植物乳杆菌ZDY 2013的耐酸机制研究及其谷氨酸脱氢酶基因的克隆表达

发布时间：2019-04-30 18:30

【摘要】：植物乳杆菌(Lactobacillus plantarum)广泛存在于发酵的蔬菜和果汁中,在食品、饲料及医疗保健等领域受到高度关注。植物乳杆菌作为人体胃肠道中重要的益生菌群,为了有效发挥益生功能,必须耐受胃液中的极端酸性环境,以较高的存活率抵达人体肠道中。因此,乳酸菌的耐酸性成为选择益生菌的标准之一。本研究以植物乳杆菌ZDY 2013为实验菌株,植物乳杆菌ATCC 8014为对照菌株,利用生理生化测定的研究方法,分析比较两株植物乳杆菌在酸性环境中的生理耐受现象,探讨其机制。此外,借助基因工程的方法,构建谷氨酸脱氢酶原核表达载体,阐释谷氨酸脱氢酶对提高工程菌耐酸性能的作用,从而为提升植物乳杆菌抵御酸胁迫的能力提供有效策略。主要研究结果如下:1、从细胞生理学水平比较植物乳杆菌ZDY 2013和ATCC 8014在酸应激中的生理变化。扫描电镜观察发现,两株植物乳杆菌酸处理后均出现形态结构的变化,其中植物乳杆菌ZDY 2013在酸性环境中可以更好地维持细胞形态和细胞膜完整性,缓解酸应激对细胞产生的损伤。对植物乳杆菌胞内pH值测定结果表明,植物乳杆菌ZDY 2013在酸性环境中较ATCC 8014保持更高的胞内pH值。此外,植物乳杆菌ZDY 2013的Na+,K+-ATPase活性和胞内ATP含量在酸性环境中分别比ATCC 8014高出35%和41%。植物乳杆菌胞内氨基酸含量测定结果表明,植物乳杆菌ZDY 2013在酸环境中具有更高的胞内精氨酸、谷氨酸、丙氨酸和组氨酸含量。精氨酸脱亚胺酶和谷氨酸脱羧酶酶活测定结果表明,植物乳杆菌ZDY 2013在酸性环境中的精氨酸脱亚胺酶和谷氨酸脱羧酶活力分别是ATCC 8014的1.2和1.3倍。2、以植物乳杆菌ZDY 2013基因组DNA为模板,PCR扩增谷氨酸脱氢酶基因,连接到表达载体pET-32a(+)上,重组质粒转入大肠杆菌BL21(DE3)中,经IPTG诱导表达和镍柱亲和层析后获得目的蛋白,活性测定显示该蛋白具有谷氨酸脱氢酶的活性。同时,对表达菌株的酸耐受性测定结果表明,工程菌株在pH 4.5的存活率提高1.4倍。
[Abstract]:Lactobacillus botanica (Lactobacillus plantarum) is widely found in fermented vegetables and juices, and has attracted much attention in the fields of food, feed and health care. Lactobacillus botanica is an important probiotics group in human gastrointestinal tract. In order to play its probiotics function effectively, it is necessary to tolerate the extreme acidic environment in the gastric juice and reach the human intestinal tract with a higher survival rate. Therefore, acid tolerance of lactic acid bacteria has become one of the criteria for selection of probiotics. In this study, Lactobacillus botanica ZDY 2013 was used as the experimental strain and Lactobacillus botanica ATCC 8014 as the control strain. The physiological tolerance of two Lactobacillus botanica strains in acidic environment was analyzed and compared by means of physiological and biochemical methods, and the mechanism was discussed. In addition, the prokaryotic expression vector of glutamic dehydrogenase was constructed by means of genetic engineering, and the effect of glutamic dehydrogenase on improving acid tolerance of engineering bacteria was explained, thus providing an effective strategy for enhancing the ability of Lactobacillus botanica to resist acid stress. The main results are as follows: 1. The physiological changes of Lactobacillus botanica ZDY 2013 and ATCC 8014 in acid stress were compared in terms of cell physiology. Scanning electron microscopy (SEM) showed that the morphological and structural changes of two strains of Lactobacillus were observed after treatment with Lactobacillus acid, in which Lactobacillus botanica ZDY 2013 could maintain cell morphology and cell membrane integrity better in acidic environment. Relieves cell damage caused by acid stress. The results of intracellular pH measurement of Lactobacillus botanica showed that Lactobacillus botanica ZDY 2013 maintained a higher intracellular pH value than that of ATCC 8014 in acidic environment. In addition, the Na, K-ATPase activity and intracellular ATP content of Lactobacillus botanica ZDY 2013 in acidic environment were 35% and 41% higher than those of ATCC 8014, respectively. The results showed that Lactobacillus botanica ZDY 2013 had higher intracellular arginine, glutamic acid, alanine and histidine contents in acid environment. The activity of arginine decarboxylase and glutamic acid decarboxylase of Lactobacillus botanica ZDY 2013 in acidic environment was 1.2 and 1.32 times higher than that of ATCC 8014, respectively, the activity of arginine decarboxylase and glutamic acid decarboxylase of Lactobacillus botanica was 1.2 and 1.32 times higher than that of ATCC 8014, respectively. Glutamic acid dehydrogenase gene was amplified by PCR using genomic DNA of Lactobacillus botanica ZDY 2013 as template, and was ligated into expression vector pET-32a (). The recombinant plasmid was transformed into E. coli BL21 (DE3). The target protein was obtained by IPTG induced expression and Ni column affinity chromatography. The activity analysis showed that the protein had the activity of glutamic acid dehydrogenase. At the same time, the acid tolerance test of the expressed strain showed that the survival rate of the engineering strain in pH 4.5 was increased by 1.4 times.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q93;Q78

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