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標題: 以枯草桿菌為宿主表達纖維素分解酶複合體之研究
The study of express cellulosome in Bacillus subtilis
作者: 蘇蕭咨
Su, Hsiao-Tzu
關鍵字: 纖維素酶複合體;cellulosome;枯草桿菌;bacillus subtilis
出版社: 生命科學系所
引用: Arai, T., S. Matsuoka, et al. (2007). Inaugural Article: Synthesis of Clostridium cellulovorans minicellulosomes by intercellular complementation. Proceedings of the National Academy of Sciences 104(5): 1456-1460. Carvalho, A. L. (2003). Cellulosome assembly revealed by the crystal structure of the cohesin-dockerin complex. Proceedings of the National Academy of Sciences 100(24): 13809-13814. Gold, N. D. and V. J. J. Martin (2007). Global View of the Clostridium thermocellum Cellulosome Revealed by Quantitative Proteomic Analysis. Journal of Bacteriology 189(19): 6787-6795. Pinheiro, Benedita A., Harry J. Gilbert, et al. (2009). Functional insights into the role of novel type I cohesin and dockerin domains fromClostridium thermocellum. Biochemical Journal 424(3): 375-384. Sakka, K., Y. Kishino, et al. (2009). Unusual binding properties of the dockerin module ofClostridium thermocellumendoglucanase CelJ (Cel9D-Cel44A). FEMS Microbiology Letters 300(2): 249-255. Miras, I., F. Schaeffer, P. Beguin, and P. M. Alzari. 2002. Mapping by site-directed mutagenesis of theregion responsible for cohesin-dockerin interaction on the surface of the seventh cohesin domainof Clostridium thermocellum CipA. Biochemistry 41:2115-9. Pages, S., A. Belaich, J. P. Belaich, E. Morag, R. Lamed, Y. Shoham, and E. A. Bayer. 1997.Species-specificity of the cohesin-dockerin interaction between Clostridium thermocell um andClostridium cellulolyticum: prediction of specificity determinants of the dockerin domain.Proteins 29:517-27. Schwarz, W. H. 2001. The cellulosome and cellulose degradation by anaerobic bacteria. Appl. Microbiol.Biotechnol. 56(5-6):634-49. Schaeffer, F., M. Matuschek, G. Guglielmi, I. Miras, P. M. Alzari, and P. Beguin. 2002. Duplicated dockerin subdomains of Clostridium thermocellum endoglucanase CelD bind to a cohesin domainof the scaffolding protein CipA with distinct thermodynamic parameters and a negativecooperativity. Biochemistry 41:2106-14. Shimon, L. J., E. A. Bayer, E. Morag, R. Lamed, S. Yaron, Y. Shoham, and F. Frolow. 1997. Acohesin domain from Clostridium thermocellum: the crystal structure provides new insights into cellulosome assembly. Structure 5:381-90. Spinelli, S., H. P. Fierobe, A. Belaich, J. P. Belaich, B. Henrissat, and C. ambillau. 2000. Crystalstructure of a cohesin module from Clostridium cellulolyticum implications for dockerinrecognition. J Mol Biol 304:189-200. Tamburini, E., A. G. Leon, B. Perito, and G. Mastromei. 2003. Characterization of bacterial pectino lytic strains involvedin the water retting process. Environ Microbiol. 5:730–6. Tavares, G. A., P. Beguin, and P. M. Alzari. 1997. The crystal structure of a type I cohesin domain at 1.7A resolution. J Mol Biol 273:701-13. Tokuda, G., I. Yamaoka, and H. Noda. 2000. Localization of symbiotic clostridia in the mixed segment of the termite Nasutitermes takasagoensis (Shiraki). Appl Environ Microbiol. 66(5):2199-207. Tsuge, K. and M. Itaya . 2001. Recombinational transfer of 100-kilobase genomic DNA to plasmid in Bacillus subtilis 168. J. Bacteriol. 183:5453-5458. Tsuge, K., K. Matsui, and M. Itaya. 2003. One step assembly of multiple DNA fragments with a designed order and orientation in Bacillus subtilis plasmid. Nucleic Acids Res. 31:e133.
Clostridium thermocellum 所產生之cellulosome(纖維素分解酶複合體) 相較目前所發現具纖維素分解能力的真菌,具有更高的纖維素分解能力,為了避開複雜與耗費較高的厭氧步驟,並提高纖維素醣化的工業生產潛力,因此利用cellulosome的概念以具大量胞外蛋白質分泌能力的枯草桿菌 Bacillus subtilis 為宿主,異源表達 C. thermocellum 產生之cellulosome相關的水解酵素與關鍵蛋白。本研究選擇cellulosome中八個主要蛋白:兩個纖維外切酵素 celK (exoglucanase), celS (exoglucanase);兩個纖維內切酵素 celA (endoglucanase), celR (endoglucanase);兩木聚醣分解酵素XynC (xylanase),XynZ (xylanase) 與cellulosome的支架蛋白CipA,及一個膜連接蛋白SdbA。本研究透過OGAB(Oderded Gene Assembly in B.subtilis method )手法構築表現在枯草桿菌,使用pGETS118質體由Pr promoter驅動,各別表達八個蛋白,藉以測試異源cellulosome構造蛋白生產能力與活性,評估枯草桿菌做為醣化酵素測試平台的可能性
未來希望透過 B.subtilis 達到建構外源cellulosom酵素的平台,建構出新的cellulosomal酵素並加以測試能力,期望能提高整體cellulosome纖維素分解能力,以大幅增加纖維素醣化效率。

Clostridium thermocellum cellulosome possess higher cellulolytic activity compared with cellulolytic fungi.Cellulosome (the cellulolytic enzymes complex) is a powerful cellulolytic complex.To produce cellulosome but avoid the complexity of manipulation and higher cost in anaerobic step can improve the potential of industrial saccharification production. As a host,Bacillus subtilis could able to secret extracellular proteins strongly, Taking Bacillus subtilis to heterologous expression C thermocellum cellulosome key hydrolytic enzymes and proteins with a high temperature tolerance and better cellulolytic activity.Cellulosome mainly consists of the following eight major proteins: two the exoglucanases enzyme celK and celS , two endoglucanases enzyme celA , celR , two xylan degradative enzymes XynC (xylanases) and XynZ (xylanase),and the cellulosome scaffold protein CipA, and a menbrane anchor protein SdbA through OGAB (Oderded Gene Assembly in B. subtilis method) techniques to build performance. Bacillus subtilis pGETS118 plasmid driven by Pr promoter,and individual expression of eight proteins to test heterologous cellulosomal protein production capacity and activity, and assess the possibility of producing saccharification enzymes in Bacillus subtilis as a test platform
Hope for the future, To creat outer source cellulosomal enzymes through the B.subtilis platform By new artificial cellulosomal enzyme costruction to improve the overall cellulosome decomposition capacity, and dramatic increase the efficiency of saccharification.
其他識別: U0005-3008201214104200
Appears in Collections:生命科學系所

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