Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3630
標題: Aeromonas caviae NCHU1發酵生產幾丁酵素之純化及特性
Purification and Characterization of Chitinases Produced by Aeromonas caviae NCHU1
作者: 洪哲瑋
Hung, Che-Wei
關鍵字: Aeromonas caviae NCHU1
Aeromonas caviae NCHU1
chitin
chitinase
purification
fermentation-modified chitin
regeneration
幾丁質
幾丁酵素
純化
發酵修飾幾丁質
再生
出版社: 化學工程學系所
引用: 參考文獻 1. Flach, J., Pilet, P.E., and Jolles, P., What’s new in chitinase research? Vol. 48. 1992: Experienta. 701-716. 2. Overdijk, B. and Van Steijn, G.J., Human serum contains a chitinase: identification of an enzyme, formerly described as 4-methylumbelliferyl-tetra-N-acetylchitotetraoside hydrolase (MU-TACT hydrolase). Glycobiology, 1994. 4(6): p. 797-803. 3. Tsujibo, H., Yoshida, Y., Miyamoto, K., Imada, C., Okami, Y., and Inamori, Y., Purification, properties, and partial amino acid sequence of chitinase from a marine Alteromonas sp. strain O-7. Canadian Journal of Microbiology 1992. 38(9): p. 891-897. 4. Roberts, R.L. and Cabib, E., Serratia marcescens chitinase: one-step purification and use for the determination of chitin. Analytical Biochemistry 1982. 127(2): p. 402-412. 5. Watanabe, T., Oyanagi, W., Suzuki, K., and Tanaka, H., Chitinase system of Bacillus circulans WL-12 and importance of chitinase A1 in chitin degradation. Journal of Bacteriology, 1990. 172(7): p. 4017-4022. 6. Ilyina, A.V., Varlamov, V.P., Tikhonov, V.E., Yamskov, I.A., and Davankov, V.A., One-step isolation of a chitinase by affinity chromatography of the chitinolytic enzyme complex produced by Streptomyces kurssanovii. Biotechnology and Applied Biochemistry, 1994. 19: p. 199-207. 7. Mellor, K.J., Nicholas, R.O., and Adams, D.J., Purification and characterization of chitinase from Candida albicans. FEMS Microbiology Letters, 1994. 119(1-2): p. 111-117. 8. Ilyina, A.V., Tikhonov, V.E., Varlamov, V.P., Radigina, L.A., Tatarinova, N.Y., and Yamskov, I.A., Preparation of affinity sorbents and isolation of individual chitinases from a crude supernatant produced by Streptomyces kurssanovii by a one-step affinity-chromatographic system. Biotechnology and Applied Biochemistry, 1995. 21: p. 139-148. 9. Escott, G.M., Hearn, V.M., and Adams, D.J., Inducible chitinolytic system of Aspergillus fumigatus. Microbiology, 1998. 144: p. 1575-1581. 10. Brine, C.J. and Austin, P.R., Chitin variability with species andmethod of preparation. Comparative Biochemistry and Physiology 1981 69B: p. 283-286. 11. Austin, P.R., Brine, C.J., Castle, J.E., and Zikakis, J.P., Chitin:New facets of research. Science, 1981(5): p. 749. 12. 李勳宜, 草蝦幾丁聚醣之製備及其應用研究. 1988: 國立台灣大學農業化學研究所碩士論文. 13. Minke, R. and Blackwell, J., The structure of alpha-chitin. Journal of Molecular Biology, 1978. 120(2): p. 167-181. 14. Hackman, R.H., Studies on chitin. Ⅳ. The occurrence of complexes in which chitin and protein are covalently linked. Journal of Biological Sciences, 1960. 13: p. 568-579. 15. Muzzarelli, R.A., Barontini, G., and Rocchetti, R., Isolation of lysozyme on chitosan. Biotechnology and Bioengineering, 1978. 20(1): p. 87-94. 16. Stanley, W.L., Watters, G.G., Chan, B.G., and Mercer, J.M., Lactose and other enzyme bound to chitin with glutaraldehyde. Biotechnology and Bioengineering, 1975. 17: p. 315-326. 17. 張鈺鱹, 草蝦頭中幾丁質類產品的製備方法、理化性質與應用. 1987: 國立台灣大學農業化學研究所碩士論文. 18. 方紹威, 幾丁質及幾丁聚醣在廢水處理、生化、食品和醫藥上之研究發展現況. 1990: 藥物食品檢驗局調查研究年報. 20-30. 19. 劉瓊淑, 幾丁質,幾丁聚醣及其相關酵素之特性與應用. 食品工業, 1994. 26: p. 26-36. 20. Suzuki, K., Mikami, T., Y., O., Tokoro, A., Suzuki, S., and Suzuki, M., Antitumor effect of hexa-N-acetylchitohexaose and chitohexaose. Carbohydrate Research, 1986. 151: p. 403-408. 21. Yang, Y., Kuramitsu, S., and Hamaguchi, K., Hydrolysis of 4-methylumbelliferyl N-acetyl-chitooligosaccharides catalyzed by human lysozyme. Journal of Biological Chemistry, 1981. 89: p. 1357-1366. 22. Hirano, S., Hiroaki, K., and Motoko, K., N-lower-fatty-derivatives of chitosan adsorbent for lysozyme andchitinase. Agricultural and Biological Chemistry, 1991. 55(6): p. 1683-1684. 23. Roby, D., Gadelle, A., and Toppan, A., Chitin oligosaccharides aselicitors of chitinase activity in melon plants. Biochemical and Biophysical Research Communication, 1987. 143(3): p. 885-892. 24. Hirano, S. and Nagao, N., Effects of chitosan, pectic acid, lysozyme and chitinase on the growth of several phytopathogens. Agricultural and Biological Chemistry, 1989. 53(11): p. 3065-3066. 25. Rupley, J.A., The hydrolysis of chitin by concentrated hydrochoric acid, and the preparation of low-molecular-weight substrates for lysozyme. Biochimical and Biophysical Acta, 1964. 83: p. 245. 26. Barker, S.A., Foster, A.B., Stacey, M., and Webber, J.M., Amino-sugars and related compounds. Part IV. Isolation and properties of oligosaccharides obtained by controlled fragmentation of chitin. Journal of the Chemical Society, 1958: p. 2218-2227. 27. Hirano, S., Tsuneyasu, S., and Konodo, Y., Hetero-geneous distribution of amino groups in partially N-acetylated derivates derivates of chitosan. Agricultural and Biological Chemistry, 1981. 45: p. 1335-1339. 28. 梁舜欣, N-乙醯幾丁寡醣之製備. 1990: 國立台灣大學農業化學研究所碩士論文. 29. Tokuyasu, K., Ono, H., Kameyama, M.O., Hayashi, K., and Mori, Y., Deacetylation of chitin oligosaccharides of dp 2-4 by chitin deacetylase from Colletotrichum lindemuthianum. Carbohydrate research. 1997. 353-358. 30. Usui, T., Hayashi, Y., Nanjo, F., Sakai, K., and Isphido, Y., Transglycosylation reaction of a chitinase purified from Nocardia orientalis. Biochimical and Biophysical Acta, 1987. 923: p. 302-309. 31. Ohtakara, A. and Izume, M., Preparation of D-glucosamine oligosaccharide by enzymatic hydrolysis of chitosan. Agricultural and Biological Chemistry, 1987. 51(4): p. 1189-1191. 32. Powning, R.F. and Irzykiewicz, H., Separation of chitin oligosaccharides by thin-layer chromatography. Journal of Chromatography, 1967. 29(1): p. 115-119. 33. Koga, D. and Kramer, K.J., Inest chitin: physical state, synthesis degradation and metabolic regulation. Insect Biochemistry 1986. 16: p. 851-877. 34. Fenton, D.M. and Eveleigh, D.E., Purification and mode of actionof a chitosanase from Penicillium islandium. Journal of General Microbiology, 1981. 126: p. 151-156. 35. Davis, B. and Eveleigh, D.E., Chitosanases: occurrence, production and immobilization. In Chitin, Chitosan and Related Enzymes, ed. J.P. Zikakis. 1984, Orlando: Academic Press. 161-179. 36. Davies, G. and Herissat, B., Structure and mechanisms of glycosyl hydrolases. Structural Chemistry, 1995. 3: p. 853-859. 37. Terwisscha, V., Scheltinga, A.C., Henning, M., and Dijkstra, B.W., The 1.8 Å resolution on structure of hevamine, a plant chitinase/ lysozyme and analysis of the conserved sequence and structure motifs of glycosyl hydrolase family 18. Journal of Molecular Biology, 1996. 262: p. 243-257. 38. Andersen, M.D., Jensen, A., Robertus, J.D., Leah, R., and Shrivers, K., Heterologous expression and characterization of wild-type and mutant forms of a 26 kDa endochitinase from barley. Biochemical Journal, 1997. 322: p. 815-822. 39. Patil, R.S., Ghormade, V.V., and Deshpande, M.V., Chitinolytic enzymes: an exploration. Enzyme and Microbial Technology, 2000. 26(7): p. 473-483. 40. Felse, P.A. and Panda, T., Production of microbial chitinases ?A revisit. Bioprocess Engineering, 2000. 23: p. 127-134. 41. Tweddell, R.J., Jabaji-Hare, S.H., and Charest, P.M., Production of Chitinases and beta-1,3-Glucanases by Stachybotrys elegans, a Mycoparasite of Rhizoctonia solani. Applied and Environmental Microbiology, 1994. 60(2): p. 489-495. 42. Tagawa, K. and Okazai, K., Isolation and some culyural conditions of Streptomyces species which produce enzymes lysing Aspergillus niger cwll wall. Journal of Fermentation and Bioengineering, 1991. 71: p. 230-236. 43. Felse, P.A. and Panda, T., Self-directing optimization of parameters for extracellular chitinase production by Trichoderma harzianum in batch mode. Process Biochemistry, 1999. 34: p. 563-566. 44. Carmi, O., A, H.B., and Ilan, C., A convenient fluorometric method for the detection of extracellular N-acetylglucosaminidase production by filamentous fungi. Journal of Microbiological Methods, 2001. 43: p. 165-169. 45. Khoury, C., Minier, M., Huynh, N.v., and Goffic, F.i., Optimal dissolved oxygen concentration for the production of chitinases by Serratia marcescens. Biotechnology Letters, 1997. 19: p. 1143-1146. 46. Vyas, P. and Deshpande, M.V., Chitinase production by Myrothecium verrucaria and its significance for fungal mycelia degradation. Journal of General and Applied Microbiology, 1989. 35: p. 343-350. 47. Skujins, J.J., Potgieter, H.J., and Alexander, M., Dissolution of fungal walls by a Streptomycete chitinase and beta-(1-3) glucanase. Archives of Biochemistry and Biophysics, 1965. 111: p. 358-364. 48. Vad, K., Mikkelsen, J.D., and Collinge, D.B., Induction, purification and characterization of chitinase isolated from pea leaves inoculated with Ascochyta pisi. Planta, 1991. 184: p. 24-29. 49. Wen, C.H., Ying, C.C., and Yaw, H.L., Chitinase activity of sweet potato. Botanical Bulletin of Academia Sinica, 1998. 39: p. 93-97. 50. Yamamoto, Y., Fukunaga, Y., Aoyagi, H., and Tanaka, H., Purification and characteristics of chitinase secreted by cultured Wasabia japonica cells. Journal of Fermentation and Bioengineering, 1995. 80: p. 148-152. 51. Romaguera, A., Tschech, A., Bender, S., Plattner, H.J., and Diekmann, H., Protoplast formation by a mycolase from Streptomyces olivaceoviridis and purification of chitinase. Enzyme and Microbial Technology, 1993. 15: p. 412-417. 52. Kumari, J.A. and Panda, T., Studies on critical analysis of factors influencing improved production of protoplasts from Trichoderma reesei mycelium. Enzyme and Microbial Technology, 1992. 14: p. 241-248. 53. Cosio, I.G., Fisher, R.A., and Carroad, P.A., Bioconversion of shellfish chitin waste: waste pretreatment, enzyme production, process design, and economic analys. Journal of Food Science, 1982. 47: p. 901-905. 54. Wu, M.L., Chuang, Y.C., Chen, J.P., Chen, C.S., and Chang, M.C., Identification and characterization of the three chitin-binding domains within the multidomain chitinase Chi92 from Aeromonas hydrophila JP101. Applied and Environmental Microbiology, 2001. 67(11): p. 5100-5106. 55. Hashimoto, M., Ikegami, T., Seino, S., Ohuchi, N., Fukada, H., Sugiyama, J., Shirakawa, M., and Watanabe, T., Expression and characterization of the chitin-binding domain of chitinase A1 from Bacillus circulans WL-12. Journal of Bacteriology, 2000. 182(11): p. 3045-3054. 56. Limon, M.C., Margolles-Clark, E., Benitez, T., and Penttila, M., Addition of substrate-binding domains increases substrate-binding capacity and specific activity of a chitinase from Trichoderma harzianum. FEMS Microbiology Letters, 2001. 198(1): p. 57-63. 57. Danielli, A., Loukeris, T.G., Lagueux, M., Muller, H.M., Richman, A., and Kafatos, F.C., A modular chitin-binding protease associated with hemocytes and hemolymph in the mosquito Anopheles gambiae. Proceedings of the National Academy of Sciences, 2000. 97(13): p. 7136-7141. 58. Tjoelker, L.W., Gosting, L., Frey, S., Hunter, C.L., Trong, H.L., Steiner, B., Brammer, H., and Gray, P.W., Structural and functional definition of the human chitinase chitin-binding domain. Journal of Biological Chemistry, 2000. 275(1): p. 514-520. 59. Chamoy, L., Nicolai, M., Ravaux, J., Quennedey, B., Gaill, F., and Delachambre, J., A novel chitin-binding protein from the vestimentiferan Riftia pachyptila interacts specifically with beta-chitin. Cloning, expression, and characterization. Journal of Biological Chemistry, 2001. 276(11): p. 8051-8058. 60. de la Cruz, J., Hidalgo-Gallego, A., Lora, J.M., Benitez, T., Pintor-Toro, J.A., and Llobell, A., Isolation and characterization of three chitinases from Trichoderma harzianum. European Journal of Biochemistry, 1992. 206(3): p. 859-67. 61. Helisto, P., Aktuganov, G., Galimzianova, N., Melentjev, A., and Korpela, T., Lytic enzyme complex of an antagonistic Bacillus sp. X-b: isolation and purification of components. Journal of Chromatography B, Biomedical Sciences and Applications, 2001. 758(2): p. 197-205. 62. Liu, R.S., Huang, H., Yang, Q., and Liu, W.Y., Purification of alpha-sarcin and an antifungal protein from mold (Aspergillus giganteus) by chitin affinity chromatography. Protein Expression and Purification, 2002. 25(1): p. 50-58. 63. Yang, Q. and Gong, Z.Z., Purification and characterization of an ethylene-induced antifungal protein from leaves of guilder rose (Hydrangea macrophylla). Protein Expression and Purification, 2002. 24(1): p. 76-82. 64. Tikhonov, V.E., Radigina, L.A., Yamskov, I.A., Gulyaeva, N.D., Ilyina, A.V., Anisimova, M.V., Varlamov, V.P., and Tatarinov, N.Y., Affinity purification of major chitinase produced by Streptomyces kurssanovii. Enzyme and Microbial Technology, 1998. 22: p. 82-85. 65. Tikhonov, V.E., Lopez-Llorca, L.V., Salinas, J., and Jansson, H.B., Purification and characterization of chitinases from the nematophagous fungi Verticillium chlamydosporium and V. suchlasporium. Fungal Genetics and Biology, 2002. 35(1): p. 67-78. 66. Ruckenstein, E. and Zeng, X., Macroporous chitin affinity membranes for lysozyme separation. Biotechnology and Bioengineering, 1998. 58(1): p. 117. 67. Zeng, X. and Ruckenstein, E., Macroporous chitin affinity membranes for wheat germ agglutinin puriRcation from wheat germ. Journal of Membrane Science, 1999. 156: p. 97-107. 68. Sakurada, M., Morgavi, D.P., Komatani, K., Tomita, Y., and Onodera, R., Purification and characteristics of cytosolic chitinase from Piromyces communis OTS1. FEMS Microbiology Letters, 1996. 137(1): p. 75-78. 69. Molano, J., Duran, A., and Cabib, E., A rapid and sensitive assay for chitinase using tritiated chitin. Analytical Biochemistry, 1977. 83(2): p. 648-656. 70. Molano, J., Polacheck, I., Cabib, E., and Duran, A., An endochitinase from wheat germ. Activity on nascent and preformed chitin. Journal of Biological Chemistry, 1979. 254(11): p. 4901-4907. 71. Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binging. Analytical Biochemistry, 1976. 72: p. 248-254. 72. 林鎧民, 應用Paenibacilius sp.發酵修飾幾丁質進行幾丁酵素之純化. 2005: 國立中興大學化學工程學系碩士學位論文. 73. Shimahara, K., Y. Takiguchi, K. Ohkouchi and O. Okada, 1984. Chemical composition and some properties of crustacean chitin prepared by use of proteolytic Pseudomonas maltophilia LC102. In: Chitin, Chitosan, and Related Enzymes. (J. P. Zikakis ed.), pp. 239-255. Academic Press, Inc. London. 74. Jung, W.J., Ju-Hee, K., Kil-Yong, K., Tae-Hwan, K. and Ro-Dong, P., Purification and characterization of chitinase from Paenibacillus illinoisensis KJA-424. Journal of Microbiological and Biotechnology, 2005. 15(2): p. 274-280. 75. Sun, C.K., Sanggyu, P., and Dong, G. L. Purification and characterization of a novel chitinase from the entomopathogenic fungus, Metarhizium anisopliae. Journal of Invertebrate Pathology. 1999. 73: p. 276-281.
摘要: 本研究之菌種篩選自台南縣下營鄉田間土壤,經食品工業發展研究所鑑定為Aeromonas caviae,並命名為Aeromonas caviae NCHU1;以幾丁質粉末當作其主要生長源,為一株具有生產幾丁酵素之菌株。所得發酵上清液經硫酸銨沉澱、透析去除鹽類、陰離子交換層析(DEAE-Sepharose)及膠體過濾層析(Gel filitration Sephadex G-75)之純化步驟。獲得單一之酵素經SDS-PAGE測得分子量為26.2kDa,其最適反應溫度、最適反應pH值、熱穩定性及pH穩定性分別為60°C、pH6、10-40°C及 pH5-10。在酵素水解產物之幾丁寡醣分析中,由HPLC圖譜得知,反應後產物為N-乙醯幾丁單醣、二醣、五醣及六醣,因此可以推測Aeromonas caviae NCHU1具有內切型幾丁酵素。 第二階段之研究內容是以Aeromonas caviae NCHU1發酵修飾片狀幾丁質為載體,利用幾丁質對幾丁酵素之親和性來進行純化,即幾丁質親和性層析。以SEM圖觀察幾丁質經發酵修飾後之表面,發現幾丁質由平滑的表面變成多孔狀的結構,推測為幾丁質的直鏈結構,因此推知,經發酵修飾之幾丁質在吸附幾丁酵素時減少了立體障礙,進而提高對酵素的吸附量。文中探討幾丁酵素吸附之最適化條件,溫度、 pH值分別為10°C及pH7。於蛋白質脫附實驗中發現,以0.05M、pH10之Glycine緩衝液可脫附幾丁酵素,純化倍率達3.97倍;接下來探討修飾後幾丁質之再生與再利用性,以0.2M、pH10之Glycine緩衝液清洗載體3次,可脫附殘留於載體上之大部分蛋白質;而經過再生之載體也具備再次吸附酵素能力。
In this investigation, a bacterial strain with chitinase activity was isolated from soil in Tainan and identified as Aeromonas caviae NCHU1. This strain can product chitinases when it is grown in a medium containing chitin power of marine waste. A chitinase was purified from culture broth of Aeromonas caviae NCHU1 by a series of purification steps, i.e. ammonium sulfate precipitation, DEAE-Sepharose, and gel filitration with Sephadex G-75. The molecular weight of NCHU1 chitinase is 26.2kDa by sodium dodecyl sulfate-polyacylamide gel electrophoresis (SDS-PAGE) analysis. The optimum temperature and pH, thermal and pH stability ranges of NCHU1 chitinase are 60°C, pH6, 10-40°C, and pH5-10, respectively. To determine the oligosaccharide contents of NCHU1 chitin hydrolysates, it was found that (GlcNAc), (GlcNAc)2, (GlcNAc)5, and (GlcNAc)6 existed. This implies that Aeromonas caviae NCHU1 may produce endo-chitinase. Furthermore, the affinity chitin chromatography with fermentation- modified chitin was employed for purification of chitinase. By observing the structural changes of chitin and fermentation-modified chitin by SEM, it was found that chitin surface became porous and branching structure, which might reveal more active sites and less the steric hinderance to the adsorption of chitinase. For chitinase adsorption capacity, factors such as loading temperature, pH and elution solutions were studied. The optimal loading conditions were found at 10℃, pH7. The elution solution was 0.05M glycine buffer. Under these conditions, a purification fold of 3.97 was obtained. Then, the modified chitin can be regeneration and reused.
URI: http://hdl.handle.net/11455/3630
其他識別: U0005-1307200715061100
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-1307200715061100
Appears in Collections:化學工程學系所

文件中的檔案:

取得全文請前往華藝線上圖書館



Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.