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文章摘要
提高产反式-4-羟脯氨酸工程菌产量的发酵策略
Strategies on Improving the trans4-Hydroxyproline Production from the Engineering Strain E. coli
  
DOI:10.3969/j.issn.1673-1689.2019.11.006
中文关键词: 反式-4-羟脯氨酸  大肠杆菌工程菌  缺陷型  透明颤菌血红蛋白  发酵优化
英文关键词: trans-4-Hydroxyproline,engineering strain E. coli,defective strain,vitreoscilla hemoglobin,fermentation optimization
基金项目:
作者单位
王晓姣 江南大学 生物工程学院江苏 无锡 214122 
杨慧敏 江南大学 生物工程学院江苏 无锡 214122 
张震宇 江南大学 生物工程学院江苏 无锡 214122 
孙付保 江南大学 生物工程学院江苏 无锡 214122 
周豪 江南大学 生物工程学院江苏 无锡 214122 
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中文摘要:
      为了实现羟脯氨酸的高产,首先以缺陷型菌株E. coli JM109ΔargB/pUC19-BH作为实验菌,通过单因素优化及正交试验,优化得到新的培养基配方及培养条件。其次通过基因工程手段引入透明颤菌血红蛋白VHB基因,构建菌株E. coli JM109ΔargB/pUC19-BHV,进行高密度发酵。结果显示,发酵培养基为:麦芽糖10 g/L,甘油5 g/L,胰蛋白胨22 g/L,K2HPO4 4 g/L,FeSO4(Fe2+∶VC=1∶1) 5 mmol/L,MgSO4·7H2O 1.7 g/L,NaCl 3 g/L,CaC l 2 0.005 g/L;培养条件为:初始pH 8.5,温度30 ℃,接种体积分数4%,转速220 r/min。此条件下,菌株E. coli JM109ΔargB/pUC19-BH羟脯氨酸产量约为1 602 mg/L,约为优化前的1.4倍. 在7 L罐发酵培养,菌株E. coli JM109ΔargB/pUC19-BHV的羟脯氨酸产量约为18.3 g/L,与JM109ΔargB/pUC19-BH相比,提高了13.2%,说明VHB 基因的引入有利于羟脯氨酸产量提高.
英文摘要:
      To enhance the yield of trans-4-Hydroxyproline from the engineering strain E. coli JM109ΔargB/pUC19-BH,optimization of fermentation medium and culture condition was carried out with a single-factor and orthogonal design experiment. Further,a recombinant E. coli JM109ΔargB/pUC19-BHV,containing the vitrescilla hemoglobin gene VHB was constructed to be desirable for high-density fermentation. Results showed that the fermentation medium was optimized as follows:maltose 10 g/L,glycerol 5 g/L,trypsin 22 g/L,K2HPO4 4 g/L,FeSO4:VC 5 mmol/L,MgSO4 1.7 g/L,NaCl 3 g/L and CaCl2 0.005 g/L. The optimal fermentation conditions were as below:inoculum size 4%(v/v),initial pH 8.5 and culture temperature 30 ℃. At 24 h of the fermentation,the yield of P4H reached 1 602 mg/L,which was about 1.4 times of that before the optimization. In a 7 L fermenter,the recombinant E. coli JM109ΔargB/pUC19-BHV yielded 18.3 g/L of P4H,13.2% higher than that of the parental strain,indicating that the introduction of VHB gene was beneficial for the high-density production of trans-4-Hydroxyproline.
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