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HCOOH和HCHO将CO2还原为CH3OH。这些脱氢酶被安排在核心蛋白或支架蛋白上，这些蛋白在纤维素酶复合物，纤维素体中被发现。核心蛋白由几个重复的内聚模块组成。一个内聚模块可以结合酶的dockerin模块，纤维素组分。这种内聚-码头蛋白的相互作用使我们能够构建人工酶复合物，有效地将CO2还原为CH3OH。作为连续酶还原CO2的驱动力，太阳能通过叶绿素通过NADH脱氢酶将NAD还原为NADH。根据近20年来厌氧菌纤维素分解基因和酶的研究数据，本研究建立并具体如下：1)从热胞梭菌（Clostridium thermocellum）、焦氏梭菌（Clostridium josui）和白色瘤胃球菌（Ruminococcus albus）等厌氧纤维素分解菌中分离到更多的纤维分解酶基因及其酶。纤维素酶复合体的组成部分，如核心蛋白组成的黏结蛋白和20多个酶的dockerin被指定，除了它们的结合性质，形成纤维素从这些厌氧菌。2)指定3种不同类型、不同结合特性的黏结蛋白，将编码2和3个黏结蛋白模块的基因连接到一个基因上，生成嵌合体黏结蛋白。由两种和三种黏结蛋白组成的嵌合体支架蛋白或纤维素酶整合蛋白（Cip）分别记为Cip2和Cip3。随后，使用BlAcore方法证实dockerin模块蛋白仅与它们的黏结蛋白对应物特异性结合。这表明具有dockerin模块的嵌合体酶可以被安置在嵌合体的cipp上。3)将dockerin模块基因与母牛分枝杆菌N10的甲酸脱氢酶基因连接，形成dockerin甲酸脱氢酶嵌合体。4)得到的嵌合体蛋白纯化后的重组大肠杆菌对Cip2的酶活性和解离常数均与游离dockerin相似，表明dockerin与甲酸脱氢酶的嵌合不影响dockerin与内聚体的结合特性和酶的性质。5)从变腥腥鱼PCC7120染色体DNA中克隆出NADH脱氢酶基因，并在大肠杆菌中表达。酶的NAD还原反应比NADH氧化反应快。在我们进一步的研究中，这种性质是可以产生NADH的。6)将NADH脱氢酶溶液与固定在纳米多孔二氧化硅中的叶绿素混合，在日光下搅拌过夜。NADH是在电子介质甲基紫紫素存在下合成的。7) NADH脱氢酶对接蛋白嵌合体已构建到嵌合体Cip2上，但其在Cip2上的排列仍在进行中。根据本研究的结果，制备了除乙醛脱氢酶-dockerin和醇脱氢酶-dockerin嵌合体外的许多构建人工酶复合物所需的元件。这将使我们能够通过利用太阳能优化酶促反应条件后将二氧化碳还原为甲酸。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