【作者】 张灿；

【导师】 金建玲；

【作者基本信息】 山东大学 ， 微生物学， 2016， 硕士

【摘要】 真菌感染在临床上波及范围广,常用的抗真菌药物有多烯大环内酯类、唑类、棘球白素类、丙烯胺类、嘧啶类,每种药物均存在一定的毒副作用,但研究发现多烯大环内酯类类相对于其它种类药物来说其毒副作用相对较低,同时也是临床上治疗真菌感染的最后用药。但长期大量使用同样具有导致肾功能衰竭和血栓性静脉炎的毒性,因此寻找更为低毒的抗生素显得尤为重要。纺锤链霉菌(Streptomycese netropsis) SD-07是本实验室2007年发现的一株新型菌株,其所产抗生素为多烯大环内酯类抗生素,相比两性霉素B,它具有更强的抗真菌活性。SD-07具有两种高产突变株163和180,为验证三种菌株经过5年传代后菌株是否仍高产,抗生素结构及活性是否有变化,本论文对三种菌株所产抗生素进行产量和活性的比较,同时对不同的纯化方法进行了活性、稳定性和毒性比较。本实验室在对链霉菌SD-07接合转移体系的建立过程中发现Ca2+能够极大地增加接合转移的频率,电镜下观察链霉菌SD-07菌体发现当Ca2+存在时其胞外多糖的产生量明显比不添加Ca2+时多。为更好的阐明胞外多糖与Ca2+以及接合转移之间的关系,本论文同时对SD-07胞外多糖的发酵和结构解析进行了相关研究。研究结果如下：1.抗生素相关研究结果纺锤链霉菌SD-07的180突变株是抗生素高产菌株,163菌株次之,相对于原始菌株SD-07,180抗生素产量是其3.33倍,163菌株是其1.28倍。高压液相(High Performance Liquid Chromatography, HPLC)。液相-质谱分析(liquid chromatography-mass spectrum, LC-MS)和抑菌实验结果显示三种抗生素主要成分之间并无大的差别。不同的制备方法表明萃取法所制备的抗生素活性最为稳定,分子筛所制备的抗生素最不稳定。相隔三个月的抑菌性实验证明菌株180所产的抗生素粗品活性最高,随着时间的延长,抑菌活性增加,推测可能与溶解性有关。就细胞毒性试验来讲中压液相层析(Fast Pressure Liquid Chromatography, FPLC)是一种提纯抗生素、降低毒性并提高抑菌性的好方法。SOS/umu-1acZ体系的致突变性检测结果表明链霉菌所产生抗生素的体外致突变性为阴性,并与突变株类型和制备方法无关。2.胞外多糖相关研究结果胞外多糖产量最高时所用发酵时间为48h,培养基为R2YE,Ca2+对多糖的产生有明显的促进作用,Cu2+的抑制作用最为明显,K+、Na+多糖产生几乎没有影响,Mg2+、Fe3+、Mn2+低浓度时促进胞外多糖的产生。胞外粗多糖经过醇沉和sevege法除蛋白后,随后经过DEAE纤维素阴离子交换柱及分子筛凝胶SephadexG-75分离得到纯化多糖。多角度激光光散射仪分析得知多糖主要成分平均分子量约为32.29kDa。单糖组分分析表明,SD-07的胞外多糖由Fuc(岩藻糖)、Gal(半乳糖)、Glc(葡萄糖)和Man(甘露糖)组成,其中Fuc:Gal:Glc:Man=1.33:1.00:8.67:27.33。红外光谱分析(Infrared Spectra Analysis, FTIR)结果证明多糖中含有甘露糖和葡萄糖,并且其构型为p-D-葡萄糖和p-D-甘露糖。本论文研究结果：筛选出稳定高产高活性菌株180,其抗生素组分及抑菌活性等性质与原始菌株SD-07无显著差异,提示180是未来抗生素生产的合适菌株。胞外多糖的产量与Ca2+的存在有关系,结构解析结果为接合转移和胞外多糖之间的关系提供了可靠数据。更多还原

【Abstract】 Fungal infections have a wide spread in clinical, the common antifungal drugs include polyene macrolides, azoles, echinocandins, allylamines and pyrimidine. However, they are toxic, and can cause serious side effects. Study found that the side effects of polyene macrolides are the lowest when compared with other types of drugs, and it is also the last drug to treat fungal infections clinically. Use a large amount for a long time can also lead to renal failure and thrombophlebitis, so it is very important to look for a antibiotic with lower toxicity.Streptomycese netropsis SD-07 is a new strain found by our laboratory in 2007, which can produce polyene macrolide antibiotics. When compared with amphotericin B, it’s has a higher antifungal activity. This strain has two high-yielding mutant strains, named 163 and 180. In order to verify three strains after five years still have high yield, where there is a change of structure or activity of antibiotics. The present study produced by the comparison with three strain’s antibiotic production and activity, meanwhile different purification methods of activity, stability and toxicity were compared.The laboratory found that Ca2+ can greatly increase the conjugation frequency of the genetic transfer systems of S.netropsis SD-07. When observed Streptomycese netropsis SD-07 by electron microscopy, we can find there are more extracellular polysaccharide (EPS) around the strain when add Ca2+. In order to better clarify the relationship between extracellular polysaccharide, Ca2+ and conjugation, the present study had the related research of the fermentation and the analysis of extracellular polysaccharide structure.The results of the study are as follows:1. The experimental results of antibiotics180, the mutant strain of Streptomycese netropsis SD-07 is a high-yielding strain of antibiotics, the mutant strain 163 is lower, compared with the original strain SD-07, 180 antibiotics yield is 3.33 times and 163 is 1.28 times.The experiment results of HPLC, LC-MS and antibacterial proved that there is no major components difference between the three kinds of antibiotics.Different methods of preparation showed that the antibiotics have the most stable activity by extraction method, and the most unstable method is made by molecular sieve.By the antibacterial experiment which conducted after three months proved that the most stable antibiotics is produced by 180 strain, antibacterial activity increased with the extended time, that may be related to the solubility.Cytotoxicity test showed FPLC is a good way to reduce toxicity and improve antibacterial activity when purified antibiotic.Mutagenicity test by SOS/umu-lacZ system showed that in vitro antibiotics produced by Streptomycese are had no mutagenicity. Regardless of the mutant strains types and preparation methods.2. The experimental results of EPSWhen the fermentation time was 48h, the yield of extracellular polysaccharide was highest. We choose medium R2YE as fermentation medium, Ca2+ has obvious promoting role in the formation of polysaccharides, while Cu2+ inhibitory effect is most obvious, K+ and Na+ have almost no impact on polysaccharides, low concentration of Mg2+、Fe3+ and Mn2+ can promote the production of extracellular polysaccharide.Through alcohol precipitation and sevege method, the protein in EPS was removed, after separating EPS by DEAE, and the molecular sieve named SephadexG-75.Multi-angle laser light scattering analysis showed that the average molecular weight of the main component with polysaccharides is about 32.29kDa.Monosaccharide composition analysis results show the EPS of SD-07 is composed by four monosaccharide:Fuc, Gal, Glc and Man, and Fuc:Gal:Glc:Man= 1.33:1.00:8.67:27.33.FTIR results show that the polysaccharide contains mannose and glucose, and their configuration is β-D-glucose and (3-D-mannose.Research results:this research screened the stable high yielding and highly active strain 180, its composition antibacterial activity and other properties of antibiotics as the same as original strain SD-07, suggesting that 180 is the suitable strains in the future when production of antibiotics. The production of EPS is related to Ca2+,and EPS structure provide the reliable data for the relationship between EPS and the genetic transfer systems.更多还原