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丙酮丁醇梭菌纤维小体组装模块的相互作用研究
姚形哲
Thesis Advisor冯银刚
2015-05
Degree Grantor中国科学院大学
Place of Conferral北京
Degree Discipline生物工程
Keyword丙酮丁醇梭菌 对接模块 粘连模块 相互作用 组装
Abstract

纤维小体是由脚架蛋白与多种纤维素/半纤维素酶等通过组装模块——粘连模块(cohesin,Coh)和对接模块(dockerin,Doc)之间的相互作用形成的超分子复合体。梭菌类微生物的纤维小体是自然界中最高效降解木质纤维素的体系之一。丙酮丁醇梭菌是能高效产生丙酮、丁醇和乙醇等生物产品的工业菌株。若能使用木质纤维素作为碳源将有利于降低丙酮丁醇梭菌的工业应用成本,但目前的研究表明丙酮丁醇梭菌不能以纤维素为唯一碳源生长。以前的研究表明,丙酮丁醇梭菌含有编码纤维小体的基因,能表达和分泌很少量的纤维小体但活性极低。丙酮丁醇梭菌纤维小体的粘连模块(cohA)和对接模块(DocA)的系统进化关系分类与热纤梭菌和解纤维梭菌等有很大差异。进一步研究丙酮丁醇梭菌纤维小体的粘连模块和对接模块之间的相互作用,可以揭示丙酮丁醇梭菌纤维小体的组装机制,发现可能影响其纤维小体活性的因素,为进一步改良丙酮丁醇梭菌利用木质纤维的能力提供新的方案。 本实验室的前期工作已经通过核磁共振方法解析了CohA和DocA的单独溶液结构,通过表面等离子共振技术对CohA-DocA之间的相互作用强度进行了测定。本论文在此基础上进一步研究了CohA-DocA之间的相互作用对纤维小体组装与组装后功能的影响。以解纤维梭菌的粘连模块(CohC)和对接模块(DocC)作为对照组。本论文的工作主要包括对所需组件的基因克隆与蛋白表达纯化,实验分析相互作用对组装的影响以及对组装后复合物协同效应的影响。 本论文研究克隆和表达的单独组件有来自丙酮丁醇梭菌的CohA和DocA;来自解纤维梭菌的CohC、DocA、以及纤维素酶Cel9G和Cel48F;来自热纤梭菌的碳水化合物结合模块(carbohydrate binding module,CBM)。融合组件包括用于检测Coh-Doc相互作用对组装影响的Cel9G-DocA、Cel9G-DocC、Cel48F-DocA、Cel48F-DocC、CohA-CBM、和CohC-CBM;以及用于检测相互作用对协同效应影响的CohA-CohA-CBM和CohC-CohC-CBM。本论文成功完成了这些蛋白模块和融合蛋白的质粒构建、高效重组表达、以及蛋白质纯化。 获得目的蛋白后通过native-PAGE分析了它们的组装效果。对于“CohA与DocA”和“CohA与酶-DocA”这两种组合,由于部分蛋白在native-PAGE上不出现条带或条带位置与其他蛋白重合,不能清楚的分析组装结果;对于“CohA-CBM与酶-DocA”的组合,结果表明他们能通过CohA-DocA正常完全组装;而组装成微型纤维小体的“CohA-CohA-CBM和Cel9G-DocA+Cel48F-DocA”,组装复合物不均一,有多种状态。这些结果表明,丙酮丁醇梭菌的粘连模块和对接模块之间的相互作用能够实现将酶组装到脚架蛋白的功能,但模块串联之后可能相互影响组装的效果。 本论文进一步比较了相互作用对组装后复合物酶活性的影响,以反映其对协同作用的影响。以DNS法测定降解微晶纤维素产生的还原糖量来表征酶活性。结果显示,以CohA-DocA为组装模块将一个酶与CBM组装以反映酶与底物的协同作用时,组装复合物协同因子和对照组CohC-DocC几乎完全一致。但将两种酶与CBM通过CohA-DocA组装在一起反映纤维小体的酶之间的协同作用时,丙酮丁醇梭菌的组装模块产生的协同作用明显比对照组解纤维梭菌的模块弱,约为对照组CohC-DocC的76%。这些结果和前面的组装实验结果一致,说明丙酮丁醇梭菌的粘连模块和对接模块之间的相互作用能够实现酶与底物的协同作用,但在实现酶与酶之间的协同作用时可能存在作用减弱的问题。 综上所述,本论文主要研究了丙酮丁醇梭菌粘连模块和对间蛋白模块之间的相互作用对组装、以及对组装后复合物协同作用的影响。发现丙酮丁醇梭菌的组装模块可以起到组装与发挥酶与底物之间协同作用的功能,但在实现酶与酶之间的协同作用时可能存在组装作用与协同作用减弱的问题。这些工作为进一步结合结构分析揭示丙酮丁醇梭菌中纤维小体的组装机制及其蛋白质工程改造提供了基础。

Other Abstract

Cellulosome, which is assembled by cohesins and dockerins, is a supramolecular complex of Scaffoldin, cellulases or hemicellulases, and other components. Cellulosome from clostridium is one of the most efficient systems to degrade lignocellulose. Clostridium acetobutylicum is a very useful strain to produce acetone, butanol, and ethanol, but it cannot grow with cellulose as the sole carbon source. Previous research shown that C. acetobutylicum has genes of cellulosome and can produce a little cellulosome with very low enzyme activity. Evolutionary of Cohesins and dockerins of celluilosome from C. acetobutylicum are found different to C. cellulolyticum and C. thermocellum through sequence analysis. Considering vital importance of cohesin and dockerin in assembling cellulosome, it is necessary to study the interaction between cohesin(CohA) and dockerin(DocA) of cellulosome from C. acetobutylicum. Our lab have purified CohA and DocA through Escherichia coli, studied their resolution structure through nuclear magnetic resonance (NMR), and tested their interaction through surface plasmon resonance (SPR), too. My work in this thesis dose further study about assembly CohA with DocA, and enzyme activity of assembled complexes. We select one cohesin(CohC) and one dockerin(DocC) from Clostridium cellulolyticum as control. The proteins used in this study are CohA and DocA from C. acetobutylicum, and CohC, DocC, Cel9G, Cel48F from C. cellulolyticum, and CBM from C. thermocellum. More fusion proteins needed to assembly cellulase with CBM are Cel9G-DocA, Cel9G-DocC, Cel48F-DocA, Cel48F-DocC, CohA-CBM, CohA-CohA-CBM and CohC-CohC-CBM are needed to assembly mini-cellulosome. We construct recombinant plasmids, and purified these proteins succesfully. Native-PAGE is used to test if cohesin and dockerin, or recombinant proteins containing cohesin or dockerin, can be assembled. The results show that CohA and DocA can be used to assemble one cellulase and one CBM well, but weaker to assemble two cellulases and one CBM at the same time. DNS method is used to determine the amount of reducing sugar which accounts for enzyme activity of free cellulases or assembled complex. Results show that the improvement of enzyme activities through assembling one cellulase and a CBM into a complex by CohA-DocA is very similarly to CohC-DocC. But when it comes to assembling two cellulase and one CBM at the same time, the improvement of CohA-DocA is only 76% of CohC-DocC. In conclusion,this work has studied the impact of the interaction between CohA and DocA on assembling different proteins into a complex, and the impact on complexes’ enzyme activity. Results show that the interaction between CohA and DocA is able to assemble cellulase and CBM into a complex, and improve enzyme activity of the complex. But when it comes to assemble two cellulases and one CBM into a complex, the effect will be decreased. This work will provide experiment foundation for further study about the interaction and assembly mechanism of cohesin and dockerin from C. acetobutylicum.

Department代谢物组学团队
Subject Area生物化学
Date Available2017-05-31
Subtype硕士 ; 学位论文
Language中文
Document Type学位论文
Identifierhttp://ir.qibebt.ac.cn/handle/337004/8105
Collection代谢物组学团队
Affiliation中国科学院青岛生物能源与过程研究所
Recommended Citation
GB/T 7714
姚形哲. 丙酮丁醇梭菌纤维小体组装模块的相互作用研究[D]. 北京. 中国科学院大学,2015.
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