碩士 / 國立臺灣大學 / 生化科學研究所 / 105 / Cellulose and hemicelluloses from plant biomass can be used to produce biofuel after enzymatic process. We have engineered Clostridium thermocellum Cel5E (CtCel5E), a bifunctional cellulase/xylanase, into a tri-functional cellulase/xylanase/mannanase, named CtCel5E-Tmloop, by replacing its flexible loop region with that in Thermotoga maritima Cel5A (TmCel5A), a homologous bifunctional cellulase/mannanase, suggesting that the Tmloop might be essential for multi-functionality.
In this thesis, the recombinant TmCel5A expressed in E. coli was purified and characterized. Surprisingly, TmCel5A also showed low xylanase activity, in addition to the known cellulase and mannanase activities. By site-directed mutagenesis, H205A, W210A, and Y198A in the loop region showed significantly reduced activities, indicating their importance in catalysis. In particular, H205 and W210 are essential for mannanase activity. Moreover, by searching the conserved Tmloop structures in other cellulases, we identified Clostridium thermocellum CelC (CtCelC) also with tri-functional cellulase/xylanase/mannanase activity.
| Identifer | oai:union.ndltd.org:TW/105NTU05103013 |
| Date | January 2017 |
| Creators | Tsung-Yi Lin, 林瑽羿 |
| Contributors | 梁博煌 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 46 |
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