臺灣博碩士論文加值系統

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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