【摘要】 谷胱甘肽是广泛存在于生物体内的生物活性三肽化合物,因其具有重要的生理功能而在医药、食品、化妆品等行业有着广泛的应用,该研究克隆了来自Listeria monocytogenes的谷胱甘肽双功能酶基因gsh F,以p ET28a(+)作为表达载体,利用大肠杆菌实现重组表达,研究表明,重组菌于37℃培养4 h后,在28℃下经0.2 mmol/L IPTG诱导表达,胞内酶活可达2 318 U/L。表达产物破胞、离心、亲和层析后,进一步考察了其酶学性质,表明该酶催化最适温度37℃,最适p H 8.5,还研究了Mg2+和ATP对其的影响以及该酶的稳定性。最终通过Gsh F体外酶催化合成谷胱甘肽,得到的最高产量为2.58 g/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。更多还原

【Abstract】 As the most abundant non-protein thiol compound in organisms,glutathione is a tripeptide formed from glutamate,cysteine and glycine,and has important biological activity. Therefore it has been widely used in medicine,food and cosmetic industry. The current production of glutathione in industry is the fermentation of Saccharomyces cerevisiae. Recently,the bifunctional enzyme Gsh F which can catalyze the two-step reaction of glutathione biosynthesis is discovered,and is expected to improve the productivity of GSH. In this work,a bifunctional glutathione synthtase( Gsh F) from Listeria monocytogenes was cloned and epressed,and the characterization of Gsh F was studied. The gene coding Gsh F in Listeria monocytogenes was cloned and linked with vector p ET28a( +),resulting in the expression vector p ET28a( +)-gsh F. By adopting E. coli BL21( DE3) to express the exogenous proteins Gsh F,the effects of different expression conditions were investigated to improve the expression level,such as induction temperature,induction timing and IPTG concentration. The highest activity of Gsh F( 2 318 U / L) in flasks was achieved when 0. 2 mmol / L IPTG was added into the cell culture( in the stage of mid-log growth phase) for 6 h induction. Due to the His-tag of Gsh F expressed in E. coli BL21( DE3) /p ET28a( +)-gsh F,Ni-NTA affinity chromatography was used to recover Gsh F,the specific activity of purified Gsh F was 14. 68 U / mg which was 15. 1 times of that in crude cell lysate,with a good recovery rate of 76%. The characterization of Gsh F was also studied,the optical catalytic p H of purified Gsh F was 8. 5,and the optimum temperature was 37 ℃,the optimum concertation of ATP and Mg2 +were 10 mmol / L and 30 mmol / L respectively. Excess ATP would inhibit the Gsh F. Finally,the highest concentration of Gsh F in those optimum conditions was 2. 58 g / L. It is of important reference on the application of the enzyme in biosynthesis of glutathione.更多还原