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研究生:蘇家彥
研究生(外文):CHIA YAN SU
論文名稱:AlterococcusagarolyticusS3PY及Cellvibriosp.AG1W之洋菜酶定性研究
論文名稱(外文):Characterization of agarases from Alterococcus agarolytics S3PY and Cellvibrio sp. AG1W
指導教授:李重義
指導教授(外文):Chung-Yi Lee
學位類別:碩士
校院名稱:東吳大學
系所名稱:微生物學系
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:132
中文關鍵詞:洋菜酶洋菜分解菌
外文關鍵詞:agarase
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本實驗室於外雙溪,篩選到一株中溫洋菜分解菌 AG1W 。經實驗發現 AG1W為桿菌、 gram-negative 、大小約 0.5 × 3.0 μm、具移動性、好氧,可以水解 starch 及 lipid , catalase 、 oxidase 為正反應,不具 tryptophanase 。AG1W 經 16S rDNA 序列比對,結果發現和 Cellvibrio sp. R4079 有 98 % 、 Cellvibrio mixtus 有 97 % 、 Cellvibrio ostraviensis 有 97 % 、 Cellvibrio fulvus 有 97 % 的相似度。因此初步鑑定中溫洋菜分解菌為 Cellvibrio 屬。高溫洋菜分解菌 S3PY 、具洋菜酶基因且可表現洋菜酶活性的 E. coli 3A 、中溫洋菜分解菌 AG1W 產生的粗洋菜酶特性比較,發現 S3PY 粗洋菜酶對溫度的穩定性最佳,在60 ℃ 處理 30分鐘還擁有 90 % 以上的殘存活性。並發現 S3PY 粗洋菜酶對 pH 值及化學物質穩定性皆最高, S3PY 粗洋菜酶在 pH 值 3 、 10 的環境中,處理 30 分鐘還具有 70 % 以上殘存活性,在 4 M Urea 、 9.5 % 酒精、 2.5 % SDS 下處理 1小時,還具有 50 % 以上殘存活性。 S3PY 和 E. coli 3A 的水解產物用 TLC 分析,發現主要水解產物為新洋菜四糖及新洋菜六糖, AG1W 主要水解產物為新洋菜二糖。S3PY 之菌液,以硫酸銨沉澱、離子交換層析法及膠體過濾層析法進行純化,獲得洋菜酶 G 、洋菜酶 E 及部分純化之洋菜酶B 。洋菜酶 G 、 B 、 E 之分子量分別為39 、128 、55 KDa,KM值分別為19.4 、11.6 、6.8 g/L。在溫度60 ℃下處理10 小時後,洋菜酶 B還具有100 % 的殘存活性,洋菜酶 G剩約70 % 的殘存活性,洋菜酶E已經幾乎完全沒有活性。以 TLC 來分析 S3PY 粗洋菜酶、洋菜酶 G 、 洋菜酶 B ( 未完全純化 ) 、洋菜酶 E 水解洋菜糖之水解產物,發現洋菜酶 G 、B 、 E 之水解產物和 S3PY 粗洋菜酶水解產物不完全相同。以 TLC 分析洋菜酶 G 、 B 、 E 水解新洋菜四、六、八、十、十二糖之水解產物,發現三種洋菜水解酶之水解產物不完全相同。
We isolated the agar-degrading bacterial strain AG1W from the Waishuangshi. After biochemical test, we found AG1W was bacillus, gram-negative, size is about 0.5 × 3.0 μm, movable, aerobe, able to hydrolyze starch and lipid, catalase、oxidase are positive reaction, doesn't have tryptophanase. AG1W after compare with 16S rDNA sequence, we found out that AG1W with Cellvibrio sp. R4079 has 98 %, with Cellvibrio mixtus has 97 %, with Cellvibrio ostraviensis has 97 %, and Cellvibrio fulvus has 97 % similarity. Therefore we defined the agar-degrading bacterial strain AG1W was belong to Cellvibrio sp. To compare agarases from the agar-degrading bacterial strain S3PY, E. coli 3A which possess agarase gene and can represent agar-degrading activity, the agar-degrading bacterial strain AG1W, found that crude agarases of strain S3PY have the best stability to temperature, we made it at 60 degree, after 30 minutes, it still have over 90 % residual activity. We also found S3PY crude agarases stability are the best at pH and chemical. S3PY crude agarases at pH value 3, 10, after 30 minutes, still owned over 70 % residual activity; S3PY crude agarases at 4 M Urea, 9.5 % ethanol, 2.5 % SDS, after 1 hour, it still have over 50 % residual activity. We analyze S3PY and E. coli 3A hydrolysis products with TLC, which the main products was neoagarotetraose and neoagarohexarose; hydrolysis products of AG1W analyze with TLC, found that the main products was neoagarobiose. The agarase G, agarase B ( which was not completely purified ), agarase E which were purified from the S3PY culture supernatant by ammonium sulfate precipitation, anion exchange and gel giltration column chromatographic methods. Estimation of the molecular mass of agarase G, agarase B, agarase E by SDS-PAGE gave values of 39, 128, 55 KDa, respectively. The KM of agarase G, agarase B, agarase E gave values of 19.4 、11.6 、6.8 g/L, respectively. To analyze S3PY crude agarases, agarase G, agarase B ( which was not completely purified ), agarase E, hydrosis products of hydrolyze agarose by thin-layer chromatogram, we find hydrolysis products of agarase G, agarase B, agarase E are all different from hydrolysis products of S3PY crude agarases.To analyze agarase G, agarase B ( which was not completely purified ), agarase E hydrolysis products of hydrolyze neoagarobiose, neoagarotetraose, neoagarohexaose, neoagarooctaose, neoagarodecaose, neoagarododecaose by thin-layer chromatogram, find that hydrolysis products of three kinds agarases has different.
中文摘要…………………………………………………………………Ⅰ
英文摘要………………………………………………………………Ⅲ
目錄………………………………………………………………………Ⅴ
表目錄…………………………………………………………………
圖目錄…………………………………………………………………XI
第一章 前言………………………………………………………………1
第一節 洋菜………………………………………………………………1
1.1 洋菜的組成…………………………………………………1
1.2 洋菜的應用…………………………………………………1
第二節 洋菜酶…………………………………………………………2
2.1 洋菜酶之基本性質…………………………………………2
2.1.1 洋菜酶之分子量……………………………………2
2.1.2 洋菜酶之最適作用溫度……………………………4
2.1.3 洋菜酶之最適作用pH值……………………………5
2.1.4 洋菜酶作用後之水解產物…………………………6
2.1.5 洋菜酶之受質特異性………………………………9
2.2 洋菜分解菌…………………………………………………9
2.2.1 洋菜分解菌之種類…………………………………9
2.2.2 具有一種以上洋菜酶之洋菜分解菌………………10
2.2.3 Cellvibrio 屬…………………………………………12
2.3 洋菜酶之純化………………………………………………13
2.3.1 硫酸銨沉澱…………………………………………13
2.3.2 離子交換層析法……………………………………13
2.3.3 膠體過濾層析法……………………………………14
2.3.4 親和性管柱層析法…………………………………15
第三節 洋菜水解產物………………………………………………15
3.1 洋菜水解產物之獲得………………………………………15
3.2 洋菜水解產物之分析………………………………………16
3.2.1薄層層析法…………………………………………16
3.2.2 高效能液相層析法…………………………………16
第四節 洋菜水解產物之應用………………………………………16
4.1 海藻多醣之生理活性………………………………………16
4.2 非消化性碳水化合物之生理活性…………………………17
第五節 洋菜酶之應用………………………………………………17
5.1 利用洋菜酶之專一性生產高純度產物……………………17
5.2 開發海藻寡醣之健康食品…………………………………18
5.3 DNA 或 RNA 分離………………………………………18
第六節 研究目的及動機……………………………………………18
第二章 材料與方法……………………………………………………20
第一節 藥品與器材…………………………………………………20
第二節 菌株…………………………………………………………20
2.1 菌株 S3PY………………………………………………20
2.2 菌株 E. coli 3A……………………………………………20
2.3 菌株 AG1W………………………………………………21
第三節 菌株活化與保存……………………………………………21
第四節 S3PY菌株培養………………………………………………21
4.1 S3PY種菌的製備…………………………………………21
4.2 S3PY 封口錐形瓶的培養…………………………………21
4.3 S3PY 醱酵槽培養…………………………………………22
第五節 培養基配製…………………………………………………22
5.1.1菌株 S3PY 液體生長培養基……………………………22
5.1.2菌株 S3PY 固體生長培養基……………………………22
5.1.3菌株 S3PY 酵素生產培養基……………………………23
5.2.1 菌株E. coli 3A生長培養基………………………………23
5.3.1 菌株 AG1W 液體生長培養基……………………………23
5.3.2 菌株 AG1W 固體生長培養基……………………………24
第六節 菌株 AG1W 16S rDNA 分析………………………………24
第七節 菌株粗洋菜酶的獲得………………………………………25
7.1 菌株 S3PY 粗洋菜酶酶獲得……………………………25
7.2 菌株 E. coli 3A 粗洋菜酶獲得……………………………25
7.3 菌株 AG1W 粗洋菜酶酶獲得……………………………25
第八節 洋菜酶活性分析……………………………………………26
8.1 偵測還原糖變化量,得知洋菜酶的活性…………………26
8.1.1緩衝液的製備:………………………………………26
8.1.2受質的製備:…………………………………………27
8.1.3 半乳糖標準溶液製備:………………………………27
8.1.4 還原糖反應劑的製備:………………………………27
8.1.5偵測還原糖變化量,得知洋菜酶活性之方法………28
8.2.1利用洋菜膠測定洋菜酶酵素活性……………………28
8.3.1利用SDS-PAGE電泳後活性染測定洋菜酶活性……29
第九節 菌株S3PY洋菜酶純化……………………………………29
9.1菌株 S3PY 粗洋菜酶酶獲得……………………………29
9.2 離子交換層析法…………………………………………29
9.3 膠體過濾層析法…………………………………………30
9.4 製備型電泳………………………………………………31
第十節 蛋白質分析…………………………………………………31
10.1 蛋白質的定量分析………………………………………31
10.2 SDS-PAGE 分析…………………………………………32
10.2.1 SDS-PAGE 的材料製備…………………………32
10.2.2 操作步驟…………………………………………34
10.3 native page分析…………………………………………34
10.4 銀染分析…………………………………………………34
第十一節 洋菜酶定性………………………………………………35
11.1 洋菜酶活性受溫度的影響………………………………35
11.2 洋菜酶對溫度穩定性分析………………………………35
11.3 洋菜酶活性受 pH 值之影響……………………………36
11.4 洋菜酶對 pH值穩定性分析………………………………36
11.5 洋菜酶活性受鹽濃度之影響……………………………37
11.6 洋菜酶活性受化學物質之影響…………………………37
11.6.1 洋菜酶活性受 Urea 之影響……………………37
11.6.2洋菜酶對 Urea 穩定性分析………………………38
11.6.3 S3PY洋菜酶受化學物質之影響…………………38
第十二節 水解產物分析……………………………………………39
12.1 水解產物獲得……………………………………………39
12.2 TLC 分析水解產物………………………………………39
12.3 利用TLC分離回收新洋菜寡糖…………………………40
第三章 結果……………………………………………………………41
第一節 AG1W 菌種鑑定…………………………………………41
第二節 粗洋菜酶的定性……………………………………………41
第三節 S3PY 洋菜酶的純化………………………………………46
第四節 純化之 S3PY 洋菜酶特性分析……………………………51
第四章 討論……………………………………………………………54
第一節 AG1W 菌種鑑定…………………………………………54
第二節 粗洋菜酶的定性……………………………………………55
第三節 S3PY 洋菜酶的純化………………………………………58
第四節 純化之 S3PY 洋菜酶特性分析……………………………59
參考文獻…………………………………………………………………62
圖表………………………………………………………………………72
附錄……………………………………………………………………117
圖 目 錄
圖一、 AG1W 在1/6 AAPY 固體培養基上,以28 ℃ 培養3天之菌落形態……………………………………………………………72
圖二、 AG1W 在1/6 AAPY 固體培養基上28 ℃ 培養5天後,利用碘液來偵測洋菜酶活性…………………………………………73
圖三、 AG1W 之16S rDNA 序列定序結果……………………………74
圖四、 S3PY 在 Bacto Marine Broth 2216 配製成的固體培養基上,以50 ℃ 培養3天之菌落形態……………………………………75
圖五、 S3PY 在 Bacto Marine Broth 2216 配製成的固體培養基上50 ℃培養3天後,利用碘液來偵測洋菜酶活性…………………76
圖六、 A. agarolyticus 菌株 S3PY 在醱酵槽培養的監測圖…………77
圖七、利用洋菜膠檢測洋菜酶之活性…………………………………78
圖八、 S3PY 、 E. coli 3A及 AG1W 之粗洋菜酶 SDS-PAGE 活性染…………………………………………………………………79
圖九、 AG1W 之粗洋菜酶 negative page 及 SDS page 活性染…………………………………………………………………80
圖十、溫度對洋菜水解酶活性的影響……………………………81
圖十一、溫度對洋菜水解酶穩定性之影響……………………………82
圖十二、溫度對S3PY 洋菜水解酶長期穩定性之影響………………83
圖十三、 pH 值對 S3PY 、 E. coli 3A 、 AG1W 洋菜水解酶活性之影響………………………………………………………………84
圖十四、 pH 值對 S3PY 、 E. coli 3A 、 AG1W 洋菜水解酶穩定性之影響……………………………………………………………85
圖十五、鹽濃度對洋菜水解酶活性之影響……………………………86
圖十六、 Urea 對洋菜水解酶活性之影響……………………………87
圖十七、 Urea 對洋菜水解酶穩定性之影響…………………………88
圖十八、 SDS 對 S3PY 洋菜水解酶穩定性的影響…………………89
圖十九、 Urea 對 S3PY 洋菜水解酶穩定性的影響…………………90
圖二十、 Urea 對 S3PY 洋菜水解酶穩定性的影響…………………91
圖二十一、以TLC 分析 S3PY 、 E. coli 3A 、 AG1W 洋菜酶水解洋菜糖之水解產物…………………………………………………92
圖二十二、以TLC 分析 S3PY 、 E. coli 3A 、 AG1W 洋菜酶水解新洋菜四、六、八、十糖之水解產物……………………………93
圖二十三、利用洋菜膠活性染測定 S3PY 粗洋菜酶離子交換層析法回收管酵素活性……………………………………………………94
圖二十四、菌株 S3PY 之粗洋菜酶使用 TOYOPEARL DEAE-650 M 膠體分離之離子交換層析圖……………………………………95
圖二十五、利用 SDS-PAGE 活性染測定 S3PY 粗洋菜酶離子交換層析法回收管酵素組成……………………………………………96
圖二十六、 S3PY粗洋菜酶 SDS-PAGE 活性染電泳圖………………97
圖二十七、 SDS-PAGE 活性染測定 S3PY 粗洋菜酶離子交換層析法回收管酵素組成…………………………………………………98
圖二十八、第 Ⅰ 組離子交換層析法回收之酵素液進行膠體過濾層析法數值圖…………………………………………………………99
圖二十九、第 Ⅰ 組離子交換層析法回收之酵素液再經過膠體過濾層析法回收之酵素液銀染及活性染……………………………100
圖三十、 S3PY之酵素 G 純化流程銀染及活性染…………………101
圖三十一、第 Ⅱ 組離子交換層析法回收之酵素液進行膠體過濾層析法數值圖………………………………………………………102
圖三十二、第 Ⅱ 組離子交換層析法回收之酵素液再經過膠體過濾層析法回收之酵素液活性染……………………………………103
圖三十三、第 Ⅲ 組離子交換層析法回收之酵素液進行膠體過濾層析法數值圖………………………………………………………104
圖三十四、第 Ⅲ 組離子交換層析法回收之酵素液再經過膠體過濾層析法回收之酵素液活性染……………………………………105
圖三十五、 S3PY 之酵素 E 純化流程銀染及活性染………………106
圖三十六、第 Ⅳ 組離子交換層析法回收之酵素液進行膠體過濾層析法數值圖………………………………………………………107
圖三十七、第 Ⅳ 組離子交換層析法回收之酵素液再經過膠體過濾層析法回收之酵素液活性染……………………………………108
圖三十八、第 Ⅴ 組離子交換層析法回收之酵素液進行膠體過濾層析法數值圖………………………………………………………109
圖三十九、第 Ⅴ 組離子交換層析法回收之酵素液再經過膠體過濾層析法回收之酵素液活性染……………………………………110
圖四十、 S3PY 粗洋菜酶經過離子交換層析法及膠體過濾層析法純化之酵素液活性染………………………………………………111
圖四十一、溫度對S3PY 粗洋菜酶、洋菜酶 G 、 B ( 未完全純化 ) 、 E長期穩定性之影響…………………………………………112
圖四十二、S3PY 粗洋菜酶在 60 ℃下處理 0、 1、 2、 3 小時後活性染電泳圖……………………………………………………113
圖四十三、S3PY 粗洋菜酶、洋菜酶 G 、 B ( 未完全純化 ) 、 E之double-reciprocal plot…………………………………………114
圖四十四、以 TLC 分析 S3PY 洋菜酶 G 、 B 、 E 水解洋菜糖之水解產物………………………………………………………115
圖四十五、以 TLC 分析 S3PY 洋菜酶 G 、 B 、 E 水解新洋菜四、六、八、十、十二糖之水解產物………………………………116
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