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响应面法优化重组大肠杆菌产谷氨酸脱氢酶培养基的研究

  

  1. 浙江大学 工业生物催化浙江省工程实验室, 杭州 310027
  • 出版日期:2019-08-18 发布日期:2019-08-18
  • 通讯作者: 杨立荣,教授,研究方向为生物催化与转化,E-mail: lryang@zju.edu.cn
  • 作者简介:张雨晴,硕士研究生,研究方向为生物催化与转化,E-mail:21528082@zju.edu.cn
  • 基金资助:
    国家自然科学基金面上项目(21476199);国家“973”计划(2011CB710800) 

Optimization of glutamate dehydrogenase production medium by recombinant Escherichia coli using response surface methodology

  1. Engineering Laboratory of Zhejiang Province, Industrial Biocatalysis, Zhejiang University, Hangzhou 310027, China
  • Online:2019-08-18 Published:2019-08-18

摘要: 通过响应面实验设计方法对重组大肠杆菌的发酵培养基进行优化,提高胺化还原反应工艺制备L-草铵膦中关键生物催化剂谷氨酸脱氢酶的生产能力。首先通过单因素实验筛选发酵培养基最适的碳源、氮源,并用Plackett-Burman设计确定发酵培养基各成分对产酶的影响程度。然后根据Box-Bohnken的中心组合设计实验和响应面分析确定最佳的培养基成分组合。优化后的培养基组成为:甘油11.3 g/L,安琪酵母粉905 16.28 g/L,MgSO4·7H2O 0.63 g/L,Na2HPO4·12H2O 17.1 g/L, KH2PO4 3 g/L,NH4Cl 1.5 g/L,NaCl 0.5 g/L。采用优化后发酵培养基获得的谷氨酸脱氢酶对2-羰基-4-(羟基甲基膦酰基)丁酸(PPO)的催化活力为129.1 U/mL,是LB培养基的2.58倍,显著降低了酶催化剂的制备成本。

关键词: 谷氨酸脱氢酶, Plackett-Burman 设计, 响应面分析, 培养基优化

Abstract: Response surface methodology was used to optimize the fermentation media of recombinant E. coli and to improve the production capacity of glutamate dehydrogenase which is key biocatalyst in reductive amination. Firstly, the single factor experiment was used to screen the optimal carbon source and nitrogen source, and then the Plackett-Burman design was used to evaluate the influencing degree for response value of related factors in original fermentation medium. The optimum medium components combination was further determined according to Box-Bohnken design and response surface analysis. The optimized medium was obtained: glycerol 11.3 g/L, yeast extract (Angel 905) 16.28 g/L, MgSO4·7H2O 0.63 g/L, Na2HPO4·12H2O 17.1 g/L, KH2PO4 3 g/L, NH4Cl 1.5 g/L, NaCl 0.5g/L. Under above conditions, the enzyme activity of glutamate dehydrogenase was 2.58 times higher than that of LB medium and reached 129.1U/mL, which significantly improved the preparing efficiency of enzyme catalyst.

Key words: glutamate dehydrogenase, Plackett-Burman design, response surface method analysis, optimization of medium

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