Glutathione production by using a cyanobacterial ATP regeneration system

使用蓝藻 ATP 再生系统生产谷胱甘肽

基本信息

批准号：
06660108
负责人：
SAWA Yoshihiro
金额：
$ 1.34万
依托单位：
Shimane University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06660108/
关键词：
Glutathione production ATP regeneration GSH-I GSH-II Cyanobacteria Phormidium 好温性ラン藻 Phormidium lapideum gshI gshII Synechococcus sp.PCC7002 pQCx GSH-I GSH-II

项目摘要

1. PCR Clonings of gsh-I and gsh-II from E.coli MV1184 strain and E.coli cells with plasmid containing those genes : PCR clonings were carried out with the primer set designed from the terminal regions of gsh-I and gsh-II,respectively, and a template DNA from MV1184 strain. The amplified genes were inserted to pUC18 vector and the plasmids containing gsh-I and II were introduced into MV1184 strain. The MV1184 with the plasmid showed significantly high GSH-I (21.5-fold) and GSH-II (475-fold) activities compared to the strain without plasmid. 2. Treatment of the cloned E.coli cells with toluene : The MV1184 strains with plasmids were treated with toluene (0-10 %) at 25ﾟC for 1 hr. The optimum concentration of toluene in the accumulated glutathione level was around 4 %. The treated cells were stable at least for 1.5 year at -20ﾟC.3. Glutathione production by the mixture of the cloned E.coli cells and cyanobacterial cells (Phormidium lapideum) : The optimum ratio (based on wet weight) of gsh-I cloned cells and gsh-II cloned cells was 30 : 1 (GSH-I activity : GSH-II activity =1 : 3). Under the optimum condition, the glutathione level was 7mumol/wet g for 6 hr. 4. Transformation of E.coli gsh-I and II genes into cyanobacterial cells : To develop an efficient cyanobacterial glutathione production, the E.coli gsh-I and II genes were preliminary transformed into cyanobacterial cells.To increase intracellular supply of the substrates (glutamate, cysteine, glycine), the enzymes involving biosynthesis of those amino acids were also studied.

1.从大肠杆菌MV1184菌株和带有含有这些基因的质粒的大肠杆菌细胞中PCR克隆gsh-I和gsh-II：用根据gsh-I和gsh-末端区域设计的引物组进行PCR克隆。分别将来自MV1184菌株的模板DNA插入到pUC18载体中，并将含有gsh-I和II的质粒引入到pUC18载体中。与没有质粒的菌株相比，带有质粒的MV1184菌株表现出显着高的GSH-I（21.5倍）和GSH-II（475倍）活性。 2.用甲苯处理克隆的大肠杆菌细胞：带有质粒的 MV1184 菌株用甲苯 (0-10%) 在 25°C 下处理 1 小时。累积谷胱甘肽水平中的甲苯含量约为 4%。在 -20°C 下，细胞可稳定至少 1.5 年。3. 克隆大肠杆菌细胞和蓝藻处理细胞（Phormidium lapideum）的混合物产生谷胱甘肽： gsh-I克隆细胞和gsh-II克隆细胞的最佳比例（基于湿重）为30：1（GSH-I活性： GSH-II活性=1:3)。在最佳条件下，谷胱甘肽水平为7μmol/湿g，持续6小时。 4.将大肠杆菌gsh-I和II基因转化到蓝藻细胞中：开发高效的蓝藻谷胱甘肽。生产时，将大肠杆菌 gsh-I 和 II 基因初步转化到蓝藻细胞中。以增加细胞内底物（谷氨酸、半胱氨酸、甘氨酸），还研究了涉及这些氨基酸的酶生物合成。