谷氨酸脱羧酶的分子改造及酶学性质研究  

Molecular Modification and Enzymatic Properties of Glutamate Decarboxylase

作　　者：周丽亚[1] 牛玉杰 郑晓冰[1] 姜艳军[1] 马丽[1] 白敬 贺莹[1] ZHOU Li-ya;NIU Yu-jie;ZHENG Xiao-bing;JIANG Yan-jun;MA Li;BAI Jing;HE Ying(School of Chemical Engineering and Technology,Hebei University of Technology,Tianjin 300130,China;College of Food Science and Biology,Hebei University of Science&Technology,Shijiazhuang 050018,China)

机构地区：[1]河北工业大学化工学院,天津300130 [2]河北科技大学食品与生物学院,石家庄050018

出　　处：《中国生物工程杂志》2023年第5期24-36,共13页China Biotechnology

基　　金：国家自然科学基金(21878068、2217083);河北省省级科技计划(20372802D);河北省自然科学基金(B2020202036)资助项目。

摘　　要：以大肠杆菌(Escherichia coli)来源的谷氨酸脱羧酶(GadB)为研究对象,通过组合突变,获得了pH适用范围拓宽、催化活力提高和稳定性增强的组合突变体M2。与野生型GadB-WT相比,组合突变体M2的pH适用范围有效拓宽,在pH6.0时催化活力比GadB-WT提高113.43%。之后对含有M2突变体基因重组菌的发酵培养基和诱导条件进行优化,优化后单位培养基酶活力比未优化时提高了104.13%。在此基础上对M2的酶学性质进行测定,测得其最适pH为5.0,最适温度为37℃。通过稳定性测定M2的pH稳定性和热稳定性与野生型GadB-WT相比都有一定程度的增强。M2的动力学参数K_(m)值为7.316μmol/L,k_(cat)为13.387 s^(-1),k_(cat)/K_(m)为1.830 L/(s·μmol)。研究获得的组合突变体M2进一步丰富了催化合成γ-氨基丁酸的GadB突变体酶库,具有良好应用前景。By combining and screening several mutations of glutamate decarboxylase(GadB)derived from E.coli,a combined mutant M2 with a wider pH range,higher catalytic activity and higher pH and thermal stabilities was obtained.Compared with the wild-type GadB-WT,the pH range of the combined mutant M2 was effectively broadened,and it produced a higher catalytic activity of 113.43%over the wild-type GadB-WT under pH6.0.After the optimization for fermentation medium and induction conditions of the recombinant bacteria,an overall 104.13%increase of enzyme activity was gained over the unoptimized medium.On this basis,the enzymatic properties of M2 were determined.The optimum pH was 5.0 and the optimum temperature was 37℃.Compared with wild-type Gad-WT,the pH stability and thermal stability of M2 were enhanced to some extent.The kinetic parameters of M2 were determined as follows:K_(m)was 7.316μmol/L,k_(cat)was 13.387 s~(-1),and k_(cat)/K_(m)was 1.830 L/(s·μmol).The combined mutant M2 obtained in this study further enriches the GAD mutant enzyme library for the synthesis ofγ-aminobutyric acid and has a promising application prospect.

关 键 词：谷氨酸脱羧酶 Γ-氨基丁酸 定点突变 发酵优化 

分 类 号：Q814[生物学—生物工程]