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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2013, Vol. 33 Issue (8): 98-104    
技术与方法     
Thermobifida fusca海藻糖合成酶基因的克隆表达及发酵优化
罗锋1,2, 段绪果1,2, 宿玲恰1,2, 吴敬1,2
1. 江南大学食品科学与技术国家重点实验室 无锡 214122;
2. 江南大学生物工程学院 工业生物技术教育部重点实验室 无锡 214122
Cloning,Expression and Fermentation Optimization of Thermobifida fusca Trehalose Synthase Gene in E.coli
LUO Feng1,2, DUAN Xu-guo1,2, SU Ling-qia1,2, WU Jing1,2
1. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China;
2. School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, Wuxi 214122, China
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摘要：

将来源于嗜热单孢菌(Thermobifida fusca)的海藻糖合成酶基因进行克隆表达,构建重组菌E.coli BL 21(DE3) {pET-24a(+)/Tres}。并对基因工程菌的发酵产酶条件进行优化,得到最优培养基成分为:甘油12 g/L,酵母粉24 g/L,蛋白胨12g/L,磷酸盐浓度60mmol/L,Zn2+ 2mmol/L。最佳培养条件为:装液量20 ml(250 ml摇瓶),发酵2 h后25 ℃诱导并添加终浓度为0.4 mmol/L的IPTG。优化后的最终发酵酶活达到28.6 U/ml,为未优化前的3.4倍,是目前国内报道大肠杆菌摇瓶发酵产海藻糖合成酶的最高表达水平,为该酶的工业化生产奠定了基础。
关键词： 嗜热单孢菌;  海藻糖合成酶;  重组大肠杆菌;  发酵优化    
Abstract:

In the present study, the trehalose synthase gene of Thermobifida fusca was cloned and expressed in Escherichia coli BL21(DE3). The optimized composition of fermentation medium and culture conditions for the recombinant E. coli were investigated in shake flasks. The optimal fermentation medium was as follows: 12 g/L glycerol, 24 g/L yeast extract, 12 g/L peptone, 60 mmol/L PO43-, 2 mmol/L Zn2+. And the optimal culture conditions were that: volume 20 ml in 250 ml shake flask, induction temperature 25℃, induced by 0.4 mmol/L IPTG at 2 h of culture. Under these conditions, the maximal enzyme activity reached 28.6 U/ml, which was 3.4 times as high as that not optimized.
Key words: Thermobifida fusca    Trehalose synthase    Recombinant E.coli    Optimization
收稿日期: 2013-05-02 出版日期: 2013-08-25
ZTFLH:  Q786  
通讯作者: 吴敬jingwu@jiangnan.edu.cn;jingwu80@hotmail.com     E-mail: jingwu@jiangnan.edu.cn;jingwu80@hotmail.com
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引用本文:

罗锋, 段绪果, 宿玲恰, 吴敬. Thermobifida fusca海藻糖合成酶基因的克隆表达及发酵优化[J]. 中国生物工程杂志, 2013, 33(8): 98-104.

LUO Feng, DUAN Xu-guo, SU Ling-qia, WU Jing. Cloning,Expression and Fermentation Optimization of Thermobifida fusca Trehalose Synthase Gene in E.coli. China Biotechnology, 2013, 33(8): 98-104.

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https://manu60.magtech.com.cn/biotech/CN/    或    https://manu60.magtech.com.cn/biotech/CN/Y2013/V33/I8/98

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