纖維素結合功能域對木質醋酸菌生產細菌纖維素之效應

Effect of cellulose-binding domains on bacterial cellulose production by Gluconacetobacter xylinus

劉人漢(Ren-Han Liu)

指導教授 ： 蘇文達

國立臺北科技大學/工程學院/化學工程研究所/碩士(2013年)

摘要

細菌纖維素生產菌株中以木質醋酸菌Gluconacetobacter xylinus 所產生之細菌纖維素量遠高於其它生產菌株，近年來有許多研究皆著重於降低發酵成本及提升細菌纖維素品質，而纖維素結合功能域 (Cellulose-binding domains)由文獻記載能促進植物纖維素生長，且能增加細菌纖維素之合成速率，本研究利用木質醋酸菌異源表現纖維素功能域與雙纖維素結合功能域 (Double cellulose-binding domains)以及外加纖維素功能域與雙纖維素結合功能域於培養基當中，期以提升細菌纖維素產量與提升細菌纖維素品質。研究結果顯示，以建構CBD與dCBD之表現質體轉殖入G. xylinus中，能成功表現出CBD，而基改G. xylinus與外加CBD實驗比較顯示以基改G. xylinus較好，在30℃靜置培養8天其細菌纖維素產量最高能達 2.78 g/l，相較於對照組能提升20.4 %；另在細菌纖維素品質方面結晶度從78.0 % 提升至 86.8 %，保水力從 83.4 % 提升至 94.7 %。因此木質醋酸菌異源表現CBD蛋白質不僅提升細菌纖維素產量更能提升品質。

關鍵字

纖維素結合功能域 ； 木質醋酸菌 ； 細菌纖維素

並列摘要

Bacterial cellulose can be produced by Gram-negative bacterium, such as rod-shaped Gluconacetobacter xylinus. As the organism is obligate aerobic, bacterial cellulose is always produced at the air/liquid interface. In recent years there have been more reports about minimizing the cost of culture and improving the quality of bacterial cellulose. Cellulose-binding domains (CBD) have previously been shown to modulate the elongation of cellulose. In this work, the cellulose-binding domains gene and double cellulose-binding domains (dCBD) were cloned and successfully expressed in G. xylinus. In this study, the feasibility of using G. xylinus to drive the expression of heterologous proteins as well as the function of expressed CBD to enhance cell growth, cellulose productivity, crystallinity, water-holding capacity and at static culture was studied. After 8 days culturing at 30℃, the maximum cell density was 1.16 g/l while bacterial cellulose density was 2.78 g/l (20.4 % higher than control). In terms of bacterial cellulose (BC) quality, the crystallinity index increased from 78.0 % to 86.8 % while water-holding capacity increased from 83.4 % to 94.7 %. The expression system in this study is very successful in that it not only increased cell density and cellulose productivity, but also increased the BC quality.

並列關鍵字

Cellulose-binding domains ； Bacterial cellulose ； Gluconacetobacter xylinus

參考文獻

[1] M. Linder, I. Salovuori, L. Ruohonen and T. T. Teeri, "Characterization of a double cellulose-binding domain : synergistic high-affinity binding to cellulose," The Journal of Biological Chemistry, vol. 271, no. 35, 1996, 21268-21272.

[3] P. Tomme, R. A. Warren and N. R. Gilkes, "Cellulose hydrolysis by bacteria and fungi," Advances in Microbial Physiology, vol. 37, 1995, pp. 1-81.

[6] H. Van-Tilbeurgh, P. Tomme, M. Claeyssens and R. Bhikhabhai, "Limited proteolysis of the cellobiohydrolase I from Trichoderma reesei: Separation of functional domains," Federation of European Biochemical Societies Letters, vol. 204, no. 1, 1986, pp. 223-227.

[8] Shoseyov, T. Hamamoto, F. Foong and R. H. Doi, "Cloning of Clostridium cellulovorans endo-1,4-beta-glucanase genes," Biochemical and Biophysical Research Communications, vol. 169, no. 2, 1990, pp. 667-672.

[10] P. Tomme, A. Boraston, B. McLean, J. Kormos, A. L. Creagh, K. Sturch, N. R. Gilkes, C. A. Haynes, R. A. Warren and D. G. Kilburn, "Characterization and affinity applications of cellulose-binding domains," Journal of Chromatography B: Biomedical Sciences and Applications, vol. 715. no. 1, 1998, pp. 283-296.