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中文論文名稱 Bacillus subtilis TKU007所生產幾丁聚醣酶及耐有機溶劑蛋白酶之純化與定性
英文論文名稱 Purification and Characterization of a Chitosanase and a Solvent Stable Protease from Bacillus subtilis TKU007
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 94
學期 2
出版年 95
研究生中文姓名 葉佩怡
研究生英文姓名 Pei-Yi Yeh
學號 693290024
學位類別 碩士
語文別 中文
口試日期 2006-06-08
論文頁數 124頁
口試委員 指導教授-王三郎
委員-陳銘凱
委員-王全祿
中文關鍵字 Bacillus subtilis  耐鹼性蛋白酶  界面活性劑安定  有機溶劑安定  蝦殼粉  幾丁聚醣酶 
英文關鍵字 Bacillus subtilis  alkaliphilic protease  surfactent stable  solvent stable  shrimp shell wastes  chitosanase 
學科別分類 學科別醫學與生命科學生物學
中文摘要 Bacillus subtilis TKU007係株以蝦殼粉為主要碳源,篩選自台灣北部土壤之蛋白酶生產菌,發酵蝦殼粉所得離心上清液具有幾丁聚醣酶及蛋白酶活性。幾丁聚醣酶較適生產條件為1.5% 蝦殼粉、0.1%
K2HPO4、0.05% MgSO4.7H2O之液態培養基(pH7)於30℃振盪培養1天。發酵所得離心上清液經硫酸銨沉澱、DEAE-Sepharose及Sephacryl-S100管柱層析後,可純化出幾丁聚醣酶。經SDS-PAGE及膠體層析測定分子量分別為25 kDa及33 kDa。TKU007幾丁聚醣酶之N末端胺基酸序列含:PQNI。其最適反應pH、最適反應溫度、pH安定性、熱安定性、Km 及Vmax分別為pH 7, 37℃, pH 4-9,25-37 ℃, 0.438 mg/mL, 0.144 U/mL;其活性會受Cu2+所抑制,而2%(v/v) Tween 40或Triton X-100則不具抑制效果。
B. subtilis TKU007生產耐界面活性劑、耐有機溶劑及對鹼性安定性高之絲胺酸蛋白酶之較適培養條件為1.5% 蝦殼粉、0.1% K2HPO4
、0.05% MgSO4.7H2O之液態培養基(pH7)於30 ℃培養2天。發酵所得離心上清液離心,經硫酸銨沉澱、DEAE-Sepharose、Phenyl Sepharose及Sephacryl S-100管柱層析後,純化出一分子量經SDS-PAGE及膠體層析測定為28 kDa及30 kDa之蛋白酶,且培養第一天時發現TKU007蛋白酶會造成幾丁聚醣酶活性降低。TKU007蛋白酶之N末端胺基酸序列為:AQSVPYGISQIKAPALGSQG。其最適反應pH、最適反應溫度、pH安定性、熱安定性、Km 及Vmax分別為pH 7-11, 50 ℃, pH 5-11, 50 ℃, 0.13 mg/mL, 0.86 U/mL;在25%有機溶劑存在下,於25℃及4℃放置10天,蛋白酶仍維持原活性80%以上。此外,蛋白酶在2% Tween 20、2% Tween 40及2 mM SDS存在下分別還維持原活性之100%、100%及69%。

英文摘要 The chitosanase and protease-producing bacterium, Bacillus subtilis TKU007, was isolated from soil in the north Taiwan. The supernatant of the culture medium contains the protease and the chitosanase activity. The optimized culture condition for production of the chitosanase was composed of 1.5% SSP,0.1% K2HPO4,0.05% MgSO4.7H2O at pH 7 and incubation in 250mL Erlenmeyer flask containing 200mL kept shaking at 30℃for one day. The chitosanase was purified from the culture supernatant by ammonium sulfate precipitation, DEAE-Sepharose, and Sephacryl-S100. The molecular mass of TKU007 chitosanase determined by SDS-PAGE and gel filtration was approximately 25 kDa and 33 kDa, respectively. The N-terminal amino acid sequence of the protease contains PQNI. The optimum pH, optimum temperature, pH stability, thermal stability, Km, and Vmax of TKU007 chitosanase were pH7, 37℃, pH4-9, 25-37℃, 0.438 mg/mL, and 0.144 U/mL, respectively. TKU007 chitosanase was inactivated by Cu2+,but not by 2%Tween 40, and 2%Triton X-100.
An extracellular serine protease with novel properties of surfactant-
stable, solvent-stable, and alkaliphilic was purified from B. subtilis TKU007. The optimized culture condition for production of the protease was composed of 1.5% SSP,0.1% K2HPO4, 0.05% MgSO4.7H2O at pH 7 and incubation in 250mL Erlenmeyer flask containing 100mL kept shaking at 30℃ for two days. The protease was purified from the culture supernatant by ammonium sulfate precipitation, DEAE-Sepharose, Phenyl Sepharose, and Sephacryl-S100. The molecular mass of TKU007 protease determined by SDS-PAGE and gel filtration was approximately 28 kDa and 30 kDa, respectively. The TKU007 protease showed suppressing effect on the chitosanase, which was apparent at the first day. The N-terminal amino acid sequence of the protease was: AQSVPYGISQIKAPALGSQG. The optimum pH, optimum temperature, pH stability, thermal stability, Km, and Vmax of TKU007 protease were pH 7-11, 50℃, pH 5-11, 50℃, 0.13 mg/mL, and 0.86 U/mL, respectively. More than 80% of the original activity was retained even after preincubation for 10 days at 25℃or 4℃ in the presence of 25% tested organic solvents. Additionally, the TKU007 protease retained 100%, 100%, and 69% of its original activity in the presence of 2% Tween 20, 2% Tween 40, or 2 mM SDS, respectively.
論文目次 封面內頁
簽名頁
授權書
中文摘要 I
英文摘要 III
誌謝 V
目錄 VI
圖目錄 XII
表目錄 XIV

第一章 緒論 1
第二章 文獻回顧 2
2.1枯草桿菌之應用………………………………………….2
2.2幾丁質、幾丁聚醣及其寡醣之製備與應用..…….……2
2.2.1幾丁質………………………………………………2
2.2.2幾丁聚醣……………………………………………3
2.2.3 N-乙醯幾丁寡醣與幾丁寡醣…………...………6
2.2.4 N-乙醯幾丁寡醣與幾丁寡醣之生理活性……….8
2.3幾丁質及幾丁聚醣水解酵素……………………….…..8
2.3.1幾丁質酶……………………………………….….….8
2.3.2幾丁聚醣酶…………………………………………….9
2.3.3其他可水解幾丁聚醣之酵素………………………….9
2.4蛋白酶…………………………………………………..12
2.4.1蛋白酶之簡介…………………………………….12
2.4.2蛋白酶之分類…………………………………….12
2.4.3蛋白酶在工業上之應用………………………….13
2.5酵素在有機溶劑中之行為……………………………..14
2.5.1蛋白酶在有機溶劑中之安定性與合成反應…….14

第三章 材料與方法………..………………..…………………….17
3.1實驗菌株………………………………………………..17
3.2實驗材料………………………………………………..17
3.3實驗儀器………………………………………………..18
3.4生產菌株之篩選………………………………………..19
3.5蛋白酶之活性測定……………………………………..19
3.6幾丁聚醣酶之活性測定………………………….…….20
3.7蛋白酶之較適培養條件探討……………………….….20
3.7.1碳源之選擇……………………………………….20
3.7.2培養基之酸鹼值……………...………….…….20
3.7.3培養溫度………………………………………….21
3.7.4培養基之通氣量………………………….………21
3.7.5較適培養時間…………………………….………21
3.8酵素之純化分離………..……………..………………21
3.8.1粗酵素液之製備…………………….……………21
3.8.2離子交換樹脂層析法………………….…………22
3.8.3疏水性層析法……………….……………………22
3.8.4膠體過濾層析法………………………….………22
3.9分子量標定……………………………...…………….23
3.10蛋白質電泳分析……………………….………………24
3.11蛋白質定量分析…………………………………….…24
3.12酵素生化特性分析………………………….…………24
3.12.1酵素最適溫度………………………………….24
3.12.2酵素熱安定…………………………………….24
3.12.3酵素最適pH……….………….……………….25
3.12.4酵素pH安定性………………………………….25
3.12.5金屬離子與抑制劑對酵素活性之影響……….25
3.12.6界面活性劑對酵素活性之影響……………….26
3.12.7酵素動力學性質………………………..…….26
3.12.8蛋白酶之基質特異性………….………………26
3.13有機溶劑對酵素活性之影響…….……………………27
3.14粗酵素液之蛋白酶及幾丁聚醣酶活性消長……….…27
3.15蛋白質轉印…………………………………………….28
3.16利用Bacillus subtilis TKU007之發酵液進行小白菜生長測試……………………………………......................28
3.16.1小白菜之預培養……………..……………….28
3.16.2促進小白菜生長試驗…………..…………….28
第四章 結果與討論………………………………………………...30
4.1蛋白酶生產菌之篩選…………………..………………30
4.2菌株特性………………………….…………………….30
4.3碳源之選擇……………………………………………..30
4.4蛋白酶較適生長條件探討……………………………..35
4.5粗酵素液之蛋白酶及幾丁聚醣酶活性關係…..………36
4.6蛋白酶之純化分離……………………..………………37
4.6.1粗酵素液製備……………………..…………….37
4.6.2離子交換樹脂層析法…..……………………….38
4.6.3疏水性層析法………………………….…………38
4.6.4膠體過濾層析法………………..……………….38
4.6.5綜合結果…………………………..…………… 39
4.7蛋白酶之分子量判定………………………..…………39
4.7.1膠體過濾層析法………………………………….39
4.7.2 SDS-PAGE………………………………………..40
4.7.3綜合結果………………………………….………40
4.8純化後蛋白酶之生化特性分析…………….………….40
4.8.1蛋白酶反應最適溫度及熱安定性……………….40
4.8.2蛋白酶之最適反應pH及pH安定性………….....41
4.8.3各種化學藥品對蛋白酶之影響…….……………41
4.8.4各種界面活性劑對蛋白酶之影響……………….41
4.8.5蛋白酶之基質特異性………………………….…42
4.8.6蛋白酶之酵素動力學…………………………….42
4.9有機溶劑對蛋白酶活性及安定性影響…………….….42
4.10蛋白酶N端序列分析………………………………....43
4.11幾丁聚醣酶之純化分離……………………………….76
4.11.1粗酵素液製備………………………………….76
4.11.2離子交換樹脂層析法……….…………………76
4.11.3膠體過濾層析法……………….………………76
4.11.4綜合結果…………………………………….…77
4.12幾丁聚醣酶之分子量判定……………..……..…….77
4.12.1膠體過濾層析法…………………………..….77
4.12.2 SDS-PAGE………………………………..……77
4.12.3綜合結果………………………………..…….78
4.13純化後幾丁聚醣酶之生化特性分析……….……..…78
4.13.1純化後幾丁聚醣酶之最適溫度及熱安定性...78
4.13.2幾丁聚醣酶之最適反應pH及pH安定性….....78
4.13.3各種化學藥品對幾丁聚醣酶之影響…..…...79
4.13.4各種界面活性劑對幾丁聚醣酶之影響.………79
4.13.5幾丁聚醣酶之酵素動力學………………..….79
4.14有機溶劑對幾丁聚醣酶活性及安定性影響…….……79
4.15幾丁聚醣酶N端序列分析……………………………..80
4.16利用B. subtilis TKU007抗腫瘤測試……………….81
4.17利用B. subtilis TKU007之發酵液進行小白菜生長測試
………………………………………………………….81

第五章 結論與未來展望…………………………………………..101
參考文獻…………………………………………………………….102
附錄………………………………………………………………….116

圖 目 錄
頁次
圖2.1幾丁質、幾丁聚醣之製備 4
圖2.2幾丁質、幾丁聚醣、纖維素之構造 5
圖4.1 Bacillus subtilis TKU007之顯微照片……………………31
圖4.2 16S rDNA部份鹼基序列分析……………………………....32
圖4.3 MIDI微生物脂肪酸組成分析鑑定系統分析結果........ .33
圖4.4 VITEK鑑定套組分析結果 .............................34
圖4.5蝦殼粉末添加對TKU007產生蛋白酶活性之影響 ..........44
圖4.6不同碳源對於TKU007產生蛋白酶活性之影響........... .45
圖4.7溫度對TKU007產生蛋白酶活性之影響................. .46
圖4.8 pH對TKU007產生蛋白酶活性之影響 ...................47
圖4.9通氣量對TKU007產生蛋白酶活性及幾丁聚醣酶之影響.....48
圖4.10不同通氣量培養一天之SDS-PAGE.................... 49
圖4.11 Bacillus subtilis TKU007培養於蝦殼培養基所產蛋白酶
及幾丁聚醣酶變化情形……………………………………50
圖4.12蛋白酶與幾丁聚醣酶於37℃下反應後之SDS-PAGE...... 51
圖4.13 Bacillus subtilis TKU007所生產蛋白酶之純化分離流程
圖 ..............................................54
圖4.14 DEAE-Sepharose CL-6B 之蛋白酶層析圖譜.......... 55
圖4.15 Phenyl Sepharose 6 Fast Flow之蛋白酶層析圖譜... 56
圖4.16 Sephacryl S-100之蛋白酶層析圖譜................ 57
圖4.17分子量標準品和蛋白酶於Sephacryl S-100之層析圖譜..59
圖4.18蛋白酶於SDS-PAGE之分子量分析…………………….....60
圖4.19蛋白酶之最適反應溫度…………………………………...61
圖4.20蛋白酶之熱安定性………………………………………...62
圖4.21蛋白酶之最適反應pH……………………………………...63
圖4.22蛋白酶之pH安定性…………………………………….....64
圖4.23蛋白酶於50℃之Lineweaver-Burk Plot………………….65
圖4.24有機溶劑對蛋白酶活性之影響…………………………...70
圖4.25蛋白酶對有機溶劑之安定性……………………………...71
圖4.26 Bacillus subtilis TKU007所生產幾丁聚醣酶純化分離流
程圖………………………………………………………..82
圖4.27 DEAE-Sepharose CL-6B 之幾丁聚醣酶層析圖譜……...83
圖4.28 Sephacryl S-100 之幾丁聚醣酶層析圖譜………………84
圖4.29分子量標準品和幾丁聚醣酶於Sephacryl S-100之層析圖
譜…………………………………………………………..86
圖4.30幾丁聚醣酶於SDS-PAGE之分子量分析……………….....87
圖4.31幾丁聚醣酶之最適反應溫度……………………………...88
圖4.32幾丁聚醣酶之熱安定性…………………………………...89
圖4.33幾丁聚醣酶之最適反應pH………………………………...90
圖4.34幾丁聚醣酶之pH安定性……………………………….....91
圖4.35幾丁聚醣酶於37℃之Lineweaver-Burk Plot…………...97
圖4.36有機溶劑對幾丁聚醣酶活性之影響………….…………..98
圖4.37幾丁聚醣酶對有機溶劑之安定性………….……………..99


表 目 錄
頁次
表2.1幾丁質、幾丁聚醣的應用…………………………………...7
表2.2幾丁質酶之來源與特性……………………………………..10
表2.3各種酵素對幾丁聚醣酶之降解反應…………..……………11
表2.4一般常用有機溶劑之logP值…………………..….……….16
表3.1蛋白質轉印所需溶液配方………………………………..…29
表4.1比較蛋白酶與幾丁聚醣酶活性關係………………………..52
表4.2模擬各階段之蛋白量、蛋白酶、幾丁聚醣酶……………..53
表4.3 Bacillus subtilis TKU007蛋白酶之純化總表………….58
表4.4各種化學藥品對蛋白酶之影響……………………………..66
表4.5各種界面活性劑對蛋白酶之影響…………………………..67
表4.6蛋白酶之基質特異性………………………………………..68
表4.7 Bacillus subtilis蛋白酶特性比較………………………69
表4.8比較Bacillus所產酵素之N端序列分析…………………...72
表4.9本研究與其他耐界面活性劑及耐有機溶劑蛋白酶之比較..74
表4.10 Bacillus subtilis TKU007幾丁聚醣酶之純化總表…..85
表4.11各種化學藥品對幾丁聚醣酶之影響…………..….………92
表4.12各種界面活性劑對幾丁聚醣酶之影響……….……………93
表4.13各種微生物生產幾丁聚醣酶之特性比較…………….…..94
表4.14 Bacillus subtilis TKU007之發酵液對小白菜生長影響.100
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