GshF在大肠杆菌中的表达及酶学性质研究  

Characterization of GshF Expressed in Escherichia coli

作　　者：陈永露[1] 吴绵斌[1] 林建平[1] 杨立荣[1] 岑沛霖[1] 

机构地区：[1]生物质化工教育部重点实验室浙江大学化学工程与生物工程系,杭州310027

出　　处：《中国生物工程杂志》2013年第12期21-28,共8页China Biotechnology

基　　金：浙江省重点科技创新团队计划资助项目(2011R50002)

摘　　要：谷胱甘肽是参与细胞活动的最普遍的非蛋白巯基化合物,是机体内重要的活性三肽,因其抗氧化作用而在医药、食品、化妆品等行业有着广泛的应用,生物合成是制备谷胱甘肽的主要方法,一直引起广泛关注。近年来发现的双功能酶GshF可同时催化谷胱甘肽生物合成的两步反应,有望提高酶法催化产谷胱甘肽的效率,但该酶的酶学性质尚未得到充分研究。克隆了来自Streptococcus thermophilus的双功能酶基因gshF,并用大肠杆菌表达,研究表明,重组菌于37℃培养4 h后经0.1 mmol/L IPTG诱导,然后在30℃下表达,胞内酶活可达44 U/L。表达产物经破胞、离心、亲和层析等步骤分离纯化后,进一步考察了其相关酶学性质,结果表明,该酶最适pH为8.0,最适温度为37℃,还研究了其最适pH和温度下的稳定性。最终通过重组菌酶法催化合成谷胱甘肽得到的最高产量为2.11g/L。这些对该酶在谷胱甘肽生物合成上的应用有重要的借鉴作用。Glutathione, an important active tripeptide, is the most common non-protein thiol compound involved in cell activity. It has a wide range of applications in the pharmaceutical, food, cosmetics and other industries because of its antioxidant effect. And improving glutathione production via biosynthesis is the main method and has always been a focus. The bifunctional enzyme GshF discorvered recently can catalyze the two- step reaction of glutathione biosynthesis simultaneously and is expected to improve the catalytic efficiency. However, the properties of the enzyme have not been sufficiently studied. A bifunctional enzyme gene gshF from Streptococcus thermophilus was cloned and expressed in E. coll. GshF reached its highest activity of 44 U/L when the recombinant was induced with O. lmmol/L IPTG at 30~C after being cultivated for 4 hours at 37~C. The related enzymatic properties of GshF were achieved after such purification steps as cell lysis, centrifugation and ＇affinity chromatography. The optimum pH and temperature was 8.0 and 37℃ respectively. The stabilities of GshF under its optimum pH and temperature were also studied. The final maximum yield of glutathione was 2.11g/L by enzymatic synthesis of recombinant bacteria. It is of important reference on the application of the enzyme in the biosynthesis of glutathione.

关 键 词：谷胱甘肽 GshF 大肠杆菌 酶学性质 酶法催化 

分 类 号：Q786[生物学—分子生物学]