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
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摘要
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 of γ-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 p GEM-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 m RNA 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.
The gene encoding glutamate decarboxylase(GAD), a key enzyme for the biosynthesis of γ-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 p GEM-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 m RNA 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.
引文
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[24]张祥,方卉,谢东芳,等.低H+-ATPase活性植物乳杆菌突变菌筛选及基因表达的相对定量分析[J].微生物学报,2017,57(2):293-303.DOI:10.13343/j.cnki.wsxb.20160304.
[25]DIANA M,TRES A,QUILEZ J,et al.Spanish cheese screening and selection of lactic acid bacteria with high gamma-aminobutyric acid production[J].LWT-Food Science and Technology,2014,56(2):351-355.DOI:10.1016/j.lwt.2013.11.027.
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[27]郁凯.以计算机辅助分子设计方法改造谷氨酸脱羧酶的研究[D].杭州:浙江大学,2013.
[28]NOLAN T,HANDS R E,BUSTIN S A.Quantification of m RNA using real-time RT-PCR[J].Nature Protocols,2006,1(3):1559-1562.DOI:10.1038/nprot.2006.236.
[29]田灵芝.高效转化生产γ-氨基丁酸重组大肠杆菌构建及其条件优化[D].无锡:江南大学,2012.
[30]LI H,CAO Y.Lactic acid bacterial cell factories for gammaaminobutyric acid[J].Amino Acids,2010,39(5):1107-1111.DOI:10.1007/s10035-005-0215-0.
[2]林亲录,王婧,陈海军.γ-氨基丁酸的研究进展[J].现代食品科技,2008,24(5):496-500.DOI:10.3969/j.issn.1673-9078.2008.05.028.
[3]曾林,刘波,许小艳,等.四川泡菜中产γ-氨基丁酸微生物的系统发育与表达能力评估[J].食品科学,2017,38(2):87-91.DOI:10.7506/spkx1002-6630-201702014.
[4]SANCHART C,RATTANAPOM O,HALTRICH D,et al.Technological and safety properties of newly isolated GABAproducing Lactobacillus futsaii strains[J].Journal of Applied Microbiology,2016,121(3):734-745.DOI:10.1111/jam.13168.
[5]VO T D L,KIM T W,HONG S H.Effects of glutamate decarboxylase and gamma-aminobutyric acid(GABA)transporter on the bioconversion of GABA in engineered Escherichia coli[J].Bioprocess and Biosystems Engineering,2012,35(4):645-648.DOI:10.1007/s00449-011-0634-8.
[6]BANCHUEN J,THAMMANRUTWASIK P,OORAIKUL B,et al.Increasing the bio-active compounds contents by optimizing the germination conditions of Southern Thai Brown Rice[J].Songklanakarin Journal of Science&Technology,2010,32(3):174-176.
[7]BHANWAR S,BAMNIA M,GHOSH M,et al.Use of Lactococcus lactis to enrich sourdough bread withγ-aminobutyric acid[J].International Journal of Food Sciences&Nutrition,2013,64(1):77-81.DOI:10.3109/09637486.2012.700919.
[8]CHO Y R,CHANG J Y,CHANG H C.Production of gammaaminobutyric acid(GABA)by Lactobacillus buchneri isolated from kimchi and its neuroprotective effect on neuronal cells[J].Journal of Microbiology&Biotechnology,2007,17(1):104.
[9]田灵芝,徐美娟,饶志明.一株重组大肠杆菌/p ET-28a-lpgad的构建及其高效生产γ-氨基丁酸转化条件的优化[J].生物工程学报,2012,28(1):65-75.
[10]乌云达来,张博润,郭雪娜,等.生物合成γ-氨基丁酸酿酒酵母谷氨酸脱羧酶基因的克隆与表达[J].食品科学,2015,36(13):131-136.DOI:10.7506/spkx1002-6630-201513025.
[11]陈琳,张充,吕凤霞,等.重组谷氨酸脱羧酶大肠杆菌合成γ-氨基丁酸条件的优化[J].食品科学,2015,36(1):158-163.DOI:10.7506/spkx1002-6630-201501030.
[12]黄燕,宿玲恰,吴敬,等.重组谷氨酸脱羧酶制备γ-氨基丁酸的工艺条件优化[J].生物技术通报,2016,32(6):199-204.DOI:10.13560/j.cnki.biotech.bull.1985.2016.06.029.
[13]SHI F,LI Y.Synthesis ofγ-aminobutyric acid by expressingLactobacillus brevis-derived glutamate decarboxylase in the Corynebacterium glutamicum strain ATCC 13032[J].Biotechnology Letters,2011,33(12):2469-2474.DOI:10.1007/s10529-011-0723-4.
[14]张天萌.谷氨酸脱羧酶的克隆表达及酶学性质研究[D].无锡:江南大学,2012.
[15]KOMATSUZAKI N,NAKAMURA T,KIMURA T,et al.Characterization of glutamate decarboxylase from a high gammaaminobutyric acid(GABA)-producer Lactobacillus paracasei[J].Bioscience Biotechnology&Biochemistry,2008,72(2):278.DOI:10.1271/bbb.70163.
[16]HUANG J,MEI L,SHENG Q,et al.Purification and characterization of glutamate decarboxylase of Lactobacillus brevis CGMCC 1306isolated from fresh milk[J].Chinese Journal of Chemical Engineering,2007,15(2):157-161.DOI:10.1016/S1004-9541(07)60051-2.
[17]PARK K,OH S.Cloning,sequencing and expression of a novel glutamate decarboxylase gene from a newly isolated lactic acid bacterium,Lactobacillus brevis OPK-3[J].Bioresource Technology,2007,98(2):312.DOI:10.1016/j.biortech.2006.01.004.
[18]李杰.生物合成γ-氨基丁酸酵母菌谷氨酸脱羧酶基因的克隆及表达[D].金华:浙江师范大学,2009.
[19]SOMASUNDARAM S,TRAN K N T,RAVIKUMAR S,et al.Introduction of synthetic protein complex between Pyrococcus horikoshii glutamate decarboxylase and Escherichia coli GABA transporter for the improved production of GABA[J].Biochemical Engineering Journal,2017,120:1-6.DOI:10.1016/j.bej.2016.12.020.
[20]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.DOI:10.1007/s00726-010-0507-5.
[21]李理,满朝新,刘少敏,等.植物乳杆菌(Lactobacillus plantarum)NDC75017产γ-氨基丁酸的影响因素及其代谢机制[J].食品科学,2015,36(7):84-89.DOI:10.7506/spkx1002-6630-201507016.
[22]齐育平,孙蕾,刘琴英,等.短乳杆菌谷氨酸脱羧酶的生物信息学分析[J].微生物学杂志,2014,34(1):5-11.DOI:10.3969/j.issn.1005-7021.2014.01.002.
[23]FAN E,HUANG J,HU S,et al.Cloning,sequencing and expression of a glutamate decarboxylase gene from the GABA producing strain Lactobacillus brevis CGMCC 1306[J].Annals of Microbiology,2012,62(2):689-698.DOI:10.1007/s13213-011-0307-5.
[24]张祥,方卉,谢东芳,等.低H+-ATPase活性植物乳杆菌突变菌筛选及基因表达的相对定量分析[J].微生物学报,2017,57(2):293-303.DOI:10.13343/j.cnki.wsxb.20160304.
[25]DIANA M,TRES A,QUILEZ J,et al.Spanish cheese screening and selection of lactic acid bacteria with high gamma-aminobutyric acid production[J].LWT-Food Science and Technology,2014,56(2):351-355.DOI:10.1016/j.lwt.2013.11.027.
[26]GRONE B P,MARUSKA K P.Three distinct glutamate decarboxylase genes in vertebrates[J].Scientific Reports,2016,6:30507.DOI:10.1038/srep30507.
[27]郁凯.以计算机辅助分子设计方法改造谷氨酸脱羧酶的研究[D].杭州:浙江大学,2013.
[28]NOLAN T,HANDS R E,BUSTIN S A.Quantification of m RNA using real-time RT-PCR[J].Nature Protocols,2006,1(3):1559-1562.DOI:10.1038/nprot.2006.236.
[29]田灵芝.高效转化生产γ-氨基丁酸重组大肠杆菌构建及其条件优化[D].无锡:江南大学,2012.
[30]LI H,CAO Y.Lactic acid bacterial cell factories for gammaaminobutyric acid[J].Amino Acids,2010,39(5):1107-1111.DOI:10.1007/s10035-005-0215-0.