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

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研究生：	許寶月 Pao-Yueh Hsu
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
指導教授：	黃雪莉 Shir-Ly Huang
口試委員:
學位類別：	碩士 Master
系所名稱：	生醫理工學院 - 生命科學系 Department of Life Science
畢業學年度：	92
語文別：	中文
論文頁數：	72
中文關鍵詞：	Actinomycetes H12 、轉麩胺酸醯胺基酶、鈷六十
外文關鍵詞：	Actinomycetes H12, transglutaminase, Co 60
相關次數：	點閱：6 下載：0
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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。

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.

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

                                

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