木聚糖水解酵素之純化與特性探討

• Main Author: 賴明良
• Other Authors: 鄭智元
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/14973115971182097291

碩士 === 國立成功大學 === 化學工程學系 === 89 === A Bacillus sp. Strain produced xylanase, but negligible quantities of cellulase, in the extracellular medium. Protease was also detected with bacterial grouth. The enzyme was purified from the culture supernatant in two steps which comprised ammonnium sulfate precipitation and cation-exchange (CM Sepharose) chromatography. The highest xylanase activity obtained in liquid culture was 3U/mL. The optimal pH and temperature for the enzyme was 6 and 50℃.The enzyme stable over a range of pH 5∼6. Sn2+ and Ag+ions showed about 50% inhibition in activity by concentration of 1mM. The molecular weight and isoelectric point of xylanase was about 29 kDa , 6.8. After treated with xylanase, beecheood released the more quantities of reducing sugar than birchwood and oat spelt xylan. The total recovery of enzyme activity was 60% with an increase of 6.1-fold in purification factor after purification. 英文摘要………………………………………………………………Ⅱ 誌謝……………………………………………………………………Ⅲ 總目錄…………………………………………………………………Ⅳ 表目錄…………………………………………………………………Ⅶ 圖目錄…………………………………………………………………Ⅷ 內文 第一章 緒論…………………………………………………1 1-1 酵素……………………………………………………………1 1-2 酵素的純化與分離……………………………………………3 1-3 木聚糖水解酵素與枯草菌……………………………………5 1-4 研究方向………………………………………………………7 第二章 實驗材料與方法……………………………………8 2-1 實驗材料………………………………………………………8 2-1-1 菌株………………………………………………….8 2-1-2 藥品……………………………………………….…8 2-1-3 儀器與裝置…………………………………………10 2-2 原理………………………………………………………….11 2-2-1 離心…………………………………………………11 2-2-2 蛋白質沉澱(鹽析)…………………………………13 2-2-3 快速蛋白質液體層析儀(FPLC)……………………14 2-2-4 蛋白質分子量測定(SDS-PAGE)……………………17 2-3 分析方法…………………………………………………….19 2-3-1 木聚糖分解酵素活性測定…………………………19 2-3-2 纖維素分解酵素活性測定…………………………21 2-3-3 快速蛋白質液體層析儀(FPLC)……………………22 2-3-4 蛋白質分子量測定(SDS-PAGE)……………………24 2-2-5 酵素活性之定性分析………………………………25 第三章 實驗步驟…………………………………………27 3-1 酵素分離步驟…………………………………………….27 3-2 鹽析……………………………………………………….28 3-3 FPLC純化酵素過程……………………………………….30 3-4 SDS-PAGE 分子量鑑定……………………………………31 3-5 電泳溶出(Electroelution)…………………………….32 第四章 結果與討論…………………………………….36 4-1 酵素特性………………………………………………….36 4-1-1 發酵槽培養………………………………………36 4-1-2 溫度對酵素活性及穩定性的影響………………38 4-1-3 pH值對酵素活性及穩定性的影響………………40 4-1-4 金屬離子及界面活性劑對酵素的影響…………42 4-1-5 酵素對木聚糖的水解情形………………………44 4-2 鹽析……………………………………………………….47 4-2-1 酵素濃縮…………………………………………47 4-2-2 粗酵素的製備……………………………………49 4-3 FPLC純化酵素過程……………………………………….50 4-3-1 FPLC系統的使用…………………………………50 4-3-2 連續式操作………………………………………52 4-4 酵素的分離與回收……………………………………….55 4-4-1 分離純化過程中木聚糖水解酵素的回收情況…55 4-4-2 各菌株酵素活性比較……………………………56 4-5 分子量測定結果………………………………………….58 4-6 酵素之等電點………………………………………………61 第五章 結論……………………………………………….63 參考文獻…………………………………………………….65