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。
Abstract
The fermentation process for the metabolic engineered recombinant bacteria with methylenetetrahydrofolate reductase gene was optimized to improve L-5-methyltetrahydrofolate accumulation in the engineering bacteria. METHODS Single-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 wereNaCl-K2HPO4-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-1, incubation temperature was 37 ℃, induction duration was 6 h, addition degree of inducer was 2. Under the optimum fermentation process, the optimization results were as following:the bacteria amount had a 36.4% enhancement, the specific activity of methylenetetrahydrofolate reductase 46.8%, the L-5-methyltetrahydrofolate accumulation 66.1%, comparing with the original fermentation process. CONCLUSION The research work provides 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
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Key words
methylenetetrahydrofolate reductase /
metabolic engineering /
L-5-methyltetrahydrofolate /
fermentation process
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中图分类号：
R944
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参考文献
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脚注
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基金
中央高校基本科研业务费专项资金资助项目(JKQ2011046)
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