1999 Fiscal Year Final Research Report Summary
Functional analysis of substrate binding domain in cellulase and hemicellulase
| Project/Area Number |
10650779
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
生物・生体工学
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| Research Institution | Nagaoka University of Technology |
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Principal Investigator |
OKADA Hirofumi Nagaoka University of Technology, Engineering, Associate Professor, 工学部, 助教授 (70233343)
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| Co-Investigator(Kenkyū-buntansha) |
NOGAWA Masahiro Nagaoka University of Technology, Engineering, Research Associate, 工学部, 助手 (10283037)
MORIKAWA Yasushi Nagaoka University of Technology, Engineering, Professor, 工学部, 教授 (50239638)
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| Project Period (FY) |
1998 – 1999
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| Keywords | Cellulase / Endoglucanase / Cellobiohydrolase / Cellulose binding domain / Trichoderma reesei / Schizosaccharomyces pombe / Arabinofuranosidase / Xylan binding domain |
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| Research Abstract |
(1) Improvement of secretion in Schizosaccharomyces pombe of Trichoderma reesei endoglucanase III (EG III) and arabinofuranosidase (AF)
To study on enzymatic characterization of the chimera enzyme of EG III with a cellulose binding domain and on functional analysis of C-terminal xylan binding domain of AF, heterologous protein secretion system in S. pombe was improved for EG III and AF expression. Among the EG III signal peptide substituted to those of other cellulases and a xylanase, the CBH II signal peptide led to 3-fold increase the EG III production than the native signal peptide. Furthermore AF substituted with CBH II signal peptide was secreted in S. pombe to 100-fold compared to its own signal peptide.
(2) Effect of cellulose binding domain (CBD) in addition to EG III for cellulose hydrolysis
To clarify the role of CBD for enzymatic cellulose hydrolysis, the chimera EG IIIs were constructed by the EG III, which consists of single catalytic domain without CBD, joined to the CBDs from other cellulases. Two chimera enzymes, the CBD of T. reesei CBH I or CBH II attached to the EG III, were expressed in S. pombe with the CBH II signal peptide. Although the chimeras increased in molecular mass because of over-glycosylation, their specific activity was unchanged before and after the addition of the CBD. Adsorption experiment of the enzymes with cellulose suggested the CBD in chimera to be functional. Either EG III with CBD increased in the hydrolysis activity for bacterial microcrystalline cellulose and in synergistic enzymatic reaction with CBH for crystalline cellulose.
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