發(fā)布時(shí)間：2018-01-09 17:54

  本文關(guān)鍵詞：拮抗性芽孢桿菌的篩選及其抗菌肽的分離和性質(zhì)分析　出處：《安徽工程大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 地衣芽孢桿菌 抗菌肽 培養(yǎng)優(yōu)化 分離純化 抗菌性質(zhì)

【摘要】：抗菌肽是自然界中廣泛存在的一種蛋白類(lèi)抗生素。幾乎所有種屬的生物都能在一定程度上產(chǎn)生某種抗菌肽。由于該類(lèi)物質(zhì)對(duì)細(xì)菌、真菌等具有廣譜的抑菌效果,一直是備受各領(lǐng)域的關(guān)注。近些年隨著傳統(tǒng)意義上的抗生素在使用過(guò)程中出現(xiàn)了某些問(wèn)題,尋找一些抑菌機(jī)理不同的新型抗生素作為傳統(tǒng)抗生素在實(shí)際應(yīng)用中的補(bǔ)充已成為某些領(lǐng)域研究的主要任務(wù)之一,而抗菌肽由于具有其獨(dú)特的抑菌性質(zhì)因而成為研究的熱點(diǎn)。除此之外,抗菌肽的某些藥理作用(如抗病毒、抗腫瘤等)被發(fā)現(xiàn),一度使得對(duì)該類(lèi)物質(zhì)的研究快速的向縱深發(fā)展。本研究從篩選得到的地衣芽孢桿菌B32中成功分離得到抗菌肽,并對(duì)其高產(chǎn)抗菌肽培養(yǎng)條件優(yōu)化,確定最佳條件。同時(shí)對(duì)抗菌肽的分離純化條件進(jìn)行了摸索,并對(duì)其穩(wěn)定性、結(jié)構(gòu)、抑菌性等性質(zhì)進(jìn)行了深入研究。研究結(jié)果可為該拮抗性芽孢桿菌及抗菌肽在醫(yī)療、食品等領(lǐng)域的應(yīng)用以及該物質(zhì)的研究提供參考。本文主要研究結(jié)果如下:(1)利用金黃色葡萄球菌作為指示菌,成功從土壤中篩選出一株對(duì)指示菌具有顯著拮抗性的菌株,命名為B32。通過(guò)菌落鑒定和分子生物學(xué)鑒定,明確該菌株為地衣芽孢桿菌。(2)通過(guò)碳源、氮源、無(wú)機(jī)鹽及生長(zhǎng)因子等培養(yǎng)基組分優(yōu)化和培養(yǎng)條件優(yōu)化,確定該地衣芽孢桿菌高產(chǎn)抗菌肽的最佳條件。結(jié)果顯示,最佳培養(yǎng)基配方為:可溶性淀粉1.5%,混合氮源(豆粕粉:硫酸銨=5:1)3.5%,硫酸錳0.1%,碳酸鈣0.2%,蛋氨酸0.025%。最佳培養(yǎng)條件為:溫度35℃,pH為6.0,接種量2%。在此條件下培養(yǎng)36 h可獲得抑菌效價(jià)最高達(dá)82.57 U/mL(對(duì)照氨芐青霉素鈉)的發(fā)酵液。(3)利用有機(jī)溶劑對(duì)發(fā)酵液中的抗菌肽進(jìn)行萃取,優(yōu)化萃取條件,確定以正丁醇為有機(jī)萃取劑,控制V有機(jī)相/V發(fā)酵液為2:1、操作溫度為45℃時(shí),可獲得最佳的萃取效益,萃取率達(dá)到44.51%左右;同時(shí)在反萃取實(shí)驗(yàn)中,影響其效果的主要因數(shù)為操作溫度和反萃取操作次數(shù),在操作溫度為45℃、V有機(jī)相/V超純水為1:1的條件下,單次反萃取為53.80%,二次反萃取率接近61.28%。利用高效液相色譜(流動(dòng)相:乙腈/超純水)對(duì)萃取所得制備樣品進(jìn)行分離,收集有抗菌活性的組分并進(jìn)行純度檢測(cè),成功獲得純度達(dá)90%左右的抗菌肽。(4)進(jìn)一步對(duì)該抗菌肽的穩(wěn)定性和抑菌性進(jìn)行測(cè)定,發(fā)現(xiàn)其具有較強(qiáng)的穩(wěn)定性,高溫(大于100℃)、酸堿變化等外界因素均不能使其抗菌效價(jià)明顯下降。利用某些蛋白酶(胃蛋白酶、胰蛋白酶、蛋白酶K)對(duì)該抗菌肽進(jìn)行處理�？傻贸龅鞍酌笇�(duì)該抗菌肽的影響并不一致,胰蛋白酶可使該物質(zhì)結(jié)構(gòu)被破壞。同時(shí)由抗菌實(shí)驗(yàn)結(jié)果可得,抗菌肽對(duì)某些革蘭氏陽(yáng)性致病菌(如金黃色葡萄球菌)具有較好的拮抗性,同時(shí)對(duì)部分真菌(如里氏木霉等)也有一定的抑制作用。(5)通過(guò)質(zhì)譜手段鑒定抗菌肽氨基酸序列,并將其在地衣芽孢桿菌(NC_006270.3)基因組蛋白庫(kù)中進(jìn)行比對(duì)和分析,并利用分子生物學(xué)方法對(duì)其編碼基因進(jìn)行克隆,最終確定該抗菌肽的序列MAVKQKKEYLIRQLAKPGRLD NLKDLSSHKLDEVRQHQL。生物信息學(xué)分析結(jié)果顯示該抗菌肽分子式為C203H345N63O58S1,相對(duì)分子量為4628.42Da,理論等電點(diǎn)為9.87,屬于可溶性蛋白。
[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.

【學(xué)位授予單位】：安徽工程大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：Q936

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本文編號(hào)：1402036