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研究生:鄭茜如
論文名稱:由AspergillusflavusNTU-FC-8生產幾丁聚醣脢之研究
論文名稱(外文):Studies on production of chitosanase from aspergillus flavus NTU-FC-8
指導教授:李敏雄李敏雄引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:農業化學研究所
學門:農業科學學門
學類:農業化學類
論文種類:學術論文
論文出版年:2000
畢業學年度:90
語文別:中文
論文頁數:99
中文關鍵詞:幾丁聚醣幾丁聚醣脢Aspergillus flavus
外文關鍵詞:chitosanase
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本研究由土壤中篩選得一株幾丁聚醣之分解菌株,經鑑定後定名為Aspergillus flavus NTU-FC-8。以100 mL液態培養基中含有 0.3 g colloidal chitosan,0.2 g yeast extract,0.2 g casamino acid,0.2 g peptone,0.2 g soytone,0.2 g tryptone,0.1 g KH2PO4,0.05 g MgSO4‧7H2O,於30oC、起始pH 3.6,裝液量200 mL下,以100 rpm振盪培養36小時,發酵液中的幾丁聚醣活性最高為1.2 unit/mL。
培養發酵液過濾去除菌體後,以硫酸銨進行區分,收集飽和度區間70~95%之沈澱物,置於蒸餾水中透析過夜後得到粗酵素液。粗酵素以DEAE-Sepharose CL-6B離子交換層析管柱進一步區分,以NaCl 0~0.5 M梯度流洗後,可收集得一具幾丁聚醣活性峰之蛋白質區間,定名為chitosanase I。chitosanase I再以Sephacryl S-200膠體過濾層析區分後,收集幾丁聚醣的活性區間,經SDS-PAGE檢測可得一單一組成之蛋白質,其分子量估計為43.5 kDa.
chitosanase I與膠態幾丁聚醣反應之最適溫度為60℃,最適反應pH值為6。反應24小時後,主要產物為幾丁二糖、幾丁三糖、幾丁四糖,其km值為0.086% chitosan/mg prot. ,Vmax值為11.470μmole/min/mg prot.。
A chitosanase-producing strain NTU-FC-8 was isolated from soil at pH 3. It was identified and designated as Aspergillus flavus NTU-FC-8. When the Aspergillus sp. NTU-FC-8 cultured in broth media containing 0.3% colloidal chitosan, yeast extract 0.2%, casamino acid 0.2%, peptone 0.2%, soytone 0.2%, tryptone 0.2%, KH2PO4 0.1%, MgSO4‧7H2O 0.05%, initial pH 4 and shaking at 100 rpm and 30℃ was investigate to be as the optimal conditions for chitosanase production.
The chitosanases from Aspergillus flavus NTU-FC-8 were preliminarily purified by means of ammonium sulfate fractionation, and collected the precipitate at 0~85% saturation of ammonia sulfate as crude chitosanase. The optimal reaction pH and temperature of crude chitosanase were 5 and 50℃, respectively. The major hydrolysis products were monomer, dimmer, trimer and tetramer. The crude chitosanase was further by purified with DEAE-Sepharose CL-6B, following with Sephacryl S-200 gel filtration chromatography, and obtained the active chitosanase fraction, and named chitosanse I. The characteristics of chitosanase I were demostrated as follows : the molecular mass was preliminarily estimated as 43.5 kDa by SDS-PAGE, and the optimal pH and temperature were respective 6.0 and 60℃. The major hydrolysates of chitosanase I reacted with colloidal chitosan were revealed as monomer、dimer、trimer and tetramer of glucosamine. The Km and Vmax of the chitosanase I is 0.086%/mg protein and 11.47μmole/min/mg protein.
目次 ....................................................Ⅰ
圖次 ....................................................Ⅱ
表次 ....................................................Ⅳ
中文摘要 ................................................Ⅴ
英文摘要 ................................................Ⅵ
壹、前言 .................................................1
貳、文獻整理 .............................................2
一、幾丁質與幾丁聚醣 ...................................2
二、N-乙醯幾丁寡醣與幾丁寡醣...........................14
三、N-乙醯幾丁寡醣與幾丁寡醣的製備.....................17
四、幾丁質脢與幾丁聚醣脢...............................19
五、幾丁聚醣脢活性的測定方法...........................23
參、材料與方法 ..........................................25
A、材料與試藥..........................................25
B、分析方法 ...........................................28
C、實驗步驟............................................37
肆、結果與討論...........................................45
一、菌株特性...........................................45
二、幾丁聚醣脢活性測定程序之設計計.....................50
三、酵素生產用培養基組成成分之探討.....................53
四、酵素之回收與純化...................................63
五、酵素性質之探討.....................................76
伍、結論.................................................91
陸、參考文獻.............................................93
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