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研究生:高逸馨
論文名稱:人類糞便中幾丁質水解菌之分離與鑑定及幾丁質酶之純化
論文名稱(外文):Isolation and identification of chitinolytic bacteria in human feces and purification of the hrolytic enzymes
指導教授:陳幸臣陳幸臣引用關係
學位類別:碩士
校院名稱:國立海洋大學
系所名稱:食品科學系
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:2002
畢業學年度:90
語文別:中文
論文頁數:87
中文關鍵詞:幾丁質酶厭氧菌糞便幾丁質結合區
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  • 被引用被引用:2
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本研究之目的在分離人類糞便中具幾丁質和 (或) 幾丁聚醣水解能力的細菌並探討其產生幾丁質和 (或) 幾丁聚醣水解酵素之培養條件並純化此酵素。由10-8糞便稀釋液在含膠體幾丁質之BHIA (37oC, 2天) 上可發現三個具透明環菌落,鑑定後該三個菌落皆為Bacteroides fragilis。因此挑選其中一株分離株與參考株 B. fragilis CCRC 10620 一起進行酵素活性試驗。不論分離株或參考株,均不需誘導即有相當高之幾丁質酶活性 (培養一天活性即可達30 unit/ml以上)。但N-乙醯葡萄糖胺酶活性皆相當低。兩菌皆以 BHI 培養液、添加 0.5% (w/v) yeast extract,在37 oC培養五天為最佳之幾丁質酶生產條件。分離株所產粗酵素液經由超過濾膜濃縮後比活性僅有未濃縮前的1/8,再經硫銨沈澱後,以hight Q cartridge 層析後蛋白質量很少。經吸附試驗發現膠體幾丁質 (CC) 及幾丁質粉末 (CP) 等會吸附本分離株之幾丁質酶,因此以此為酵素快速純化之依據。粗酵素液經硫酸銨沈澱後以 1% CP 吸附並去除未吸附之蛋白質,於50oC水解24小時後,經native-PAGE分析後以CBR-250及4-MU-(GlcNAc)2活性染色後可見單一的蛋白質區帶;經SDS-PAGE判斷,此幾丁質酶為多個分子量為 31.8 kDa 的次單元所構成,比活性為433.96 unit/ml,純化倍率為2.57。
目 錄
頁次
中文摘要 I
英文摘要 II
第一章 前言 1
第二章 文獻整理 3
一、腸道微生物 3
1. 腸道菌相 4
2. 腸內菌對宿主健康的影響 4
3. Bacteroides fragilis 的特性 4
二、幾丁質及其衍生物的結構與應用 5
1. 幾丁質、幾丁聚醣的分子結構 5
2. N-乙醯幾丁寡醣及幾丁寡醣的分子結構 6
3. 幾丁質、幾丁聚醣的應用 6
4. 幾丁質及幾丁聚醣水解物的應用 6
4-1. 抑菌方面 6
4-2. 其他方面 7
三、幾丁質酶系統 8
1. 幾丁質酶的分佈、種類及水解幾丁質模式 8
1-1. 幾丁質酶的分佈 (動物、植物、微生物) 8
1-2. 幾丁質酶的種類 8
1-3. 水解幾丁質的模式 9
2. 影響微生物產生幾丁質酶的因子 10
3. 幾丁質酶的結構分析 10
3-1. 催化區 (catalytic domain) 10
3-2. 幾丁質結合區 (chitin-binding domains, ChBDs) 11
3-3. 纖維網蛋白第三功能部位 (Fibronectin type III-like domain) 12
3-4. Cadherin-like domain 12
四、動物消化幾丁質、幾丁聚醣及幾丁寡醣的情形 12
1. 幾丁質、幾丁聚醣在動物消化道的變化 13
2. 動物體消化道中幾丁質酶的分佈 13
3. 腸道微生物的消化作用 14
五、幾丁質酶活性測定及純化方法 16
1. 細菌幾丁質酶的純化 16
2. 幾丁質酶活性測定的探討 17
2-1. 傳統檢測法 17
2-2. 快速檢測法 18
第三章實驗方法與步驟 20
一、材料 20
1. 菌株來源 20
2. 糞便來源 20
3. 化學試藥 20
3-1. 有機溶劑 20
3-2. N-乙醯幾丁寡醣標準品 20
3-3. 酵素基質 21
3-4. 電泳試劑 21
3-5. 其他 21
4. 設備與器材 22
4-1. 厭氧菌培養 22
4-2. HPLC 之設備及管柱規格 22
4-3. FPLC 之設備 23
4-4. 蛋白質電泳 23
4-5. 其他 23
二、步驟 24
1. 糞便中幾丁質、幾丁聚醣水解菌之分離 24
1-1. 糞便取樣 24
1-2. 菌株分離 24
1-3. 菌落計數與菌株單離 25
1-4. 菌株鑑定 25
1-5. 菌株保存 25
1-6. 接種源的製備 25
2. B. fragilisg生產幾丁質酶最適培養條件 26
2-1. 不同培養基對幾丁質酶及N-乙醯葡萄糖胺酶活性之影響 26
2-2. 膠體幾丁質濃度對幾丁質酶活性及菌數之影響 26
2-3. 在BHI、37 oC 培養下幾丁質酶活性、菌數、pH的變化 26
2-4. 培養基中yeast extract濃度對幾丁質酶活性之影響 27
2-5. 不同培養溫度對幾丁質酶活性之影響 27
3. 幾丁質酶與其受質的吸附作用 27
3-1. 吸附作用對酵素活性之影響 27
3-1. 幾丁質粉末吸附幾丁質酶後水解產物分析 27
4. 分離株硫酸銨沈澱酵素幾丁質酶最適反應條件 28
4-1 最適反應pH的測定 28
4-2 最適反應溫度的測定 28
4-3 酵素熱穩定性的測定 29
5. 幾丁質酶的純化 29
5-1. 粗酵素液 (CES) 之製備 29
5-2. 超過濾膜濃縮粗酵素 29
5-3. 酵素之硫銨區分 29
5-4. Econo-Pac high Q Cartridge 層析法 30
6. 幾丁質酶的快速純化 30
三、方法 30
1. 幾丁聚醣的製備 30
2. 膠體幾丁質 (colloidal chitin;CC) 的製備 31
3. 膠體幾丁聚醣 (colloidal chitosan;CS) 的製備 31
4. 培養基的配製 31
4-1. Brain heart infusion broth (BHI) 31
4-2. Basal medium (PY broth-Peptone Yeast Extract) 32
4-3. 含幾丁質、幾丁聚醣培養基 (BCCA及BCSA) 的製備 33
5. 厭氧稀釋液 (Anaerobic dilution blanks, ADB) 33
6. 幾丁質酶活性分析 34
6-1. 反應試劑的配製 34
6-2. 酵素的反應步驟 34
7. Native 和 SDS-PAGE 電泳分析 35
7-1. 膠體的製備 35
7-2. Native-PAGE 分析 38
7-3. SDS-PAGE 分析 37
7-4. 膠片的保存 38
8. 蛋白質的定量 38
9. 統計分析 39
第四章 結果與討論 40
一、糞便中幾丁質、幾丁聚醣水解菌之分離 38
二、B. fragilisg生產幾丁質酶之最適培養條件 41
1. 不同培養基對幾丁質水解酵素活性的影響 41
2.添加不同濃度之CC對幾丁質酶活性及菌株生長的影響 42
3. 培養溫度對幾丁質酶活性的影響 42
4. Yeast extract添加量對幾丁質酶活性的影響 42
5. 兩菌株在BHI、37 oC 培養下幾丁質水解酵素活性、菌數、pH的變化 43
三、分離株幾丁質酶的純化 43
1 粗酵素液經濃縮、硫銨沈澱等粗步純化的結果 43
2. High Q-cartrige層析 44
3. 幾丁質酶與其受質的吸附作用 44
3-1. 分離株幾丁質酶與其受質吸附特性的測試 45
3-1-1. CC、CP對粗酵素液幾丁質酶活性之影響 45
3-1-2. CC、CP吸附幾丁質酶後水解產物分析 45
3-2. 其他多醣類的吸附作用 46
•α-cellulose及 cellulose對粗酵素液活性之影響 46
4. 分離株硫酸銨沈澱酵素幾丁質酶之最適反應條件 46
4-1. 硫酸銨沈澱酵素之最適反應pH及溫度 46
4-2. 硫銨沈澱酵素之熱穩定性 47
5. 幾丁質酶的快速純化 47
第五章 結論 71
第六章 參考文獻 72
圖目錄
頁次
圖一 在含 0.3% (W/V) CC 之 BHIA 平板上,B. fragilis CCRC 10620 (A) 及一號分離株 (B) 的菌落形態 (37oC,2天)。 51
圖二 不同培養液中一號分離株 (A) 及 B. fragilis CCRC 10620 (B) 的幾丁質酶的活性。 52
圖三 不同培養液中一號分離株 (A) 及 B. fragilis CCRC 10620 (B) 的 GlcNAcase 的活性。 53
圖四 培養液中不同膠體幾丁質濃度對一號分離株 (A) 及 B. fragilis CCRC 10620 (B) 幾丁質酶活性之影響。 54
圖五 在培養液中不同膠體幾丁質濃度對一號分離株 (A) 及 B. fragilis CCRC 10620 (B) 生長之影響。 55
圖六 不同培養溫度對一號分離株 (A) 及B. fragilis CCRC 10620 (A) 幾丁質酶活性之影響。 56
圖七 在培養液中不同yeast extract濃度對一號分離株 (A) 及B. fragilis CCRC 10620 (B) 幾丁質酶活性之影響。 57
圖八 一號分離株 (A) 及 B. fragilis CCRC 10620 (B) 於BHI中培養,菌數 (A1、B1)、pH (A2、B2) 及幾丁質酶活性 (A3、B3) 之變化情形。 58
圖九 一號分離株生產之粗酵素液經超過濾濃縮、硫酸銨沈澱後在High Q cartridge 管柱層析圖。 60
圖十 一號分離株生產之粗酵素液以 1% CP 吸附後 (4 oC, 靜置1小時) 於42.5oC 水解之幾丁質水解產物 HPLC 分析圖譜。 62
圖十一 pH對硫銨沈澱酵素幾丁質酶活性之影響。 64
圖十二 溫度對硫銨沈澱酵素幾丁質酶活性之影響。 65
圖十三 硫銨沈澱酵素幾丁質酶之熱穩定性。 66
圖十四 一號分離菌所產幾丁質酶經快速純化之SDS-PAGE電泳圖。 67
圖十五 硫銨沈澱的酵素及快速純化的幾丁質酶在native-PAGE之活性染色。 68
圖十六 以SDS-PAGE檢測分子量之檢量曲線。 69
表目錄
表一 分離株以 API 20 A 套組鑑定之結果。 49
表二 分離株以 API 32 A 套組鑑定之結果。 50
表三 硫酸銨沈澱酵素活性與蛋白質區分表。 59
表四 一號分離株生產之粗酵素液以 CC 及 CP 在不同條件進行吸附試驗後之酵素活性。 61
表五 一號分離株生產之粗酵素液以 cellulose 及 α-cellulose 在不同條件進行吸附試驗後之酵素活性。 63
表六 分離株幾丁質酶之快速純化摘要。 70
第六章 參考文獻
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