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題名 | PEG-鹽兩水相萃取與SDS-聚丙烯醯胺凝膠電泳系統影響α型澱粉水解酵素的分離與檢測之研究=The Influence of PEG-Salt Aqueous Two-Phase Extraction and SDS-Polyacrylamide Gel Electrophoresis on the Separation and Detection of α-Amylases |
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作者姓名(中文) | 陳文章; 周士農; | 書刊名 | 臺北科技大學學報 |
卷期 | 34:2 2001.09[民90.09] |
頁次 | 頁11-21 |
分類號 | 460.025 |
關鍵詞 | 兩水相萃取; 凝膠電泳; α型澱粉水解酵素; 生化分離; Aqueous two-phase extraction; Gel electrophoresis; α-Amylase; Bioseparation; |
語文 | 中文(Chinese) |
中文摘要 | 本文提出探討PEG-無機鹽兩水相萃取與SDS-聚丙烯醯胺凝膠電泳系統兩種方法應用於分離與檢測生化物質的可行性,並嘗試以α型澱粉水解酵素作為目標蛋白質,我們探討萃取分離與純化的控制因素,包括聚乙二醇(PEG)濃度、磷酸鹽緩衝劑濃度、最初酵素劑量及萃取操作條件等,並經由凝膠電泳實驗的比對結果得知,使用越多級的萃取,隨著酵素之溶解於PEG,使之分配於由PEG與磷酸鹽所構成的上層水溶液中,而不純添加物及雜質被萃離出,可以提高酵素之純度,且富PEG萃取層有利於保護蛋白質分子。SDS-PAGE電泳過程確實檢測出α型澱粉水解酵素在富PEG層中的分子量組成,至於超音波和還原劑的前處理程序也會對酵素之蛋白質亞單位與聚集體兩者間產生明顯的氧化還原影響。 |
英文摘要 | In this work, the two methods for separation and detection of biological materials-PEG-salt aqueous two-phase extraction and SDS-polyacrylamide gel electrophoresis was investigated. Using α-amylases as a model enzyme, we present the factors that influence the extraction separation and purification: the concentration of polyethylene glycol (PEG), the concentration of phosphate buffer, the initial enzyme dose, and the extraction conditions. SDS-PAGE experiments showed that α-amylases strongly prefer the top phase (PEG-rich) in aqueous two-phase extraction systems. Two-step extractions can be applied to purify the enzyme leaving the impurities and other proteins in the salt-rich solution. The electrophoretogam of the gel pattern revealed that the molecular masses of α-amylases are very sensitive to the sonication and reducing conditions. |
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