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

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

作　　者：陈子晗 周海胜[1] 尹新坚 吴坚平[1] 杨立荣[1] 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)

机构地区：[1]浙江大学化学工程与生物工程学院生物工程研究所

出　　处：《中国生物工程杂志》2019年第10期58-66,共9页China Biotechnology

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

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,KH2PO43g/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(9066±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。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,KH2PO43g/L,NH4Cl 1.5g/L.At last,the fermentation enzyme activity of AxyGDH is(9066±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.

关 键 词：谷氨酸脱氢酶 异源表达 比酶活 稳定性 发酵 

分 类 号：Q815[生物学—生物工程]