一株产γ-氨基丁酸植物乳杆菌谷氨酸脱羧酶基因的克隆与表达

摘要/Abstract

摘要： 以一株产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对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能力进行评估。结果表明：植物乳杆菌中谷氨酸脱羧酶基因大小为1410 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，初步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2387 mg/L。

关键词: 植物乳杆菌BC114, γ-氨基丁酸, 谷氨酸脱羧酶, 克隆, 表达, 生物信息学

Abstract: Glutamate decarboxylase (GAD) which is a key enzyme for the biocatalysis of y-aminobutyric acid (GABA) from Lactobacillus plantarum BC114 possessing GABA producing capacity was studied in this work. The GAD gene from L. plantarum BC114 was amplified and obtained using PCR technology, and bioinformatics of the gene was further analyzed. Then, the target GAD gene was subcloned into vector and hetreogenously expressed by E. coli BL21 (DE3). Real-time reverse transcription PCR (real-time RT-PCR), polyacrylamide gelelectrophoresis and high performance liquid chromatography (HPLC) technology were used to measure the expression value of GAD gene, protein expression and GABA production, respectively. Results showed that the fragment of GAD gene from L. plantarum BC114 was 1410 bp and encoded 469 amino acids. The predicted secondary structure of GAD contained of 35.73% α-helixes, 12.79% β-fold and 51.48% random curl, meanwhile, the three dimensional structures was also predicted by using homologous modeling method. The recombinant GAD E. coli was obtained successfully. The highest expression level of GAD gene was achieved after induction for 6 h, while the maximum protein expression level which lagged behind the gene expression was achieved at 8 h. Meanwhile, the highest production of GABA reached to 2387 mg/L at 8 h of fermentation.

Key words: Lactobacillus plantarum BC114, γ-aminobutyric acid, Glutamate decarboxylase, Clone, Expression, Bioinformatic

中图分类号:

TQ920.6

谭霄张庆. 一株产γ-氨基丁酸植物乳杆菌谷氨酸脱羧酶基因的克隆与表达[J]. 食品科学, 0, (): 0-0.

Xiao Tan Zhang Qing. Cloning and expressing of glutamate decarboxylase gene from Lactobacillus plantarum with capability of producing γ-aminobutyric acid[J]. FOOD SCIENCE, 0, (): 0-0.

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