臺灣博碩士論文加值系統

本研究擬比較微生物來源之纖維素分解酵素活性之差異性，採用Thermomonospora fusca (Actinomycete)、Cellulomonas uda (Bacterium)、Trichoderma reesei (Fungus)等三種菌株，對維纖素(cellulose)、甲基纖維素( methyl cellulose)、醋酸纖維素(cellulose acetate)和微結晶纖維素(Avicel, PH 101)等商品纖維；以及稻草、芒草和牧草等天然纖維進行水解作用。分別測定其纖維素分解酵素在有氧和厭氧活性及產物之薄層色層分析。
根據實驗結果得知，無論商品纖維或天然纖維均能有效被三種菌株所利用，以真菌分解纖維酵素活性最強，其次為放線菌以及細菌。先經過混入真菌於24℃培養四天後之各種cellulose碳源，再經過三種菌株處理均呈現出良好之糖化轉換率。由TLC色層分析圖中得知還原糖種類以葡萄糖及纖維二糖為主。

In order to understand the divergence of induced cellulase activity among Thermomonospora fusca (actinomycete), Cellulomonas uda (bacterium) and Trichoderma reesei (fungus), the commercial fiber: cellulose, methyl cellulose, cellulose acetate and Avicel PH 101; and the natural fiber: rice straws, awns and herbages were taken as carbon source for incubating the three strains in this study. The enzyme activity was monitored by reduction sugar release from carbon source under the experimental condition, aerobic or anaerobic. And thin-layer chromatogram, TLC, was employed to analyze the hydrolysates.
According to results, the induced cellulase activity the three strains were found to be Tri. reesei> Ther. fusca> Cellulo. uda under experimental conditions. After treating with Tri. reesei 4 days at 24℃, the commercial fiber and natural fiber were showing a good saccharification conversion rate. In TLC pattern, that derived form the digestion of commercial fiber and natural fiber by various enzymes the major released reducing sugar were glucose and cellobiose.

中文摘要………………………………………………………………1
英文摘要………………………………………………………………2
壹、前言………………………………………………………………3
貳、文獻整理…………………………………………………………5
一、纖維素的構造………………………………………………‥5
二、纖維素分解酵素種類之介紹…………………………………8
(一)內切型纖維分解酵素…………………………………………8
(二)外切型纖維分解酵素………………………………………8
(三)β-葡萄糖苷酵素……………………………………………9
三、微生物生產纖維酵素的種類…………………………………10
四、纖維分解酵素的作用機制……………………………………20
(一)早期的C1-CX假說…………………………………………20
(二)修正的C1假說………………………………………………20
(三)纖維分解酵素之產物迴饋抑制作用………………………25
五、纖維分解酵素之協同作用……………………………………26
六、微生物分解酵素活性之天然纖維素及經鹼處理……………27
參、材料與方法………………………………………………………29
一、材料……………………………………………………………29
(一) 菌種…………………………………………………………29
(二) 培養基………………………………………………………29
(三)化學藥品………………………………………………29
二、 商品碳源及天然碳源的製備………………………………30
(一)商品碳源……………………………………………………30
(二)天然碳源……………………………………………………30
三、主要儀器設備及器材…………………………………………31
四、培養方法……………………………………………………31
(一)菌種的活化…………………………………………………31
(二)菌株的保存…………………………………………………31
(三)菌種之繼代培養……………………………………………31
(四)增殖培養……………………………………………………32
(五)誘導纖維分解酵素生產用培養基…………………………32
(六)、有氧與厭氧狀態下誘導纖維分解酵素生產用培養基…32
五、纖維分解酵素之活性測定……………………………………33
六、薄層色層分析法測定纖維分解酵素水解產物………………34
肆、結果與討論………………………………………………………35
一、菌種之活化與增殖……………………………………………35
(一)三種菌株之培養方式………………………………………35
(二)三種菌株之生長測定及結果………………………………35
二、有氧與厭氧狀態下三種菌株對不同碳源活性的影響……36
(一)商品纖維在有氧與厭氧性中的活性………………………36
(二)天然纖維在有氧與厭氧性中的活性………………………38
三、三種菌株對纖維所產生之還原糖量及糖化轉換率…………40
(一)三種株菌株對商品纖維產生之還原糖量及糖化轉換率…40
(二)三種株菌株對天然纖維產生之還原糖量及糖化轉換率…42
四、薄層色層分析纖維酵素之水解產物…………………………44
(一)商品纖維水解後產物之薄層色層分析……………………44
(二)天然纖維水解後產物之薄層色層分析……………………45
五、培養基經二次接種三種菌株培養後之最終pH值…………46
伍、結論………………………………………………………………48
陸、參考文獻…………………………………………………………49

王鳳英。1991。利用固態發酵以玉米穗軸生產纖維分解酵素。國立台灣大學農業化學研究所博士學位論文。台北。

王三郎。梁瑞麟。蔡育達。1995。氫氧化鈉前處理對酵素糖化農業廢棄物之影響。中國環境工程學刊5: 309-316。

西澤俊一。1974。纖維素總論。南山堂出版社。

余碧。陳乃菁。曾浩洋。1993。Trichoderma reesi 羧甲基纖維素酶之部份特性與純化。中國農業化學會誌。31: 614-622。

林君泰。2002。微生物混合固體醱酵對纖維分解酵素活性之影響。國立屏東科技大學食品科學研究所碩士學位論文。屏東。

林澤揚。1996。Trichoderma longibrachiatum 185聚木糖酶之特性探討半纖維素碳源對酵素誘導之影響。國立中興大學食品科學研究所碩士學位論文。台中。

周姿佑。1999。農業纖維素誘導嗜高溫菌生產纖維素酶之探討。國立台灣大學食品科學研究所碩士學位論文。台北。

郭堂彬。2000。鹼處理農產基質對嗜高溫菌生產纖維酶之影響。國立台灣大學食品科學研究所碩士學位論文。台北。

曾秀月。1992。用鹼液萃取稻穀半纖維素。中華生質能源學會會誌。11(1-2): 31-41。

黃妃君。1997。纖維素分解酵素生產菌的篩選培養條件及糖化反應之探討。國立屏東科技大學食品科學研究所碩士學位論文。屏東。

詹峰順。陳國樹。楊盛行。黃景軍。1998。纖維素酶活性及對稻草堆肥品質之影響。中國農學會報。185:58-71。

廖惠。1975。微生物纖維素分解酵素之生產與利用研究。私立文化大學家政學研究所碩士學位論文。台北。

廖彩娟。2000。微生物間之纖維分解酵素的協同作用。國立屏東科技大學食品科學研究所碩士學位論文。台北。
廖啟成。袁國芳。李福臨。王伯徹。2000。細菌目錄。財團法人食品工業發展研究所。第一版。新竹。

廖啟成。袁國芳。李福臨。王伯徹。2000。真菌目錄。財團法人食品工業發展研究所。第一版。新竹。

趙乃昕。王尊哲。蔡文城。馬麥生。1999。醫學細菌詞彙及分類鑑定。九州圖書文物有限公司，第一版。台北。

葉丁源。1997。嗜高溫放線菌纖維素分解酵素之探討。國立台灣大學農業化學研究所碩士學位論文。台北。

賴敏男。1977。微生物纖維分解酵素之生產與利用。食品工業9(2):20-29。

蔡芷季。呂玉梅。蔡珊珊。1991。利用酵素將鹼處理過之稻草糖化。中國農業化學會誌。29：145-153。

闕麗卿。李洪朝。1986。以纖維素物質生產丙酮丁醇。1.纖維素物質之酵素水解。中國農業化學會誌。24：320-329。

戴曉蕾。2001。Aspergillus fumigatus 纖維分解酵素之純化與生化特性探討。國立屏東科技大學食品科學碩士學位論文。屏東。

Abd El-Nasser, N. H., Helmy, S. N., El-Gammal, A. A. 1997. Formation of enzymes by biodegredation of agricultural waste with white rot fungi. Polymer Degradation and stability.Journal. 55: 249-255.

Aiello, C., Ferrer, A., and Ledesma, A. 1996. Effect of alkaline treatment at various temperatures on cellulase and biomass production using submerged sugarcane bagasse fermentation with Trichoderma reesei QM9414. Biores. Technol. 57: 3-18.

Arai, M., Sadamoto. R., and Murao, S. 1987. Enzymatic properties of two avicelase form Aspergillus aculeatus No. F-50. Agric. Biol. Chem. 51: 627-633.

Beguin, P. and Aubert, J. P. 1992. La de’gradation de la cellulose parles microorganisms Amm. Inst. Pasteur/Actualit’es 3: 91-115.

Beguin, P. and Aubert, J. P. 1994. The biological degradation of cellulose FEMS Microbiol. 13: 25-58.

Bisaria, V. S. and Ghose. 1981. Biodegradation of cellulosic materials: Substrates, microorganisms enzymes and products. Enzyme Microb. Technol. 3: 90-104, 123.

Candace, H. H. and Paul, J. W. 1991. Biosynthesis and Biodegradation of Cellulose. Marcel Dekker, New York.

Chen, S., and Wayman, M. 1991. cellulase production induced by carbon source derived form waste newspaper. Process Biochem. 25: 93-100.

Claseyssens, M. Tilbeurgh, H. V., Tomme, P., wood, T. M., and Mcrae, S. I. 1989. Fungal cellulase system comparison of the specificities of the cellobiohydrolases isolated form Penicillium pinophilum and Trichoderma reesi. Biochem. J. 261: 819-825.

Claseyssens, M., and Aerts, G. 1992. Characterization of cellulolytic activities in commercial Trichoderma reesei preparations: an approach using small, chromogenic substrates. Biores. Technol. 39: 143-146.

Correa, M. G., Portal, L., Moreno, P., Tengerdy, R.P. 1999. Mixed culture solid substrate fermentation of Trichoderma reesei With Aspergillus niger on sugar cane bagasse. Biores. Technol. 68: 173-178

Curreli, N., Fadda, M. B., Rescigno, A., Rinaldi, A. C., Soddu, G., Sollsi, F., Vaccargiu, S., Sanjust, E., and Tinalde, A. 1997. Mild alkaline/oxidative pretreatment of wheat straw. Process Biochem. 32: 665-670.

David, N. S. H. 1991. Wood and cellulosic chemistry. Marcel Dekker, New York.

Emtiazi, G., Nahvi, I. 2000. Multi-enzyme production by Cellulomonas sp. grown on wheat straw. Bioma. and Bioene. 19: 31-37.

Ghose, T. K. 1987. Measurement of cellulase activities. Pure and Appl. Chem. 59: 257-268.

Han, Y, W., and Callihan, C. D. 1974. Cellulose fermentation: effect of substrate pretreatment on microbiol growth. Appl. Microbiol. 27: 159-165

Hudson, J. A., Morgan, H. W., Daniel, R. M. 1991. The cellulase activity of an extreme thermophile. Appl. Microbiol. Biotechnol. 35: 270-273.

Knapp, J. S. and Legg, M. 1986. The effect of different cellulosic growth substrates and pH on the production for cellulolytic enzyme by Trichoderma reesei. J. Appl. Bacteriol. 61: 319-329.

Kubicek, C. P. 1987. Involvement of a conidial endoglucanase and a plasma-memberane bound glucosidase in the induction of endoglucanase synthesis by cellulose in Trichoderma reesei. J. Gen. Microbiol. 133: 1481-1487.

Liaw, E. T. 1994. The relative active activity of the cellulase enzyme system of Trichoderma reesei QM 9491 with native and modified cellulosic substrates. Ph. D. Dissertation, Oregon state University.

Lrwin, D. C., Spezio, M. Walder, L. P. and Wilson, D. B. 1993. Activity studies of eight purified cellulases : specificity, synergism, and binding domain effects. Biotechnol. Bioeng. 42 : 1002-1013.

Mandels, M. and Sternberg, D. 1976. Recent advances in Cellulase technology. J. Ferment. Technol. 54: 267-286.

Mandels, M. and Andreotti, R. E. 1978. Problems and challenges in the cellulose to cellulase fermentation. Process Biochem. 13: 6-13.

Miller, G. L. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31: 426-428.

Reese, E. T., Sin, R. G. and Levinson, H. 1950. The biological degradation of soluble cellulose derivatives and its relationship to the mechanism of cellulose hydrolysis. J. Bacteriol. 59: 485-493.

Roa, M. Gaikwad, S., Mishra, C., and Deshpande, V. 1988. Induction and catabolite repression of cellulase in Penicillium funiculosum Appl. Biochem. Biotechnol. 19: 129-101.

Ryu, D. D., Kim, C., and Mandels, M. 1984. Competitive adsorption of cellulase components and its significance in a synergistic mechanism. Biotechnol. Bioeng. 26: 488-496.

Suto, M. and Tomita, F. 2001. Review: Induction and catabolite repression of mechanisms of cellulase in fungi. Journal of bioscience and bioengineering. 92(4): 305-311.

Virendra, S. B. and Mishra, S. 1989. Regulatory aspects of cellulase biosynthesis and secretion. Crit. Rev. Biotech. 9: 61-103.

William, M. F. 1983. Mirobial enzyme and biotechnology. Applied Science Publishers, New York.

Woodward, J. and Arnold, S. L. 1981. The inhibition of β-glucosidase
activity inTrichoderma reesei C30 cellulase by derivatives and isomers of glucose. Biotechnol. Bioeng. 23: 1553-1562.

Wood, T. M. and McCrae, S. I. 1986. The Cellulase of Penicillium pinophilum: Synergism bewteen enzyme components in solubilizing cellulose with special reference to the involvement of two immunologically distinct cellulobiohydrolases. Biochem. J. 234: 93-99.

Zhang, S., Lao, G., and Wilson, K. B. 1995. Characterization of a Thermomonospora fusca exocellulase. Biochem. 34: 3386-3395.