探討蛋白質沉澱劑對自萃取雞冠玻尿酸最適條件之探討 林曉億、陳明造
E-mail: 9707199@mail.dyu.edu.tw
摘 要
玻尿酸是體內的多醣類高分子聚合物，具有親水性與黏彈性，其製備方法有二種：一是由動物組織中萃取，主要來源是人 類臍帶、雞冠和牛眼玻璃體等；二是微生物醱酵方法，主要是存在鏈球菌(streptococci)莢膜中。在業界所選用的原料中，以 雞冠為主要來源，經萃取及純化後而得，但在純化過程常用到氯仿去除雜質，而氯仿屬於列管中化學毒性物質，具有毒性 高、單價高的缺點。故本篇論文主要以不同的蛋白質沉澱劑列出作為變因，找出是否有其他試劑可以取代目前常用的物質
，並探討純化後產品之產率及物理性質。結果顯示控制組所得到玻尿酸產率為0.661%、純度為每克粗萃取物中含有0.201 mg的玻尿酸、黏度為5.04 cps、蛋白質含量為0.064 mg/mL，由上述結果得知控制組純化出的玻尿酸產物其物性和氯仿有相 似的結果，故控制組用來取代氯仿，不但可以節省成本還可達到減少環境污染之效果。
關鍵詞 : 玻尿酸;去蛋白劑;蛋白質沉澱
目錄
1. 前言1 2. 文獻回顧2 2.1 玻尿酸發現2 2.2 玻尿酸之結構2 2.2.1 玻尿酸一級與二級結構2 2.2.2 玻尿酸三級結構3 2.3 玻尿酸 之生理機制4 2.3.1 玻尿酸之生化合成4 2.3.2玻尿酸之異化作用4 2.4 玻尿酸物化特性8 2.4.1 玻尿酸水膠之膨潤性8 2.4.2 玻尿 酸之黏彈性9 2.5 玻尿酸萃取來源10 2.5.1 動物組織10 2.5.2 微生物來源11 2.6 玻尿酸純度分析11 2.7 蛋白質分離-沉澱法12 2.8 有機溶劑之選擇14 3. 材料與方法18 3.1 材料18 3.2 實驗器材與儀器設備19 3.3 雞冠玻尿酸的萃取19 3.3.1 粗萃取液製 備21 3.3.2 第一次去除雜蛋白純化方法21 3.3.3 第二次雜蛋白去除之溶劑萃取法24 3.4分析方法25 3.4.1 己醣醛酸測定25 3.4.2 玻尿酸之蛋白質含量分析26 3.4.2.1試劑配製26 3.4.2.2實驗流程26 3.4.3 玻尿酸之黏度性質測定27 3.4.4 玻尿酸之高效能液相 層析儀(HPLC)純度分析27 3.4.5 玻尿酸之離子交換層析28 3.4.6 玻尿酸之核磁共振(nuclear magnetic resonance, NMR)分析28 4. 結果與討論29 4.1 不同蛋白質沉澱方法對玻尿酸粗萃取物產率的影響29 4.2 不同蛋白質沉澱方法對玻尿酸粗萃取物純度 的影響29 4.3 不同蛋白質沉澱方法對玻尿酸粗萃取物黏度的影響30 4.4 不同蛋白質沉澱方法對玻尿酸粗萃取物蛋白質含量 的影響30 4.5 高效能液相層析儀分析31 4.6 玻尿酸之離子交換層析31 4.7 玻尿酸核磁共振分析33 5. 結論42 參考文獻43 圖目 錄 圖2.1 玻尿酸之ㄧ級結構4 圖2.2 雙醣單位之間的糖?鍵4 圖2.3 雙醣單位間的氫鍵5 圖2.4 玻尿酸分子在溶液中的三度空間 型態6 圖3.1 實驗流程圖20 圖4.1 不同蛋白質沉澱劑對玻尿酸粗萃取物產率影響之比較34 圖4.2 不同蛋白質沉澱劑對玻尿酸 粗萃取物純度之比較35 圖4.3 不同蛋白質沉澱劑對玻尿酸萃取物黏度影響之比較36 圖4.4 不同蛋白質沉澱劑對玻尿酸粗萃 取物蛋白質含量之比較37 圖4.5 不同蛋白質沉澱劑對玻尿酸粗萃取物高效能液相層析儀分析結果之比較38 圖4.6 玻尿酸控 制組之離子交換層析圖39 圖4.7 核磁共振分析之氫光譜圖(A)Control, (B)Standard40 圖4.8 核磁共振分析之碳光譜
圖(A)Control,(B)Mytilus galloprovincialis (C)Mytilus galloprovincialis 45-110 ppm41 表目錄 表2.1 常用的沉澱蛋白質之方法15 表2.2 Hofmeister Series蛋白質沉澱劑16 表2.3 常用有機溶劑之log P值17
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