臺灣博碩士論文加值系統

本研究以樟芝Antrodia camphorata BCRC 35396於攪拌式發酵槽培養下，探討不同通氣量對於樟芝發酵生產脂解酵素的影響，並將最適條件之發酵液進行酵素的分離純化。由實驗結果顯示，0.3 vvm的最大脂解酵素活性為1.151 U/ml，而1.0 vvm的最大酵素活性為2.588 U/ml，大約提升了2.25倍，所以通氣量的增加有利於脂解酵素活性的提升。而YL/P(specific activity)顯示，0.8 vvm有較高的比活性(29.493 U/mg)。在蛋白質的生成方面，0.3 vvm的最大蛋白質含量為0.061 mg/ml，而1.0 vvm的最大蛋白質含量為0.120 mg/ml，大約提升1.97倍，所以提升通氣量對於蛋白質含量有提高的效果。
脂解酵素的純化步驟是將發酵澄清液利用冷凍乾燥再經由DEAE Sepharose陰離子交換管柱層析分離純化。經過冷凍乾燥處理的脂解酵素活性及比活性分別為341.6 U及29.19 U/mg。而通過陰離子交換管柱層析後，純化倍率為4.50倍，回收率為8.28％。最後由SDS-PAGE電泳分析，發現最後純化的脂解酵素分子量是60.0 kDa。

This work studied the production and purification of lipase fermented from Antrodia camphorata BCRC 35396 by aeration rate in the stired tank. Shown by the experimental result, the more aeration rate can enhance the lipase activity. The biggest lipase activity of 1.0 vvm was 2.25 times higher than that of 0.3 vvm. The specific activity YL/P show the 0.8 vvm has the higher quality than other aeration rate. The biggest protein of 1.0 vvm was 1.97 times higher than one of 0.3 vvm.
Lipase fermented from Antrodia camphorata BCRC 35396 was purified by lyophilization as concentration, follow by anion exchange chromatography(DEAE-Sepharose). The specific activity of the purified enzyme was 4.5 times higher than that of the crude broth after lyophilization. The lipase had a molecular weight of 60.0 kDa when examined by SDS-polyacrylamide gel electrophoresis.

摘要……………………………………………………………………...I
目錄……………………………………………………………………...Ⅱ
表索引…………………………………………………………………...Ⅵ
圖索引…………………………………………………………………...Ⅶ

第一章 緒論………………………………………………………….....1
1-1研究動機…………………………………………………….. .....1
1-2研究目的…………………………………………………….. .....2
第二章 文獻回顧…………………………………………………….....3
2.1菇類…………….…………………………………………….. .....3
2.2 樟芝…………………………………………………………........3
2.2.1 分類…………….………………………………………. .....3
2.2.2 型態…………………………………..……………......4
2.2.3 成分……………..…………………………………......5
2.2.4 功用…………………………………………………......5
2.3 酵素………………………………………………………......6
2.3.1 脂解酵素………………………………………………....9
2.3.1.1 脂解酵素簡介……………………………………….9
2.3.1.2 脂解酵素應用…..……………………………….....14
2.4 酵素之分離與純化………………………………………….18
2.4.1 濃縮方法………………………………………………..18
2.4.1.1 離心………………………………………………...18
2.4.1.2超微薄膜過濾法…….……………………………...19
2.4.1.3 其他濃縮方法……………………………………...20
2.4.1.4 層析法……………………………………………...20
2.4.1.4.1 離子交換管柱層析…………………………....21
2.4.1.4.2疏水性作用管柱層析………………………….22
2.4.1.4.3親和性作用管柱層析………………………….22
2.4.1.4.4膠體過濾管柱層析…………………………….23
2.4.2 快速蛋白質液相層析儀(FPLC)……………………….23
第三章 實驗材料與方法……………………………………………..25
3.1 實驗材料…………………………………………………….25
3.1.1 實驗菌株………………………………………………..25
3.1.2 實驗藥品………………………………………………..25
3.1.3 實驗儀器及其他設備…………………………………..26
3.1.4 實驗裝置......................................27
3.2 實驗方法…………………………………………………...27
3.2.1 菌株保存………………………………………………27
3.2.2 培養基組成.…………………………………………….28
3.2.3 操作方法………………………………………………29
3.2.4分析方法…………………………………………………30
1. 發酵液處理流程……………………………………………30
2. 菌重分析……………………………………………………32
3. 葡萄糖殘量分析…………………………………………....32
4. 冷凍乾燥……………………………………………………33
5. 管柱層析…………………………………………………....33
陰離子交換管柱層析……………………………………...33
6. 酵素活性分析……………………………………………….35
7. 蛋白質含量測定...................................36
8. SDS膠體電泳（SDS-PAGE）……………………………...37
第四章 結果與討論………………………………………………….41
4.1樟芝菌絲型態…………………………………………………41
4.1.1固態培養………………………………………………….41
4.1.2 搖瓶培養…………………………………………………42
4.1.3 攪拌式發酵槽……………………………………………43
4.2 不同通氣量攪拌槽批次實驗………………………………...44
4.2.1發酵時間曲線比較……………………………………….44
4.2.2不同通氣量對於菌體生長的影響…………… ………....47
4.2.3不同通氣量對於生產脂解酵素的影響………………….49
4.2.4 不同通氣量對於生產蛋白質的影響……………………51
4.3 脂解酵素的純化……………………………………………...53
4.3.1 發酵澄清液………………………………………………53
4.3.2 陰離子交換管柱層析……………………………………54
4.3.3 SDS膠體電泳（SDS-PAGE）…………………………55
第五章 結論與未來展望…………………………………………58
5-1結論…………………………………………………………...58
5-2未來展望………………………………………………. .59
參考文獻………………………………………………………………..60

Abramic, M., Lescic, I., Korica, T., Vitale, L., and Saenger, W., ( 1999) “Purification and properties of extracellular lipase from Streptomyces rimosus”, Enzyme and Microbial Technology, 25, 522-529.
Brzozowski, A. M., U. Derewenda, Z. S. Derewenda, G. G. Godson, D. M. Lawson, J. P. Turkenburg, F. Bjorkling, B. Huge-Jensen, S. A Patkar, L. Thim, (1991)“A model for interfacial activation in lipases from the structure of a fungal lipase-inhibitor complex”, Nature, 351, 491-494.
Cardenas, F., Alvarez, E., M.S. de Castro-Alvarez, Sanchez-Montero, J.M., Valmaseda, M., Elson, S.W., Sinisterra, J.V., (1991)“Screening and catalytic activity in organic synthesis of novel fungal and yeast lipases”, Journal of Molecular Catalysis B: Enzymatic, 14, 111–123.
Chang, H. L., Chao, G. R., Chen, C. C., and Mau, J. L., (2001) “Non-volatile taste components of Agaricus blazei, Antrodia camphorata and ordyceps militaris mycelia”, Food Chem., 74, 203-207.
Chang, S. T., and Miles, P.G., “Recent trends in word production of cultivated mushrooms”, Mushroom Journal, 1991, 503, 15-18.
Chaplin, M.F., and Bucke, C., “Enzyme technology”, © 1990 Cambridge University Press.
Chen, C. H., and Yang, S. W., (1995)“New steroid acids from Antrodia cinnamomea a fungal parasite of Cinnamomum microanthum”, J. Nat. Prod, 58, 1655-1661.
Chen, J.Y., Wen, C.M., and Chen, T.L., (1999)“Effect of Oxygen Transfer on Lipase Production by Acinetobacter radioresistens”, Biotechnology and Bioengineering, 62, 311-316.
Chen, S.J., Cheng, C.Y., and Chen, T.L., (1998)“Production of an Alkaline Lipase by Acinetobacter radioresistens”, Journal of Fermentation and Bioengineering, 86, 308-312.
Crueger, W., and A. Crueger, (1984)“Biotechnology: a textbook of industrial microbiology”, Science Tech, Madison, WI, 177-178.
Cunha, A. E., M. de Fatima Silva Lopes, Clemente, J. J., Teixeira Carrondo, M. J., and Barreto Crespo, M. T., (1999)“Influence of environmental factors on lipase production by Lactobacillus plantarum”, Applied Microbiology and Biotechnology, 51, 249-254.
Dalmau, E., Montesinos, J.L., Lotti, M., and Casas, C., (2000)“Effect of different carbon sources on lipase production by Candida rugosa”, Enzyme and Microbial Technology, 26, 657–663
Fredrik, B., E. G. Sven, and K. Ole, (1991)“The future impact of industrial lipases”, TIBTECH, 9, 360-363.
Frost, G. M., and D. A. Moss, “Production of enzymes by fermentation”, 1987, Biotechnology, Rehm, H. J., Reed, G., Eds. VCH Publishers: New York, 7a, 113-121.
Genovefa, Z., Wenzig, E., and Mersmann, A., (1994)“Improvement of lipase production by addition of catalase during fermentation”, Applied Microbiology and Biotechnology, 40, 650-652.
Gerhartz, W., “Enzyme in Industry”, © VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990.
Gordillo, M.A., Obradors, N., Montesinos, J.L., Valero, F., Lafuente, J., and Sola, C., 1995 (1995)“Stability studies and effect of the initial oleic acid concentration on lipase production by Candida rugosa”, Applied Microbiology and Biotechnology, 43, 38-41.
Gupta, N., Rathi, P., and Gupta, R., (2002)“Simplified para-nitrophenyl palmitate assay for lipases and esterases”, Analytical Biochemistry, 311, 98-99.
Hawksworth, D.L., Kirk, P.M., Sutton, B.C., and Pegler, D.N., (1996)“Dictionary of the fungi”, Internal Mycological Institute.
Janson, J.C., and Ryden, L., “Protein purification”, © 1998 by John Wiley ＆ Sons, Inc.
Kordel, M., Hofmann, B., Schomburg, D., and Schmid, R. D.,( 1991) “Extracellular Lipase of Pseudomonas sp. Strain ATCC 21808: Purification, Characterization, Crystallization, and Preliminary X-Ray Diffraction Data”, Journal of Bacteriology, 173, 4836-4841.
Krieger, N., Taipa, M.A., Melo, E.H.M., Lima-Filho, J.L., Aires-Barros, M.R., and Cabral, J.M.S., (1999)“Purification of a Penicillium citrinum lipase by chromatography processes”, Bioprocess Engineering, 20, 59-65.
Large, K. P., Mirjalili, N., Osborne, M., Peacock, L.M., Zormpaidis, V., Walsh M., Cavanagh M.E., Leadlay P.F., and Ison A.P., (1999) “Lipase activity in Streptomycetes”, Enzyme and Microbial Technology, 25, 569-575.
Lotrakul, P., and Dharmsthiti, S., (1997)“Purification and characterization of lipase from Aeromonas sobria LP004”, Journal of Biotechnology, 54, 113-120.
Maia, M.M.D., Heasley, A., M.M. Camargo de Morais, Melo, E.H.M., Morais Jr., M.A., Ledingham, W.M., and Lima, Filho J.L., (2001) “Effect of culture conditions on lipase production by Fusarium solani in batch fermentation”, Bioresource Technology, 76, 23-27.
Pencreac’h, G., and Baratti, J. C., (1997)“Activity of Pseudomonas cepacia lipase in organic media is greatly enhanced after immobilization on a polypropylene support”, Applied Microbiology and Biotechnology, 47, 630-635.
Pencreac’h, G., and Baratti, J. C., (1996)“Hydrolysis of p- nitrophenyl palmitate in n-heptane by the Pseudomonas cepacia lipase: A simple test for the determination of lipase activity in organic media”, Enzyme and Microbial Technology, 18, 417-422.
Pratt, J., Cooley, J. D., Purdy, C.W., Straus, D.C., (2000)“Lipase Activity from Strains of Pastrurella multocida”, Current Microbiology, 40, 306-309.
Rapp, P., (1995)“Production, regulation, and some properties of lipase activity from Fusarium oxysporum f. sp. vasinfectum”, Enzyme and Microbial Technology, 17, 832-838.
Sarkar, S., Sreekanth, B., Kant, S., Banerjee, R., and Bhattacharyya, B.C., (1998)“Production and optimization of microbial lipase”, Bioprocess Engineering, 19, 29-32.
Saxena, R.K., Sheoran, A., Giri, B., and Davidson, W. S., (2003) “Purification strategies for microbial lipases”, Journal of Microbiological Methods, 52, 1 – 18
Scopes, R.K., “Protein Purification”, © 1994 Springer-Verlag New York, Inc.
Sharma, R., Chisti, Y., and Banerjee, U.C., (2001)“Production, purification, characterization, and applications of lipases”, Biotechnology Advances, 19, 627-662.
Sharon, C., Furugoh, S., Yamakido, T., Ogawa, H.I., and Kato, Y.,( 1998) “Purification and characterization of a lipase from Pseudomonas aeruginosa KKA-5 and its role in castor oil hydrolysis”, Journal of Industrial Microbiology & Biotechnology, 20, 304–307
Shuler, M.L., and Kargi, F., “Bioprocess Engineering”, © 2002 by Prentice Hall PTR, 57-58.
Smith, G. M., (1995)“The nature of enzymes”, Biotech., 9, 7-72.
Sokolovska, I., Albasi, C., Riba, J.P., and Bales, V., (1998)“Production of extracellular lipase by Candida cylindracea CBS 6330”, Bioprocess Engineering, 19, 179-186.
Vulfson, E.N., (1994)“Industrial applications of lipases”, In Woolley, P., and Perersen, S.B., Lipase, Cambridge University Press, Cambridge, UK 271-288.
Walsh, G., and Headon, D., (1994)“Protein biotechnology”, John Wiley & Sons, New York, 302-336.
Winkler, U.K., and Stuckmann, M., (1979)“Glycogen, hyaluronate, and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens”, Journal of Bacterial, 138, 663-670.
水野卓、川合正允，“菇類的化學，生化學”，1997，賴慶亮譯，國立編譯館。
王伯徹，“具開發潛力食藥用菇介紹”， 食品工業，2000，32：1-17。
王惠民，“以正十六烷回收Acinetobacter radioresistens 脂肪酵素之研究”， 國立成功大學化學工程研究所，博士論文，2002。
王超群，“固定Pseudomonas fluorescens 脂肪酵素於幾丁聚糖之研究”，國立清華大學化學工程研究所，碩士論文，2001。
田蔚城，“生物技術的發展與應用”，1997，九州圖書文物有限公司。
朱建儒，“探討通氣量對於樟芝發酵生產生物鹼之影響”，國立中央大學化學工程與材料工程研究所，碩士論文，2003。
江文德，“脂肪脢在油脂加工上的應用”，1999，食品工業月刊，31， 10-19。
徐維富，“以基因轉殖菌株生產creatinase：醱酵、分離純化及酵素物性與化性探討”，國立成功大學化學工程研究所，碩士論文，2000。
高曉薇，“台灣靈芝屬新種樟芝之三帖類成份研究”，台北醫學院天然物醫學研究所，碩士論文，1991。
張東柱，“牛樟之病害”，牛樟生物學及育林技術研討會論文集，林業叢刊第72號，1997，127-131。
張淑微，“以反應曲面法研究酵素合成己醇酯類之最優化”，大業大學食品工業研究所，碩士論文，2002。
莊榮輝網頁系統 (Juang Web) juang.bst.ntu.edu.tw/
黃鈴娟，“樟芝與姬松茸之抗氧化物質及其多醣組成分析”，國立中興大學食品科學研究所，碩士論文，2000。
溫智敏，“攪拌與通氣對以Acinetobacter radioresistens發酵生產脂肪分解酵素之影響，碩士論文，1997。
蔡雁暉，“樟芝深層培養液及其多醣體之抗氧化特性”， 國立中興大學食品科學研究所，碩士論文，2002。
戴宇昀，“樟芝菌絲體與子實體對四氯化碳及酒精誘導之慢性及急性肝損傷之保肝功能評估”，2001。
簡秋源、姜宏哲、陳淑貞，“牛樟菇培養性狀及其三帖類成分分析之研究”，牛樟生物學及育林技術研討會論文集，林業叢刊第72號，133-137。