生G。为了从GSH纯化S-LG，对反应混合物进行高效液相色谱（HPLC）。通过加入TCA（1%）和离心从反应混合物中除去蛋白质后，将上清液施加到Dowex 1x 8（HCOOH形式）柱上。S-LG通过增加HCOOH浓度梯度来洗脱。通过蒸发收集反浓缩洗脱液，然后将浓缩物上样到反相HPLC（柱; μ Bondelements are 5 μ C-18 100 A/μ C）上。用5%甲醇/9.5%HCOOH/85.5%水（v/v/v）洗脱，流速为1 ml/min，洗脱液为单峰，经薄层层析和元素分析，其熔点、Rf值与标准品一致，并对其物理性质进行了分析。S-LG抑制伴刀豆球蛋白A和/或化合物48/80诱导的大鼠腹腔肥大细胞组胺释放。抗炎活性与吲哚美辛和2-氨基嘌呤相当。少