大肠杆菌L-苏氨酸脱氢酶的高效表达及其酶学性质分析  

Efficient Expression and Enzymatic Properties of L-Threonine Dehydrogenase from Escherichia coli

作　　者：刘欣欣 王瑶 史红玲[1] 姚伦广[1] 王贤 唐存多[1,2,3] LIU Xinxin;WANG Yao;SHI Hongling;YAO Lunguang;WANG Xian;TANG Cunduo(College of Life Science and Agricultural Engineering,Nanyang Normal University,Nanyang 473061,China;Postdoctoral Innovation Practice Base,She Dian Lao Jiu Co.Ltd.,Nanyang 473300,China;College of Food Science and Technology,Henan Agricultural University,Zhengzhou 450002,China)

机构地区：[1]南阳师范学院生命科学与农业工程学院,河南南阳473061 [2]赊店老酒股份有限公司博士后创新实践基地,河南南阳473300 [3]河南农业大学食品科学技术学院,河南郑州450002

出　　处：《食品科学》2023年第18期85-92,共8页Food Science

基　　金：国家自然科学基金青年科学基金项目(31900916);河南省高校科技创新人才项目(21HASTIT041);河南省青年人才托举工程项目(2021HYTP036)。

摘　　要：为提高L-苏氨酸脱氢酶(L-threonine dehydrogenase,L-TDH)催化L-苏氨酸脱氢合成L-2-氨基乙酸乙酯效率,通过基因挖掘的手段挖掘出来自大肠杆菌(Escherichia coli)的L-TDH,再借助pACYCDuet-1表达系统将其在E. coli BL21(DE3)中进行可溶性表达及鉴定。结果表明,L-TDH在E. coli BL21(DE3)中实现了高水平的可溶性表达,其裂解液中的酶活力为19.13 IU/mL,约为E. coli BL21(DE3)本底表达水平的79倍。纯化后的比活力为12.77 IU/mg,它的最适反应温度为45℃,最适反应pH值为9.0,在35℃和40℃保温120 min,残留酶活力仍能达到90%以上。此外,Ec TDH动力学参数也优于其他已报道的L-TDH,在转化L-苏氨酸合成2,5-二甲基吡嗪(2,5-dimethylpyrazine,2,5-DMP)的过程中具有更大优势,也为实现转化L-苏氨酸合成2,5-DMP的工业化生产奠定了理论基础。Our aim was to improve the catalytic efficiency of L-threonine dehydrogenase(L-TDH)on L-threonine dehydrogenation to synthesize ethyl L-2-aminoacetate.An L-TDH gene from Escherichia coli was mined and solubly expressed in E.coli BL21(DE3)through pACYCDuet-1 expression system.The expressed enzyme was purified and characterized.The results showed that high-level soluble expression of L-TDH was achieved in E.coli BL21(DE3),and the enzyme activity in the lysate was 19.13 IU/mL,which was about 79 times higher than that the level of E.coli BL21(DE3)background expression.The specific activity of the purified L-TDH was 12.77 IU/mg;its optimal reaction temperature was 45℃,and its optimal reaction pH was 9.0.The residual enzyme activity was still more than 90%after being held at 35 or 40℃for 120 min.In addition,the kinetic parameters of EcTDH were better than those of other reported L-TDHs,and EcTDH was superior in the synthesis of 2,5-dimethylpyrazine(2,5-DMP)by converting L-threonine.Our findings could lay a theoretical foundation for the industrial production of 2,5-DMP.

关 键 词：L-苏氨酸 L-苏氨酸脱氢酶 大肠杆菌 表达 生物转化 

分 类 号：Q814.9[生物学—生物工程]