E.coli谷氨酸脱羧酶高产菌株选育及发酵条件研究  

BREEDING OF HIGH GAD PRODUCING E.coli STRAINS AND OPTIMIZATION OF FERMENTATION CONDITIONS

作　　者：左斌[1] 刘唐兴[2] 丰来[1] 谭显胜[3] 孟桂元[4] 

机构地区：[1]湖南农业大学生物科技学院,湖南长沙410128 [2]湖南生物机电职业技术学院,湖南长沙410127 [3]湖南农业大学生物安全科学技术学院,湖南长沙410128 [4]湖南农业大学农学院,湖南长沙410128

出　　处：《核农学报》2009年第5期789-793,811,共6页Journal of Nuclear Agricultural Sciences

基　　金：国家自然科学基金(30570351);教育部新世纪优秀人才计划项目(NCET-06-0710)

摘　　要：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。优化试验显示: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分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5g/L,蛋白胨10g/L,氯化钠3g/L,L-谷氨酸0.5g/L,葡萄糖1.5g/L,磷酸二氢钾2g/L,硫酸镁0.7g/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。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, KH2PO4 and MgSO4 , 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 10 g/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.

关 键 词：大肠杆菌 谷氨酸脱羧酶(GAD) 溴甲酚绿 高产突变株 发酵条件 

分 类 号：TQ925[轻工技术与工程—发酵工程] Q93[生物学—微生物学]