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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MV 1184菌株和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：用分别从gsh-I和gsh-II的末端区域设计的引物对和来自MV 1184菌株的模板DNA进行PCR克隆。将扩增的基因插入pUC 18载体，将含有gsh-Ⅰ和gsh-Ⅱ的质粒导入MV 1184菌株。与没有质粒的菌株相比，具有质粒的MV 1184显示出显著高的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：用甲苯（0- 10%）在25 ℃下处理带有质粒的MV 1184菌株1小时。经处理的细胞在-20 ℃下至少稳定1.5年。通过克隆的大肠杆菌细胞和蓝细菌细胞（石藻）的混合物生产谷氨酸：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，谷胱甘肽的水平为7 μ mol/湿g，反应时间为6 h。大肠杆菌gsh-Ⅰ和gsh-Ⅱ基因转化蓝藻的研究：为了开发高效的蓝藻谷胱甘肽生产技术，将大肠杆菌gsh-Ⅰ和gsh-Ⅱ基因初步转化蓝藻细胞，并对参与这些氨基酸生物合成的酶进行了研究，以增加细胞内底物（谷氨酸、半胱氨酸、甘氨酸）的供应。