重组大肠杆菌生产多形拟杆菌肝素酶I的发酵优化  

Optimization of fermentation conditions for the Heparinase I of Bacteroides thetaiotaomicron production by recombinant Esherichia coli

作　　者：张川[1] 刘敏敏 刘耀天 张悦[1] 李中媛 宋亚囝 罗学刚[1] ZHANG Chuan;LIU Minmin;LIU Yaotian;ZHANG Yue;LI Zhongyuan;SONG Yajian;LUO Xuegang(Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education,College of Bioengineering,Tianjin University of Science and Technology,Tianjin 300457)

机构地区：[1]工业发酵微生物教育部重点实验室暨天津市工业微生物重点实验室天津科技大学生物工程学院

出　　处：《食品科技》2019年第6期1-8,共8页Food Science and Technology

基　　金：国家重点研发计划项目(2017YFD0400303);国家863计划项目(2012AA021505)

摘　　要：以实验室前期构建的N端融合SUMO标签的多形拟杆菌肝素酶Ⅰ(SUMO-Bt-Hep Ⅰ)生产菌株重组大肠杆菌Rosetta(DE3)/pE-SUMO-Bt-HepⅠ为研究对象,通过单因素确定其最佳表达条件:诱导温度25℃、诱导剂浓度0.4mmol/L、诱导时间12 h;首先通过Plackett-Burman试验,筛选出培养基中3个主要影响酶活的因素:葡萄糖、酵母提取物和蛋白胨。并通过响应面试验确定最佳培养基为:葡萄糖9.52g/L,酵母提取物9.61g/L,蛋白胨19.92g/L,硫酸铵4g/L,磷酸氢二钠18g/L,磷酸二氢钾3g/L,硫酸镁5g/L。在此最佳培养基下发酵酶活预测可达55133IU/L,3次验证试验获得的SUMO-Bt-HepI酶活力平均为56961 IU/L,与理论预测值接近,比优化前(24215.9 IU/L)提高了2.3倍。通过5L发酵罐的分批补料扩大培养得到的最高发酵酶活为3.937×10^5IU/L,比摇瓶提高了6.91倍,这是目前报道发酵生产肝素酶I的最高水平,对工业化应用具有一定的指导意义。The Escherichia coli Rosetta(DE3)/pE-SUMO-Bt-HepⅠ preserved in our lab was used as the Bt-HepⅠ producing strain. The optimal express condition was obtained by sihgle factor experiments:induction temperature 25 ℃, inducer concentration 0.4 mmol/L, induction time 12 h. The optimal enzyme producing condition was studied by Plackett-Burman. The most important factors were glucose, yeast extract, peptone of medium. Then, response surface methodology was used to optimize the medium,with the optimum concentration glucose 9.52 g/L, yeast extract 9.61 g/L, peptone 19.92 g/L, ammonium sulfate 4 g/L, sodium hydrogen phosphate 18 g/L, monopotassium phosphate 3 g/L, magnesium sulfate5 g/L. Under the above optimum fermentation conditions the enzyme activity of Heparinase I can be enhanced to 55133 IU/L, increasing 2.3 fold than before optimization. The average activity of heparinase I was 56961 IU/L by repeat three times, closed to the theoretical predicted value. The maximum fermentation enzyme activity obtained by expanding culture of 5 L fermenter was 3.937×10^5 IU/L,increasing 6.91 fold than shake flask. This is the highest level of fermentative production of Heparinase I,which has certain guiding significance for industrial application.

关 键 词：重组大肠杆菌 多形拟杆菌 肝素酶Ⅰ 响应面 发酵优化 

分 类 号：TS201.3[轻工技术与工程—食品科学]