产GABA发酵乳杆菌的筛选、发酵条件优化及其谷氨酸脱羧酶基因的克隆

从酸菜汁中分离到一株具有谷氨酸脱羧酶活力的乳酸菌YS2,经过16S rDNA序列分析鉴定为发酵乳杆菌（Lactobacillus fermentum）.YS2在含l％L-谷氨酸钠的MRS培养基中,GABA的产量达到4.37 g/L.对YS2产GABA的碳源、氮源、初始pH进行了初步优化,确定最佳条件为：以蔗糖为主要碳源,以蛋白胨和牛肉膏为氮源,初始pH 6.0,在此条件下,GABA产量达到5.68 g/L.通过PCR顺利地扩增出了YS2菌株的谷氨酸脱羧酶gadB基因,将PCR产物与表达载体pEASY-E1连接.测序结果表明,YS2的谷氨酸脱羧酶与Lactobacillus fermentum...

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Published in广东农业科学 Vol. 41; no. 8; pp. 192 - 197
Main Author 林谦 姜康怡 韦素娟 郭丹妮 农石蓉 高敏
Format Journal Article
LanguageChinese
Published 玉林师范学院生命科学与技术学院,广西玉林,537000 2014
Subjects
γ-氨基丁酸
克隆
发酵乳杆菌
序列分析
谷氨酸脱羧酶
序列分析
克隆
谷氨酸脱羧酶
γ-氨基丁酸
发酵乳杆菌
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ISSN1004-874X

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Summary:从酸菜汁中分离到一株具有谷氨酸脱羧酶活力的乳酸菌YS2,经过16S rDNA序列分析鉴定为发酵乳杆菌（Lactobacillus fermentum）.YS2在含l％L-谷氨酸钠的MRS培养基中,GABA的产量达到4.37 g/L.对YS2产GABA的碳源、氮源、初始pH进行了初步优化,确定最佳条件为：以蔗糖为主要碳源,以蛋白胨和牛肉膏为氮源,初始pH 6.0,在此条件下,GABA产量达到5.68 g/L.通过PCR顺利地扩增出了YS2菌株的谷氨酸脱羧酶gadB基因,将PCR产物与表达载体pEASY-E1连接.测序结果表明,YS2的谷氨酸脱羧酶与Lactobacillus fermentum F-6的谷氨酸脱羧酶序列一致性达到99％.重组酶与组氨酸标签融合表达,经镍柱亲和层析纯化出融合蛋白,电泳显示出56 kDa的目的条带,纯化的重组酶表现出了酶活性（1.06 U/mg）.
Bibliography:44-1267/S
LIN Qian, JIANG Kang-yi, WEI Su-juan, GUO Dan-ni, NONG Shi-rong, GAO Min （School of Life Science and Technology, Yulin Normal University, Yulin 537000, China）
Lactobacillus fermentum; r-Aminobutyric acid; Glutamate decarboxylase; cloning; sequence analysis
A strain of lactic acid bacteria, YS2, capable of production of r-aminobutyric acid （GABA）, was isolated from pickled vegetable juice, and was identified as Lactobacillus fermentum by 16S rDNA sequence analysis. When YS2 was grown in MRS medium containing 1% L-monosodium glutamate （MSG）, GABA yield reached 4.37 g/L. The optimal growth condition for GABA production was determined as follows： sucrose as carbon source, peptone plus beef extract as nitrogen source, initial pH at 6.0. Under the condition, GABA yield reached 5.68 g/L. The gene encoding glutamate decarboxylase, gadB, was amplified by PCR, and inserted into pEASY-E1, an expression vector for E. coll. Sequence analysis showed that the GAD from YS2 had 99% identity with that of Lactobacillus f
ISSN:1004-874X