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系統識別號 U0002-2307200914163600
中文論文名稱 Pseudomonas sp.TKU008 所生產蛋白酶之純化定性及應用
英文論文名稱 Purification, Characterization and Application of a Protease from Pseudomonas sp. TKU008
校院名稱 淡江大學
系所名稱(中) 生命科學研究所碩士班
系所名稱(英) Graduate Institute of Life Sciences
學年度 97
學期 2
出版年 98
研究生中文姓名 劉珮廷
研究生英文姓名 Pei-Ting Liu
學號 696180289
學位類別 碩士
語文別 中文
第二語文別 英文
口試日期 2009-07-14
論文頁數 54頁
口試委員 指導教授-王三郎
委員-王全祿
委員-顏裕鴻
中文關鍵字 Pseudomonas sp.  蛋白酶  去蛋白 
英文關鍵字 Pseudomonas sp.  protease  deproteinization 
學科別分類 學科別醫學與生命科學生物學
中文摘要 本研究以蝦頭殼粉(SHP)為主要碳/氮源,經TKU008發酵後生產蛋白酶,純化並進行生化性質分析及其應用。TKU008篩選自台灣南部土壤,具蛋白酶及幾丁質酶生產能力,經鑑定為Pseudomonas sp.。在培養條件為1% SHP、0.1% K2HPO4、0.05%MgSO4.7H2O於pH7、100mL之液態培養基在30℃下震盪培養5天後,蛋白酶活性最佳。其發酵上清液經硫酸銨沉澱、DEAE-Sepharose及Phenyl Sepharose 6 Fast Flow等層析步驟,可分離出分子量約23kDa的蛋白酶,其最適反應pH為7、最適反應溫度為60℃、pH安定性為pH6-9及熱安定性為<50℃。此蛋白酶活性受EDTA完全抑制,為金屬型蛋白酶。對酪蛋白、彈性蛋白、人類白蛋白、血紅蛋白具不同程度的水解能力;在測試的界面活性劑中均相當穩定。應用方面則探討TKU008蛋白酶對蝦頭粉(SHP)、烏賊殼粉(SPP)的去蛋白能力,培養六天後其蛋白質去除率可達75%和85%。
英文摘要 This study reports the purification, characterization and application of a protease from Pseudomonas sp. TKU008, which takes shrimp head shells powder (SHP) as main carbon nitrogen source. TKU008 was isolated from Taiwan south soil, which can produce chitinase and protease. The optimized condition for protease production was found when the culture was shaken at 30℃ for 5 days in 100mL of medium (pH7) containing 1% SHP, 0.1% K2HPO4, 0.05%MgSO4.7H2O. One protease was purified by chromatography procedures of DEAE-Sepharose, and Phenyl Sepharose 6 Fast Flow. The molecular mass of the protease determined by SDS-PAGE was approximately 23 kDa. The optimum pH, optimum temperature, pH stability, and thermal stability of protease were pH7, 60℃, pH6-9 and <50℃, respectively. The protease was characterized as a metalloprotease, due to it was inactivated by EDTA. And it had different specific proteolytic activity with casein, elastin, human albumin, hemoglobin as the substrates. When the protease was treated of various surfactants, its activity was stable. We also discuss the deproteinization ability of this enzyme when grown in different mediums such as SHP, squid shell powder (SPP). The percent of protein removal for SHP and SPP after 6-day incubation were 75% and 85%, respectively.

論文目次 目錄
封面內頁
簽名頁
授權書
誌謝
中文摘要...I
英文摘要...II
目錄...V
圖目錄...IX
表目錄...X
第一章 緒論...1
第二章 文獻回顧…2
2.1 水產廢棄物之微生物再利用…2
2.2 蝦、蟹殼與烏賊軟骨廢棄物的組成…2
2.3 蛋白酶…5
2.3.1 蛋白酶之分類依據與其命名…9
2.3.2 蛋白酶之特性…11
2.3.3 蛋白酶的應用…12
2.4 Pseudomonas sp…13
第三章 材料與方法…15
3.1 實驗菌株…15
3.2 實驗儀器與材料…15
3.2.1 實驗儀器…15
3.2.2 實驗材料…16
3.3 實驗方法…17
3.3.1 懸浮態幾丁質之製備…17
3.3.2 蛋白酶活性之測定…17
3.3.3 幾丁質酶活性測定…17
3.3.4 蛋白酶之較適培養條件探討…18
3.3.5 蛋白酶之純化分離…18
(1) 粗酵素液的製備…18
(2) 硫酸銨沉澱…19
(3) 離子交換樹脂層析法…19
(4) 疏水性層析...19
(5) 蛋白質電泳分析…19
(6) 蛋白質定量分析…20
3.3.6 純化蛋白酶之生化特性分析…20
(1) 蛋白酶之最適溫度測定…20
(2) 蛋白酶之熱安定性測定…20
(3) 蛋白酶最適pH的測定…20
(4) 蛋白酶pH安定性的測定…21
(5) 金屬離子與抑制劑對酵素活性之影響…21
(6) 界面活性劑對酵素活性之影響…21
(7) 蛋白酶之基質特異性…21
3.3.7 抗氧化特性分析…22
(1) DPPH自由基清除能力之測定…22
(2) 金屬離子螯合能力之測定…22
(3) 還原力之測定…23
(4) 總酚類化合物之定量分析…23
3.3.8 液態培養去除蛋白質...24
第四章 結果與討論…25
4.1 Pseudomonas sp. TKU008之特性與培養條件探討…25
4.2 蛋白酶之純化分離…27
4.2.1 粗酵素液製備…28
4.2.2 陰離子交換層析…28
4.2.3 疏水性層析…30
4.2.4 综合結果…32
4.3 蛋白酶分子量測定…33
4.4 純化後蛋白酶之生化特性分析…34
4.4.1最適反應溫度及熱安定性之測定…34
4.4.2 最適反應pH和pH安定性之測定…36
4.4.3 金屬離子與抑制劑對蛋白酶活性的影響…38
4.4.4 界面活性劑對蛋白酶活性的影響…39
4.4.5 蛋白酶之基質特異性…40
4.5 利用Pseudomonas sp. TKU008發酵蝦頭粉與烏賊軟骨粉生產蛋白酶及其蛋白質去除率之探討…43
4.6 利用Pseudomonas sp. TKU008發酵蝦頭粉與烏賊軟骨粉之抗氧化實驗探討…46
第五章 結論…49
參考文獻…50

圖目錄
圖2.1 α-幾丁質之構造…………………………………………………..3
圖2.2 β-幾丁質之構造…………………………………………………..4
圖4.1 不同來源之碳氮源與培養天數對酵素產量之影響…………..26
圖4.2 TKU008所生產蛋白酶之純化分離流程圖……………………27
圖4.3 TKU008蛋白酶之DEAE-Sepharose CL-6B離子性層析圖譜………………………………………………………………………..29
圖4.4 TKU008蛋白酶於Phenyl Sepharose 6 Fast Flow疏水性層析之圖譜………………………………………………………………………..31
圖 4.5 TKU008蛋白酶之SDS-PAGE電泳分析圖…………………….33
圖4.6 TKU008蛋白酶之最適反應溫度………………………………35
圖4.7 TKU008蛋白酶之熱安定性……………………………………35
圖4.8 TKU008蛋白酶之最適反應pH………………………………..37
圖4.9 TKU008蛋白酶之pH安定性………………………………….37
圖4.10 液態培養TKU008對SHP之蛋白質去除率………………….44
圖4.11 液態培養TKU008對SPP之蛋白質去除率…………………44
圖4.12 TKU008之總酚含量測定……………………………………..47
圖4.13 TKU008之還原力測定………………………………………..47
圖4.14 TKU008之鐵離子螯合測定…………………………………..48
圖4.15 TKU008之DPPH清除率測定………………………………..48

表目錄
表2.1 含幾丁質水產加工廢棄物之微生物再利用…………………….6
表2.2 蛋白酶的應用範圍……………………………………………….7
表2.3 不同來源的蛋白酶及特性……………………………………….8
表2.4 不同微生物所產金屬型蛋白酶之比較………………………...14
表4.1 TKU008蛋白酶之純化概要…………………………………….32
表4.2 化學藥品對蛋白酶活性之影響………………………………...38
表4.3 界面活性劑對蛋白酶活性之影響……………………………...39
表4.4 蛋白酶之基質特異性…………………………………………...40
表4.5 不同來源之Pseudomonas屬所生產蛋白酶特性之比較……...41
表4.6 不同來源微生物去蛋白率之比較……………………………..45


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