【作者】 李国强；

【导师】 田黎；

【作者基本信息】 青岛科技大学 ， 生物工程， 2016， 硕士

【摘要】 环肽类化合物是微生物产生的重要活性物质的一大类,具有广泛的生理活性和研究价值,而芽孢杆菌是该类化合物的重要来源。本文对实验室保存的分离自海洋具有抗茄链格孢菌活性的芽孢杆菌进行产环肽类化合物产量筛选;利用紫外线和亚硝酸两种方式对筛选出的产环肽类化合物菌株进行诱变处理,通过小样发酵以及稳定性测试,获得高产稳定菌株,菌株发酵条件通过正交试验进行了优化。(1)芽孢杆菌是重要的农药活菌制剂资源菌,其在植物体表现的抑菌活性除了与本身的抑菌作用有关外,与产生的各种酶活性也有一定的关联,而酶活性往往与其代谢密切相关,试验发现酶活性与菌株活性与目标化合物的产量成正相关,得到了3株有较高酶活和抑菌活性的菌株BL10-02、BS10-15和BS10-19。(2)本实验对来自海洋的菌株进行分离纯化获得单菌落的菌株,菌株发酵液进行环肽产量测试和抑菌活性测试,得到了2株活性较高环肽产量较高的菌株BL10-02,BM10-01。BL10-02经紫外和亚硝酸诱变后获得17株高活性菌株,其中7株菌对真菌茄链格孢菌孢子萌发抑制效果提高了4倍;BM10-01同样经紫外和亚硝酸诱变后,获得5株抑菌活性提高了2倍的菌株。BL10-02的诱变菌株BL10-02-7环肽产量比出发菌株产量提高了49%。(3)BL10-02-7该菌株通过培养基条件进行优化,环肽类化合物产量比原始菌株提高了57%。对该突变株的培养条件初步优化结果表明,该菌的发酵培养基的最优配方为:蛋白胨7g/L,酵母粉4g/L,葡萄糖0.5 g/L,NaCl 30g/L,MgCl2·6H2O 2.3g/L,KCl 0.3g/L,FePO4 0.02g/L,pH7.0,最适培养温度25℃。(4)本实验通过收集菌株B9987的4种不同形态的菌落,研究其形态与生理特性及环肽产量的关系。四种菌落形态相互比较得出,分散光滑的菌株在传代稳定和环肽产量方面有较明显的优势,同时有较高的抑菌活性,对于以后菌种选育提供一定的帮助。更多还原

【Abstract】 Cyclic peptides are important substances produced by microbes, with a wide range of physiological activity and research value. Many of them are derived from bacillus. In this paper, bacillus producing cyclic peptide compounds derived from marine and this strains may be screened by cyclic peptide production.These strains can be mutagenic ultraviolet and nitrite to get the strains producing high cyclic peptide,and strain fermentation conditions were optimized by orthogonal experiment.(1)Bacillus is important pesticide formulations of viable resource bacteria, antibacterial activity at its plant in addition to the performance itself inhibitory effect related, and produce a variety of activity also have some relevance, and its metabolic activity is often closely related activity and yield was found that the activity of strains of the target compound is positively correlated。Got three strains(BL10-02、BS10-15 and BS10-19)have high activity and antibacterial activity.(2)The experimental strains were isolated and purified from the ocean to obtain single colonies of strain, fermentation liquid was tested in cyclic peptide production and antibacterial activity to get two strains(BL10-02 and BM10-01)having high cyclic peptide production and high antibacterial activity.The strain of BL10-02 was mutagenized by UV and nitrous acid treatment to get 17 high activated strains.Among them 7 strians bacteria Alternaria solani fungus spore germination inhibiting effect by the 7 strains from the 17 high activated increased four-fold.BM10-01 same after UV mutagenesis and nitrous acid mutagenesis, get 5 inhibitory activity was increased 2-fold strain.Select BL10-02 high activity strain cyclic peptide production tests, cyclic peptide production by up to more than the original strain is BL10-02-7 production increased 49%。(3)After the culture conditions were optimized,the results showed that the mutant strain BL10-02-7was stable and the cyclic peptide yield of which increased by 57% than the original strain. Preliminary optimization of culture conditions of the mutant showed that the optimal fermentation medium included peptone(7g/L), yeast extract(4g/L), glucose(0.5 g/L), Na Cl(30g/L), Mg Cl2·6H2O(2.3g/L), KCl(0.3g/L), Fe PO4(0.02g/L), the appropriate p H was 7.0, the optimal incubation temperature was 25 ℃.(4)In this study, four different forms colonies are gathered to study the relationship between morphological and physiological characteristics and yield of cyclic peptides.The results showed that dispersed smooth strains subculture in stability and cyclic peptide production aspects obvious advantage and havea higher antibacterial activity, for future breeding strains provide some help.更多还原