| 摘要: |
| 采用响应面法对重组大肠杆菌生物合成别藻蓝蛋白(holo-apcA) 的发酵条件进行优化, 提高了蛋白的表达量。以对重组别藻蓝蛋白α 亚基在大肠杆菌中表达量影响较大的几种因素用于中心组合设计; 中心组合设计试验结果表明诱导温度、培养基初始pH 和诱导时机对诱导结果影响显著; 经Design Expert 8.0 软件优化后的最佳诱导条件为: 诱导温度28 ℃, 培养基初始pH 8.5, IPTG 终浓度0.1 mmol/L, 以及诱导时机3 h。最后验证试验得到的蛋白表达量在已有文献报道结果的基础上提高了70%~580%。中心组合设计-响应面法能够在有限的实验次数下, 对影响生物过程的因子进行优化及对其交互作用进行评价, 蛋白表达量达20.4 mg/L; IPTG 用量由原来的1 mmol/L 减少至现在的0.1 mmol/L, 降低了发酵成本。 |
| 关键词: 重组别藻蓝蛋白α 亚基 诱导表达 优化 响应面法 中心组合设计 |
| DOI：10.11759/hykx20131021002 |
| 分类号: |
| 基金项目:国家海洋公益性行业科研专项经费项目(201205027-2);国家863 计划项目(2014AA093505, 2014AA039501); 国家自然科学基金项目(41276164) |
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| Optimization of fermentation conditions for production of Holo-α-allophycocyanin (holo-apcA) by recombinant Escherichia coli using response surface methodology |
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| Abstract: |
| Response surface methodology was employed to optimize the fermentation conditions for holo-α-allophycocyanin (holo-apcA) production in recombinant metabolically engineered Escherichia coli, by which the expression level of protein indeed increased. In the design of response surface experiment, four variables, i.e. initial pH of culture medium, IPTG concentration, post-induction temperature, and induction start time, were selected for investigation of the individual and interactive effects between them and on holo-apcA production by E.a coli. The experimental results showed that post-induction temperature, initial pH of culture medium, and induction start time had significant individual effects on holo-apcA production, and the best possible induction condition optimized by Design Expert 8.0 software is: a post-induction temperature of 28℃, pH of culture medium of 8.5, IPTG concentration of 0.1 mmol/L, and induction start time of 3 h after starting cultivation. The protein expression level of final verification experiment was increased 70%～580% based on the reported results. The response surface methodology was used to evaluate the interactions among the factors under a limited number of trials. An expression level of 20.4 mg/L was obtained under optimized conditions. The IPTG consumption was decreased from 1 mmol/L to 0.1 mmol/L, which reduced the costs of fermentation. |
| Key words: Holo-α-allophycocyanin (holo-apcA) induce and express condition optimization response surface methodology central composite design |
