彭春龙,黄俊,赵伟睿,胡升,梅乐和,姚善泾

• 1:浙江大学化学工程与生物工程学院
• 2:浙江科技学院生物与化学工程学院
• 3:浙江大学宁波理工学院生物与化学工程学院

摘要(Abstract)：

谷氨酸脱羧酶(GAD)能催化L-谷氨酸(Glu)脱羧生成γ-氨基丁酸(GABA),而GAD和位于细胞膜上的Glu/GABA反向转运蛋白的协同作用则和一些细菌的耐酸机制有关。短乳杆菌GAD系统主要含有两个谷氨酸脱羧酶(Gad A和Gad B)和一个Glu/GABA反向转运蛋白(Gad C)。研究以Lactobacillus brevis CGMCC NO.1306为材料,在不同培养条件下,采用实时定量PCR的方法研究了酸胁迫对gad A、gad B和gad C表达的影响。实验结果表明,gad B和gad C组成操纵子,在菌体生长进入对数后期以后,低p H环境能极大地促进gad CB的表达,菌体的GAD活力主要由Gad B提供且在菌体生长进入稳定期时达到最大值;相对而言,gad A的表达水平几乎不受p H环境和细胞周期的影响。以上结果将为短乳杆菌GABA发酵工艺的优化和研究酸胁迫对该菌株耐酸能力的影响提供理论指导。

关键词(KeyWords)： GAD系统;实时定量PCR;酸胁迫;操纵子

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(21176220,31240054);; 浙江省自然科学基金重点项目(Z13b060008);; 宁波市重大科技攻关项目(2011C11023)

作者(Author): 彭春龙,黄俊,赵伟睿,胡升,梅乐和,姚善泾

参考文献(References)：

• [1]Kim H W,Kashima Y,Ishikawa K,et a1.Purification and characterization of the first archaeal glutamate decarboxylase from Pyrococcus horikoshii[J].Biosci Biotechnol Bio Chem,2009,73(1):224-227.
• [2]Foster J W.Escherichia coli acid resistance:tales of an amateur acidophile[J].Nat Rev Microbiol,2004,2(11):898-907.
• [3]Sanders J W,Leenhouts K,Burghoorn J,et al.A chloride-inducible acid resistance mechanism in Lactococcus lactis and its regulation[J].Mol Microbiol,1998,27(2):299-310.
• [4]Higuchi T,Hayashi H,Abe K.Exchange of glutamate and gaminobutyrate in a Lactobacillus strain[J].J Bacteriol,1997,179(10):3362-3364.
• [5]Benjamin C S,Ju¨rgen B,Rudi F V.Role of GAD system in hop tolerance of Lactobacillus brevis[J].Eur Food Res Technol,2013,237(2):199-207.
• [6]FAN En-yu(范恩宇).Cloning and expression of glutamate decarboxylase gene from Lactobacillus brevis(短乳杆菌谷氨酸脱羧酶基因的克隆与表达)[D].Hangzhou(杭州):Zhejiang University(浙江大学),2011.
• [7]XIA Jiang(夏江).Breeding ofγ-aminobutyric acid-producing Lactobacillus and optimization of fermentation conditions(产γ-氨基丁酸段乳杆菌的筛选和发酵条件的优化)[D].Hangzhou(杭州):Zhejiang University(浙江大学),2006.
• [8]FAN En-yu,HUANG Jun,HU Sheng,et al.Cloning,sequencing and expression of a glutamate decarboxylase gene from the GABA-producing strain Lactobacillus brevis CGMCC 1306[J].Ann Microbiol,2012,62(2):689-698.
• [9]HUANG Jun(黄俊),MEI Le-he(梅乐和),HU Sheng(胡升),et al.Optimization ofγ-aminobutyric acid liquid fermentation conditions and its fed-batch fermentation(γ-氨基丁酸液体发酵过程的条件优化及补料研究)[J].J Chem Eng of Chinese Univ(高校化学工程学报),2008,22(4):618-623.
• [10]Ueno Y,Hayakawa K,Takahashi S,et al.Purification and characterization of glutamate decarboxylase from Lactobacillus brevis IFO 12005[J].Biosci Biotechnol Biochem,1997,61(7):1168-1171.
• [11]Huang J,Mei L H,Xia J.Application of artificial neural network coupling particle swarm optimization algorithm to biocatalytic production of GABA[J].Biotechnol Bioeng,2007,96(5):924-931.
• [12]Kenneth J L,Thomas D S.Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△Ct method[J].Methods,2001,25(3):402-408.
• [13]Kim J Y,Lee M Y,Ji G E,et al.Production of gamma-aminobutyric acid in black raspberry juice during fermentation by Lactobacillus brevis GABA100[J].Int J Food Microbiol,2009,130(1):12-16.
• [14]Huang G D,Li C B,Cao Y S.Proteomic analysis of differentially expressed proteins in Lactobacillus brevis NCL912 under acid stress[J].FEMS Microbiol Lett,2011,318(2):177-182.
• [15]Cotter P D,Gahan C G,Hill C.A glutamate decarboxylase system protects Listeria monocytogenes in gastric fluid[J].Mol Microbiol,2001,40(2):465-475.
• [16]HUANG Gui-dong(黄桂东),LI Chao-bo(李超波),CAO Yu-sheng(曹郁生).Study on acid resistance of Lactobacillus brevis NCL912(短乳杆菌NCL912耐酸性研究)[J].F&FI(食品与发酵工业),2010,36(12):58-63.
• [17]Nomura M,Nakajima I,Fujita Y et al.Lactococcus lactis contains only one glutamate decarboxylase gene[J].Microbiology.1999,145(6):1375-1380.
• [18]Mazzoli R,Pessione E,Dufour M,et al.Glutamate-induced metabolic changes in Lactococcus lactis NCDO 2118 during GABA production:combined transcriptomic and proteomic analysis[J].Amino Acids,2010,39(3):727-737.
• [19]Jakobs C,Jaeken J,Gibson K M.Inherited disorders of GABA metabolism[J].J Inherit Metab Dis,1994,16(4):704-715.
• [20]Wong C G,Bottiglieri T,Snead O C 3rd.GABA,gamma-hydroxybutyric acid,and neurological disease[J].Ann Neurol,2003,54(Suppl 6):S3-S12.
• [21]Oh C H,Oh S H.Effects of germinated brown rice extracts with enhanced levels of GABA on cancer cell proliferation and apoptosis[J].J Med Food,2004,7(1):19-23.
• [22]Cotter P D,Hill C.Surviving the acid test:responses of gram-positive bacteria to low p H[J].Microbiol Mol Biol Rev,2003,67(3):429-453.