【作者】 陈凝；

【导师】 卢雪梅；

【作者基本信息】 山东大学 ， 微生物学， 2011， 硕士

【摘要】 哈氏噬纤维菌(Cytophaga hutchinsonii)是广泛存在于自然界中的一类可彻底降解结晶纤维素的好氧细菌,在降解纤维素方面有着快速、彻底等特点。哈氏噬纤维菌降解纤维素的机制十分特殊,与真菌和厌氧细菌的纤维素降解机理明显不同。哈氏噬纤维菌降解纤维素的首要条件是与纤维素的直接接触。其菌体表面不存在纤维小体,也不分泌胞外纤维素酶。所编码的绝大部分纤维素酶类都不含有碳水化合物结合结构域(CBMs),也不含有类似于纤维小体中docking domain的结构域。这些特点显示哈氏噬纤维菌吸附降解结晶纤维素具有独特性,此类研究目前在国内外几乎都是空白。为了揭示哈氏噬纤维菌的纤维素吸附机制,本文对影响其吸附纤维素的各种因素进行了初步研究,并推测参与吸附的主要成分：包括菌体对不同底物吸附能力的比较,菌体代谢活性对纤维素吸附的影响,表面蛋白及多糖对吸附的影响,以及纤维素类似物对吸附的抑制作用等。研究结果发现哈氏噬纤维菌对结晶纤维素的吸附具有很强的特异性,不受菌体代谢活性的影响,吸附作用也不受纤维二糖和羧甲基纤维素的竞争性抑制；而菌体表面蛋白与纤维素吸附密切相关；多糖成分对吸附作用影响较小。实验结果表明,哈氏噬纤维菌中可能存在有特异性的微晶纤维素吸附蛋白。基于这些研究结果,本文开展了哈氏噬纤维菌细胞表面与纤维素吸附相关的蛋白的进一步研究。鉴于蛋白在吸附过程中的重要作用,本文通过直接提取的方法,寻找哈氏噬纤维菌中纤维素结合蛋白,这类蛋白的分离鉴定对于研究哈氏噬纤维菌吸附纤维素的机理具有重要意义。经SDS-PAGE分离吸附在微晶纤维素上的天然膜蛋白,并通过蛋白质谱鉴定,总共得到13种哈氏噬纤维菌的纤维素结合蛋白,包括三种滑动相关蛋白,五种假定蛋白,一种外膜脂蛋白,一种β-葡萄糖苷酶,一种ATP合成酶亚基,一种热激蛋白GroEL以及一种肽脯氨酰顺反异构酶,并对这些蛋白进行生物信息学的分析,包括其信号肽及跨膜结构预测等。为实现对哈氏噬纤维菌纤维素结合蛋白的功能性研究,基于在大肠杆菌中难以获得活性表达,本文考虑构建哈氏噬纤维菌表达体系,通过同源表达并分离纯化纤维素结合蛋白,进而开展更深入的研究。由于哈氏噬纤维菌遗传操作系统的不完善,本文通过实验室构建的自主复制质粒来建立哈氏噬纤维菌的表达体系。该质粒以红霉素为筛选标记,以绿色荧光蛋白或p-半乳糖苷酶为报告基因,实验证明了这种质粒能在哈氏噬纤维菌中表达。同时,比较几种不同的启动子启动蛋白表达的能力强弱,发现其中CHU 1284的启动子是最强的启动子。本文从通过研究比较不同处理对哈氏噬纤维菌吸附纤维素的影响出发,分离鉴定了十三种在吸附过程中起着重要的作用纤维素结合蛋白。成功构建了哈氏噬纤维菌表达体系,为其遗传操作体系的提供重要补充,为进一步研究哈氏噬纤维菌的吸附降解纤维素的机制提供了实验依据。更多还原

【Abstract】 Cytophaga hutchinsonii is a widespread aerobic cellulose-degrading bacterium which can degrade crystalline cellulose thoroughly and rapidly.Cytophaga hutchinsonii exhibits a specific cellulose degrading mechanism different from the classic mechanisms reported previously in fungi or anaerobic bacteria. To digest cellulose, Cytophaga hutchinsonii requires the direct contact with the substrate. It does not produce cellulosomes on cell surface or secrete extracellular cellulolytic enzymes. Most of the cellulases encoded by Cytophaga hutchinsonii contain neither carbohydrate-binding modules （CBMs） nor docking domain-like structures. All these characteristics show that cellulose degradation in Cytophaga hutchinsonii is unique and distinctive. Up to now,research on the mechanism of cellulose degradation in Cytophaga hutchinsonii remains to be blank at home and abroad.To uncover the mechanism of cellulose adhesion in Cytophaga hutchinsonii, a preliminary study on factors influencing cellulose adhesion was applied, such as metabolism activity of bacterium, membrane proteins or polysaccharides, and cellulose analogs.We also investigate the specificity of bacterial adhesion as well as the main constituent involved in the adhesion process.The research results showed that the adhesion between Cytophaga hutchinsonii and crystalline cellulose is highly specific. The adhesion is not affected by bacterial metabolism activity and competitive inhibition ofcellobiose and sodium carboxymethyl cellulose as well. The progress is closely related to cell surface proteins, but little affected by cell surface polysaccharides. Specific proteins for crystalline cellulose binding were supposed to exist on cell surface. Based on these results, further study on cell surface proteins related to adhesion is applied.Because of the important role of proteins play in the adhesion progress, we searched for the cellulose binding proteins （CBPs） of Cytophaga hutchinsonii by direct isolation and identification. To study the mechanism of Cytophaga hutchinsonii adhered to cellulose, the identification of these proteins has a very important significance. Nature membrane proteins which binding to cellulose were isolated by SDS-PAGE, and identified by MALDI-TOF mass spectrometry.13 CBPs were obtained together, contains three gliding motility-related proteins, five hypothetical proteins, an outer membrane lipoprotein, aβ-glucosidase, an ATP synthase, a heat shock protein GroEL, and a peptidyl-prolyl cis/trans isomerase. Bioinformatics analyses were carried out, such as prediction of their potential signal peptides and trans-membrane domains.To study the function of cellulose adhesion proteins, we firstly tried to express these proteins inE.coli but hardto acquire active expression. So we considered building the expression system of Cytophaga hutchinsonii for isolation and purification of CBPs and for depth research.The genetic manipulation system is still incomplete in Cytophaga hutchinsonii, so we build the expression system based on self-replicated plasmids that laboratory possessed.Erythrocin resistance gene was used as selective marker gene, and the green fluorescent protein orβ-galactosidase was used as reporter gene in this plasmid. Results showed that it can replicate and express in Cytophaga hutchinsonii. At the same time, the activities of some promoters have been compared, and find out that the promoter of CHU₁284 exhibits the maximal activity.In this paper, we started the research by comparing the cellulose adhesion influenced by different treats;thirteen three cellulose binding proteins were isolated which play an important role in binding process. The expression system ofCytophaga hutchinsonii was built successfully as an important supplement of genetic manipulation system and provides experiment basis for the further study of cellulose adhesion and degradation in Cytophaga hutchinsonii.更多还原