利用L-谷氨酰胺高产菌株产新型靛蓝色素的方法及条件优化

发布时间：2018-07-07 10:32

本文选题：L-谷氨酰胺 + 高通量筛选方法　；参考：《内蒙古农业大学》2017年硕士论文

【摘要】：本研究首先通过分子克隆、大肠杆菌表达和蛋白纯化获得了BPSA蛋白,对BPSA蛋白进行动力学表征显示,测定其Km值和Kcat值。然后利用BPSA蛋白催化谷氨酰胺合成蓝色化合物indigoidine的特性构建了 L-谷氨酰胺高通量检测方法。以此为基础,成功构建了谷氨酰胺高产菌株的高通量筛选方法,为今后高产菌株的筛选和工业化生产提供理论基础。研究结果如下:动力学实验完成了BPSA蛋白的表征,其Km值为0.79±0.667mM,Kcat值为5.658mM。当发酵液中L-谷氨酰胺浓度在0.2mM至5mM时,可以通过该方法准确检测到L-谷氨酰胺的含量。本课题通过克隆及在谷氨酸棒杆菌中共表达bpsA和sfp基因,首次实现了在谷氨酸棒杆菌中生产新型靛蓝染料indigoidine。通过对L-谷氨酰胺的添加量(0、0.73、4.38、11.68)g/L、IPTG 添加量(0.1、0.2、0.4、0.8、1.5、2)mM、诱导时间(48、72)小时及诱导温度(18℃、25℃、30℃)的条件优化,最终确定,当L-谷氨酰胺添加为11.68g/L,IPTG浓度为0.8mM,18℃诱导48小时后indigoidine产量最高可达到 1.75g/L。
[Abstract]:In this study, BPSA protein was obtained by molecular cloning, expression of Escherichia coli and protein purification. The kinetic characterization of BPSA protein showed that the Km value and Kcat value were determined. Then, the high throughput assay method of L- glutamine was constructed by using BPSA protein to catalyze the synthesis of blue compound indigoidine from glutamine. Based on this, it was formed. A high throughput screening method for high yield strain of glutamine was constructed to provide a theoretical basis for the screening and industrial production of high-yielding strains in the future. The results were as follows: the kinetic experiment completed the characterization of BPSA protein, its Km value was 0.79 + 0.667mM, and the Kcat value was 5.658mM. when the concentration of L- glutamine in the fermentation liquid was from 0.2mM to 5mM. The content of L- glutamine was accurately detected by this method. By cloning and expressing bpsA and SFP genes in Corynebacterium glutamibacillus, the production of new indigo dye indigoidine. in Corynebacterium glutamias was first realized through the addition of L- glutamine (0,0.73,4.38,11.68) g/L, IPTG addition (0.1,0.2,0.4,0.8,1.5,2) mM. The conditions were optimized for time (48,72) hours and induction temperature (18 C, 25 C, 30 C). Finally, when L- glutamine was added to 11.68g/L, IPTG concentration was 0.8mM, and 18 C was induced for 48 hours, the highest indigoidine yield could reach 1.75g/L.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78;Q936

【参考文献】