溶氧对杀菌肽-X发酵工艺的影响

Effect of Dissolved O_2 on the Fermentation of Recombinant Cecropin-X by Escherichia coli

本研究采用 30L自动控制发酵罐研究了重组杀菌肽 X工程菌的基本发酵条件。经 12h发酵培养 ,发酵培养基中氨苄青霉素浓度为 0和 10 0 μg mL时 ,包涵体得率基本一致 ,干重分别为 1 2 4和 1 2 0g L ;控制溶氧为 2 0 %～30 %和溶氧自然变化 (转速分别为 2 5 0和 15 0r min)的条件下 ,包涵体得率有较大差异 ,干重分别为 0 0 5、0 71和1 2 4g L。在较优化的发酵条件下 ,目的融合蛋白的表达量占菌体总蛋白的 4 5 %～ 5 0 %。

Cecropin-X is an amphipathic peptide of 35 acid residues with an absence of methionine and cystein. Besides its broad-spectrum antibacterial activity, Cecropin-X also have activity to inhibit the growth of some tumor cell lines. Cecropin-X gene was fused at the 3′-terminus of TNFb gene and high fusion expression was achieved in E.coli system. The fusion protein was presented as inclusion body and biological activated Cecropin-X was obtained after being cleaved by BrCN. A fed-batch fermentation process was carried out for production of recombinant cecropin-X by Escherichia coli in large amount. The essential parameters during the fermentation were studied and optimized in a 30L-fermentor. It nearly made any difference to the yield of inclusion body while the ampicillin was absent or not. Dissolved O 2 played a key role in the fermentation. By keeping the dissolved O 2 level at 20%～30% saturation, the inclusion body was hardly produced. By keeping the stirring speed at 250r/min and 150r/min throughout the fermentation, leaving the dissolved O 2 changing spontaneously, the inclusion body obtained were 0.71g/L and 1.24g/L respectively. The content of recombinant fusion protein was as high as 45%～50% of the total protein content. Since Escherichia coli is aerobic bacteria, enough dissolved O 2 (20%～60% on the average) has been employed during fermentation in the previous reports. In the case of the engineered strain in this test, high expression was achieved only in the condition of relatively low dissolved O 2, the yield of inclusion body decrease with the increasing of dissolved O 2.