芽孢杆菌B1胞外活性物质的分离鉴定及溶藻机制的研究

发布时间：2018-03-01 11:39

本文关键词： 赤潮芽孢杆菌B1 溶藻活性物质分离与鉴定技术溶藻机制　出处：《暨南大学》2014年硕士论文　论文类型：学位论文

【摘要】：球形棕囊藻是我国珠江口海域典型的优势藻种之一，近年来爆发日益频繁，对我国经济和海洋生态环境造成了严重的威胁。采用物理法和化学法治理赤潮，由于受到成本和二次污染等因素制约，经济、高效、环保的生物法治理赤潮尤其以菌控藻已成为赤潮治理领域的一个新的研究热点。基于课题组已从珠海球形棕囊藻赤潮爆发的海域中分离筛选得到一株溶藻芽孢杆菌B1，本论文对B1菌的溶藻方式及溶藻效果，溶藻活性物质的分离鉴定及溶藻机制进行了研究。主要结果如下：（1）B1菌是通过分泌胞外活性物质的间接方式对球形棕囊藻、中肋骨条藻、东海原甲藻及赤潮异弯藻起溶藻作用，，溶藻效果与B1无菌滤液的投加量成正相关。当B1无菌滤液的投加量为4%（V无菌滤液/V藻液=1/25）时，对四种赤潮藻的溶藻效果是：球形棕囊藻＞中肋骨条藻＞东海原甲藻＞赤潮异弯藻，除藻率分别为：92.3%、91.8%、89.4%和84.4%。（2）采用ODS-A柱层析法和HPLC对B1菌分泌的胞外活性物质进行分离纯化，最终得到四种对球形棕囊藻除藻率分别为46.9%、50.3%、55.0%和55.7%的活性物质。对这四种活性物质进行Q-TOF-MS测定，采用PeakViewTM软件分析Q-TOF-MS测得的数据，推断出溶藻活性物质分别为L-组氨酸、o-酪氨酸、N-乙酰组胺和咪唑丙烯酸。（3）B1菌分泌的胞外活性物质经硅胶柱层析、Sephadex G-15柱层析和HPLC分离纯化，得到了两种除藻率为59.5%和54.6%的溶藻活性物质。采用Q-TOF-MS和PeakViewTM软件对其进行结构测定和分析，发现这两种溶藻活性物质为脯氨酰蛋氨酸和次黄嘌呤。（4）对已鉴定出的L-组氨酸等六种溶藻活性物质进行了溶藻实验，结果显示L-组氨酸仅对球形棕囊藻有溶藻效果，而o-酪氨酸、N-乙酰组胺、咪唑丙烯酸、脯氨酰蛋氨酸和次黄嘌呤对球形棕囊藻、东海原甲藻、赤潮异弯藻和中肋骨条藻均有溶藻效果，说明B1菌分泌的胞外活性物质具有较好的广谱杀藻性。（5）溶藻机理研究表明，溶藻活性物质通过抑制藻体内超氧化物歧化酶（SOD）和过氧化氢酶（CAT）的活性，致使藻体内有害活性氧自由基（ROS）不断积累，丙二醛（MDA）含量不断上升，细胞膜脂质过氧化程度加剧，最终导致细胞膜系统和功能的破坏，细胞因溶解而死亡。即芽孢杆菌B1分泌的胞外活性物质是通过扰乱细胞抗氧化系统来抑制藻类的生长繁殖。
[Abstract]:Phaeocystis globosa is one of the typical dominant algae species in the Pearl River Estuary in China. It has become more and more frequent in recent years, which poses a serious threat to the economic and marine ecological environment of our country. The physical and chemical methods are used to control the red tide. Because of the restriction of cost and secondary pollution, the biological control of red tide, especially algae control by bacteria, has become a new research hotspot in the field of red tide control, which is economical, efficient and environmentally friendly. Based on the isolation and screening of a strain of Bacillus alginolyticus B1 from the sea area of red tide outbreak of Phaeocystis globosa in Zhuhai by our research group, the way of algae dissolution and the effect of algae dissolution on B1 bacteria were studied in this paper. The separation and identification of algae-soluble active substances and the mechanism of algolysis were studied. The main results were as follows:. Phaeocystis spp. 1 is an indirect way of secreting extracellular active substances, which dissolves the algae of Phaeocystis globosa, Ribella costatum, Prorocentrum of the East China Sea and Heteropodium of the Red Tide. The effect of algae dissolution was positively correlated with the amount of aseptic filtrate B1. When the dosage of aseptic filtrate B1 was 4V aseptic filtrate / / V algal fluid / / 25), The effect of algae dissolution on four species of red tide algae was as follows: Phaeocystis globosa > Chlorophyta costatum > Prorocentrum sinensis > Chlorophyta albicans, the removal rates of which were 89.4% and 84.4%, respectively. (2) the extracellular active substances secreted by B1 were separated and purified by ODS-A column chromatography and HPLC. Finally, four bioactive substances were obtained, which were 46.9% and 55.0% and 55.7% respectively. The four active substances were determined by Q-TOF-MS. The data obtained from Q-TOF-MS were analyzed by PeakViewTM software. The results showed that the active substances of algae-lytic acid were L-histidine N-acetyl histamine and imidazole-acrylic acid respectively. The extracellular active substances secreted by the bacteria were separated and purified by silica gel column chromatography Sephadex G-15 column chromatography and HPLC. Two algae-soluble active substances with removal rates of 59.5% and 54.6% were obtained, and their structures were determined and analyzed by Q-TOF-MS and PeakViewTM software. Prolyl methionine and Hypoxanthine were found to be the two active substances. The results showed that L-histidine only had algaecitic effect on Phaeocystis globosa, but otyrosine N-acetyl histamine, imidazolyl acrylic acid, and so on. The results showed that L-histidine had only algaecitic effect on Phaeocystis globosa, but otyrosine N-acetyl histamine, imidazolyl acrylic acid, and so on. Prolyl methionine and Hypoxanthine could dissolve the algae against Phaeocystis sphaeroides, Prorocentrum East China Sea, Heterophyllum Red Tide and Streptococcus costatum, which indicated that the extracellular active substances secreted by B1 had good broad-spectrum algicide. Studies on the mechanism of algolysis showed that by inhibiting the activities of superoxide dismutase (SOD) and catalase (CAT) in algae, the harmful active oxygen species (Ros) and the content of malondialdehyde (MDAs) in algae were continuously accumulated, and the contents of malondialdehyde (MDAs) were increased. The degree of lipid peroxidation of the cell membrane is intensified, resulting in the destruction of the cell membrane system and function, and the cell death due to dissolution. That is, the extracellular active substance secreted by Bacillus spp B1 inhibits the growth and reproduction of algae by disrupting the antioxidant system of the cell.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：X172;X55

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