高产γ-氨基丁酸的富锌乳酸菌培养基优化及GAD基因的克隆和序列分析

doi:10.7506/spkx1002-6630-201019066

食品科学 ›› 2010, Vol. 31 ›› Issue (19): 304-308.doi: 10.7506/spkx1002-6630-201019066

高产γ-氨基丁酸的富锌乳酸菌培养基优化及GAD基因的克隆和序列分析

谢晓阳，陈瑶，刘志文，郭晓燕，程新，高小玉，徐波*

南昌市发酵应用技术重点实验室，江西农业大学生物科学与工程学院

收稿日期:2010-06-22 修回日期:2010-09-25 出版日期:2010-10-15 发布日期:2010-12-29
通讯作者: 徐波 E-mail:xubo583@sohu.com
基金资助:
国家自然科学基金项目(31060208)；江西省科技厅科技支撑项目(2009BNB05704)；
江西农业大学博士科研启动基金项目(2001)

Medium Optimization for Producing Gamma-aminobutyric Acid by the Fermentation of Lactobacillus brevis BS2 with Zinc-enriching ability and Cloning and Sequencing of Its Glutamate Decarboxylase Gene

XIE Xiao-yang，CHEN Yao，LIU Zhi-wen，GUO Xiao-yan，CHENG Xin，GAO Xiao-yu，XU Bo*

(Nanchang Key Laboratory of Fermentation Application Technology, College of Biology Science and Engineering,
Jiangxi Agricultural University, Nanchang 330045, China)

Received:2010-06-22 Revised:2010-09-25 Online:2010-10-15 Published:2010-12-29
Contact: XU Bo E-mail:xubo583@sohu.com

摘要/Abstract

摘要：

为了检测和提高乳酸菌γ- 氨基丁酸(GABA)产量，本研究采用纸层析预染法分析Lactobacillus brevis BS2最佳发酵产GABA 条件为：pH5.0，以葡萄糖为碳源，以大豆蛋白胨和牛肉膏为复合氮源，碳氮比为1:1，底物质量分数1%，GABA 产量可达到6.32g/L。采用CTAB 法提取该菌株基因组，并以此基因组为模板应用降落PCR扩增出1407bp 的谷氨酸脱羧酶基因gad，将gad 克隆到T 载体后经测序分析发现与L. brevis strain BH2 的gad 具有高度的同源性，同源性达98%，证明该菌株为高产GABA 的富锌短小乳酸杆菌，在工业应用方面具有很大潜力。

关键词: γ- 氨基丁酸, Lactobacillus brevis BS2, 谷氨酸脱羧酶基因, 培养基优化, 序列分析

Abstract:

The objectives of this work were to optimize the medium composition for improved production ofγ-aminobutyric acid (GABA) by the fermentation of Lactobacillus brevis BS2 with Zinc-enriching ability and to clone and sequence the glutamate decarboxylase (GAD) gene of this strain. GABA quantification was performed using pre-staining paper chromatography method. The optimal medium composition was determined as follows: carbon source, glucose; nitrogen source, soybean peptone plus beef extract; carbon/nitrogen ratio, 1:1; and substrate concentration, 1%, and the maximum GABA production reached up to 6.32 g/L under such conditions. CTAB method was used for the extraction of genomic DNA from stain BS2, and the extracted genomic DNA was used as the template for the amplification of a 1407 bp gad gene by touch down PCR. The gad gene was finally cloned into the T vector. The sequencing analysis showed that the gene had 98% homology with that of Lactobacillus brevis BH2, suggesting that the obtained target gene is the gad gene of Lactobacillus brevis.

Key words: Gamma-aminobutyric acid, Lactobacillus brevis BS2, glutamate decarboxylase, medium optimization, sequence analysis

中图分类号:

Q936

谢晓阳，陈瑶，刘志文，郭晓燕，程新，高小玉，徐波*. 高产γ-氨基丁酸的富锌乳酸菌培养基优化及GAD基因的克隆和序列分析[J]. 食品科学, 2010, 31(19): 304-308.

XIE Xiao-yang，CHEN Yao，LIU Zhi-wen，GUO Xiao-yan，CHENG Xin，GAO Xiao-yu，XU Bo*. Medium Optimization for Producing Gamma-aminobutyric Acid by the Fermentation of Lactobacillus brevis BS2 with Zinc-enriching ability and Cloning and Sequencing of Its Glutamate Decarboxylase Gene[J]. FOOD SCIENCE, 2010, 31(19): 304-308.

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高产γ-氨基丁酸的富锌乳酸菌培养基优化及GAD基因的克隆和序列分析

Medium Optimization for Producing Gamma-aminobutyric Acid by the Fermentation of Lactobacillus brevis BS2 with Zinc-enriching ability and Cloning and Sequencing of Its Glutamate Decarboxylase Gene

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