【作者】 李颖杰；

【导师】 刘巍峰； 陈冠军；

【作者基本信息】 山东大学 ， 微生物学， 2009， 硕士

【摘要】 随着社会经济的发展,人类面临着资源匮乏的巨大挑战。预测地球上可利用的石油将在今后几十年内耗竭,从长远看,能源短缺将是困扰人类发展的大问题。然而,地球上最为丰富的可再生资源—纤维素却没有得到充分利用,常常因为利用不当而造成环境污染。因此,纤维素类资源的有效利用,已引起世界各国的高度重视,成为世界各国普遍重视的研究课题。虽然纤维素材料廉价并且高产,但是纤维素的降解效率却很低,尤其是结晶纤维素难以降解,造成纤维素生物转化过程成本过高,这也是纤维素利用的主要限制性因素。Cytophaga hutchinsonii具有独特的纤维素降解机制,高效的纤维素降解能力。因此,对其纤维素降解机制的阐明,将对开发新的纤维素生物降解策略以利用纤维素这一可再生资源方面有重大意义。因此本论文从C.hutchinsonii基因组出发,围绕其高效吸附及降解纤维素的能力,初步研究了纤维素酶的催化特性及纤维素结合蛋白—碳水化合物结合模块（CBM）的结合特性。本论文主要的工作内容及结果如下:1、C.hutchinsonii基因组中与纤维素降解相关基因序列的生物信息学分析利用生物信息学数据库和生物软件,对C.hutchinsonii基因组中相关的糖苷水解酶进行了序列及结构分析,得到的纤维素降解相关酶分布在糖苷水解酶家族GH3,GH5和GH9。碳水化合物结合模块（CBM）在纤维素降解过程中发挥着重要的作用。对C.hutchinsonii基因组中CBMs序列分析显示,仅存在三种CBMs,分别是CBM4,CBM6和CBM9,但这三种CBMs都没有与纤维素酶串联,大多数存在于半纤维素降解相关的酶中。根据目前的研究发现,只有CBM9可以结合结晶纤维素,因此我们进一步对CBM9的序列进行分析,最终在C.hutchinsonii中得到10个基因编码的蛋白质含有CBM9结构域。序列同源性分析发现,这10个CBM9又可以分为三小类,分别命名为CBM9-A,CBM9-B和CBM9-C。2、C.hutchinsonii纤维素降解酶的异源表达、分离纯化及功能初步分析由于纤维素酶的异源表达一直是纤维素酶结构功能表征的限制性因素,缺少相对成熟的表达体系,因此我们通过采用包括大肠杆菌、枯草芽孢杆菌和毕赤氏酵母等不同的表达体系,对C.hutchinsonii内切纤维素酶CHU₁280及其它的纤维素降解相关酶进行了异源表达。结果显示,CHU₁280在大肠杆菌表达体系中成功表达,但表达量低,没有得到纯化的纤维素酶,仅能够通过粗酶液酶活的测定来初步研究CHU₁280的催化特性。CHU₁280粗酶液酶活测定结果显示,CHU₁280有很高的CMCase,同时具有纤维寡糖酶的活性,可以将四糖降解成二糖,将五糖降解成二糖和三糖,但对于二糖和三糖没有明显的降解作用。3、C.hutchinsonii碳水化合物结合模块-CBM9对不溶性多糖、可溶性多糖、单糖及寡糖吸附作用的研究,初步确定了CBM9活性位点的2个氨基酸残基的作用。CBM是纤维素酶及纤维素吸附结合蛋白的重要组成部分,在纤维素酶的降解过程中发挥着重要的作用。目前只有两篇详细介绍CBM9吸附特性的文章,一篇研究了CBM9与各种糖的吸附特性,另一篇通过晶体结构解析介导其吸附的活性位点。通过异源表达C.hutchinsonii CBM9,对CBM9的吸附特性进行了初步分析,发现其可以与不溶性木聚糖和微晶纤维素结合,其中与不溶性木聚糖的结合能力更强,同时CBM9还能够与可溶性的木聚糖结合。C.hutchinsonii CBM9虽然具有三个Ca²⁺结合位点,但是Ca²⁺浓度对CBM9结合能力并没有影响,由此推测Ca²⁺没有参与CBM9与糖的结合,而是起到一个稳定蛋白质结构的作用。进一步运用生物学软件对C.hutchinsonii CBM9序列进行模建。模板与C.hutchinsonii CBM9的重叠表明二者在内部片层骨架上重合较好,发生变化的部分主要是无规卷曲形成的表面loop区。同时,与模板比较,C.hutchinsonii CBM9在结合活性位点区域112位多出了一个Tyr。CBM9定点突变结果显示,Tyr75和Tyr112同时突变会使CBM9与不溶性糖的结合能力降低,推测可能是由于C.hutchinsonii CBM9相对于模板来说,loop环的差异导致活性位点两个芳香族氨基酸残基Tyr75和Trp183的芳香环平面不平行,疏水作用减弱,进而由Tyr75和Tyr112共同作用参与糖的结合。更多还原

【Abstract】 With the development of the word economy,global resource crisis is being more and more serious.It is supposed that available petroleum will run out in the coming decades,and resource crisis has already been a general and urgent problem.Cellulose, the most abundant component of plant biomass,is not fully utilized.Therefore,efficient bioconversion of cellulose has attracted universal attention in recent years.Although cellulosic biomass has distinct advantages in costs and scale-up production,its hydrolysis is very difficult,especially for crystalline cellulose.The low efficiency of cellulase has been one of limiting factors responsible for high costs of cellulose hydrolysis.Cytophaga hutchinsonii use a novel strategy for cellulose utilization,so the research of its cellulolytic mechanism is meaningful to utilization of renewable resources.In this thesis,we studied catalytic characteristics of cellulase enzyme expressed in E.coli and binding characteristics of carbohydrate binding module （CBM）.The major results of the thesis are as follows.1.Bioinformatic analysis of cellulolytic enzymes encoding genes in C. hutchinsonii genomeAll sequences related to cellulolytic enzymes were analyzed by various bioinformatic softwares.Results showed that C.hutchinsonii glycohydrolases that may be involved in cellulose digestion belong to glycohydrolase family 3,5 and 9.Meanwhile,there are only three kinds of carbohydrate binding modules（CBMs）, CBM4,CBM6 and CBM9 in C.hutchinsonii genome.Surprisingly,none of proteins related to endo-1,4-glucanases contain CBMs domains and most of CBMs are found on glycohydrolases which may be involved in hemicellulose digestion.According to current research,only CBM9 could bind to crystalline cellulose,so we analyzed all CBM9 sequences detailedly.Our work revealed that there are 10 CBM9 in the genome of C.hutehinsonii,which could be divided into three classes named CBM9-A,CBM9-B and CBM9-C.2.Heterologous expression and primary characterization of cellulolytic enzymes of C.hutchinsoniiBecause cellulolytic enzymes’ expression is difficult,we tried to use three expression systems,including E.coli,Pichia and Bacillus subtillis systems.Endo-1,4 - glucanases CHU₁280 was successfully expressed in E.coli system.The production of CHU 1280 was low and could not be purified,so we tested its catalytic characteristics by mixture.We found that CHU₁280 could hydrolyze CMC efficiently and degrade tetrasaccharides to disaccharides,pentasaccharides to disaccharides and trisaccharides, but it could not degrade disaccharides and trisaccharides.3.Characterization of binding properties of CBM9 in C.hutchinsoniiCBMs,as an important component in cellulose binding proteins,play a great role in cellulose degradation.Only two reports studied CBM9 binding characteristics.One was by biochemical methods,and the other was to analyze its crystal structure.In this thesis,we expressed CBM9 in E.coli.After purification,we studied its binding characteristics and found that it could bind to insoluble xylan and Avicel,and combining capacity to xylan was stronger.CBM9 could also bind to soluble xylan.Though CBM9 has three Ca²⁺ binding sites,Ca²⁺ concentration did not affect CBM9 to bind to sugars.Therefore,we speculated Ca²⁺ did not enjoy in combination, but played a role to stable protein structure.The three dimensional structure of CBM9 was constructed by homology modeling. From the final modeled structure,it is obvious that CBM9 in C.hutchinsonii could overlay with template well in sheet skeletons,but the loops in the active site were different with an additional Tyr112.Further site-directed mutagenesis revealed that simultaneous mutations of Tyr75 and Tyr112 resulted in a decrease in the binding capacity.We presumed that aromatic tings of two aromatic amino residues in CBM9 active site did not perfectly parallel the sugar ring which weakens the hydrophobic interaction between CBM and the sugar chain while Tyr75 and Tyr112 coordinate to ensure the efficient binding.更多还原