瑞氏木霉内切葡聚糖酶1及其CBD的噬菌体表面展示

【作者】 张曦；

【导师】 曲音波；

【作者基本信息】 山东大学 ， 微生物学， 2005， 硕士

【摘要】 瑞氏木霉内切葡聚糖酶Ⅰ（Endoglucanase Ⅰ,EGI）在降解纤维素的过程中是一种十分重要的酶。该酶作用于纤维素内部的非结晶区,随机水解β-1,4-糖苷键。EGI包含两个结构域,一个是纤维素的催化结构域（catalytic domains,CD）,一个是纤维素的吸附结构域（cellulose-binding domains,CBD）,二者由一个连接区（Linker）连接。 本文意图在不改变EGI的CD和Linker的前提下,对CBD进行随机突变,通过筛选找到与野生型相比纤维素吸附能力改变的CBD,再将其与CD和Linker结合构建杂合酶,比较研究杂合酶的性质,进一步阐明CBD与CD和Linker相互作用降解纤维素的机理。 噬菌体表面展示技术（Phage display）是一种新型的筛选方法,它使表达的外源肽（或者蛋白质的结构域）以融合蛋白的形式展现在噬菌体的表面,并保持相对独立的空间结构和生物活性。与传统筛选手段相比,该技术更加高效,尤其是在亲和筛选方面可以很方便地得到与配体结合能力不同的受体,而且筛选库的容量很大,是一种很适合本研究的筛选方法。近年来,噬菌体表面展示技术亦开始被应用于酶学定向进化的研究。目前,国内外未见有纤维素酶噬菌体展示的报道。如果可以将EGI成功地展示在噬菌体表面,无疑将会对应用该技术进行纤维素酶研究提供有力的帮助,这也是本文的一个工作内容。 本文首先将EGI展示在丝状噬菌体表面,分别用酶联免疫吸附试验（Enzyme-linked immunosorbent assay,简称ELISA）和Somogyi方法对其活性进行验证。被展示在噬菌体表面的EGI依然具有纤维素的吸附能力和内切酶活力,其CMC（carboxymethyl cellulose）酶活为7.0×10^-16 IU/pfu,说明EGI被成功展示在丝状噬菌体表面。 然后再将EGI的CBD展示在噬菌体表面,通过ELISA方法验证被展示的CBD依然具有对纤维素的吸附特性。利用易错PCR的方法对CBD基因进行随机突变,建立了一个5×10⁵大小的、可用来进行噬菌体表面筛选的CBD基因突变菌种库。为进一步合理解释吸附后CBD是如何与CD和Linker相互作用降解更多还原

【Abstract】 Endoglucanase I (EGI) is an important enzyme of Trichoderma reesei, it cleaves internal glycosidic bonds in cellulose chains. EGI consists of two domains joined together by a glycosylated interdomain linker peptide. The larger catalytic domain contains the active site, which carries out general acid catalyzed hydrolysis of the β-1,4-glycosidic bonds in cellulose. The smaller cellulose-binding domain (CBD) improves binding of enzymes onto cellulose and enhances its enzymatic degradation.Although the hydrolysis mechanism and the substrate specificity of EGI, while it is cleaving the β-l,4-glycosidic bonds in cellulose, have been basically clarified, how CBD module cooperates with CD and Linker modules in the process of cellulose hydrolysis has not been reasonably explained yet.In this thesis, we hope generate a series of hybrid endoglucanases, which have the same CD module and Linker module with EGI except CBD module. An Error-PCR library of the gene of CBD from EGI has been constructed. It would be very hard to use the traditional screening method to obtain the genes of CBD of different cellulose-binding ability. Compared with the traditional screening method, phage display has more advantages.Phage display is a powerful method for selecting and engineering polypeptides with desired binding specificities. The method relies on the fact that if gene fragments encoding polypeptides are fused to M13 coat protein genes, these ’fusion genes’ can be incorporated in bacteriophage particles that also display the heterologous proteins on their surfaces. In this way, a physical linkage is established between phenotype and genotype. Using simple molecular biology techniques, large and diverse phage-displayed polypeptide libraries can be generated. Phage displaying polypeptides with a desired binding specificity can be selected from library pools by binding to an immobilized ligand, and the sequences of selected polypeptides can be deduced from the sequence of the encapsulated DNA.In addition, phage display application to the directed evolution of enzymes is beginning to emerge. Until now, the research of phage-display cellulase has not beenpublished yet. If a cellulase is successfully displayed on the surface of phage, the result will be clearly helpful to the research of phage display as a tool for the directed evolution of cellulase. This is another emphasis in this paper.A phage enzyme of EGI has been constructed for proving the feasibility of phage display in cellullolytic enzymes. The phage enzyme EGI has proved to be active, which not only has the ability of binding cellulose by enzyme -linked immunosorbent assay (ELISA), but also has catalytic efficiency on insoluble substrate. The value of CMCase activity is 7.0 * 10"16IU /pfu. The results showed the phage display used in cellullolytic enzymes was feasibile.Despite considerable efforts, the exact role and function of the CBDs in the enzymatic action of cellulases are not well understood. In this study, CBD has been displayed on phage surface, which proved to have the ability of binding cellulose by ELISA. Then a 5xl05 size phage library displaying the CBDs derived from the gene of CBDEGiby Error-prone PCRhas been constructed. This library will be screened for obtaining different CBDs of binding ability to cellulose by phage display, then a series of recombinant enzymes will be constructed. Compare their performances with the corresponding wild-type enzyme on soluble and cystalline substrates, and then discuss the role of CBD in cellulose degradation.Furthermore, the EGI and CBD recombinant phages infected E.coll HB2151 cells. IPTG induced expression of EGI and CBD in E.coll’ HB2151 cells. After assaying the supernantant, periplasmic extract, and whole cell extract, that soluble EGI and CBD were found to be produced and concentrated in the supernantant and confirmed by SDS-PAGE. Some efforts of purifying CBD protein have also been done. The purified CBD will be used to measure the kinetic parameters for getting more useful information.更多还原