重组大肠杆菌产普鲁兰酶的高密度发酵工艺研究

Production of Pullulanase by High Cell Density Cultivation of Recombinant Escherichia coli

以表达普鲁兰酶的重组大肠杆菌作为出发菌株,在摇瓶培养的基础上,建立了大肠杆菌工程菌产普鲁兰酶的高密度发酵工艺。通过测定细胞密度、细胞干重、分离菌体可溶性成分与不溶性成分及SDS-PAGE电泳,分析重组大肠杆菌的生长和普鲁兰酶的表达情况。摇瓶试验使用合成培养基和LB培养基,重组大肠杆菌在合成培养基生长较慢,诱导5h的普鲁兰酶表达量高于LB培养基,包涵体比例低于LB培养基。重组大肠杆菌的高密度发酵使用合成培养基,补料阶段采用指数流加的工艺,在设定细胞的比生长速率为0.12的前提下,限制补料中碳源的供应,以阻止乙酸的产生。当细胞密度OD600达到70.0开始诱导,最终细胞密度为每升53.3g细胞干重,细胞内可溶性普鲁兰酶为每升1.35g。

The recombinant Escherichia coli producing pullulanase was studied.After analysis of the data in the shake flask culture,the strategy of producing of pullulanase by high cell density cultivation of recombinant E.coli was investigated.The data of the cell optical density,cell dry mass,separation of soluble and insoluble cell fractions,SDS-PAGE were analyzed cell growth and production of pullulanase.Shake flask experiments were done in the defined medium and LB medium.After 5h of induction,the specific pullululanase concentration obtained on defined medium was considerably higher than that on LB medium,whereas the cell growth was slower.The pullulanase produced on defined medium accumulated in the insoluble cell fraction less than that on LB medium.A pre-determined feeding（μset=0.12） strategy was chosen to maintain carbon-limited growth using a defined medium.Feeding was carried out to increase the cell mass concentration exponentially in the bioreactor in order to prevent the accumulation of acetic acid.Expression of pullulanase was induced when cell concentration OD600 was 70.0.Applying the feeding strategy,final cell concentration of 53.3g per liter dry cell weight（g/L DCW） and production of pullulanase in the soluble cell fraction in a volumetric concentration of 1.35 g per liter were obtained.