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

该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，这些脱氢酶计划排列在纤维素酶复合物中的核心蛋白或支架蛋白上。核心蛋白由多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，这种内聚力-dockerin相互作用可以让我们构建人工酶复合物，以有效地将CO2还原为CH3OH。对于CO2连续酶还原的驱动力，利用叶绿素将NAD还原为NADH。 NADH脱氢酶。本研究根据过去20年厌氧细菌纤维素分解基因和酶的研究数据，制定并具体化了如下计划： 1) 纤维分解新酶基因及其酶是从厌氧纤维素分解细菌（如热纤梭菌、Josui 梭菌和白色瘤胃球菌）中提取和表征的。超过 20 种具有 dockerin 的酶被指定，此外它们还具有从这些酶形成纤维素体的结合特性2)指定具有不同结合特性的三种不同类型的粘连蛋白，并将编码2和3个粘连蛋白模块的基因连接至一个基因以产生嵌合粘连蛋白，嵌合支架蛋白或纤维素酶整合蛋白(Cip)由两种和一种组成。三个coheshin分别记为Cip2和Cip3。此后，通过BlAcore方法证实，dockerin模块蛋白仅与这表明具有dockerin模块的嵌合酶可以排列在嵌合体Cips上。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以形成dockerin甲酸嵌合体。 4)所得嵌合蛋白纯化的重组大肠杆菌显示出正常的酶活性和相似的解离常数5)从多变鱼腥藻PCC7120染色体DNA中克隆NADH脱氢酶基因，并在其中表达。大肠杆菌酶的 NAD 还原反应比 NADH 氧化反应更快，这一特性对于产生 NADH 是可以接受的。 6) 将NADH脱氢酶溶液与固定在纳米多孔二氧化硅上的叶绿素混合，在电子介体“甲基紫精”存在下，在日光下搅拌过夜，合成NADH。 7) NADH脱氢酶-dockerin嵌合体。构建了排列在嵌合体 Cip2 上，但其在 Cip2 上的排列仍在进行中。从本研究获得的这些结果来看，构建人工酶的许多必需元素复合物，除了制备了甲醛脱氢酶-dockerin和醇脱氢酶-dockerin的嵌合体之外，这将使我们能够在优化酶反应条件后利用太阳能将CO2还原为甲酸。

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