Grant-in-Aid for Scientific Research (A)
|Allocation Type||Single-year Grants|
|Research Institution||Meijo University(2004)|
OHMIYA Kunio Meijo University, Professor, 農学部, 教授 (60023488)
ICHIHARA Shigeyuki Meijo University, Professor, 農学部, 教授 (30092993)
SAKKA Kazuo Meijo University, Professor, 生物資源学部, 教授 (20154031)
KIMURA Tetsuya Meijo University, Associate Professor, 生物資源学部, 助教授 (00281080)
KAJINO Tsutomu Toyota Central R&D Labs INC, Senior Researcher, 主任研究員 (10394636)
|Project Period (FY)
2002 – 2004
Completed(Fiscal Year 2004)
|Budget Amount *help
¥53,430,000 (Direct Cost : ¥41,100,000、Indirect Cost : ¥12,330,000)
Fiscal Year 2004 : ¥13,520,000 (Direct Cost : ¥10,400,000、Indirect Cost : ¥3,120,000)
Fiscal Year 2003 : ¥13,520,000 (Direct Cost : ¥10,400,000、Indirect Cost : ¥3,120,000)
Fiscal Year 2002 : ¥26,390,000 (Direct Cost : ¥20,300,000、Indirect Cost : ¥6,090,000)
|Keywords||cellulosome / cohesin / dockerin / nano-porous silica / chlorophyll / immobilizatiom / NADH synthesis / organization of global warming gas / セルラーゼ複合体 / Clostridium thermocellum / 炭酸ガス固定 / 人工酵素複合体 / 表面プラズモン共鳴 / 人口酵素複合体|
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
olated and characterized
from 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