表达重组谷氨酸脱羧酶工程菌发酵条件的优化

目的对表达谷氨酸脱羧酶(Glutamate decarboxylase,EC4.1.1.15,简称GAD或GDC)的重组大肠杆菌的发酵条件进行优化。方法通过单因素试验优化表达GAD的重组大肠杆菌B3C1的诱导剂IPTG添加时间、IPTG浓度、诱导时间、诱导温度,再经响应面分析法优化工程菌的发酵条件(氯化钠、胰化蛋白胨、酵母粉、摇床转速、初始pH值、培养时间、接种量、装液量)。结果工程菌的最佳诱导条件为:IPTG添加时间为工程菌接种后4 h,IPTG浓度为0.8 mmol/L,诱导时间为4 h,诱导温度为35℃;工程菌的最佳发酵条件为:酵母粉1.01%,氯化钠0.6%,胰化蛋白胨1.5%,培养时间11.25 h,摇床转速250 r/min,初始pH值6.5,接种量3.0%,装液量11.70 ml。工程菌在该条件下发酵培养,GAD酶活达1 227.33 U,比优化前提高了12.29%。结论已成功对表达GAD的重组大肠杆菌的发酵条件进行了优化,为其工业化生产奠定了基础。

Optimization of fermentation condition of recombinant E.coli for expression of glutamate decarboxylase

Objective To optimize the fermentation condition of recombinant E.coli for expression of glutamate decarboxylase(GAD).Methods The time point,IPTG concentration,time duration and temperature for induction of recombinant E.coli B3C1 for expression of GAD were optimized by single factor test,while the fermentation condition,including the contents of sodium chloride,peptone,yeast powder,shaking speed of incubator shaker,initial pH value,culture time,MOI and liquid volume,by response surface assay.Results The optimal time point,concentration,time duration and temperature for induction with IPTG were 4 h after inoculation,0.8 mmol / L,4 h and 35℃ respectively.The optimal contents of yeast powder,sodium chloride,peptone,culture time,shaking speed of incubator shaker,initial pH value,MOI and liquid volume for fermentation of recombinant E.coli B3C1 were 1.01%,0.6%,1.5%,11.25 h,250 r / min,6.5,3.0% and 11.70 ml respectively.The activity of GAD expressed in E.coli under the fermentation condition reached 1 227.33 U,which increased by 12.29% as compared with that before optimization.Conclusion The fermentation condition of recombinant E.coli for expression of GAD was successfully optimized,which laid a foundation of industrial production of GAD.