基因工程酶法结合酵母能量耦联高效合成L-谷氨酰胺的研究

High Efficiency of L-Glutamine Production by Coupling Genetic Engineered Bacterial Glutamine Synthetase with Yeast Alcoholic Fermentation System

通过PCR方法从Bacillussubtilis基因组DNA中扩增出谷氨酰胺合成酶基因 (glnA) ,克隆至表达载体pET2 8b ,经测序鉴定后转化大肠杆菌BL2 1(DE3) ,用IPTG及乳糖诱导表达。SDS PAGE分析表明 ,所表达的谷氨酰胺合成酶 (glutaminesynthetase ,简称GS)为可溶性蛋白 ,约占总菌蛋白的 80 %。利用表达的GS蛋白N端的 6×His Tag对GS进行亲和层析 ,将获得的纯蛋白进行酶活性测定。结果表明 ,纯化的GS合成反应的最适温度为 6 0℃ ,最适pH为 6 5 ,Mn2 + 能明显提高GS的活性和稳定性。工程菌BL2 1(DE3) pET2 8b glnA粗提物中GS的比活是宿主菌本身的 84倍。以谷氨酸、NH4 Cl和ATP为底物的转化实验表明谷氨酸的转化率达 95 %以上。经筛选获得一株高效能量耦联酵母菌株 ,命名为YC0 0 1;通过能量耦联表明 ,该系统对谷氨酸的转化率高达 80 % ,平均谷氨酰胺产量为2 2g L。

Glutamine is an important conditionally necessary amino acid in human body. The effort is to establish a new and high efficient L-glutamine production system instead of traditional fermentaion. In this paper, high efficiency of L-glutamine production is obtained by coupling genetic engineered bacterial glutamine synthetase(GS) with yeast alcoholic fermentation system. Glutamine Synthetase gene(glnA) was amplified from Bacillus subtilis genomic DNA with primers designed according to sequences reported in EMBL data bank, then it was inserted into expression vector PET28b, the sequence of glnA was proved to be the same as that reported in the data bank by DNA sequencing. After transformation of this recombinant plasmid PET28b-glnA into BL-21(DE3)strain, Lactose and IPTG were used to induce GS expression at 37℃ separately. Both of them can induce GS expression efficiently. The induced protein is proved to be soluble and occupies about 80% of the total proteins by SDS-PAGE analysis. The soluble GS was purified by Ni 2+ chelating sepharose colum. After purification, the purified enzyme was proved active. Results reveal that the optmum temperature of this enzyme is 60℃ and optmum pH is 6.5 in biosynthetic reaction by using glutamate, ammonium choloride and ATP as substrates. After induction, the enzyme activity in crude extract of BL-21/PET28b-glnA is 83 times higher than that of original BL-21 extract. Mn 2+ can obviously increase the activity and stability of this enzyme. Experiments show that the transformation efficiency of glutamate to glutamine is more than 95%. Because of the high cost from ATP, a system coupling GS with yeast for ATP regenaration was established. In this system, GS utilizes ATP released by yeast fermentation to synthesize L-glutamine. Yeast was treated by 2% toluence to increase its permeability and a yeast named YC001 with high yield of glutamine by coupling with recombinant GS was obtained. The good efficiency was achieved with the presence of 250 mmol/L glucose and 200 mmol/L phosphate, the transformation efficiency of glutamate to glutamine in this system is more than 80%, the average yield of glutamine is about 22g/L. This provides the basis for future large scale production of L-gluta^mine.