屎肠球菌纤维素结合域谷氨酸脱羧酶构建及其酶学性质  

Construction and characteristics of recombinant cellulose-binding domain-glutamate decarboxylase of Enterococcus faecium

作　　者：杨胜远 林谦 赖丽萍 黄婷婷 YANG Shengyuan;LIN Qian;LAI Liping;HUANG Tingting(School of Chemistry and Chemical Engineering,Lingnan Normal University,Zhanjiang 524048,China;College of Biology and Pharmacy,Yulin Normal University,Yulin 537000,China)

机构地区：[1]岭南师范学院化学化工学院,广东湛江524048 [2]玉林师范学院生物与制药学院,广西玉林537000

出　　处：《食品与发酵工业》2019年第21期22-30,共9页Food and Fermentation Industries

基　　金：广东省自然科学基金项目(2014A030307039);岭南师范学院科研专项项目(ZL1602)

摘　　要：为了开发可用于高效固定化的优质谷氨酸脱羧酶(glutamate decarboxylase,GAD,EC 4.1.1.15),以Enterococcus faecium GDMCC60203为gadB基因供体,对纤维素结合域(cellulose-binding domain,CBD)GAD融合酶(CBD-GAD)的构建及其酶学性质进行了探讨。结果显示:构建的含pRPOCB-EfagadB重组质粒的Escherichia coli GDMCC 60445可高效表达CBD-GAD,120 mL发酵液制备的纤维素固定化CBD-GAD的活力为(347.93±27.63)U/g;在无氨苄青霉素LB培养基中连续培养5批,E.coli GDMCC 60445仍可稳定表达CBD-GAD;经一步纯化,CBD-GAD在SDS-PAGE电泳上呈单一条带,分子质量约为74.02 kDa;CBD-GAD最适反应pH和温度分别为4.6和60℃,在pH 4.8~5.6和4~40℃较稳定,仅对L-谷氨酸具有催化活性,K m和V max分别为10.58 mmol/L和3.83μmol/(mL·min),其催化反应不受底物和产物抑制。重组菌株稳定,CBD-GAD酶学性质优良,有望应用于γ-氨基丁酸和D-谷氨酸工业化生产。In order to develop high-quality glutamate decarboxylase(GAD,EC4.1.1.15)for efficient immobilization,the construction of cellulose-binding domain(CBD)GAD fusion enzyme(CBD-GAD)and its enzymatic properties were investigated using Enterococcus faecium GDMCC60203 as gadB donor.The results showed that the recombinant Escherichia coli GDMCC60445 carrying pRPOCB-EfagadB could efficiently express CBD-GAD,and the activity of the cellulose immobilized CBD-GAD prepared from 120 mL fermentation broth was 347.93±27.63 U/g.When continuously transferred five batches in LB medium without ampicillin,the capability of CBD-GAD expression in the recombinant E.coli GDMCC60445 was still stable.The CBD-GAD purified by one-step purification showed a single band on SDS-PAGE electrophoresis with a molecular weight of 74.02 kDa.The purified CBD-GAD showed maximum activities at pH 4.6 and 60℃,respectively,and was stable in the range of pH 4.8 to 5.6 and temperature from 4 to 40℃.CBD-GAD reacted only with L-glutamic acid with apparent Km and V max of 10.58 mmol/L and 3.83μmol/(mL·min),respectively,and the reaction activities were not inhibited by substrate and product.Hence,this strain has the potential to be applied in industrial production of gamma-aminobutyric acid and D-glutamic acid due to its high stability and excellent enzymatic properties.

关 键 词：纤维素结合域 屎肠球菌 谷氨酸脱羧酶 表达 酶学性质 

分 类 号：R74[医药卫生—神经病学与精神病学]