重组亚甲基四氢叶酸还原酶代谢工程菌发酵工艺的优化  

Optimization of Fermentation Process for Metabolic Engineered Recombinant Bacteria with Methylenetetrahydrofolate Reductase Gene

作　　者：邵飞[1] 卞筱泓[1] 阙哲夫[1] 许激扬[1] 尤晓清[1] 赵玉成[1] 刘为营[1] 

机构地区：[1]中国药科大学生物化学教研室,南京210009

出　　处：《中国药学杂志》2013年第21期1808-1814,共7页Chinese Pharmaceutical Journal

基　　金：中央高校基本科研业务费专项资金资助项目(JKQ2011046)

摘　　要：目的优化重组亚甲基四氢叶酸还原酶代谢工程细菌株的发酵工艺,以提高此菌株中L-5-甲基四氢叶酸的累积量。方法通过单因素实验和正交实验研究此菌株的最优发酵工艺。结果此菌株的最优发酵工艺为:蔗糖为3%,酵母浸粉为1.0%,无机盐为NaCl-K2HPO4-MgSO4(1.26%∶0.42%∶0.02%),磷酸二氢铵为1.0%,接种量为5%,初始pH为7.5,诱导时机为2.5 h,诱导剂浓度为1.4 mmol·L-1,诱导温度为37℃,诱导时间为6 h,诱导剂添加次数为2次。优化后与优化前相比,此菌株的细菌生长量提高了36.4%,亚甲基四氢叶酸还原酶比活力提高了46.8%,L-5-甲基四氢叶酸的累积量提高了66.1%。结论本实验为此菌株的发酵罐中试放大提供了实验依据。OBJECTIVE The fermentation process for the metabolic engineered recombinant bacteria with methylenetetrahydrofo- late reduetase gene was optimized to improve L-5-methyhetrahydrofolate accumulation in the engineering bacteria. METHODS Sin- gle-factor and orthogonal experiments were employed to investigate the optimum fermentation process of the bacteria. RESULTS The optimum fermentation process of the bacteria was as following：sucrose was 3% , yeast extract was 1.0% , inorganic salts wereNaC1- K2HPOa-MgSO4 = 1.26% ： 0. 42% ： 0. 02% , ammonium dihydrogen phosphate was 1.0% , inoculum amount was 5% , initial pH value was 7. 5, induction time point was 2. 5 h, inducer concentration was 1.4 mmol ~ L - i, incubation temperature was 37 ~C, induction duration was 6 h, addition degree of inducer was 2. Under the optimum fermentation process, the optimization results were as follow- ing：the bacteria amount had a 36. 4% enhancement, the specific activity of methylenetetrabydrofolate reduetase 46. 8% , the L-5-meth- yltetrahydrofolate accumulation 66. 1 % , comparing with the original fermentation process. CONCLUSION The research work pro- vides experimental evidence for amplifying of the bacteria in agitating fermentor.

关 键 词：亚甲基四氢叶酸还原酶 代谢工程 L-5-甲基四氢叶酸 发酵工艺 

分 类 号：R587.2[医药卫生—内分泌]