【作者】 张莉；

【导师】 陆兆新；

【作者基本信息】 南京农业大学 ， 发酵工程， 2013， 硕士

【摘要】 芽孢杆菌能产生多种非核糖体合成的抗菌脂肽,具有很强的广谱抗菌作用,因而在食品、医药、环保、畜牧、石油开采等领域有良好的应用潜力。其中以表面活性素(surfactin)研究最为深入。Surfactin分子量约为1000Da,是目前公认最好的生物表面活性剂,具有良好的乳化和发泡性能。芽孢杆菌发酵产抗菌脂肽surfacitn的产量通常较低,一般低于1.0g/L,无法满足工业生产的需要,因此提高脂肽的产量尤为重要。基因组改组(Genome shuffling)作为近年来发展起来的微生物育种新技术,是最重要也最为有效的增产手段。本研究以本实验室保藏枯草芽孢杆菌(Bacillus subtilis) BS1为出发菌株,采用多种选育方式提高其产surfactin的能力,以期得到性质稳定的高产菌株,为今后该领域的分子生物学研究及抗菌脂肽.surfactin的工业化生产奠定基础。主要研究结果如下：1.通过理化诱变选育枯草芽孢杆菌(Bacillus subtilis) BS1surfactin高产菌株。分别采用紫外线、60Co γ射线、NTG对枯草芽孢杆菌(Bacillus subtilis) BS1进行诱变处理。通过致死率及突变率的统计,确定紫外诱变的条件为：紫外灯功率20w,垂直照射距离15cm,照射时间240s；60Co γ射线辐照剂量300Gy; NTG终浓度0.4mg/mL,37℃保温处理30min。经抑菌实验初筛、摇瓶发酵复筛后获得高产菌株11株,其中菌株UV21surfactin产量较出发菌株提高54%。2.通过复合诱变选育枯草芽孢杆菌(Bacillus subtilis)BS1surfactin高产菌株。先通过单因素诱变确定链霉素、硫酸阿米卡星、青霉素三种抗生素的作用剂量,再将LiCl溶液和种子液1：1混合,30℃保温处理30min,吸取1mL处理液与NTG溶液1：1混合,30℃保温处理30min后涂布在三种抗生素平板上,进行初筛和复筛。通过致死率及突变率的统计,确定三种抗生素的最适诱变浓度为：链霉素2.0mg/L；硫酸阿米卡星40.0U/L；青霉素钠3.0U/L。LiCl溶液浓度30%；NTG终浓度0.4mg/mL。经抑菌实验初筛、摇瓶发酵复筛后获得高产菌株14株,其中菌株PNG27surfactin产量较出发菌株提高63.2%。经过诱变育种共选育出surfactin产量提高40%以的菌株25株,选择其中的UV21、NTG14、SM182、PNG27作为电融合实验的亲本菌株。3.对枯草芽孢杆菌(Bacillus subtilis) BS1进行原生质体制备、再生及灭活条件的研究。原生质体制备的最佳条件为：对数期种子液二次转接后培养5h收集菌体,以0.2mg/mL溶菌酶37℃酶解15min,原生质体形成率达98%。优化再生培养基,优化后mol/L丁二酸钠作为培养基的高渗稳定剂,原生质体再生率达到70%。原生质体热灭活和紫外灭活的最佳条件分别为：100℃沸水浴,煮沸20min；紫外灯管30w,照射距离5cm,照射时间20min。4.分别采用化学试剂融合和电融合的方法进行多亲本原生质体递推融合,采用亲本原生质体灭活的方法检出合子。PEG结合Ca2+诱导原生质体融合的最适条件为：pH9.0,37℃条件下,40%PEG6000融合5min,融合率为3.36×10-6,化学试剂融合选用8株诱变菌株作为融合亲本,但未能筛选到稳定融合子。电融合的参数为：脉冲强度9KV/cm,脉冲宽度50us,脉冲个数2个,融合率为8.51×10-5,是化学融合法的25.3倍。电融合选用UV21、NTG14、SM182、PNG27及原始菌株BS1作为融合亲本,经过两轮基因组改组,获得遗传性能稳定的高产突变株F2-174,surfactin产量是原始菌株的2.24倍。更多还原

【Abstract】 Bacillus strains produce kinds of bioactive lipopeptides with broad-spectrum antibacterial function.that are not ribosomally synthesized. Thus they have well-recognized potential applications in areas such as food, medicine, environmental protection, animal husbandry and oil exploration. Among these compounds, the lipopeptide surfactin is well characterized. It is currently recognized as the best bio-surfactant with excellent emulsifying and foaming properties.The production of antimicrobial lipopeptides in Bacillus strains is generally lower than1.0g/L, which can hardly meet the needs of industrial production, thereby increasing production of antimicrobial lipopeptides is particularly important. Genome shuffling is the most important and most effective way to increase production of secondary metabolites.The research chose Bacillus subtilis BS1as the original strain, using a variety of selection ways to improve its production of surfactin, in order to receive stable high-yielding strains to lay foundation for the research of molecular biology of this field and the industrialization of lipopeptide production. Main results of the study are as follows:1. Breeding high-yielding strains of surfactin by physical and chemical mutagens. BS1cells were mutagenized with ultraviolet irradiation,60Co gama-ray irradiation or nitrosoguanidine. Through the measurement of the death rate and mutation rate, determining the following conditions of mutations:UV lamp power20w, veitical distance15cm, irradiation time240s;60Coγ-ray irradiation dose300Gy;0.4mg/ml NTG,37℃for30minutes. After preliminary screening and shake flask fermentation screening,11high-yielding strains were screened, and the maximum mutation rate of UV21reached up to54%。2. Breeding high-yielding strains of surfactin by compound mutation. At first the doses of three antibiotics (streptomycin, amikacin sulfate and penicillin sodium) were determined through single-factor mutagenesis, and then mixed the LiCl solution with seeds solution at1:1, keeped in30℃water for30minutes. Then absorbed1.0mL handling liquid and mixed it with NTG solution at1:1, keeped in30℃for another30minutes. Coat the mixture on the surface of the tablets mixing different types of antibiotics for screening. Through the measurement of the death rate and mutation rate, determining the following doses of the three antibiotics:streptomycin2.0mg/L; amikacin sulfate40.0U/L; penicillin sodium3.0U/L. LiCl solution concentration30%; NTG solution0.4mg/mL. After preliminary screening and shake flask fermentation screening,14high-yielding strains were screened, and the maximum mutation rate of PNG27reached up to63.2%。 Through mutation breeding,25high-yielding strains were selected, and we chose UV21、 NTG14、SM182、 PNG27as the parental strains of electric fusion。3. The conditions of Bacillus subtilis BS1protoplast preparation, regeneration and inactivation were studied. The optimized conditions for protoplast preparation were as follows:seeds solution cultured for5h to collect cells,0.2mg/mL lysozyme enzyme at37℃for15minutes to allow cell wall lysis, and the protoplast formation rate reached98%. Through optimization of regeneration medium,with0.1mol/L sodium succinate as medium osmotic stabilizer, protoplast regeneration rate reached up to70%. The best comditions of UV-inactivated and heat-inactivated were:100℃boiling water for20minutes;UV lamp power30w, veitical distance5cm, irradiation time20minutes.4. Using chemical reagents fusion method and electric fusion method respectively for multi-parental protoplast fusion, inactivated the parental protoplasts to detect regenerated protoplasts. The optimum conditions of chemical reagents fusion were:both inactivated protoplasts were fused by40%PEG6000, at pH9.0and37℃for5minutes. The fusion rate was3.36×10-6. Chemical reagents fusion chose8strains as parental strains, but failed to receive stable strains. The parameters of electric fusion were:pulse intensity9KV/cm, pulse width50us,2of the number of pulses. The fusion rate was8.51×10-5, which exhibited25.3-fold increases than chemical reagents fusion. Electric fusion chose UV21、 NTG14. SM182、PNG27and the original strain BS1as parental strains, after two rounds of genome shuffling, a high-yielding recombinant F2-174was obtained, the surfactin yield is2.24times that of the original strain BS1.更多还原