短乳杆菌NM-1谷氨酸脱羧酶基因的克隆表达及其酶活力  

Gene cloning and expression of glutamic acid decarboxylase and its activity from Levilactobacillus brevis NM-1

作　　者：李文杰 谭琛 刘昊 张春枝[1] LI Wenjie;TAN Chen;LIU Hao;ZHANG Chunzhi(School of Biological Engineering,Dalian Polytechnic University,Dalian 116034,China)

机构地区：[1]大连工业大学生物工程学院,辽宁大连116034

出　　处：《大连工业大学学报》2025年第2期98-102,共5页Journal of Dalian Polytechnic University

基　　金：辽宁省教育厅高等学校基本科研项目(重点项目)(LJIKZZ20220060)。

摘　　要：谷氨酸脱羧酶(GAD)能将底物L-谷氨酸(L-Glu)转化成γ-氨基丁酸(GABA),谷氨酸脱羧酶的酶活力是影响GABA合成效率的关键因素。为了提高GABA的产量,本实验从自然界中筛选出一株产谷氨酸脱羧酶的乳酸菌,初步鉴定为短乳杆菌,命名为短乳杆菌NM-1。分别克隆短乳杆菌NM-1中编码GAD的基因gadA和gadB,连接到pET-30a(+)表达质粒,构建重组质粒,并将重组质粒通过热激法转入E.coli BL21(DE3),构建得到基因工程菌E.coli BL21(DE3)/pET-30a(+)-gadA和E.coli BL21(DE3)/pET-30a(+)-gadB(分别简称GadA-30a和GadB-30a)。结果表明,在相同培养条件下,GadB-30a全细胞催化能力远高于GadA-30a,提示短乳杆菌NM-1的GAD活性依赖于gadB基因。GadB-30a的GAD酶活力为6.52 U/mL,比活为9.59 U/mg。Glutamate decarboxylase (GAD) can convert the substrate L-glutamic acid (L-Glu) into γ-aminobutyric acid (GABA), and the enzyme activity of GAD is a key factor affecting the efficiency of GABA synthesis. To increase the production of GABA, a lactic acid bacteria producing glutamic acid decarboxylase was screened from nature and preliminarily identified as Levilactobacillus brevis , named Levilactobacillus brevis NM-1. The GAD encoding genes gadA and gadB in Levilactobacillus brevis NM-1 were cloned and ligated into the pET-30a(+) expression plasmid, then the recombinant plasmids were transferred into E.coli BL21(DE3) by heat shock to create the genetically engineered E.coli BL21(DE3)/pET-30a(+)- gadA and E.coli BL21(DE3)/pET-30a(+)- gadB (GadA-30a and GadB-30a). The results showed that the whole cell catalytic capacity of GadB-30a was much higher than that of GadA-30a at the same culture conditions, suggesting that the GAD activity of Levilactobacillus brevis NM-1 depending on the gadB gene. The GAD activity of GadB-30a was 6.52 U/mL and the specific enzyme activity was 9.59 U/mg.

关 键 词：谷氨酸脱羧酶 短乳杆菌 L-谷氨酸 Γ-氨基丁酸 

分 类 号：Q936[生物学—微生物学]