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

纤维素酶复合物的纳米排列分析及其在固定碳酸生产甲醇中的应用

基本信息

批准号：
14206038
负责人：
OHMIYA Kunio
金额：
$ 34.2万
依托单位：
Meijo University (2004)Mie University (2002-2003)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

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

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CH 3OH。这些纤维素酶被计划排列在纤维素酶复合物纤维素体中发现的核心蛋白或支架蛋白上。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锚定蛋白模块，多纤维素体组分。这种内聚-锚定蛋白相互作用可能使我们能够构建人工酶复合物，用于有效地将CO2还原为CH 3OH。本研究在总结近20年来厌氧细菌纤维素分解基因和酶研究成果的基础上，提出了利用太阳能通过叶绿素将NAD还原为NADH作为连续酶还原CO_2的动力，并建立了以下几个方面的研究方案：1）纤维素分解新酶基因及其酶的筛选; 2）纤维素分解新酶基因的筛选; 3）纤维素分解新酶基因的筛选; 4）纤维素分解新酶基因的筛选; 5）纤维素分解新酶基因的筛选; 6）纤维素分解新酶基因的筛选; 7）纤维素分解新酶基因的筛选; 8）纤维素分解新酶基因的筛选; 9）纤维素分解新酶基因的筛选; 10）纤维素分解新酶基因的 ...更多信息从厌氧纤维素分解细菌如热纤梭菌、梭菌和白色瘤胃球菌中分离和表征。确定了纤维素酶复合物的组成成分纤维素体，如核心蛋白，由cohesin和20多种具有dockerin的酶组成，以及它们与纤维素酶复合物形成纤维素体的结合特性。2）确定了3种不同结合特性的cohesin，并将编码2个和3个cohesin模块的基因连接到一个基因上，产生嵌合cohesin蛋白。将由2个和3个粘着蛋白构成的嵌合体支架蛋白或纤维素酶整合蛋白（Cip）分别记为Cip 2和Cip 3，之后，通过BlAcore方法确认了dockerin模块蛋白仅与其对应的粘着蛋白特异性结合。3）将Dockerin模块基因与来自母牛分枝杆菌N10的甲酸脱氢酶基因连接，形成Dockerin甲酸脱氢酶嵌合体。在大肠杆菌中，锚定蛋白与Cip 2的解离常数与游离锚定蛋白相近，说明锚定蛋白与甲酸脱氢酶的嵌合不会影响锚定蛋白的结合特性和酶的性质。杆菌该酶的NAD还原反应比NADH氧化反应快。这种性质在我们进一步的研究中是可接受的，以产生NADH。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，并在日光下搅拌过夜。7）构建了NADH脱氢酶-dockerin嵌合体，并将其排列在嵌合体Cip 2上，但其在Cip 2上的排列仍在进行中。根据这些结果，除了甲醛脱氢酶-dockerin嵌合体和乙醇脱氢酶-dockerin嵌合体外，制备了许多构建人工酶复合物所必需的元件。这将使我们能够通过使用太阳能优化酶促反应条件后将CO2还原为甲酸。少

项目成果

期刊论文数量（10）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Interaction between a Type-II Dockerin Domain and a Type-II Cohesin Domain from Clostridium thermocellum Cellulosome

DOI：
10.1271/bbb.68.924
发表时间：
2004-01
期刊：
Bioscience, Biotechnology, and Biochemistry
影响因子：
0
作者：
Sadanari Jindou;T. Kajino;M. Inagaki;S. Karita;P. Béguin;Tetsuya Kimura;K. Sakka;K. Ohmiya
通讯作者：
Sadanari Jindou;T. Kajino;M. Inagaki;S. Karita;P. Béguin;Tetsuya Kimura;K. Sakka;K. Ohmiya

酵素配列複合体及び固体化酵素配列複合体とそれらの製造

酶序列复合物、固化酶序列复合物及其制备