E.coli谷氨酸脱羧酶高产菌株选育及发酵条件研究

以普通大肠杆菌(E. coli)为出发菌株，以L-谷氨酸、溴甲酚绿(pH3.8~5.4)为筛选及指示剂，经亚硝基胍(NTG)、UV诱变处理，得到了L-谷氨酸脱羧酶活性变异菌株，其L-谷氨酸脱羧酶（GAD）活性大幅度提高，比出发菌株酶活性提高了2.22倍。优化实验显示：pH值、发酵时间、诱导剂L-谷氨酸、硫酸镁对GAD产酶有较大影响。通过对产酶发酵培养基中几种成份单因素变动及正交优化实验，对该菌株产GAD的诱导剂L-谷氨酸、营养要求和发酵条件进行了研究，优化后的发酵培养基组成为牛肉膏5 g/L，蛋白胨10 g/L，氯化钠3 g/L，L-谷氨酸0.5 g/L，葡萄糖1.5 g/L，磷酸二氢钾2 g/L，硫酸镁0.7 g/L。发酵条件是：pH6.8，接种菌龄20h，发酵时间20h。在优化条件下突变菌株GAD活性可达6790.7U，是出发菌株的2.76倍。

Breeding of High Gad Producing E. coli Strains and Optimization of Fermentation Conditions

By using indicators of bromocresol green and L-glutamate(pH3.8～5.4),a high L-glutamate decarboxylase(GAD)producing mutant was screened from gener Escherichia coli by N-methyl-N'-nitro-N-nitrosoguanidine(NTG)and UV.The enzyme activity of mutation strain was 2.22 times more than that of starting strains.The results showed that pH,fermentation time,L-glutamic acid,glucose,KH_2PO_4 and MgSO_4,all had effect on GAD production.The optimum medium and fermentation conditions were optimized by single factor experiments and orthogonal experiment.The optimum medium was as follows:beef extract 5 g/L,peptone 10g/L,NaCl 3 g/L,L-glutamic acid 0.5 g/L,glucose 1.5 g/L,KH_2PO_4 2 g/L,MgSO_4 0.7 g/L.The optimum conditions were:temperature 37℃,pH 6.8,incubation time 20h,the inoculated strain age 20h.The mutant strain GAD production was 2.76 times of starting strains in optimum conditions,GAD activity of mutant strains Was up to 6790.7U.