<i>Amphibacillus xylanus</i>谷氨酸脱氢酶基因工程菌培养条件优化 <sup>*</sup>

doi:10.13523/j.cb.20191007

中国生物工程杂志

2019, Vol. 39

Issue (10): 58-66 DOI: 10.13523/j.cb.20191007

技术与方法

Amphibacillus xylanus谷氨酸脱氢酶基因工程菌培养条件优化 ^*

陈子晗,周海胜,尹新坚,吴坚平,杨立荣(

)

浙江大学化学工程与生物工程学院生物工程研究所杭州 310027

Optimizing the Culture Conditions for Amphibacillus xylanus Glutamate Dehydrogenase Gene Engineering Bacteria

CHEN Zi-han,ZHOU Hai-sheng,YIN Xin-jian,WU Jian-ping,YANG Li-rong(

)

College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China

全文: PDF(2045 KB) HTML

摘要：

首先构建了5株表达NADH依赖型谷氨酸脱氢酶的大肠杆菌基因工程菌,获得来源于Amphibacillus xylanus的谷氨酸脱氢酶AxyGDH。其最适温度为60℃、最适pH为8.0,该条件下比酶活达到(903.1±24.6)U/mg,酶活半衰期为167h。其次,确定了表达AxyGDH的大肠杆菌基因工程菌E.coli BL21(DE3)/pET-28a(+)-AxyGDH的产酶条件:诱导剂IPTG浓度为0.7mmol/L、诱导温度为25℃;优化后的培养基组成为:甘油11.3g/L,酵母粉16.3g/L,MgSO₄·7H₂O 0.62g/L,NaCl 0.5g/L,Na₂HPO₄·12H₂O 17.1g/L,KH₂PO₄ 3g/L,NH₄Cl 1.5g/L。最后,在10L发酵罐中控制比生长速率为0.2h ^-1进行补料分批发酵,所得AxyGDH的发酵酶活为(9 066±45)U/ml,是LB摇瓶发酵酶活的51.1倍,为谷氨酸脱氢酶的低成本生产奠定了基础。

关键词： 谷氨酸脱氢酶; 异源表达; 比酶活; 稳定性; 发酵

Abstract:

Firstly,five expressing NADH-dependent glutamate dehydrogenase E.coli engineering bacterias are constructed,get a glutamate dehydrogenase AxyGDH from Amphibacillus xylanus.The optimum temperature is 60℃ and pH is 8.0,the specific enzyme activity is (903.1±24.6)U/mg,the half-life is 167h at this condition.Then, decide the enzyme production conditions of E.coli BL21(DE3)/pET-28a(+)-AxyGDH: Inducer IPTG concentration 0.7mmol/L,induction temperature 25℃.The optimized medium composition: Glycerol 11.3g/L, yeast powder 16.3g/L, MgSO₄·7H₂O 0.62g/L,NaCl 0.5g/L, Na₂HPO₄·12H₂O 17.1g/L,KH₂PO₄ 3g/L,NH₄Cl 1.5g/L.At last,the fermentation enzyme activity of AxyGDH is (9 066±45)U/ml in the 10L tank fermentation when controlling the specific growth rate is 0.2h ^-1,which is 51.1 times than that of LB medium.It lays the foundation for glutamate dehydrogenase low cost production.

Key words: Glutamate dehydrogenase Heterologous expression Specific enzyme activity Stability Fermentation

收稿日期: 2019-03-12 出版日期: 2019-11-12

ZTFLH:

Q815

基金资助: * 国家自然科学基金面上项目(21476199);国家973计划(2011CB710800)

通讯作者: 杨立荣 E-mail: lryang@zju.edu.cn

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引用本文:

陈子晗,周海胜,尹新坚,吴坚平,杨立荣. Amphibacillus xylanus谷氨酸脱氢酶基因工程菌培养条件优化 ^*[J]. 中国生物工程杂志, 2019, 39(10): 58-66.

CHEN Zi-han,ZHOU Hai-sheng,YIN Xin-jian,WU Jian-ping,YANG Li-rong. Optimizing the Culture Conditions for Amphibacillus xylanus Glutamate Dehydrogenase Gene Engineering Bacteria. China Biotechnology, 2019, 39(10): 58-66.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20191007 或 https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I10/58

图1 谷氨酸脱氢酶诱导表达电泳图

图2 粗酶液酶活(a)和稳定性(b)

图3 AxyGDH纯化电泳图

图4 AxyGDH酶最适温度(a)和pH(b)

表1 AxyGDH在不同温度下的酶活半衰期

图5 诱导剂浓度(a)和诱导温度(b)对酶活的影响

图6 不同碳源(a)和氮源(b)对酶活和菌体生长的影响

表2 响应面分析试验因素水平表

图7 X1、X2(a),X2、X3(b),X1、X3(c)对酶活的响应面及等高线图

图8 不同比生长速率对发酵产酶的影响(a)及控制比生长速率为0.20h-1时的生长过程和产酶曲线(b)

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