谷胱甘肽双功能合成酶的克隆表达、酶学性质及其应用的研究

Cloning,Expression,Characteristics and Application of Bifunctional Glutathione Synthetase

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。

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 GshF 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（GshF） from Listeria monocytogenes was cloned and epressed, and the characterization of GshF was studied. The gene coding GshF in Listeria rnonocytogenes was cloned and linked with vector pET28a（ ＋ ） , resulting in the expression vector pET28a（ ＋ ）-gshF. By adopting E. coli BL21 （DE3） to express the exogenous proteins GshF, 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 GshF（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 GshF expressed in E. coli B121 （ DE3 ）/ pET28a（ ＋ ）-gshF,Ni-NTA affinity chromatography was used to recover GshF,the specific activity of purified GshF 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 GshF was also studied, the optical catalytic pH of purified GshF was 8.5, and the optimum temperature was 37 ℃, the optimum coneertation of ATP and Mg^2＋ were 10 mmol/L and 30 mmol/L respectively. Excess ATP would inhibit the GshF. Finally,the highest concentration of GshF in those optimum conditions was 2.58 g/L. It is of important reference on the application of the enzyme in biosynthesis of glutathione.