产抗菌肽PSI的重组乳酸克鲁维酵母高密度发酵及产物的分离纯化研究

摘要

以实验室构建的产抗菌肽PSI的重组乳酸克鲁维酵母基因工程菌作为出发菌株,在摇瓶培养的基础上,建立了100 L发酵罐中高密度发酵工艺及产物分离纯化方法.研究了不同培养基、菌体密度、诱导物及其添加方式等对产物的影响.结果表明,用BSM培养基发酵培养至菌体密度达到190 g/L时开始添加乳糖诱导,乳糖添加量为5 kg,补料流加速度为1 L/h,诱导25 h,菌体细胞湿重达280 g/L,发酵液中蛋白产量达27.8 mg/L,抗菌肽PSI效价达到2.36×109 U/L.采用多种分离纯化方法对发酵液中抗菌肽PSI进行分离纯化,结果表明,阴离子交换色谱纯化效率最高,纯化后蛋白效价为8.83×109 U/L,其次为丙酮沉淀、超滤、硫酸铵分级沉淀,纯化后蛋白效价依次为6.35×109、4.56×109、3.28×109 U/L.%A high density fermentation technology for the production of antimicrobial peptide PSI in a 100 L fermentor using a recombinant Kluyveromyces lactis strain was reported in this paper.The effects of mediums,cell density,as well as the inducer lactose and its fed-batch models,on the production of PSI were investigated.Results showed that 5 kg inducer lactose should be added with feeding velocity of 1 L/h when the cell density reach 190 g/L during the fermentation using BSM medium.After 25 h of induction culture,the cell wet weight,the protein yield,and the titer of PSI reached 280 g/L,27.8 mg/L,and 2.36 × 109 U/L,respectively.The separation and purification of the PSI in the fermentation broth were also investigated.The purification efficiency of anion exchange chromatography,ultrafiltration,precipitation of ammonium sulphate or acetone were 8.83 × 109,6.35 × 109,4.56 × 109,and 3.28 × 109 U/L,respectively,which indicated that the most efficient technique for purification of PSI was anion exchange chromatography.