重组毕赤酵母高密度发酵表达肽抗生素hPAB-β及其抑菌活性初步鉴定

Expression and biological activity analysis of peptide antibiotics hPAB-βin recombinant Pichia pastoris by high-density fermentation

目的探讨hPAB-β工程菌的高密度发酵方法,鉴定发酵产物中目的肽的活性。方法采用补料分批培养方法,在3.7 L发酵罐中对重组酵母菌进行高密度发酵,温度控制在30℃,pH值范围为4.95～5.05,在菌体湿重达到200～250 g/L后开始诱导,经过约50 h甲醇诱导后结束发酵。对发酵上清进行反相层析、分子筛层析以及反相高效液相色谱纯化,通过抑菌实验对纯化产物进行活性鉴定。结果3次高密度发酵在菌体湿重分别达到235.0 g/L、210 g/L和264.8g/L时进行诱导表达,发酵上清中目标肽的表达量分别达73.7 mg/L、76.6 mg/L和74.3 mg/L。发酵上清通过反相层析、分子筛层析以及反相高效液相色谱纯化,获得重组肽抗生素hPAB-β,该重组蛋白经抑菌实验显示了较好的抑菌活性。结论成功实现了酵母工程菌的高密度发酵,为下一步规模化制备hPAB-β奠定了基础。

To evaluate the expression of hPAB-βin recombinant Pichia pastori by the high density fermentation and the biological activity of hPAB-β.Engineering bacteria was fermented in fed-batch high-density fermentation in a 3.7 L fermentor at temperature 30℃and pH 4.95-5.05.When the wet weight achieved 200-250g/L,engineering bacteria was induced by methanol.After more than 50 hours induction,fermentation was terminated and the supernatant was purified by reverse phase chromatogram,gel filtration chromatogram,and high performance liquid chromatogram.The biological activity was identified by bacteriostasis assay.Engineering bacteria was induced when the wet weight achieved 235.0 g/L,210 g/L and 264.8 g/L in 3 fermentations,and the yields of target peptide were 73.7 mg/L,76.6 mg/L, and 74.3 mg/L in supernatant,respectively.The recombinant peptide hPAB-βpurified from fermentation supernatant showed fine bacteriostasis activity.All data imply that engineering bacteria was fermented in fed-batch high-density fermentation successfully,and which made a foundation for the large-scale production of hPAB-β.