1株产γ-氨基丁酸植物乳杆菌谷氨酸脱羧酶基因的克隆与表达

Cloning and Expressing of Glutamate Decarboxylase Gene from Lactobacillus plantarum Producing γ-Aminobutyric Acid

以1株产γ-氨基丁酸(γ-aminobutyric acid,GABA)植物乳杆菌(Lactobacillus plantarum)BC114谷氨酸脱羧酶为研究对象,通过聚合酶链式反应(polymerase chain reaction,PCR)技术获得该酶基因,对其进行生物信息学分析,并转入大肠杆菌BL21(DE3)中实现异源表达。采用实时荧光定量PCR、聚丙烯酰胺凝胶电泳和高效液相色谱分别测定重组菌不同发酵时间点谷氨酸脱羧酶基因的表达量、蛋白表达量以及产GABA能力。结果表明:植物乳杆菌中谷氨酸脱羧酶基因大小为1 410 bp,编码469个氨基酸,蛋白二级结构由α-螺旋(32.2%)、β-折叠(11.5%)和无规则卷曲(56.3%)构成,完成了该酶的三维结构同源建模;成功构建了重组谷氨酸脱羧酶大肠杆菌,谷氨酸脱羧酶基因在诱导6 h相对表达量最大,而蛋白表达量较基因在转录水平表达量有一定滞后,在8 h达最大值,此时GABA产量也达最高,为2 387 mg/L。

The gene encoding glutamate decarboxylase(GAD),a key enzyme for the biosynthesis ofγ-aminobutyric acid(GABA),from GABA-producing Lactobacillus plantarum BC114 was amplified by using PCR.Bioinformatics analysis of this gene was performed.Then,the target gene was subcloned into the expression vector pGEM-T-gad and the recombinant plasmids were transformed into competent Escherichia coli BL21(DE3)for heterogeneous expression.Real-time reverse transcription PCR(RT-PCR),polyacrylamide gel electrophoresis(PAGE)and high performance liquid chromatography(HPLC)were used to measure the mRNA and protein expression of GAD and GABA production,respectively.Results showed that the size of the GAD gene from L.plantarum BC114,encoding 469 amino acids,was 1 410 bp.The predicted secondary structure of the GAD protein contained 32.2%α-helix,11.5%β-sheet and 56.3%random coil.Meanwhile,the three dimensional structure was also predicted by using homologous modeling method.The recombinant E.coli harboring the gad gene was obtained successfully.The highest expression level of the gad gene was achieved after induction for 6 h,while the maximum protein expression level appeared 2 h later and the highest production of GABA of 2 387 mg/L was also obtained at this time.