臺灣博碩士論文加值系統

果寡醣具有促進雙岐桿菌 (Bifidobacteria) 增殖生理機能。工業上大多利用麴菌 Aspergillus niger 所產生的酵素 β-喃果糖 (β-fructofruanosidase) 來製造，但是必須在高蔗糖濃度下才能進行反應，所以於果寡醣漿商品中仍含有大量的蔗糖，需從產物中去除單糖及雙糖，才可以得到高純度的果寡糖。本研究期望能從菊科植物中之牛蒡當中找出ㄧ個取代 Aspergillus niger 所產生的酵素 β-喃果糖，製造高純度的聚果糖，應用在食品上。
於牛蒡粗萃取液中含有未知寡醣，經過初步鑑定主要為一個葡萄糖和三個果糖所構成的聚果醣。粗酵素液經由 20% ~ 65% 硫酸銨沉澱、 Sephadex G-75 管柱層析及 Sephadex G-200 管柱層析純化。粗酵素液最適 pH 約為 6.0 ，最適溫度為 25 ℃ ，以蔗糖為基質，求得牛蒡果糖基轉移之 Km ＝ 0.26 M ， Vmax ＝ 13.00 μmole kestose / mL hr。 Hg2+及 Cu2+ 離子為強的抑制劑，而 Na+ 、 K+ 、 Fe2+ 及 Ca2+ 離子為較弱的抑制劑，添加 Iodoacetic acid 和尿素也會抑制酵素的活性。 Mg2+ 和 Zn2+ 離子會促進果糖基轉移酵素的活性，添加Phenylmethanesulfonyl fluoride 和 EDTA 也會增加果糖基轉移的活性。

In the food industry, people use β-fructofruanosidase from Aspergillus niger to produce oligosaccharide which enhance the growth Bifidobacteria. The reaction starts only at the high sucrose concentration and there are still large amount sucrose in the product. The monosaccharide and disaccharide must be removed to obtain high concentration of oligosaccharide. In this research, we wished to find an enzyme from the burdock (Arctium lappa L.) to replace β-fructofruanosidase from Aspergillus niger to produce high concentration of oligosaccharides to be utilized in the food industry.
An unknown oligosaccharide was found in the burdock extract and was identified as a tetrasaccharide which consisted of two fructosyl units linked to sucrose. The 20 ~ 65% of ammonium sulfate fraction of the enzyme was collected, and applied to a G-75 gel filtration column. Later, the partially purified enzyme was applied to another Sephadex G-200 gel filtration column. The optimum pH of the enzyme was about 6.0. The optimum temperature for enzyme reaction was 25 ℃. The Km value and the Vmax value of fructosyltransferase were determined to be 0.26 M and 13.00 μmole kestose / mL hr using sucrose as a substrate. Hg2+ and Cu2+ were apparent inhibitors, and Na+, K+, Fe2+ and Ca2+ were mild inhibitors. Iodoacetic acid and urea were also inhibitors. However, Mg2+ , Zn2+ , EDTA and phenylmethanesulfonyl fluoride were activators.

略字解…………………………………………………….……………....i
中文摘要……………………………………………...……………….…ii
英文摘要…………………………………………….…………………. iii
壹、緒論………...……………………………………………..……...…..1
貳、文獻整理………………………………………………..……...…….3
一、牛蒡簡介……….………………………………………….....……..3
二、聚果醣簡介………………………………………………...…..…...4
（一）聚果醣在植物生理學上的角色………………………….....….4
（二）聚果醣之生合成…………………………….……….....………5
（三）聚果醣之結構……….………………..……...………………..10
（四）果糖基轉移之研究……………………..………...………...11
（五）影響果糖基轉移活性之因子..………………………..…....13
1.pH值…………………………………………………………13
2.溫度…………………………………………………………..13
3.金屬離子…………………………………………………..…13
4.化學修飾劑…………………………………………………..14
（六）聚果醣之分子生物學………..…………………………….….14
（七）聚果醣的功用…………………..………………………...…...17
參、材料與方法…………………………...………………..……...……18
一、試驗材料…………………..…………...……………….…………18
（一）材料…….…………….………………………………………..18
（二）藥品.………………….…………………………….………….18
（三）酵素純化緩衝液………………………………………………20
（四）酵素反應液……………………………….……………….…..20
（五）SDS 蛋白質電泳試劑溶液配製……………………….…….20
（六）Folin-酚測定法試劑………………...………………………..22
（七）檸檬酸-磷酸鹽緩衝溶液之製備……………………………..24
二、儀器設備………..…………………………………...…………….24
三、試驗方法…………………………………………………...……...25
（一）未知聚醣之分析……………………………………...……….25
（二）果糖基轉移純化之步驟…………………………...……….25
（三）酵素液中蛋白質含量之測定……………………...………….28
（四）牛蒡中粗酵素液之濾紙層析………………………………....29
（五）新舊牛蒡中果糖基轉移之活性比較………………………29
（六）pH 對粗酵素液中果糖基轉移活性的影響……………….29
（七）溫度對粗酵素液中果糖基轉移活性的影響………………31
（八）蔗糖濃度對牛蒡果糖基轉移活性的影響…………………31
（九）化學修飾劑及金屬離子對牛蒡粗酵素液中果糖基轉移活性之影響…………………………………………………………31
1.不同的化學修飾劑對果糖基轉移活性之影響……………31
(1)硫醇及二硫蘇糖醇對果糖基轉移安定性之研究……31
(2)不同濃度的化學修飾劑對果糖基轉移活性之影響…33
2.不同濃度的金屬離子對果糖基轉移活性之影響…………33
(1) 鎂離子對粗酵素液中果糖基轉移活性的影響………33
(2) 鋅離子對粗酵素液中果糖基轉移活性的影響……....33
3.不同濃度的金屬離子及化學修飾劑對果糖基轉移活性之影 響………………………………………………………………34
肆、結果與討論…………………………………………………………36
一、牛蒡中未知聚醣之分析……..……………………………….…….36
二、果糖基轉移之純化………..……………………………………..36
（一）不同硫酸銨沈澱對酵素活性之影響…………………………36
（二） Sephadex G-75 膠體管柱層析……………………………...41
（三） Sephadex G-200 膠體管柱層析…..………………………...44
（四） SDS－聚丙烯醯胺凝膠電泳…………...………...……….…44
三、新舊牛蒡中果糖基轉移之活性比較……………………………48
四、pH 對粗酵素液中果糖基轉移的影響………………………….48
五、溫度對粗酵素液中果糖基轉移的影響…………………………51
六、蔗糖濃度對牛蒡果糖基轉移活性的影響………………………51
七、化學修飾劑及金屬離子對牛蒡粗酵素液中果糖基轉移活性之影響……………………………………………………………………55
（一）不同的化學修飾劑對果糖基轉移活性之影響…………....55
1.硫醇及二硫蘇糖醇對果糖基轉移安定性之研究…………55
2.不同濃度的化學修飾劑對果糖基轉移活性之影響………55
（二）不同濃度的金屬離子對果糖基轉移活性之影響…………58
1.鎂離子對粗酵素液中果糖基轉移活性的影響……………60
2.鋅離子對粗酵素液中果糖基轉移活性的影響……………60
（三）不同濃度的金屬離子及化學修飾劑對果糖基轉移活性之影響………………………………………………………………60
伍、結論………………………..………………………..……...……….66
參考文獻………………………………………………………………..68
圖 次
圖一、植物中合成聚果醣之反應過程……………………………..……7
圖二、聚果醣於植物中合成的模式……………………………………..9
圖三、牛蒡…………….………………………………………...………19
圖四、牛蒡粗萃取液之 HPLC 分析圖……………………………......37
圖五、牛蒡粗萃取液之濾紙層析圖……………...………………...…..38
圖六、未知聚醣水解 2 小時 HPLC 的分析圖…………….…..….....39
圖七、不同硫酸銨沈澱之沈澱物、上清液的酵素活性……….……….40
圖八、65％ 硫酸銨沈澱酵素反應之 HPLC 分析圖…………………42
圖九、 Sephadex G-75 膠體管柱層析圖……………...…….………...43
圖十、 Sephadex G-200 膠體管柱層析圖………………...…………..45
圖十一、 Sephadex G-75 管柱收集之酵素液的SDS電泳圖………...46
圖十二、 pH 對果糖基轉移活性的影響…………………………...50
圖十三、溫度對果糖基轉移活性的影響……………………………52
圖十四、蔗糖濃度對牛蒡果糖基轉移活性的影響…………………53
圖十五、蔗果三糖生成反應蔗糖受質之雙倒數作圖…………………54
圖十六、粗酵素液中果糖基轉移之安定性…………………………56
圖十七、鎂離子濃度對粗酵素液中果糖基轉移活性的影響………61
圖十八、鋅離子濃度對粗酵素液中果糖基轉移活性的影響………62
圖 次
圖一、植物中合成聚果醣之反應過程……………………………..……7
圖二、聚果醣於植物中合成的模式……………………………………..9
圖三、牛蒡…………….………………………………………...………19
圖四、牛蒡粗萃取液之 HPLC 分析圖……………………………......37
圖五、牛蒡粗萃取液之濾紙層析圖……………...………………...…..38
圖六、未知聚醣水解 2 小時 HPLC 的分析圖…………….…..….....39
圖七、不同硫酸銨沈澱之沈澱物、上清液的酵素活性……….……….40
圖八、65％ 硫酸銨沈澱酵素反應之 HPLC 分析圖…………………42
圖九、 Sephadex G-75 膠體管柱層析圖……………...…….………...43
圖十、 Sephadex G-200 膠體管柱層析圖………………...…………..45
圖十一、 Sephadex G-75 管柱收集之酵素液的SDS電泳圖………...46
圖十二、 pH 對果糖基轉移活性的影響…………………………...50
圖十三、溫度對果糖基轉移活性的影響……………………………52
圖十四、蔗糖濃度對牛蒡果糖基轉移活性的影響…………………53
圖十五、蔗果三糖生成反應蔗糖受質之雙倒數作圖…………………54
圖十六、粗酵素液中果糖基轉移之安定性…………………………56
圖十七、鎂離子濃度對粗酵素液中果糖基轉移活性的影響………61
圖十八、鋅離子濃度對粗酵素液中果糖基轉移活性的影響………62
表 次
表一、於不同維管束植物純化之蔗糖：蔗糖果糖基轉移性質….….12
表二、 SDS-PAGE標準品分子量……………..………....…………….23
表三、 HPLC 分離條件…………………………...…….....…………..26
表四、 HPLC 分離條件…………………………………...…...………30
表五、 HPLC 分離條件………………………………….……..…...…32
表六、 HPLC 分離條件…………………………………...……….…..32
表七、三因子實驗設計…………..…….……….………...…….………35
表八、牛蒡果糖基轉移分離純化的結果……………...…………….47
表九、新舊牛蒡果糖基轉移活性之比較……………………………49
表十、化學修飾劑對牛蒡粗酵素液中果糖基轉移活性之影……....57
表十一、金屬離子對牛蒡粗酵素液中果糖基轉移活性之影響…....59
表十二(A)、金屬離子及化學修飾劑對牛蒡粗酵素液中果糖基轉移活性之影響………………………………………………63
表十二(B)、金屬離子及化學修飾劑對牛蒡粗酵素液中果糖基轉移活性之影響………………………………………………65

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