Actinomycetes H12所產轉麩胺酸醯胺基酶之生產、特性及加工製程之探討

以作者查詢圖書館館藏

、以作者查詢臺灣博碩士

、以作者查詢全國書目

、勘誤回報

、線上人數：27

、訪客IP：3.137.174.52

姓名

許寶月(Pao-Yueh Hsu) 查詢紙本館藏

畢業系所

生命科學系

論文名稱

Actinomycetes H12所產轉麩胺酸醯胺基酶之生產、特性及加工製程之探討
(Production, characterization and procession of transglutaminase produced by Actinomycetes H12)

相關論文

★ 陰離子界面活性劑sodium dodecylbenzene sulfonate分解菌篩選與脫磺酸酵素研究	★ 鄰苯二酚加氧酵素的熱穩定性提昇研究
★ Triton X-100 分解菌之分離和分解酵素之特性研究	★ Triton X-100加氧酵素之純化與定性
★ Lactobacillus reuteri於酸性與膽鹽環境中之蛋白質體研究	★ 蕃茄根部受銅逆境之基因調控
★ Pseudomonas nitroreducens TX1 異化辛基苯酚聚氧乙基醇之功能性蛋白質體學：以二維電泳法分析等電點4-8之蛋白質表現	★ Pseudomonas nitroreducens TX1之具耗氧活性之麩胺酸合成酶之單離
★ 人類細胞株生產含多種亞型的干擾素-a之蛋白質體學研究	★ 辛基苯酚之分解:分解菌和生物復育之菌相研究
★ 分解辛基苯酚聚氧乙基醇之耗氧酵素（二氫硫辛醯胺脫氫酶）的純化與定性	★ AtNPR1轉殖番茄之性狀分析及抗病機制研究
★ Pseudomonas putida TX2分解辛基苯酚聚氧乙基醇及其具雌激素活性代謝物之研究	★ 以功能性蛋白質體學研究Pseudomonas nitroreducens TX1生長於辛基苯酚聚氧乙基醇之代謝與逆境反應
★ 以功能性蛋白質體學研究Pseudomonas putida TX2生長於辛基苯酚聚氧乙基醇與辛基苯酚之代謝與逆境反應	★ 以功能性基因體學研究細菌異化辛基苯酚聚氧乙基醇及抗逆境之基因

檔案

[Endnote RIS 格式]

[Bibtex 格式]

[相關文章]

[文章引用]

[完整記錄]

[館藏目錄]

[檢視]

[下載]

本電子論文使用權限為同意立即開放。
已達開放權限電子全文僅授權使用者為學術研究之目的，進行個人非營利性質之檢索、閱讀、列印。
請遵守中華民國著作權法之相關規定，切勿任意重製、散佈、改作、轉貼、播送，以免觸法。

摘要(中)

摘要
本研究由50多個土壤樣品中分離出15,000株放線菌，其中以Actinomycetes H12有最高之轉麩胺酸醯胺基酶酵素活性。Actinomycetes H12於最適化液態培養條件下，培養可得轉麩胺酸醯胺基酶產量2.7 U/ml，較原始培養(TSB)條件下之轉麩胺酸醯胺基酶產量提高1.4倍，較S. ladakanum CCRC 12422之轉麩胺酸醯胺基酶高1.8倍，而與日本生產轉麩胺酸醯胺基酶之菌株Streptoverticillium sp. S-8112 產量2.5 U/ml相似。
Actinomycetes H12 之轉麩胺酸醯胺基酶蛋白質特性分析:其最適作用溫度40℃，熱穩定性在50℃下加熱30分鐘仍保有50%活性，最適作用pH 6-8。此轉麩胺酸醯胺基酶活性會受到Cu2+、 Zn2+明顯之抑制，但一價離子、Fe3+與Ca2+則不影響其活性。
Actinomycetes H12發酵槽液態培養至40-48小時，此時其酵素分泌至胞外比率仍低，利用此特性將發酵液高速離心後，可直接回收帶有酵素之菌泥，不經純化下其收率為85%，經冷凍乾燥後其活性不變，但發現有Escherichia coli，因之再輔以Co 60-10kGy照射殺死病原菌及生產菌，然其活性下降至65%，即使照射時加入抗氧化劑(β-carotene, ascorbic acid)亦不能改善提高其殘留活性。

摘要(英)

We isolated 15,000 Actinomycetes strains from over 50 soil samples, one isolated strain H12 have the highest TGase activity. The TGase activity (2.7U/ml) under the optimal conditions was about 1.4-fold than the TSB medium condition and 1.8-fold than the TGase from S. ladakanum CCRC 12422, but it is similar to the enzyme from Streptovertecillium sp. S-8112 (2.5 U/ml).
The crude TGase from Actinomycetes H12 had the optimum pH and temperature being pH 6-8 and 40℃, respectively. The stable pH range was 5-9 and thermal stability of the crude TGase remained 50% activity after treatment at 50℃ for 30 min. The metal ions, Cu2+ and Zn2+, inhibited the activity of TGase. The addition of monovalence, Fe3+ and Ca2+ did not affect the activity of TGase.
When Actinomycetes H12 was cultured on fermentor after 40-48 hr, the extracellular TGase still low. So we centrifuged the culture fluid in a single step and with high yields (85%), then the TGase activity didn’t decrease after freeze-dry. There were contaminants in products, therefore we treatment with Co 60-10 kGy killing germs and Actinomycetes H12, but the enzyme activity is decreased (65%). It didn’t improve to raise the activity, even thought added β-carotene and ascorbic acid.

關鍵字(中)

★ Actinomycetes H12
★ 轉麩胺酸醯胺基酶
★ 鈷六十

關鍵字(英)

★ Actinomycetes H12
★ transglutaminase
★ Co 60

論文目次

目錄
中文摘要.................................................Ι
英文摘要.................................................Ⅱ
目錄.....................................................Ⅲ
圖目錄...................................................Ⅵ
表目錄...................................................Ⅷ
第一章、前言..............................................1
第二章、文獻整理..........................................3
2.1轉麩胺酸醯胺基酶的作用機制 ........................3
2.2轉麩胺酸醯胺基酶之種類及其特性.....................3
2.3微生物轉麩胺酸醯胺基酶之篩選.......................5
2.4微生物轉麩胺酸醯胺基酶之純化.......................6
2.5轉麩胺酸醯胺基酶之胺基酸序列.......................6
2.6微生物轉麩胺酸醯胺基酶之基因轉殖與表現.............7
2.7微生物轉麩胺酸醯胺基酶在食品加工上之應用. .........9
第三章、材料與方法........................................10
3.1實驗材料... .......................................11
3.2實驗方法... .......................................13
3.2.1 從土壤中分離菌株之方法.. .................... 13
3.2.2 篩選具TGase活性之菌株. ..................... 13
3.2.3 基本分析方法.. .............................. 14
3.2.4搖瓶液態發酵生產TGase最適化條件..............15
3.2.5發酵槽批次式液態發酵培養條件............. .... 16 3.2.6粗TGase之特性分析......... ..................16
3.2.7回收及加工製程................................17
第四章、結果與討論 .......................................19
4.1 土壤中具TGase放線菌之篩選 .......................19
4.1.1 選擇性培養基分離微生物................ ...... 19
4.1.2 篩選具TGase活性之菌株..... ................. 20
4.1.3液態培養基之選擇........... .................. 21
4.1.4 基礎培養狀態....... ......................... 22
4.2 TGase生產條件之單因子探討 .................. .....23
4.2.1 培養溫度.................. .................. 23
4.2.2 起始pH値............. ...................... 23
4.2.3 碳源及碳源濃度.............................. 24
4.2.4 氮源及氮源濃度................. ............. 25
4.3 發酵槽液態培養生產TGase條件探討......... ........ 26
4.4 粗TGase之特性分析....................... ........ 27
4.4.1 最適作用溫度及熱穩定性....................... 27
4.4.2 最適pH値及pH値穩定性...................... 28
4.4.3 金屬鹽類對TGase之影響....... ............... 28
4.5 TGase回收製程.. ....................... .......... 29
4.6 加工製程... ...................................... 30
第五章、結論與建議........................................32
圖....................................................... 34
表....................................................... 55
參考文獻................................................. 66
圖目錄
圖2-1. 轉麩胺酸醯胺基酶所催化的反應 ....................34
圖2-2. 轉麩胺酸醯胺基酶之純化過程... .....................35
圖3-1. 實驗流程圖........................................13
圖3-2. Folk之colorimetric hydroxamate method呈色之原理... ..34
圖4-1. 利用三種選擇性培養基篩選土壤中之放線菌............36
圖4-2. 利用96 well plate 篩選具TGase菌株之結果............37
圖4-3. Actinomycetes H12生長狀態.............. ...........38
圖4-4. Actinomycetes H12與 S. ladakanum CCRC 12422於TSB medium培養下測其TGase酵素活性表現曲線圖................39
圖4-5. Actinomycetes H12於Ando medium培養過程中TGase酵素活性、菌體乾重之變化......................................40
圖4-6. Actinomycetes H12與 S. ladakanum CCRC 12422於Tsai medium 培養下測其TGase酵素活性表現曲線圖................41
圖4-7. Actinomycetes H12於Tsai medium培養過程中酵素活性、菌體乾重及pH値之變化..................................... 42
圖4-8. 溫度對Actinomycetes H12所產TGase酵素活性之影響.......................................................43
圖4-9. 不同起始pH値對Actinomycetes H12所產TGase酵素活性之影響..................................................... 44
圖4-10. 不同起始pH値對Actinomycetes H12培養期間之pH値變化曲線..................................................... 45
圖4-11. 不同碳源及其濃度對Actinomycetes H12 發酵液TGase酵素活性之影響............................................... 46
圖4-12. 不同氮源及其濃度對Actinomycetes H12 發酵液TGase酵素活性之影響............................................... 47
圖4-13. 1% peptone 培養液中綜合維他命及核苷酸之添加對Actinomycetes H12發酵液TGase酵素活性之影響.............. 48
圖4-14. 消泡劑添加量對Actinomycetes H12發酵液TGase酵素相對活性之影響............................................... 49
圖4-15. 不同攪拌速度及通氣量對Actinomycetes H12於100 L發酵槽中TGase酵素活性之影響...................................50
圖4-16. 起始pH 6.5對Actinomycetes H12於100 L發酵槽中TGase酵素活性、pH値及菌體乾重之影響...........................51
圖4-17. 起始pH 8對Actinomycetes H12於100 L發酵槽中TGase酵素活性、pH値及菌體乾重之影響.............................52
圖4-18. 不同溫度對轉麩胺酸醯胺基酶活性之影響. ........... 53
圖4-19. 不同pH對轉麩胺酸醯胺基酶活性之影響 ............ 54
表目錄
表2-1. 轉麩胺酸醯胺基酶之種類及其特性....................56
表2-2. 微生物TGase之應用................................57
表3-1. 本實驗所使用之菌體... ............................58
表3-2. 本實驗所採用分離放線菌之培養基....................59
表4-1. Actinomycetes H12所產TGase 在菌體內之分佈..........60
表4-2. 轉麩胺酸醯胺基酶特性分析..........................61
表4-3. 金屬離子對放線菌屬TGase活性之影響................62
表4-4. 鈣離子對不同來源轉麩胺酸醯胺基酶之影響 .. .........63
表4-5. 起始 pH 6.5於100 L發酵槽培養72小時後其回收製程對
TGase活性之影響............. ............................64
表4-6. 起始 pH 6.5於100 L發酵槽培養48小時後其回收製程對
TGase活性之影響.......................... ...............65
表4-7. 起始 pH 8於100 L發酵槽培養48小時後其回收製程對
TGase活性之影響.................... .....................66
表4-8. 起始 pH 8於100 L發酵槽培養40小時後其回收製程對
TGase活性之影響.................... .....................67

參考文獻

參考文獻
潘子明。2002。食品生物技術介紹。台灣農業化學會，pp.1-36。
聶方珮，謝榮峰，江善宗。2003。微生物轉麩胺酸醯胺基酶之生產條件及對大豆蛋白成膠之影響。台灣農業化學與食品科學，41(1): 37-44。
朱文深。1998。微生物轉榖氨醯胺酶之開發與應用。食品工業月刊，30(4): 30-39。
朱文深。2003。微生物轉榖氨醯胺酶之開發。中國農業化學會，113-130。
張紅城，彭志英，越謀明，邱慧霞。1998。轉榖氨醯胺酶在食品中的應用。食品與發酵工業，24(3): 73-76。
葉雅玟。2000。Streptoverticillium kentuckense CCRC 12429生產之麩醯基轉移酶純化、生化特性及應用。國立中興大學畜產系研究所碩士論文。
吳介文，蔡國珍，江善宗。1996。轉麩胺酸醯胺基酶生產菌株之篩選及影響其產量因子之探討。中國農業化學會誌，34(2): 228-240。
Ando, H., Adachi, M., Umeda, K., Matsuura, A. & Nonaka, M. (1989). Purification and characteristics of novel transglutaminase derived from microorganisms. Agric. Biol. Chem. 53: 2613-2617.
Brunner, F., Rosahl, S., Lee, J., Geiler, C., Kauppinen, S. & Scheel, D. (2002). Pep-13, a plant defense-inducing pathogen associated pattern from Phytophthora transglutaminase. EMBO J. 21: 6681-6688.
Candi, E., Melino, G., Mei, G., Tarcsa, E., Chung, S. I., Marekov, L. N. & Steinert, P. M. (1995). Biochemical, structural, and transglutaminase substrate properties of human loricrin, the major epidermal cornified cell envelope protein. J. Biol. Chem. 270: 26382-26390.
Dallabrida, S. M., Falls, L. A. & Farrell, D. H. (2000). Factor XⅢa supports microvascular endothelial cell adhesion and inhibits capillary tube formation in fibrin. Blood. 95: 2586-2592.
Day, N. & Keillor, J. W. (1999). A continuous spectrophotometric linked enzyme assay for transglutaminase activity. Anal. Biochem. 274: 141-144.
de Jong, G. A., Wijngaards, G., Boumans, H., Koppelman, S. J. & Hessing, M. (2001). Purification and substrate specificity of transglutaminases from blood and Streptoverticillium mobaraense. J. Agric. Food Chem. 49: 3389-3393.
Falcone, P. D., Serafini-Fracassini. D. & Duca, S. D. (1993). Comparative studies of transglutaminase activity and substrates in different organs of Helianthus tuberosus. J. Plant Physiol. 142: 265-273.
Folk, J. E. (1980). Transglutaminase. Annu. Rev. Biochem. 49: 517-531.
Folk, J. E. & Cole, P. W. (1966). Mechanism of action of Guinea Pig Liver transglutaminase. J. Biol. Chem. 241: 5518-5525.
Gerber, U., Jucknischke, U., Putzien, S. & Fuchsbauer, H. L. (1994). A rapid and simple method for the purification of transglutaminase from Streptoverticillium mobaraense. Biochem. J. 299: 825-829.
Ho, M. L., Leu, S. Z., Hsien, J. F. & Jiang, S. T. (2000). Technical approach to simplify the purification method and characterization of microbial transglutaminase produced from Streptoverticillium ladakanum. J. Food Sci. 65: 76-80.
Ikura, K., Yoshikawa, M., Sasaki, R. & Chiba, H. (1981). Incorporation of amino acids into food proteins by transglutaminase. Agric. Biol. Chem. 45: 2587-2592.
Ikura, K., Nasu, T., Yokota, H., Tsuchiya, Y., Sasaki, R. & Chiba, H. (1988). Amino acid sequence of guinea pig liver transglutaminase from its cDNA sequence. Biochemistry 27: 2898-2905.
Jiang, S. T. & Lee, J. J. (1992). Purification, characterization, and utilization of Pig plasma factor XⅢa. J. Agric. Food Chem. 40: 1101-1107.
Kanaji, T., Ozaki, H., Takao, T., Kawajiri, H., Ide, H., Motoki, M. & Shimonishi, Y. (1993). Primary structure of microbial transglutaminase from Streptoverticillium sp. strain s-8112. J. Biol. Chem. 268：11565-11572.
Kawai, M., Takehana, S. & Takagi, H. (1997). High-level expression of the chemically synthesized gene for microbial transglutaminase from Streptoverticillium in Escherichia coli. Biosci. Biotechnol. Biochem. 61: 830-835.
Kikuchi, Y., Date, M., Yokoyama, K., Umezawa, Y. & Matsui, H. (2003). Secretion of active-form Streptoverticillium mobaraense transglutaminase by Corynebacterium glutamicum: processing of the pro-transglutaminase by a cosecreted subtilisin-Like protease from Streptomyces albogriseolus. Appl. Environ. Microbiol. 69: 358-366.
Kim, S. Y., Chung, S. I. & Steinert, P. M. (1995). Highly active soluble processed forms of the TGase Ιenzyme in epidermal keratinocytes. J. Biol. Chem. 270: 18026-18035.
Kobayashi, K., Suzuki, S. I., Izawa, Y., Miwa, K. & Yamanaka, S. (1998). Transglutaminase in sporulating cells of Bacillus subtilis. J. Gen. Appl. Microbiol. 44: 85-91.
Labeda, D. P. & Shearer, M. C. (1986). Isolation of actinomycetes for biotechnological applications.
Lilley, G. R., Skill, J., Griffin, M. & Bonner, P. L. (1998). Detection of Ca2+-dependent transglutaminase activity in root and leaf tissue of monocotyledonous and dicotyledonous plants. Plant Physiol 117: 1115-1123.
Matheis, G. & Whitaker, J. R. (1987). Areview: enzymatic cross-linking of proteins applicable to foods. J. Food Biochem. 29: 309-327.
Margosiak, S. A., Dharma, A., Gonzales, A. P., Louie, D., & Kuehn, G. D. (1990). Identification of the large subunit of Ribulose 1,5- biophosphate Carboxylase/Oxygenase as a substrate for transglutaminase in Medicago sativa L. Plant physiol. 92: 88-96.
Mottahedeh, J. &Marsh, R. (1998). Characterization of 101-kDa transglutaminase from Physarum polycephalum and identification of LAV1-2 as substrate. J. Biol. Chem. 273: 29888-29895.
Nonaka, M., Tanaka, H., Okiyama, A., Motoki, M . & Matsuura, A. (1989). Polymerization of several proteins by Ca2+- independent transglutaminase derived from microorganism. Agric. Biol. Chem. 53: 2619-2623.
Ohtsuka, T., Umezawa, Y., Nio, N. & Kubota, K. (2001). Comparison of deamidation activity of transglutaminases. J. Food Sci. 66: 25-29.
Radek, J. T., Jeong, J. M., Murthy, S. N., Ingham, K. C. & Lorand, L. (1993). Affinity of human erythrocyte transglutaminase for a 42-kDa gelatin-binding fragment of human plasma fibronectin. Proc. Natl. Acad. Sci. 90: 3152-3156
Serafini-Fracassini, D., Del Duca, S. & Beninati, S. (1995). Plant transglutaminases. Phytochemistry 40: 355-365.
Singh, H. (1991) Modification of food proteins by covalent crosslinking. Trends in Food Science and Technology (review) pp196-200.
Tsai, G. J., Lin, S. M. & Jiang, S. T. (1996). Transglutaminase from Streptoverticillium ladakanum and application to minced fish product. J. Food Sci. 61: 1234-1238.
Washizu, K., Ando, K., Koikeda, S., Hirose, S., Matsuura, A., Takagi, H., Motoki, M. & Takeuchi, K. (1994). Molecular cloning of the gene for microbial transglutaminase from Streptoverticillium and its expression in Streptomyces lividans. Biosci. Biotechnol. Biochem. 58: 82-87.
Yasueda, H., Kumazawa, Y. & Motoki, M. (1994). Purification and characterization of a tissue-type transglutaminase from Red Sea Bream (Pagrus major). Biosci. Biotechnol. Biochem. 58: 2041-2045.
Yokoyama, K. I., Nakamura, N., Seguro, K. & Kubota, K. (2000). Overproduction of microbial transglutaminase in Escherichia coli, in vitro refolding, and characterization of the refolded form. Biosci. Biotechnol. Biochem. 64: 1263-1270.
Zhu, Y., Bol, J., Rinzema, A. & Tramper, J. (1995). Microbial transglutaminase- a review of its production and application in food processing. Appl. Microbiol. Biotechnol. 44: 277-282.
Zhu, Y., Bol, J., Rinzema, A., Tramper, J. & Wijngaards, G. (1999). Transglutaminase as a potential tool in developing novel protein foods. Agro-Food Industry Hi-Tech- pp8-10.

指導教授

黃雪莉(Shir-Ly Huang)

審核日期

2004-1-28

推文