<em>Thermobifida fusca</em>海藻糖合成酶基因的克隆表达及发酵优化

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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

作者信息 +

Cloning,Expression and Fermentation Optimization of Thermobifida fusca Trehalose Synthase Gene in E.coli

LUO Feng^1,2, DUAN Xu-guo^1,2, SU Ling-qia^1,2, WU Jing^1,2

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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。

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 PO₄^3-, 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.

导出引用

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[J]. China Biotechnology, 2013, 33(8): 98-104

中图分类号： Q786

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