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研究生:梁文星
研究生(外文):Liang, Wen-Sing
論文名稱:無內毒素大腸桿菌轉殖株-XYLII所產β-1,3-聚木醣酶之特性及水解β-1,3-聚木醣所得木寡醣之生理活性探討
論文名稱(外文):Studies on Characterization of β-1,3-Xylanase XYLII from E. coli ClearColi BL21(DE3)-xylII and Bioactivities of β-1,3-Xylooligosaccharides Produced from β-1,3-Xylan Hydrolyzed by β-1,3-Xylanase XYLII
指導教授:潘崇良
指導教授(外文):Pan, Chorng-Liang
口試委員:周正俊顏聰榮殷儷容蔡國珍吳彰哲
口試委員(外文):Chou, Cheng-chunYan, Tsong-RongYin, Li-JungTsai, Guo-JaneWu, Chang-Jer
口試日期:2015-12-18
學位類別:博士
校院名稱:國立臺灣海洋大學
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:2015
畢業學年度:104
語文別:中文
論文頁數:158
中文關鍵詞:海葡萄β-13-聚木醣酶β-13-木寡醣Caco-2 細胞吸收運輸抗氧化活性抗發炎活性
外文關鍵詞:Caulerpa lentilliferaβ-13-xylanaseβ-13-xylooligosaccharideCaco-2 cell absorption and transportationAntioxidation activityAntiinflammatory activity
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本研究目的為 Pseudomonas vesicularis MA103 xylII 基因轉殖至 E. coli ClearColi BL21(DE3) 表現β-1,3-聚木醣酶XYLII水解β-1,3-聚木醣所得β-1,3-混合木寡醣之生理活性探討。本研究將P. vesicularis MA103 xylII基因轉殖至 E. coli ClearColi BL21(DE3) 表現系統中,以 Isopropyl β-D-1-thiogalactopyranoside (IPTG) 可誘導表現β-1,3-聚木醣酶XYLII,再由反應曲面法得知以 0.0359 mM IPTG、18℃、與 23 hr 條件可以獲得最高的β-1,3-聚木醣酶XYLII 比活性表現,接著,經 Ni-affinity 管柱純化後可得到單一β-1,3-聚木醣酶XYLII。SDS-PAGE 分析β-1,3-聚木醣酶XYLII分子量為91 kDa,經胺基酸序列比對分析屬於Glycoside Hydrolase Family 26 (GH 26) 以及 C 端具有 2 個相同 Family 31的 Carbohydrate-binding modules (CBM)。在酵素特性上,β-1,3-聚木醣酶XYLII只對β-1,3-聚木醣有酵素活性。生化測試結果得知β-1,3-聚木醣酶XYLII之最適 pH 值與溫度分別為 7.5 和 35℃;酵素活性在 pH 值 6.0-8.0 與 0-35℃ 之間有較好的安定性。Hg2+會使此酵素失去活性;Cu2+和Mn2+可提升此酵素活性。經 High performance liquid chromatography (HPLC) 分析結果得知,β-1,3-聚木醣酶XYLII可將β-1,3-聚木醣進行水解產生木糖、β-1,3-木二糖、與β-1,3-木三糖,其分子量分別為 150.0、282.1、與 414.1 Da。綠藻海葡萄 (Caulerpa lentillifera) 經鹼萃取後所獲得β-1,3-聚木醣產率為 24.93%,接著經酵素水解後,由超過濾系統收集獲得 < 3 kDa β-1,3-混合木寡醣產物 (XOSmix) 之產率為 46.07%,在硫酸基含量測定方面β-1,3-聚木醣與 XOSmix 分別為 0.69% 與 0.74%,並且經 Fourier transform infrared spectrometer (FT-IR) 分析下具有硫酸基團 (S=O) 之吸收波峰 (1,247 cm-1),且在 899 cm-1 吸收波峰顯示出β-1,3-聚木醣與 XOSmix 是以β-糖苷鍵 (β-Glycosidicbonds) 鍵結而成,其兩者主要單糖組成為葡萄糖與木糖。在抗氧化試驗中,10 mg/mL XOSmix 的劑量上所展現 2,2-Diphenyl-1-pikryl-hydrazyl (DPPH) 清除力可高達 79.7%,其相當於 8.7 μg/mL Trolox 的當量濃度,在相同劑量其總抗氧化活性相當於146.4 μg/mL Trolox。螯合亞鐵離子能力與還原力表現上 20 mg/mL XOSmix 分別呈現相當於 64.3 μg/mL Ethylenediaminetetraacetic acid (EDTA) 與 115.1 μg/mL Trolox。抗凝血活性表現上 XOSmix 可延遲 APTT 凝集時間。15 mg/mL XOSmix 具有最高抑制血管收縮素轉換酶 (Angiotensin I converting enzyme, ACE) 活性達 78.67%。在 Caco-2 細胞運輸試驗中,XOSmix 能通過 Caco-2 細胞單層膜運輸至下層液
,當反應至 0-270 min 時,上層液總糖量從 19.46 mg/mL 降至 13.11 mg/mL,下層液則會從 0 mg/mL 提升至 4.74 mg/mL,推估當反應 270 min 後 XOSmix Caco-2 細胞運輸率為 24.3%。在抗發炎試驗中,500 μg/mL XOSmix 具有降低脂多醣 (Lipopolysaccharide, LPS) 誘導 RAW 264.7 cell 中 Nitric oxide (NO)、Superoxide anion radical (O2-)、Tumour necrosis factor-α (TNF-α)、及 Interleukin-6 (IL-6) 之分泌量,並與控制組呈現顯著差異。綜合實驗結果顯示,在未來可進一步將 XOSmix 開發成具有保健功能性之食品添加物的應用。

The aim of this study is to observe cloning and expression of xylII gene from Pseudomonas vesicularis MA103 to Escherichia coli ClearColi BL21(DE3) and bioactivities of β-1,3-xylooligosaccharides produced from β-1,3-xylan hydrolyzed by β-1,3-xylanase XYLII. The DNA encoding β-1,3-xylanase XYLII from P. vesicularis MA103 was cloned into pET-39b(+) expression vector and transformed into E. coli ClearColi BL21 (DE3). After isopropyl β-D-thiogalactopyranoside induction, the soluble recombinant β-1,3-xylanase XYLII was expressed in E. coli ClearColi BL21(DE3). Response surface methodology to know that β-1,3-xylanase XYLII express the highest specific activity used 0.0359 mM IPTG, at 18℃ and in 23 hr. The β-1,3-xylanase XYLII with a molecular mass of 91 kDa was purified to electrophoretical homogeneity after nickel affinity chromatography. The domain catalytic structure of the β-1,3-xylanase XYLII belongs to Family 26 of glycoside hydrolases, followed by two Family 31 of carbohydrate-binding modules at the C terminus. It had an optimal temperature and pH of 35℃ and 7.5, respectively. It was highly inhibited by Hg2+, but activated by Mn2+ and Cu2+. High performance liquid chromatography (HPLC) results showed that the major hydrolysis products of the β-1,3-xylanase XYLII from β-1,3-xylan were xylose, β-1,3-xylobiose, and β-1,3-xylotriose. In the liquid chromatography electrospray ionization mass spectrometer (LC-ESI-MS) spectrum, the m/z ratios of xylose, β-1,3-xylobiose, and β-1,3-xylotriose were 150.0, 282.1, and 414.1, respectively. The β-1,3-xylan was alkali extracted from the green alga Caulerpa lentillifera; this compound was hydrolyzed by β-1,3-xylanase XYLII to produce mixed < 3 kDa β-1,3-xylooligosaccharide (XOSmix). The yield of β-1,3-xylan and XOSmix were 0.69% and 0.74%, respectively. The sulfate content of β-1,3-xylan and XOSmix were 24.93% and 46.07%, respectively. The fourier transform infrared spectrometer (FT-IR) spectrum of β-1,3-xylan and XOSmix revealed peaks at 1,247 cm−1 and 899 cm−1 were S=O group and β-glycosidic bonds, respectively. The monosaccharide composition indicated that β-1,3-xylan and XOSmix were mainly composed of glucose and xylose. The results of the antioxidative tests showed that the 10 mg/mL XOSmix exhibited the highest 2,2-Diphenyl-1-pikryl-hydrazyl (DPPH) scavenging activities activity (79.7%) was equivalent to that of 8.7 μg/mL Trolox. The total antioxidant status assay of 10 mg/mL XOSmix was equivalent to that of 146.4 μg/mL Trolox. The 20 mg/mL XOSmix exhibited the ferrous ion (Fe2+) chelating activity and reducing power were equivalent to that of 64.3 μg/mL ethylenediaminetetraacetic acid (EDTA) and 115.1 μg/mL Trolox, respectively. Regarding the anticoagulant activity, XOSmix delayed the activated partial thromboplastin time. When the 15 mg/mL XOSmix was exhibited to the highest Angiotensin I converting enzyme (ACE) inhibition rate at 78.67%. XOSmix was transported by Caco-2 cell, after 270 min reaction, XOSmix contents were changed in the apical fluid and in the basolateral fluid from 19.46 to 13.11 mg/mL and 0 to 4.74 mg/mL, respectively. After 270 min reacting with Caco-2 cell, the transportation of XOSmix was 24.3%. XOSmix have anti-inflammatory activity on lipopolysaccharides (LPS) induced RAW 264.7 cell, 500 μg/mL concentration they could significantly decrease Nitric oxide (NO), Superoxide anion radical (O2-), Tumour necrosis factor-α (TNF-α), and Interleukin-6 (IL-6) production compared to control group had. These results suggest that XOSmix exhibits potential for application in the food industry.
中文摘要 I
Abstract III
目錄 V
圖目錄 XIII
表目錄 XVI
附錄目錄 XVII
第一章、總論 1
壹、研究背景與目的 2
貳、文獻整理 4
一、海藻簡介 4
二、蕨藻簡介 4
2-1. 海葡萄 5
2-2. 海葡萄營養組成 5
三、海藻多醣 6
3-1. 海藻多醣簡介 6
3-2. 海藻多醣生理活性 6
3-2-1. 抗氧化 7
3-2-2. 抗凝血 8
3-2-3. 抗發炎 8
四、海藻寡醣生理活性 9
4-1. 抗氧化 9
4-2. 抗凝血 9
4-3. 抗發炎 10
4-4. 抗高血壓 10
4-5. Caco-2 細胞之醣類吸收模式 11
五、聚木醣 12
5-1. 組成與結構 12
5-2. 聚木醣萃取 13
5-3. 聚木醣降解 13
5-4. 木寡醣應用 13
六、聚木醣水解酵素 14
6-1. 聚木醣與聚木醣水解酵素 14
6-2. 聚木醣水解酵素之利用 15
七、無內毒素大腸桿菌 15
八、反應曲面法 (Response surface methodology, RSM) 之介紹 16
參、實驗設計與規劃 17
第二章、無內毒素大腸桿菌轉殖株-XYLII 所產β-1,3-聚木醣酶XYLII之表現及水解β-1,3-聚木醣所得β-1,3-木寡醣之分離純化 18
壹、前言 19
貳、實驗設計 21
參、材料與方法 22
一、實驗材料 22
1-1. 原料 22
1-2. 實驗菌株 22
1-3. 實驗藥品 22
1-4. 儀器設備 24
二、實驗方法 25
2-1. 菌株保存與活化 25
2-1-1. 菌株保存 25
2-1-2. 菌株活化 25
2-2. P. vesicularis MA103 β-1,3-xylanase (xylII) 基因分析比對 25
2-3. P. vesicularis MA103 xylII 基因之表現系統構築 26
2-4. 轉殖株E. coli ClearColi BL21(DE3) pET-39b(+)-xylII (CCBL21-xylII) 之生長曲線 26
2-5. 以反應曲面法 (Response surface methodology, RSM) 之最佳化組合反應條件誘導 E. coli ClearColi BL21(DE3) pET-39b(+)-xylII表現β-1,3-聚木醣酶XYLII 活性 26
2-5-1. 反應曲面法實驗設計 26
2-5-2. 誘導 CCBL21-xylII 表現重組β-1,3-聚木醣酶XYLII 活性 27
2-6. 純化重組β-1,3-聚木醣酶XYLII 28
2-7. 蛋白質濃度的定量 28
2-8. 十二烷基硫酸鈉聚丙烯醯胺膠電泳 (Sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE) 蛋白質分析 28
2-9. β-1,3-聚木醣酶XYLII之活性測定 29
2-9-1. β-1,3-聚木醣萃取 29
2-9-2. β-1,3-聚木醣酶XYLII活性測定 29
2-10. 重組β-1,3-聚木醣酶XYLII之生化特性分析 30
2-10-1. 最適反應pH值 30
2-10-2. pH值安定性 30
2-10-3. 最適反應溫度 30
2-10-4. 溫度安定性 31
2-10-5. 金屬離子與化學物質對重組β-1,3-聚木醣酶XYLII的影響 31
2-10-6. 不同基質對重組β-1,3-聚木醣酶XYLII的特異性 31
2-10-7. 反應時間對重組β-1,3-聚木醣酶XYLII水解β-1,3-聚木醣的影響 31
2-10-8. 以 Thin layer chromatography (TLC) 測定反應時間對重組β-1,3-聚木醣酶XYLII水解β-1,3-聚木醣的寡醣產物分析 31
三、統計分析 32
肆、結果與討論 33
一、P. vesicularis MA103 β-1,3-聚木醣酶XYLII基因分析比對 33
二、P. vesicularis MA103 xylII 基因之表現系統構築 34
三、E. coli ClearColi BL21(DE3) pET-39b(+)-xylII之生長曲線與反應曲面法 (Response surface methodology, RSM) 之最佳化誘導表現β-1,3-聚木醣酶XYLII 活性探討 34
四、E. coli ClearColi BL21(DE3) pET-39b(+)-xylII之表現與純化重組β-1,3-聚木醣酶XYLII 35
五、重組β-1,3-聚木醣酶XYLII之 pH 值與溫度的影響 36
六、金屬離子、化學物質、與不同基質對重組β-1,3-聚木醣酶XYLII的影響 37
伍、結論 38
第三章、無內毒素大腸桿菌轉殖株-XYLII 所產β-1,3-聚木醣酶XYLII水解β-1,3-聚木醣所得β-1,3-混合木寡醣之生理活性探討 39
壹、前言 40
貳、實驗設計 41
參、材料與方法 42
一、實驗材料 42
1-1. 原料 42
1-2. 實驗菌株 42
1-3. 實驗藥品 42
1-4. 儀器設備 44
二、實驗方法 45
2-1. 菌株保存與活化 45
2-1-1. 菌株保存 45
2-1-2. 菌株活化 45
2-2. β-1,3-聚木醣酶XYLII製備 45
2-3. β-1,3-混合木寡醣 (XOSmix) 之製備 45
2-4. β-1,3-木寡醣硫酸基含量測定 46
2-5. 總酚含量測定 46
2-6. High performance liquid chromatography (HPLC) 分析β-1,3-聚木醣與 XOSmix 單糖組成 46
2-7. 不同聚合度β-1,3-木寡醣之製備 47
2-8. 傅立葉轉換紅外線 (Fourier transform infrared, FT-IR) 光譜分析 47
2-9. 液相層析電噴灑離子質譜 (Liquid chromatography electrospray ionization mass, LC-ESI-MS) 分析 47
2-10. 核磁共振 (Nuclear magnetic resonance, NMR) 光譜分析 47
2-11. 抗氧化活性測定 47
2-11-1. 清除2,2-Diphenyl-1-Pikryl-hydrazyl (DPPH)自由基能力之測定 47
2-11-2. 清除2,2- Azino-bis-3-ethylbenz-thiazoline-6-sulfonic acid (ABTS) 自由基能力之測定 48
2-11-3. 亞鐵離子螯合能力之測定 48
2-11-4. 還原力之測定 49
2-12. 抗凝血活性測定 49
2-12-1. 活化部分凝血活酶時間 (Activited partial thromboplastin time, APTT) 49
2-12-2. 凝血酶原時間 (Prothrombin time, PT) 49
2-12-3. 凝血酶時間 (Thromboplastin time, TT) 49
2-13. 血管收縮素轉換酶 (ACE) 抑制能力測定 50
肆、結果與討論 51
一、β-1,3-混合木寡醣 (XOSmix) 之製備與分析 51
二、高效能液相層析 (HPLC) 分析β-1,3-聚木醣與 XOSmix 單糖組成 51
三、高效能液相層析 (HPLC) 分析 XOSmix 聚合度分佈 52
四、傅立葉轉換紅外線 (FT-IR) 光譜分析 53
五、液相層析電噴灑離子質譜 (LC-ESI-MS) 分析 53
六、核磁共振 (NMR) 光譜分析 54
七、清除 DPPH 自由基能力之測定 54
八、清除 ABTS 自由基能力之測定 55
九、螯合亞鐵離子能力之測定 55
十、還原力測定 56
十一、抗凝血活性 56
11-1. 活化部分凝血活酶時間 (APTT) 56
11-2. 凝血酶原時間 (PT) 57
11-3. 凝血酶時間 (TT) 57
十二、血管收縮素轉換酶 (ACE) 抑制能力測定 58
伍、結論 59
第四章、無內毒素大腸桿菌轉殖株-XYLII 所產β-1,3-聚木醣酶XYLII水解β-1,3-聚木醣所得β-1,3-混合木寡醣之細胞生理活性探討 60
壹、前言 61
貳、實驗設計 62
參、材料與方法 63
一、實驗材料 63
1-1. 原料 63
1-2. 實驗菌株 63
1-3. 實驗細胞株 63
1-4. 實驗藥品 63
1-5. 儀器設備 65
二、實驗方法 66
2-1. 菌株保存與活化 66
2-1-1. 菌株保存 66
2-1-2. 菌株活化 66
2-2. β-1,3-聚木醣酶 XYLII 製備 66
2-3. β-1,3-混合木寡醣 (XOSmix) 之製備 66
2-4. β-1,3-混合木寡醣 (XOSmix) 於腸道細胞吸收與運輸試驗 66
2-4-1. Caco-2 細胞株活化 66
2-4-2. Caco-2 細胞株培養方法及繼代方法 67
2-4-3. Caco-2 細胞株之冷凍保存 67
2-4-4. XOSmix 對 Caco-2 細胞株之毒性試驗 67
2-4-5. Transwell Insert 培養法 68
2-4-6. Caco-2 細胞於吸收運輸裝置其單層膜完整性測試 68
2-4-7. β-1,3-混合木寡醣 (XOSmix) 於腸道細胞吸收及運輸測定 68
2-4-8. 測定運輸液及細胞中總糖量 69
2-4-9. 測定運輸液之 HPLC 分析 69
2-5. β-1,3-混合木寡醣 (XOSmix) 之抗發炎活性測定 69
2-5-1. RAW 264.7 細胞株培養方法及繼代方法 69
2-5-2. RAW 264.7 細胞株之冷凍保存 69
2-5-3. XOSmix 對 RAW 264.7 細胞株之毒性試驗 70
2-5-4. 一氧化氮 (Nitric oxide, NO) 生成量試驗 70
2-5-5. 吞噬球呼吸爆 (Respiratory burst) Nitroblue-tetrazolium
(NBT) 還原分析試驗 70
2-5-6. 細胞激素 Tumor necrosis factor-α(TNF-α 濃度測定 71
2-5-7. 細胞激素 Interleukin-6 (IL-6) 濃度測定 71
三、統計分析 72
肆、結果與討論 73
一、β-1,3-混合木寡醣 (XOSmix) 於 Caco-2 細胞吸收運輸之變化 73
1-1. Caco-2 細胞形態之變化 73
1-2. XOSmix 對 Caco-2 細胞株之毒性試驗 73
1-3. 運輸液與細胞中總糖分析 73
1-4. 運輸液之 HPLC 分析 74
二、β-1,3-混合木寡醣 (XOSmix) 之抗發炎活性分析 74
2-1. XOSmix 對 RAW 264.7 細胞株之毒性試驗 74
2-2. 一氧化氮 (NO) 生成量試驗 74
2-3. 吞噬球呼吸爆 NBT 還原分析試驗 75
2-4. 細胞激素 TNF-α、和 IL-6 之分泌量分析 76
伍、結論 77
陸、參考文獻 78
柒、附錄 151 
圖目錄
圖 1.β-1,3-聚木醣酶XYLII分子結構 101
圖 2.不同來源β-1,3-聚木醣酶之多重胺基酸序列比對 102
圖 3.pET-39b(+)-xylII 表現載體之圖譜 103
圖 4.轉殖株 E. coli ClearColi BL21 pET-39b(+)-xylII 在 LM-Miler 培養液中於 37℃ 下培養 36 hr 之生長曲線 104
圖 5.誘導劑濃度和誘導溫度的相關性在培養 6 hr 之對應 E. coli ClearColi BL21 pET-39b-xylII 表現β-1,3-聚木醣酶XYLII之
等高線圖 105
圖 6.誘導劑濃度和誘導溫度的相關性在培養 15 hr 之對應 E. coli ClearColi BL21 pET-39b-xylII 表現β-1,3-聚木醣酶XYLII之
等高線圖 106
圖 7.誘導劑濃度和誘導溫度的相關性在培養 24 hr 之對應 E. coli ClearColi BL21 pET-39b-xylII 表現β-1,3-聚木醣酶XYLII之
等高線圖 107
圖 8.誘導劑濃度和誘導溫度的相關性在培養 23 hr 之對應 E. coli ClearColi BL21 pET-39b-xylII 表現β-1,3-聚木醣酶XYLII之
等高線圖 108
圖 9.粗酵素液與純化重組β-1,3-聚木醣酶XYLII之 SDS-PAGE 電泳分析圖 109
圖 10.pH 對重組β-1,3-聚木醣酶XYLII活性與安定性的影響 110
圖 11.溫度對重組β-1,3-聚木醣酶XYLII活性與安定性的影響 111
圖 12.反應時間對重組β-1,3-聚木醣酶XYLII水解β-1,3-聚木醣的影響 112
圖 13.混合 6 種單糖標準品 (A) 與單糖標準品 (B) 之高效能液相層析圖 113
圖 14.β-1,3-聚木醣單糖組成之高效能液相層析圖 114
圖 15.β-1,3-混合木寡醣 (XOSmix) 單糖組成之高效能液相層析圖 115
圖 16.β-1,3-混合木寡醣 (XOSmix) 聚合度分佈之高效能液相層析圖 116
圖 17.β-1,3-聚木醣 (A) 與β-1,3-混合木寡醣 (XOSmix) (B) 之傅立葉轉換紅外線光譜圖 117
圖 18.β-1,3-xylotriose (M3) 區分物之正離子模式 (A) 與負離子模式 (B) 之 LC-ESI-MS 圖譜 118
圖 19.β-1,3-xylobiose (M2) 區分物之正離子模式 (A) 與負離子模式 (B) 之 LC-ESI-MS 圖譜 119
圖 20.Xylose (M1) 區分物之正離子模式 (A) 與負離子模式 (B) 之 LC-ESI-MS 圖譜 120
圖 21.β-1,3-xylotriose (M3) 區分物之 13C NMR (A) 與 1H NMR (B) 之核磁共振圖譜 121
圖 22.β-1,3-xylobiose (M2) 區分物之 13C NMR (A) 與 1H NMR (B) 之核磁共振圖譜 122
圖 23.Xylose (M1) 區分物之 13C NMR (A) 與 1H NMR (B) 之核磁共振圖譜 123
圖 24.不同濃度β-1,3-混合木寡醣 (XOSmix) 之 DPPH 清除能力 124
圖 25.不同濃度β-1,3-混合木寡醣 (XOSmix) 之 ABTS 清除能力 125
圖 26.不同濃度β-1,3-混合木寡醣 (XOSmix) 之螯合亞鐵離子能力 126
圖 27.不同濃度β-1,3-混合木寡醣 (XOSmix) 之還原力 127
圖 28.不同濃度β-1,3-混合木寡醣 (XOSmix) 之兔血漿活化部分凝血活酶時間 (APTT) 之變化 128
圖 29.不同濃度β-1,3-混合木寡醣 (XOSmix) 之兔血漿凝血酶原時間
(PT) 之變化 129
圖 30.不同濃度β-1,3-混合木寡醣 (XOSmix) 之兔血漿凝血酶時間 (TT) 之變化 130
圖 31.人類結腸癌細胞株 Caco-2 於 100 mm 培養盤中於 37℃ 與 5% CO2 中生長 (A) 0 天、(B) 1 天、(C) 2 天、與 (D) 3 天之細胞生長形態 131
圖 32.人類結腸癌細胞株 Caco-2 培養於 Transwell Insert 測試裝置中於 37℃ 與 5% CO2 中生長 (A) 0 天、(B) 2 天、(C) 7 天、與 (D) 15 天之細胞生長形態 132
圖 33.Caco-2 細胞於 Transwell Insert 測試裝置中培養 0-15 天期間跨膜電阻 TEER 值之變化 133
圖 34.不同濃度β-1,3-混合木寡醣 (XOSmix) 對 Caco-2 細胞之細胞毒性試驗 134
圖 35.β-1,3-混合木寡醣 (XOSmix) 於 Caco-2 細胞吸收運輸裝置 (Transwell Insert) 中不同時間點上層液 (A) 與下層液 (B) 中總糖含量之變化 135
圖 36.β-1,3-混合木寡醣 (XOSmix) 於 Caco-2 細胞吸收運輸裝置 (Transwell Insert) 中不同時間點上層液 (A) 與下層液 (B) 之 高效能液相層析圖 136
圖 37.不同濃度β-1,3-混合木寡醣 (XOSmix) 對小鼠巨噬細胞株 (RAW 264.7 cells) 之細胞毒性試驗 137
圖 38.不同濃度β-1,3-混合木寡醣 (XOSmix) 經 LPS 誘導下對小鼠巨噬細胞株 (RAW 264.7 cells) 一氧化氮量之影響 138
圖 39.不同濃度β-1,3-混合木寡醣 (XOSmix) 經 LPS 誘導下對小鼠巨噬細胞株 (RAW 264.7 cells) 超氧陰離子生成之影響 139
圖 40.不同濃度β-1,3-混合木寡醣 (XOSmix) 經 LPS 誘導下對小鼠巨噬細胞株 (RAW 264.7 cells) TNF-α 分泌量之影響 140
圖 41.不同濃度β-1,3-混合木寡醣 (XOSmix) 經 LPS 誘導下對小鼠巨噬細胞株 (RAW 264.7 cells) IL-6 分泌量之影響 141 
表目錄
表 1.Psedomonas vosicularis MA103 聚木醣酶基因定序與比對現有酵素資訊 25
表 2.三因子在反應曲面法之 3 階層數值範圍 (Actual levels) 與編碼 (Coded levels: -1, 0, 1) 27
表 3.P. vesicularis MA103 XYLII 與不同來源β-1,3-聚木醣酶胺基酸序列比較 142
表 4.最適化條件誘導 E. coli ClearColi BL21-pET 39b-xylII 生產β-1,3-聚木醣酶XYLII之反應曲面法實驗設計 143
表 5.誘導E. coli ClearColi BL21-pET 39b-xylII生產β-1,3-聚木醣酶XYLII回歸所得之變異數分析 144
表 6.誘導 E. coli ClearColi BL21-pET 39b-xylII 產生β-1,3-聚木醣酶XYLII之回歸係數顯著性分析 145
表 7.E. coli ClearColi BL21(DE3) 純化重組β-1,3-聚木醣酶XYLII 146
表 8.金屬離子與化學物質對重組β-1,3-聚木醣酶XYLII的影響 147
表 9.不同基質特異性對重組β-1,3-聚木醣酶XYLII相對酵素活性
之影響 148
表 10.β-1,3-聚木醣與β-1,3-混合木寡醣 (XOSmix) 之產率、硫酸基含量、總酚含量、與總蛋白量 149
表 11.不同濃度β-1,3-混合木寡醣 (XOSmix) 對血管收縮素轉換酶之抑制活性 150 
附錄目錄
附錄 1.β-1,3-聚木醣 (A) 與 β-1,4-聚木醣 (B) 之結構 151
附錄 2.β-1,3-聚木醣之 13C NMR 之核磁共振圖譜 152
附錄 3.(A) 一般革蘭氏陰性菌的脂多醣結構,(B) 無內毒素E. coli ClearColi BL21 Lipid IVA 結構 153
附錄 4.腎素-血管收縮素轉換系統 154
附錄 5.製備β-1,3-混合木寡醣 (XOSmix) 之流程圖 155
附錄 6.細胞電阻電壓歐姆儀示意圖 156
附錄 7.不同來源β-1,3-聚木醣酶的生化特性 157
附錄 8.著作目錄 158
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