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研究生:賴惠琳
研究生(外文):Lai, Hui-Lin
論文名稱:適酸耐熱葡萄糖異構酵素之生產與純化
論文名稱(外文):Produciton and purification of a thermostable glucose isomerase having a relatively low optimum pH
指導教授:李重義
學位類別:碩士
校院名稱:東吳大學
系所名稱:微生物學系
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:1999
畢業學年度:87
語文別:中文
論文頁數:146
中文關鍵詞:耐熱酵素耐熱適酸酵素葡萄糖異構酵素葡萄糖異構酵素純化酵素純化
外文關鍵詞:thermostableglucose isomerasethermophilicpurificaiton
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本研究目的在於深入探討一株嗜熱嗜酸細菌49-1及其所生產的葡萄糖異構酵素的特性。菌株49-1為革蘭氏陽性菌產胞桿菌,菌體寬為0.7到0.8μm,長度為2到3μm。其胞子為膨脹,橢圓形,位於中間或一邊端。菌株49-1生長溫度範圍為45到75℃,生長pH值範圍為2.5到6.0,能夠利用木質糖與其他多種碳源尤其是山梨糖醇與醋酸鈉,其G+C含量為61.5%,依照目前資料,應屬Bacillus acidocaldarius。菌株49-1生產的葡萄糖異構酵素具有較低的最適作用pH值,其最適作用溫度為75到80℃,在75℃下最適作用pH值為7.0,於80℃下最適作用pH值為6.5。將菌株49-1以氣舉式醱酵槽培養,回收菌體經由超音波打破處理,再將粗酵素液經由Sephadex G-150管柱的膠體過濾、DEAE-cellulose管柱的離子交換及t-Butyl HIC管柱的斥水性交互作用等純化步驟後,獲得比活性提高19倍,回收率為22%的部分純化葡萄糖異構酵素,其在80℃的最適作用pH值為6.0。

The purpose of the present study is to make more precise investigation on the characterization of a thermoacidophilic strain 49-1 and the glucose isomerase produced by this bacterium. Strain 49-1 was gram- positive, spore-forming rods with a width of 0.7 to 0.8 μm and a length of 2 to 3 μm. The spores were swollen, ellipsoidal, and central to subterminal. The temperature range for growth was 45℃to 75℃. The pH range for growth was 2.5 to 6.0. It can utilize xylose and many other carbon source, especially sorbitol and sodium acetate. The G+C contents was 61.5%. According to the present data, this strain was identified as Bacillus acidocaldarius. The glucose isomerase of strain 49-1 possessed a relatively low optimum pH. The optimum temperature of crude enzyme was 75℃to 80℃. After incubating strain 49-1 in an air-lift fermenter, harvesting cells, distrupting cells by sonication, purifying the crude enzyme in a proceduce comprising gel filtration in Sephadex G-150 column, ion exchanging in DEAE-cellulose column, and hydrophobic interaction in t-Butyl HIC column, a partial purified glucose isomerase with an incaeasing of 19 fold of specific activity and 22% of yield was obtained. The optimum pH of purified enzyme at 80 ℃was 6.0.

目 錄
中文摘要*********************..II
英文摘要********************..*III
目錄***********************.IV
表目錄**********************..VIII
圖目錄*********************..*X
第一章 續言. ******************..*1
第一節 葡萄糖異構酵素*************.1
1.1 木質糖異構酵素的利用***********1
1.2 生產耐熱葡萄糖異構酵素的微生物******1
1.3 葡萄糖異構酵素的特性**********.*3
1.4 篩選生產嗜酸耐熱葡萄糖異構酵素之菌株**.*9
1.5 高溫嗜酸性Bacillus************..10
第二節 葡萄糖異構酵素的純化. **********10
2.1 純化的三階段***************10
2.2 酵素純化之前的處理***********.*11
2.2.1 酵素的萃取方式***********..*11
2.2.2 濃縮酵素液**************..15
2.3 純化三階段使用的層析管柱********.*18
2.3.1.1 膠體過濾層析***********.18
2.3.1.2 緩衝液與凝膠的關係*********.19
2.3.2 離子交換層析法************.20
2.3.2.1 凝膠***************..*30
2.3.2.2 緩衝液**************..*31
2.3.3 斥水性交互作用層析法**********31
2.3.4親和力層析法*************.*37
第三節 研究動機與目的*************..37
第二章 材料與方法****************.*40
第一節 藥品與藥劑***************..40
第二節 菌株******************..40
第三節 培養基的配制**************..40
3.1 Bacillus acidocaldarius CCRC 14685培養基**..*40
3.2 Bacillus acidocaldarius CCRC 14685固體培養基*.41
3.3 鑑定用培養基***************..41
3.4 鑑定用固定培養基*************..42
3.5 Allen培養基***************..*42
3.6 Bacillus coagulans CCRC 10272培養基****..*43
第四節 菌株的保存***************..44
4.1冷凍保存******************44
第五節 菌株的培養***************..44
5.1準備種菌*****************.*44
5.2 封口錐形瓶的培養**************45
5.3氣舉式醱酵槽的培養*************45
5.4測定菌量乾重****************45
第六節 菌種鑑定****************..46
第七節 超音波打破菌體*************..46
第八節 酵素活性分析**************..47
8.1 葡萄糖異構酵素活性的分析*********..47
8.2 果糖異構酵素活性的分析***********47
第九節 葡萄糖異構酵素的純化**********.51
9.1 膠體過濾層析法***************51
9.2 離子交換層析法***************52
9.3 斥水性交互作用層析法************52
第十節 蛋白質分析***************..53
10.1 蛋白質分析***************..*54
10.2 SDS-PAGE分析**************..54
第十一節 儀器設備***************..55
第三章 結果********************56
第一節 菌株49-1的鑑定與生理特性的探討*****.56
1.1 菌株49-1的鑑定**************.56
1.2 菌株49-1生理特性的探討*********.*56
1.3 菌株49-1的葡萄糖異構酵素特性*******57
第二節 大量生產酵素**************..71
2.1 菌體的大量培養***************71
2.2 酵素的獲得*****************72
第三節 修改葡萄糖異構酵素的純化步驟******..81
3.1 先前的純化步驟***************81
3.2 純化步驟的修改***************81
3.3 純化之葡萄糖異構酵素純化步驟******..*91
第四節 純化之葡萄糖構酵素的特性******.**92
4.1純化之葡萄糖異構酵素最適pH值******.*92
第四章 討論*******************.*110
參考文獻*********************.114

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