摘要
为了开发谷氨酸脱羧酶(glutamate decarboxylase,GAD),以Enterococcus faecium为gadB基因供体、纤维素结合域(cellulose-binding domain,CBD)为亲和标签,利用内含肽DnaB自剪切作用分离GAD,对融合酶CBD-DnaB-GAD的构建、表达、GAD纯化及其酶学性质进行了探讨。结果表明:重组Escherichia coli GDMCC60446可高效表达CBD-DnaB-GAD,适宜自剪切液为0.2 mol/L Na_(2)HPO_(4)-NaH 2PO_(4)缓冲液(pH 6.5,含NaCl 0.5 mol/L、EDTA 1 mmol/L);经一步纯化和自剪切制备的GAD在SDS-PAGE电泳上呈单一条带,分子质量约为55.68 kDa,仅有1个亚基;GAD最适反应pH和温度分别为5.0和55℃,在pH 4.8~5.8和-25~55℃较稳定;5 mmol/L的NaCl、CaCl_(2)和乙二醇对GAD酶活力影响不大,而KCl、EDTA-2Na、ZnSO_(4)、CuSO_(4)、MnSO_(4)、MgSO_(4)、FeCl_(2)、FeCl_(3)、AlCl_(3)、AgNO 3和Pb(CH_(3)COO)_(2)对GAD酶活力具有不同程度的抑制作用;GAD仅催化L-谷氨酸发生脱羧反应,K m和V max分别为10.51 mmol/L和3.41μmol/(mL·min),催化反应无底物和产物抑制作用。GAD纯度和酶学性质优良,制备工艺简便,该技术有望应用于工业化生产。
To develop high-quality glutamate decarboxylase(GAD),the affinity of cellulose-binding domain(CBD)and the self-cleavage of DnaB(an intein)were used for the construction of CBD-DnaB-GAD fusion enzyme using Enterococcus faecium as gadB donor,and the purification of GAD and its enzymatic properties were also investigated.The results showed that recombinant Escherichia coli GDMCC60446 carrying pRPOCDN-EfagadB could efficiently express CBD-DnaB-GAD,and the optimal solution for self-cleavage of DnaB was 0.2 mol/L phosphate buffer saline(pH 6.5,containing 0.5 mol/L NaCl and 1 mmol/L EDTA).The GAD,prepared by one-step purification and self-cleavage,showed a single band on SDS-PAGE electrophoresis with a molecular weight of 55.68 kDa,and consists of only one subunit.The purified GAD showed maximum activity at pH 5.0 and 55℃,and was stable at the range of pH 4.8 to 5.8 and-25 to 55℃.Its activity was hardly affected by 5 mmol/L of NaCl,CaCl_(2) or ethylene glycol,but was inactivated by 5 mmol/L of KCl,EDTA-2Na,ZnSO_(4),CuSO_(4),MnSO_(4),MgSO_(4),FeCl_(2),FeCl_(3),AlCl_(3),AgNO_(3) or Pb(CH_(3) COO)_(2) at different levels.The purified GAD only catalyzed the decarboxylation of L-glutamic acid with apparent K m and V max at 10.51 mmol/L and 3.41μmol/(mL·min)respectively,and the reaction activities were not inhibited by the substrate and product.The purity and enzymatic properties of GAD were excellent,and the preparation process was simple.Due to the high purity and excellent enzymatic properties of GAD,as well as the simple preparation process,this technology has potential to be applied to industrial production.
作者
杨胜远
林谦
刘淑敏
苏巧云
黄慧玲
YANG Shengyuan;LIN Qian;LIU Shumin;SU Qiaoyun;HUANG Huiling(College of Food Science and Engineering,Lingnan Normal University,Zhanjiang 524048,China;College of Biology and Pharmacy,Yulin Normal University,Yulin 537000,China)
出处
《食品与发酵工业》
CAS
CSCD
北大核心
2021年第5期28-34,共7页
Food and Fermentation Industries
基金
农业农村部水产品加工重点实验室开放基金项目(NYJG201903)
广东省自然科学基金项目(2014A030307039)
岭南师范学院科研专项(ZL1602)。
关键词
屎肠球菌
谷氨酸脱羧酶
纤维素结合域
内含肽
酶学性质
Enterococcus faecium
glutamate decarboxylase
cellulose-binding domain
intein
enzymatic property
