Nano-arrangement analysis of cellulase complex and its application to methanol production by fixing carbonic acid.

本项目的最终目的是利用甲酸脱氢酶、甲醛脱氢酶和乙醇脱氢酶，通过甲酸和甲醛将二氧化碳还原为CH3OH。这些脱氢酶计划排列在纤维素酶复合体-纤维素体中的核心蛋白或支架蛋白上。核心蛋白由几个重复的凝聚力模块组成。一个凝聚模块可以结合酶的Dockerin模块，纤维素体组件。这种凝聚力-Dockerin的相互作用可能使我们能够构建人工酶复合体，有效地将二氧化碳还原为CH3OH。本研究根据近20年来对厌氧细菌纤维素分解基因和酶的研究数据，提出了利用光能通过叶绿素将NAD还原为NADH的研究方案。1)新的纤溶酶基因及其酶…更多的是来自厌氧纤维分解细菌，如热细胞梭菌、若斯氏梭菌和白色瘤胃球菌。确定了纤维素酶复合体、纤维素体的组成，如由粘附素组成的核心蛋白和20多种含有Dockerin的酶的结合特性，以及从这些厌氧菌形成纤维素体的结合特性。2)指定了三种不同类型的粘附素，并将编码2和3个粘附素模块的基因连接到一个基因上，以产生嵌合体粘附素蛋白。由两个和三个粘连蛋白组成的嵌合体支架蛋白或纤维素酶整合蛋白(Cip)分别被命名为Cip2和Cip3，随后用BlAcore方法证实了Dockerin模块蛋白只与其相应的粘连蛋白结合。3)将Dockerin模块基因连接到母牛分枝杆菌N10的甲酸脱氢酶基因上，构建了Dockerin甲酸脱氢酶的嵌合体。4)纯化的重组大肠杆菌具有正常的酶活性和与游离Dockerin类似的解离常数，说明Dockerin和甲酸脱氢酶的嵌合不影响Dockerin与Dockerin的结合特性和酶性质。该酶的NAD还原反应比NADH氧化反应快。6)将NADH脱氢酶溶液与固定化在纳米多孔二氧化硅中的叶绿素混合，在日光下搅拌过夜。7)构建了NADH脱氢酶-Dockerin嵌合体，将其排列在嵌合体Cip2上，但其在Cip2上的排列仍在继续，根据本研究的结果，制备了除乙醛脱氢酶-Dockerin和乙醇脱氢酶-Dockerin嵌合体外，构建人工酶复合体的许多必要元件。这将使我们可以在优化了酶反应条件后，利用太阳能将二氧化碳还原为甲酸。较少

The final purpose of this project is the reduction of CO2 to CH3OH via HCOOH and HCHO by using formic acid dehydrogenase, formaldehyde dehydrogenase and alcohol dehydrogenase. These dehydrogenases are planed to arrange on a core protein or scaffolding proteins which is found in the cellulase complexes, cellulosomes. The core protein consists of several repeats of cohesion modules. One cohesion module can bind a dockerin module of an enzyme, cellulosome component. This cohesion-dockerin interaction may allow us the construction of artificial enzyme complex for the effective reduction of CO2 to CH3OH. For the driving force of the sequential enzymic reduction of CO2, solar energy via chlorophyll is employed to reduce NAD to NADH by NADH dehydrogenase.These plans were established and concreted as follows in this study based on the data obtained from the studies of anaerobic bacterial cellulolytic genes and enzymes for past 20 years.1) Fibrolytic novel enzyme genes and their enzymes were is … More olated and characterizedfrom anaerobic cellulolytic bacteria such as Clostridium thermocellum, Clostridium josui, and Ruminococcus albus. The components of cellulase complex, cellulosomes, such as core proteins consisting of cohesin and more than 20 enzymes having dockerin were specified, in addition to their binding properties to form cellulosome from these anaerobes.2) Three different types of cohesin with different binding properties were specified and the genes encoding 2 and 3 cohesin modules were ligated to one gene to produce chimera cohesin protein. The chimera scaffolding proteins or cellulase integrating proteins (Cip) consists of two and three coheshins were denoted as Cip2 and Cip3, respectively.Thereafter, it was confirmed by using a BlAcore method that the dockerin module proteins bounded only to their counterpart of cohesin, specifically. This suggested that the chimera enzymes having a dockerin module could be arranged on the chimera Cips.3) A dockerin module gene was ligated to a formic acid dehydrogenase gene from Mycobacterium vaccae N10 to form a chimera of dockerin formic acid dehydrogenase.4) The resulted chimera protein purified recombinant E. coli revealed both normal enzyme activity and dissociation constant similar to that of free dockerin to Cip2, indicating that chimerization of dockerin and formic acid dehydrogenase did not affect on both binding properties of cohesion to dockerin and enzyme properties.5) A NADH dehydrogenase gene was cloned from chromosomal DNA of Anabaena variabilis PCC7120 and expressed in E. coli. The NAD reducing reaction of the enzyme was faster than the NADH oxidizing reaction. This property was acceptable to produce NADH in our further studies.6) The NADH dehydrogenase solution was mixed with chlorophyll immobilized in the nano porous silica and stirred overnight under the daylight. NADH was synthesized under the presence of electron mediator "methyl viologen".7) The NADH dehydrogenase-dockerin chimera was constructed to arrange on chimera Cip2, but its arrangement on Cip2 is still going.From these results obtained in this study, many of the essential elements to construct artificial enzyme complex, except chimeras of form aldehyde dehydorogenase-dockerin and of alcohol dehydrogenase-dockerin were prepared. This will allows us to reduce CO2 to formic acid after optimization of the enzymatic reaction conditions by using solar energy. Less