(3.236.228.250) 您好!臺灣時間:2021/04/20 00:45
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果

詳目顯示:::

我願授權國圖
: 
twitterline
研究生:邱雅婷
研究生(外文):Ya-Ting Chiou
論文名稱:溫泉菌Tepidimonas taiwanensis I1-1蛋白酶 的純化及特性探討
論文名稱(外文):Purification and characterization of protease from a hot spring microorganism of Tepidimonas taiwanensis I1-1
指導教授:郭建民郭建民引用關係
指導教授(外文):Jen-Min Kuo
口試委員:吳建輝林穎聖
口試委員(外文):Chien-Hui WuYing-Sheng Lin
口試日期:2014-07-26
學位類別:碩士
校院名稱:國立高雄海洋科技大學
系所名稱:水產食品科學研究所
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:124
中文關鍵詞:Tepidimonas taiwanensis I1-1蛋白酶純化
外文關鍵詞:Tepidimonas taiwanensis I1-1proteasepurification
相關次數:
  • 被引用被引用:0
  • 點閱點閱:463
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:70
  • 收藏至我的研究室書目清單書目收藏:0
本研究目的為探討溫泉菌Tepidimonas taiwanensis I1-1分泌蛋白酶之分離純化及生化特性。Ⅰ1-1菌株生長溫度範圍為30~60 ℃,菌株生長pH值範圍為pH6~8。I 1-1分泌蛋白酶之最適培養溫度為50℃,pH為7.3;培養時間為10-12小時,培養基濃度為0.9%。將Tepidimonas taiwanensis I1-1培養10小時後,離心過濾獲得之上清液為粗酵素液。將粗酵素液經減壓濃縮、10K MW透析膜後,接著進行Q FF陰離子管柱層析及Superdex 200膠過濾管柱分離純化,獲得之酵素液蛋白酶純化倍率14.2倍,回收率9.8%。純化酵素的Km為0.091mM,Vmax為285.71 U/mg-min。本研究菌株的蛋白酶最適反應溫度為75℃,75℃以下酵素活性穩定,最適作用pH值為7,最適反應時間為1.5小時。該蛋白酶受EDTA(Ethylenediaminetetraacetic acid)、 Mn2+、Fe2+、Ba2+抑制,於SDS(Sodium dodecyl sulphate)、Triton X-100、DMSO(dimethyl sulfoxide)、acetonitrile、DTT(Dithiothreitol)、β-mercaptoethanol存在下蛋白酶活性穩定。I1-1分泌之蛋白酶貯藏於-20℃、4℃及25℃下三十天後,仍維持85.2%以上的酵素活性。將I1-1分泌之蛋白酶與市售商業酵素Alcalase、Flavourzyme進行水解能力比較,結果顯示水解魚肉魚鱗能力略優於商業酵素Flavourzyme,可進一步探討做為工業發展之應用。
The objective of the present study is to purify and characterize the protease from Tepidimonas taiwanensis I1-1which was newly identified and isolated from a hot spring in southern Taiwan.I1-1is able to grow between 30-60℃and pH 6.0-8.0. The optimal growth conditions of I 1-1 for producing protease were 50℃, pH 7.3, incubating time 10-12 h and media concentration of 0.9%.The 10-h culture of I 1-1was centrifuged at 15,000rpm and the supernatant was used as crude protease. Crude enzyme was concentrated and purified with QFF ion exchange chromatography and Superdex 200 gelfiltration. The purification fold was 14.2 with recovery of 9.8%,and Km and Vmax was 0.091mM and 285.71 U/mg-min, respectively, using casein as substrate.The optimal pH and temperature of the enzyme was 7.0 and75℃, respectively, and stable at temperature below 75℃. Ethylene diamine tera acetates (EDTA),Mn2+, Fe2+,Ba2+ could slight inhibitthe enzyme activityof.I 1-1 protease. However, this enzyme was stable in the presence of sodium dodecyl sulphate (SDS),Triton X-100,dimethyl sulfoxide,acetonitrile, dithiothreitol (DTT),and β-mercaptoethanol.The crude I 1-1 enzyme was further compared with alcalase, flavourzyme for their efficiency on hydrolysis of protein. I 1-1 enzyme exhibited higher efficiency on the hydrolysis of fish scales and milkfish when compared with flavourzyme. while less efficiency when compared with alcalase. After storage at 4, 25, or -20℃ for one month, the I 1-1enzyme all retained 85.2% of enzyme activity.
中文摘要 I
英文摘要 II
致謝 III
目錄 IV
附表目錄 IX
附圖目錄 X
表目錄 XI
圖目錄 XII
壹、緒論 1
一、前言 1
二、研究目的 2
貳、文獻回顧 3
一、Tepidimonas taiwanensisI1-1之介紹 3
(一) 菌種命名 3
(二) 大小、形態及生化特性 3
(三) 適合生長環境 4
二、蛋白酶 5
(一) 蛋白酶簡介 5
(二) 蛋白酶的命名 10
(三) 蛋白酶分類 11
(四) 蛋白酶應用 19
叁、實驗架構 28
肆、材料方法 30
一、材料 30
(一) 菌株來源 30
(二) 培養基 30
(三) 基質 31
(四) 商業酵素 32
(五) 藥品與儀器設備 32
二、方法 37
(一) 菌株之保存及活化 37
(二) 菌株培養 38
(三) 粗酵素液的製備 40
(四) 蛋白質濃度測定[Dye-binding (Bradford) assay] 40
(五) 蛋白酶活性測定(Lowry method) 42
(六) TFF System (Tangential Flow Filtration system ) 44
(七) 透析實驗 45
(八) 陰離子交換樹脂層析 45
(九) 膠過濾管柱層析 46
(十) 電泳分析 47
(十一) 酵素生化特性測定 58
(十二) 酵素動力學 62
(十三) 蛋白酶貯藏安定性 62
(十四) 統計分析: 63
伍、結果與討論 64
一、Tepidimonas taiwanensis I1-1蛋白酶菌株生長之特性 64
(一) Tepidimonas taiwanensis I1-1菌株大小、形態及特性 64
(二) Tepidimonas taiwanensis I1-1菌株之生長曲線及分泌蛋白酶最適培養時間 65
(三) Tepidimonas taiwanensis I1-1菌株分泌蛋白酶最適培養pH值 65
(四) Tepidimonas taiwanensis I1-1菌株分泌蛋白酶最適培養溫度 66
(五) Tepidimonas taiwanensis I1-1菌株分泌蛋白酶最適培養基濃度 66
二、Tepidimonas taiwanensis I1-1蛋白酶之純化 71
(ㄧ) 粗酵素液的製備 71
(二) 濃縮透析 71
(三) 陰離子交換樹脂 71
(四) Superdex 200膠過濾層析 72
(五) 電泳分析 73
(六) Tepidimonas taiwanensis I1-1蛋白酶純化倍率表 78
三、Tepidimonas taiwanensis I1-1蛋白酶之生化特性 80
(ㄧ) 蛋白酶作用最適pH值 80
(二) 蛋白酶作用最適溫度 80
(三) 蛋白酶作用最適時間 81
(四) 蛋白酶 pH安定性及熱安定性 81
(五) 蛋白酶抑制劑對對蛋白酶活性影響 87
(六) 金屬離子對對蛋白酶活性影響 87
(七) 界面活性劑、有機溶劑、還原劑對蛋白酶活性影響 88
(八) 酵素動力學探討 94
四、工業應用條件初步探討 98
(一) Tepidimonas taiwanensis I1-1蛋白酶與商業酵素分解魚肉魚鱗能力比較 98
(二) Tepidimonas taiwanensis I1-1蛋白酶保存試驗 98
陸、結論 101
柒、參考文獻 102

王育廷。(2010)。愛玉子瘦果幾丁質酶之純化與生化性質研究。靜宜
大學食品營養學系碩士論文。台中,台灣
王三郎。(1999)。海洋未利用生物資源之回收再利用。生物資源生物技術。1:1-8。
呂鋒洲、林仁混。(1991)。基礎酵素學。聯經出版,台北。
林奇霓。(2012)。Aquimarina sp. 分泌蛋白酶的特性。國立高雄海洋科技大學水產食品科學系碩士論文。高雄,台灣。
林雀月。(2007)。Meiothermus I40角蛋白酶之純化與特性。國立高雄海洋科技大學水產食品科學系碩士論文。高雄,台灣。
林昇慧、陳婉伶、郭武東。(2007)。微生物發酵產業微生物與健康產業。415 :22-27。
陳建誌。(2011)。鱸魚魚鱗膠原胜肽製備及功能性。國立高雄海洋科技大學水產食品科學系碩士論文。高雄,台灣。
陳明秀。(2013)。海洋菌Aquimarina salinaria 蛋白酶的純化及特性探討。國立高雄海洋科技大學水產食品科學系碩士論文。高雄,台灣。
陳天來。(2007)。南臺灣溫泉之嗜熱性鹼性蛋白酶生產菌Tepidimonas
taiwanensissp.nov.之研究。中興大學土壤環境科學系所碩士論文。台中,台灣。
陳國誠。(1989)。微生物酵素工程學。藝軒出版社,台北。
張文重(1976)蛋白質分解酵素(構造功能進化及應用)。國立編譯館,台北。
黃裕仁。(2009)。Vogesellasp.分泌之魚鱗水解酵素之特性及純化。
國立高雄海洋科技大學水產食品科學系碩士論文。高雄,
台灣。
黃子瑜。(2010)。秋刀魚魚肉水解液之製備及應用。國立高雄海洋科技大學水產食品科學系碩士論文。高雄,台灣。
鄭春田。(2003)。Aspergillus sp. FC-10(ATCC MYA-2469)鹼性蛋白酶之研究。國立臺灣大學農業化學研究所碩士論文。台北,台灣。
蘇遠志。(1999)。應用微生物學。國立編譯館,華香園出版社。台北。
蘇睿綺。(2006)。枯草菌屬角蛋白酶之純化與性質研究。靜宜大學

Anbu P, Gopinath SCB, Hilda A, Lakshmi priya T, Annadurai G.
2005.Purification of keratinase from poultry farm isolateScopulariopsis brevicaulis and statistical optimization of enzyme activity. Enzyme and Microbial Technology 36:639-647.
Adler-Nissen, J. (1986) Enzymatic hydrolysis of food proteins. pp. 19-20.Elselier Applied Science Publ. London and New York.
Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K.(2002) Molecular Biology of the Cell. Taylor and Francis. USA.
Bressollier, P., Letourneau, F., Urdaci, M. and Verneuil, B. (1999)Purificationand characterization of a keratinolytic serineprotease from Streptomyces albidoflavus.Applied andEnvironmental Microbiology 65 :2570–2576.
Barrett, A. J. and Rawlings, N. D., Tolle, D. P., (2004) Evolutionary families of peptidase inhibitors.Biochem J 378:705-16.
Bergmann, M., & Fruton, J. S. (1941).The specificity of proteinases.Advances in Enzymology, 1, 63-98.
Bergmann, M. (2009).A classification of proteolytic enzymes. Advances in Enzymology (eds. FF Nord, CH Werkman), 2, 49-67.
Bressollier P, Letourneau F, Urdaci M, Verneuil B. (1999). Purification and characterization of a keratinolytic serine proteinase from Streptomyces albidoflavus. Applied and Environmental Microbiology 65:2570-2576.
Barata, R. A., Andrade, M. H. G., Rodrigues, R. D., Castro,I. M. (2002) Purification and Characterization of an Extracellular Trypsin-Like Protease of Fusarium oxysporum var. Mini. Journal Of Biosciencaen Dbioengineering 94 (4)304-308.
Bockle B, Muller R. (1997). Reduction of disulfide bonds by Streptomyces pactum during growth on chicken feather. Applied and Environmental Microbiology 63:790-795.
Bockle B, Galunsky B, Muller R. (1995). Characterization of a keratinolytic serine proteinase from Streptomyces pactum DSM 40530. Applied and Environmental Microbiology 61:3705-3710.
Chen, T. L., Chou, Y. J., Chen, W. M., Arun, B., & Young, C. C. (2006). Tepidimonas taiwanensis sp. nov., a novel alkaline-protease-producing bacterium isolated from a hot spring. Extremophiles, 10(1), 35-40.
Cabral, C. M., Cherqui, A., Pereira, A. and Simoes, N. (2004) Purification and characterization of two distinct metalloproteases secreted by the entomopathogenic bacterium Photorhabdus sp. strain Az29.Microbiology. 70, 3831-3838.
Christianson, T. M., Goddette, D. W., Maurer, K. H., Paech, C. G., Tang, M. R., Weiss, A., & Wilson, C. R. (1998). U.S. Patent No. 5,801,039. Washington, DC: U.S. Patent and Trademark Office.

Dozie INS, Okeke CN, Unaeze NC. (1994). A thermostable, alkaline-active, keratinolytic proteinase from Chrysosporium keratinophilum. World Journal of Microbiology & Biotechnology 10:563-567.
Friedrich AB, Antranikian G. (1996). Keratin degradation by Fervidobacterium pennavorans, a novel thermophilic anaerobic species of the order Thermotogales. Applied and Environmental Microbiology 62:2875-2882.
Friedrich J, Gradisar H, Vrecl M, Pogacnik A. (2005). In vitro degradation of porcine skin epidermis by a fungal keratinase of Doratomyces microsporus. Enzyme and Microbial Technology 36:455-460.
Friedrich J, Kern S. (2003). Hydrolysis of native proteins by keratinolytic protease of Doratomyces microsporus. Journal of Molecular Catalysis 21:35-37.
Farag AM, Hassan MA. 2004. Purification characterization and immobilization of a keratinase from Aspergillus oryzae. Enzyme and Microbial Technology 34:85-93.
Gupta,R., Beg ,Q.K., Lorenz, P. (2002) Bacterial alkaline proteases: molecular approaches and industrial applications . Appl
Kumar, C.G., Takagi, H. (1999) Microbial alkaline proteases: from abioindustrial viewpoint. BiotechnolAdv17:561–594.
Kumar, C. G. 2002. Purification and characterization of a thermostable alkalind protease from alkalophilic Bacillus pumilus.Lett Appl Microbiol. 34:13-17.
Kuo, J.M., Hwang, A., and Yeh, D.B.(1997)Purification, Substrate Specificity and Products of a Ca2+-Stimulating Lipoxygen-ase from Sea Algae (Ulva lactuca) J. Agric. Food Chem 45(6)2055-2060.
Kalisz, H. M.(1988) Microbial proteinases. Adv. Biochem. Eng. Biot.36:1-65.
Kim,W., K Choi, Y Kim, H Park, J Choi, Y Lee, H Oh, I Kwon, and S Lee.(1996). Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. strain CK 11-4 screened from Chungkook-Jang. Appl. Environ. Microbiol. 62(7): 2482-2488.
Karbalaei-Heidari, H. R., Ziaee A.A. ,Schaller, J., Amoozegar, M. A.(2007) Purification and characterization of an extracellular haloalkaline protease produced by the moderately halophilic bacterium,Salinivibrio sp.strain AF-2004 Enzyme and MicrobialTechnology 40 (2,4) 266–272.
Leber, T. M., Balkwill, F. R. (1997) Zymography: a single-step staining
method for quantitation of proteolytic activity on substrate gels. Analytical Biochemistry 249(1)24-28.
Leber TM, Balkwill FR. 1997. Zymography: a single-step staining method for quantitation of proteolytic activity on substrate gels. Analytical Biochemistry 249:24-28.
Lund, T., and Granum, P. E. (1999) The 105-kDa protein component of Bacillus cereus non-haemolytic enterotoxin (Nhe) is metalloprotease with gelatinolytic and collagenolytic activiy. FEMSMicrobiol Lett 178 : 355-61.
Matsushita, O., Yoshihara, K., Katayama, S., Minami, J. and Okabe, A.
(1994) Purification and characterization of a Clostridium perfringens 120-kDa collagenase and nucleotide sequence of thecorresponding gene. J Bacteriol 176 : 149-156.
Maurer, K. H. (2004). Detergent proteases. Current opinion in Biotechnology,15(4), 330-334.
Macedo, A. J., da Silva, W. O. B.,Gava, R., Driemeier, D., Henriques, J. A. P. and Termignoni, C.(2005)Novel keratinase from Bacillus subtilis S14 exhibitingremarkable dehairing capabilities. Appl. Environ. Microbiol.71(1):594-596.
Miyaji, T., Otta, Y., Nakagawa, T., Niimura, Y. and Tomizuka, N. (2005). Purfication and molecular characterization of subtilisin-like alkaline protease BPP-A from Bacillus pumilus strain MS-1. Lett appl microbiol.42:242-247. 35
Nagodawithana,Tilak,and Gerald Reed.,(1993)Enzymes in food processing. No.Ed.3.Academic Press Inc.Segel, I. H. (1975). Enzyme kinetics (Vol. 360).Wiley, New York.Nakanishi T, Yamamoto T. (1974).Action and specificity of a Streptomyces alkalophilic proteinase. Agricultural and Biological Chemistry 38:2391-2397.
Nam G, Lee WD, Lee HS, Lee NJ, Kim BC, Choe EA, Hwang JK, Suhartono MT, Pyun YR. 2002. Native-feather degradation by Fervidobacterium islandicum AW-1, a newly isolated keratinase- producing thermophilic anaerobe. Archives of Microbiology 178:538-547.
Ogino, H., Otsubo, T. & Ishikawa, H. (2008).Screening, purification, and characterization of a leather-degrading protease. Biochemical Engineering Journal 38 : 234-40.
Patel , R., Dodia , M ., Singh , S . P .(2005) Extracellular alkaline protease from a newly isolated haloalkaliphilic Bacillus sp.:Production and optimization. Process Process Biochemistry 40 (11) 3569–3575.
Riessen S, Antranikian G. (2001). Isolation of Thermoanaerobacter keratinophilus sp. nov., a novel thermophilic, anaerobic bacterium with keratinolytic activity. Extremophiles 5:399-408.
Rao MB, Tanksale AM,Ghatge MS, Deshpande VV (1998)Molecular and biotechnological aspects of microbial proteases.MicrobiolMolBiol Rev 62:597–635
Reed, G. Enzymes in food processing. 2nd ed pp. 263. Academic Press.
Riffel A, Lucas F, Heeb P, Brandelli A. 2003. Characterization of a new keratinolytic bacterium that completely degrades native feather keratin. Archives of Microbiology 179:258-265.
Shih JCH, Lee CG. (1993). Method and composition for maintaining animals on a keratin-containing diet. US Patent N., 5186971.
Simeonova, L. S. and Dalev, P. G.(1996)Utilization of a leather industry waste;technical note. Waste Manage. 16(8):765-769.
Takeuchi, S., Murase, K., Kaidoh, T., Maeda, T. (2000) A metalloprotease is common to swine, avian and bovine isolates of Staphylococcushyicus. Veterinary Microbiology 71 (1-2) 169-174.
Takami H, Akiba T, Horikoshi K. (1990). Characterization of an alkaline protease from Bacillus sp. no. AH-101. Applied Microbiology and Biotechnology 33:519-523.
Tien-Lai Chen, Yi-JuChou , Wen-Ming Chen,BhagwathArun , Chiu-Chung Young. (2006) Tepidimonastaiwanensissp.nov., a novel alkaline-protease-producing bacterium isolated from a hot spring.Extremophiles 10:35–40
Tang, X. M., Lakay, F. M., Shen, W., Shao, W. L., Fang. H. Y., Prior, B. A., Wang., Z. X. and Zhuge, J. (2004). Purification and characterization of an alkaline protease used in tannery industry from Bacillus licheniformis. Biotechnol Lett. 26: 1421-1424.
Uesugi, Y., Arima, J., Usuki, H., Iwabuchi, M. & Hatanaka, T. 2008.Two bacterial collagenolytic serine proteases have different topological specificities. Biochimica et Biophysica Acta 1784 : 716-26.
Varela, H., Ferrari, MD., Belobradjic, L., Vazquez, A. And Loperena, ML. (1997)Skin unhairing proteases of Bacillus subtilis. Biotechnol.Lett.189 : 755-758.
Williams, C. M., Richter, C. S., Mackenzie, J. M., & Shih, J. C. (1990).Isolation, identification, and characterization of a feather-degrading bacterium.Applied and Environmental Microbiology, 56(6), 1509-1515.
Williams, C. M., Lee, C. G., Garlich, J. D., & Shih, J. C. (1991).Evaluation of a bacterial feather fermentation product, feather-lysate, as a feed protein. Poultry Science, 70(1), 85-94.
Watanabe, K. (2004) Collagenolytic proteases from bacteria.AppliedMicrobiology And Biotechnology 63 : 520-6.

QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
系統版面圖檔 系統版面圖檔