拮抗性芽孢杆菌的筛选及其抗菌肽的分离和性质分析

发布时间：2018-01-09 17:54

本文关键词：拮抗性芽孢杆菌的筛选及其抗菌肽的分离和性质分析　出处：《安徽工程大学》2017年硕士论文　论文类型：学位论文

【摘要】：抗菌肽是自然界中广泛存在的一种蛋白类抗生素。几乎所有种属的生物都能在一定程度上产生某种抗菌肽。由于该类物质对细菌、真菌等具有广谱的抑菌效果,一直是备受各领域的关注。近些年随着传统意义上的抗生素在使用过程中出现了某些问题,寻找一些抑菌机理不同的新型抗生素作为传统抗生素在实际应用中的补充已成为某些领域研究的主要任务之一,而抗菌肽由于具有其独特的抑菌性质因而成为研究的热点。除此之外,抗菌肽的某些药理作用(如抗病毒、抗肿瘤等)被发现,一度使得对该类物质的研究快速的向纵深发展。本研究从筛选得到的地衣芽孢杆菌B32中成功分离得到抗菌肽,并对其高产抗菌肽培养条件优化,确定最佳条件。同时对抗菌肽的分离纯化条件进行了摸索,并对其稳定性、结构、抑菌性等性质进行了深入研究。研究结果可为该拮抗性芽孢杆菌及抗菌肽在医疗、食品等领域的应用以及该物质的研究提供参考。本文主要研究结果如下:(1)利用金黄色葡萄球菌作为指示菌,成功从土壤中筛选出一株对指示菌具有显著拮抗性的菌株,命名为B32。通过菌落鉴定和分子生物学鉴定,明确该菌株为地衣芽孢杆菌。(2)通过碳源、氮源、无机盐及生长因子等培养基组分优化和培养条件优化,确定该地衣芽孢杆菌高产抗菌肽的最佳条件。结果显示,最佳培养基配方为:可溶性淀粉1.5%,混合氮源(豆粕粉:硫酸铵=5:1)3.5%,硫酸锰0.1%,碳酸钙0.2%,蛋氨酸0.025%。最佳培养条件为:温度35℃,pH为6.0,接种量2%。在此条件下培养36 h可获得抑菌效价最高达82.57 U/mL(对照氨苄青霉素钠)的发酵液。(3)利用有机溶剂对发酵液中的抗菌肽进行萃取,优化萃取条件,确定以正丁醇为有机萃取剂,控制V有机相/V发酵液为2:1、操作温度为45℃时,可获得最佳的萃取效益,萃取率达到44.51%左右;同时在反萃取实验中,影响其效果的主要因数为操作温度和反萃取操作次数,在操作温度为45℃、V有机相/V超纯水为1:1的条件下,单次反萃取为53.80%,二次反萃取率接近61.28%。利用高效液相色谱(流动相:乙腈/超纯水)对萃取所得制备样品进行分离,收集有抗菌活性的组分并进行纯度检测,成功获得纯度达90%左右的抗菌肽。(4)进一步对该抗菌肽的稳定性和抑菌性进行测定,发现其具有较强的稳定性,高温(大于100℃)、酸碱变化等外界因素均不能使其抗菌效价明显下降。利用某些蛋白酶(胃蛋白酶、胰蛋白酶、蛋白酶K)对该抗菌肽进行处理。可得出蛋白酶对该抗菌肽的影响并不一致,胰蛋白酶可使该物质结构被破坏。同时由抗菌实验结果可得,抗菌肽对某些革兰氏阳性致病菌(如金黄色葡萄球菌)具有较好的拮抗性,同时对部分真菌(如里氏木霉等)也有一定的抑制作用。(5)通过质谱手段鉴定抗菌肽氨基酸序列,并将其在地衣芽孢杆菌(NC_006270.3)基因组蛋白库中进行比对和分析,并利用分子生物学方法对其编码基因进行克隆,最终确定该抗菌肽的序列MAVKQKKEYLIRQLAKPGRLD NLKDLSSHKLDEVRQHQL。生物信息学分析结果显示该抗菌肽分子式为C203H345N63O58S1,相对分子量为4628.42Da,理论等电点为9.87,属于可溶性蛋白。
[Abstract]:Antibacterial peptide is a protein antibiotics widely exists in the nature. Almost all species of organisms can produce some antibacterial peptides in a certain extent. Because of such substances of bacteria, fungi and other broad-spectrum antibacterial effect, and have been paid attention in various fields. In recent years with the traditional sense of the emergence of some antibiotics the problems in the process of using, the search for new antibiotics different antibacterial mechanism of traditional antibiotics as a supplement in the practical application has become one of the main tasks of research in certain areas, and the antibacterial peptide has its unique antibacterial properties and thus has become a hot research topic. In addition, some pharmacological effects (such as antiviral, antibacterial peptides anticancer) was found, the study on this kind of material fast to develop in depth. This study from Bacillus licheniformis B32 screened successfully isolated Antibacterial peptide, antibacterial peptide and its high yield culture condition optimization, to determine the best conditions. At the same time the separation of antimicrobial peptide purification conditions were explored, and the stability of structure, such as antibacterial properties were studied. The research results can provide the Antagonistic Bacillus and antimicrobial peptides in health care, provide reference for the application of food field and study of the material. The main results of this paper are as follows: (1) using Staphylococcus aureus as indicator bacteria, screened a strain on the indicator bacteria has significant antagonistic strains from soil, named B32. through bacterial identification and molecular biology identification clear the strain of Bacillus licheniformis. (2) the carbon source, nitrogen source, medium component optimization and optimization of culture conditions for culture of inorganic salts and growth factors, determine the optimum conditions of the Bacillus licheniformis high yield of antimicrobial peptides. The results showed that the optimum medium formula : soluble starch 1.5%, mixed nitrogen sources (soybean powder: 3.5% =5:1 ammonium sulfate), manganese sulfate 0.1%, calcium carbonate 0.2% 0.025%., the optimum culture conditions for methionine: temperature 35, pH 6, H 36 can get antibacterial titer up to 82.57 U/mL 2%. cultured under these conditions (control of ampicillin the fermentation liquid of sodium). (3) for extraction of antimicrobial peptides in fermentation broth by organic solvent, optimizing the extraction conditions, determined using n-butanol as organic extractant, control V organic phase fermentation broth of /V 2:1, the operating temperature is 45 degrees centigrade, can obtain the best extraction efficiency, extraction rate reached about 44.51% at the same time; in the anti extraction experiment, the main factor affecting the operating temperature and stripping operation times, the operating temperature is 45 DEG C, V organic phase /V ultra pure water under the condition of 1:1, the single back extraction was 53.80%, two times the extraction rate is close to 61.28%. by using high performance liquid chromatography (flow Mobile phase: acetonitrile / ultra pure water) on extracted samples were collected from antibacterial active components and purity detection, successfully obtained purity of antibacterial peptides of about 90%. (4) for the further determination of stability and antibacterial activity of antibacterial peptide, its stability is strong, high temperature (greater than 100 DEG C), the external factors are not the acid-base changes antibacterial potency decreased significantly. The use of certain proteases (trypsin, pepsin, proteinase K) treatment of the antibacterial peptide can be obtained. The effects of protease on antimicrobial peptides is not consistent, trypsin can make the material structure was destroyed by antibiotics. At the same time the experimental results, the antibacterial peptide of some gram positive pathogens (Staphylococcus aureus) has better antagonistic, and some of the fungi (such as Trichoderma etc.) also inhibited (5) by means of mass spectrometry. Identification of antimicrobial peptides and its amino acid sequence in Bacillus licheniformis (NC_006270.3) comparison and analysis of the genome in the protein database, and using molecular biology method to clone the gene encoding, ultimately determine the antibacterial peptide sequence MAVKQKKEYLIRQLAKPGRLD NLKDLSSHKLDEVRQHQL. bioinformatics analysis results showed that the antibacterial peptide molecular formula C203H345N63O58S1, molecular weight 4628.42Da, isoelectric point was 9.87, belonging to the soluble protein.

【学位授予单位】：安徽工程大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q936

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