Bacillus cereus TKU044 係篩選自花蓮土壤之幾丁聚醣酶生產菌，以含有1%烏賊軟骨粉、0.1% K2HPO4、0.05% MgSO4．7H2O之100 mL液態培養基，於37℃進行搖瓶 (150 rpm)培養2天後，可得較高幾丁聚醣酶活性 (2.66U/mL)。
　　培養所得發酵上清液經硫酸銨沉澱、DEAE-Sepharose 及 Macro-Prep® DEAE Cartridge 陰離子交換層析等步驟，以285.9倍之純化度以及16%之回收率，純化出經 SDS-PAGE電泳測得分子量約為43 KDa 之一種幾丁聚醣酶。此幾丁聚醣酶的最適反應pH為7，pH安定性為6~7，最適反應溫度為40℃，而熱安定性則是低於50℃，其活性會受到Na+、Fe2+、Ba2+、Cu2+、Zn2+及EDTA所抑制。TKU044粗酵素液水解水溶性幾丁聚醣所得寡糖，具有抗氧化活性。

The chitosanase-producing strain TKU044 was isolated from the soils of Hualien with squid pen power (SPP) as the sole carbon/nitrogen source and identified as Bacillus cereus. A chitosanase was purified from the culture supernatant by ammonium sulfate precipitation, DEAE-Sepharose and Macro-Prep® DEAE Cartridge. The molecular mass of chitosanase determined by SDS-PAGE was approximately 43 kDa. The optimum pH, optimum temperature, pH stability, and thermal stability of the TKU044 chitosanase were pH 7, 40℃, pH 6~7 and under 50℃, respectively. The chitosanase activity was inhibited by Na+, Fe2+, Ba2+, Cu2+, Zn2+ and EDTA. The oligosaccharides prepared by hydrolyzing soluble chitosan with the crude chitosanase showed antioxidant activity.

目錄

中文摘要	I
英文摘要	II
目錄	III
表目錄	VIII
圖目錄	IX
縮寫表	XI
第一章　緒論	1
第二章　文獻回顧	2
2.1臘狀芽孢桿菌（Bacillus cereus）之簡介	2
2.2 幾丁質 (chitin)與幾丁聚醣 (chitosan)	3
2.3 幾丁聚醣酶 (chitosanase)	6
2.4 N-乙醯幾丁寡醣和幾丁寡醣	8
2.5 抗氧化能力	8
2.6 DPPH 自由基清除率測定	9
第三章　材料與方法	10
3.1 實驗菌株	10
3.2 實驗材料	10
3.3 實驗儀器	11
3.4 酵素生產菌株之篩選	12
3.5 革蘭氏染色	12
3.6 API (Analytical Profile Index)之鑑定	13
3.7 幾丁聚醣酶活性測定	13
3.8 蛋白酶活性測定	14
3.9 幾丁聚醣酶較適生產條件探討	14
3.9.1碳/氮源種類	14
3.9.2不同碳源濃度	15
3.9.3不同培養體積	15
3.9.4不同溫度	15
3.10 幾丁聚醣酶之分離與純化	16
3.10.1粗酵素液的製備	16
3.10.2陰離子交換層析 (一)	16
3.10.3陰離子交換層析 (二)	16
3.11 蛋白質電泳分析	17
3.12 酵素之特性分析	18
3.12.1酵素最適反應溫度	18
3.12.2酵素熱安定性	18
3.12.3酵素最適反應pH	18
3.12.4酵素pH安定性	19
3.12.5金屬離子及化學藥品對酵素活性之影響	19
3.12.6界面活性劑對酵素活性之影響	20
3.12.7酵素之基質特異性	20
3.13 幾丁聚醣酶水解基質	20
3.13.1基質水解	20
3.13.2還原糖量測定	21
3.13.3總糖含量測定	21
3.13.4幾丁寡醣之製備	22
3.14 DPPH自由基清除能力之測定	22
 
第四章　結果與討論	23
4.1幾丁聚醣酶生產菌種之鑑定	23
4.2 幾丁聚醣酶生產條件之探討	25
4.2.1不同碳/氮源之探討	25
4.2.2不同碳源濃度探討	26
4.2.3培養體積之探討	27
4.2.4培養溫度之探討	27
4.2.5 B. cereus TKU044生產幾丁聚醣酶與蛋白酶之探討	27
4.2.6較適培養條件結果與比較	30
4.3幾丁聚醣酶純化與分離	32
4.3.1 粗酵素液的製備	33
4.3.2離子交換樹脂層析 (一) 	33
4.3.3離子交換樹脂層析 (二) 	35
4.3.4分離與純化綜合結果	37
4.3.5幾丁聚醣酶分子量之測定	38
4.4 幾丁聚醣酶之特性分析	40
4.4.1 酵素最適反應溫度及熱安定性	40
4.4.2 幾丁聚醣酶之最適反應pH及pH安定性	41
4.4.3 金屬離子及化學藥品對幾丁聚醣酶活性之影響	42
4.4.4 界面活性劑對幾丁聚醣酶活性之影響	43
4.4.5 幾丁聚醣酶之基質特異性	45
4.5 幾丁聚醣酶水解基質	46
4.5.1 還原糖和總糖含量分析	46
4.6抗氧化活性測定	47
4.6.1 DPPH自由基清除能力之測定	47
第五章　結論	51
參考文獻	52
 
表目錄


表 2.1 B. cereus 於真菌抑制之應用	2
表 2.2幾丁質與幾丁聚醣酶應用	5
表 3.1 緩衝溶液種類	19
表 3.2 DNS試劑之組成	21
表 4.1 TKU044之16S rDNA 部分基因序列	24
表 4.2 API鑑定結果	24
表 4.3 B. cereus TKU044所生產酵素之較適培養條件	30
表 4.4 以幾丁質水產廢棄物作唯一碳/氮源的幾丁聚醣酶/蛋白酶生產菌之較適培養條件比較	31
表 4.5 B. cereus TKU044幾丁聚醣酶之純化總表	37
表 4.6金屬離子和化學藥品對B. cereus TKU044所產生的幾丁聚醣
酶之影響	42
表 4.7界面活性劑對 B. cereus TKU044所生產之幾丁聚醣酶之影
響	44
表 4.8 B. cereus TKU044所生產幾丁聚醣酶基質特異性	45
表 4.9幾丁寡醣之DPPH自由基清除率之ED50	48

圖目錄


圖 2.1 纖維素、幾丁質和幾丁聚醣之結構	4
圖 2.2 幾丁聚醣的種類和作用位置	7
圖 2.3 幾丁聚醣的作用模式	7
圖 2.4 DPPH 結構	9
圖 2.5 抗氧化物清除 DPPH 自由基之反應	9
圖 4.1 Bacillus cereus TKU044之顯微鏡圖	23
圖 4.2碳/氮源對B. cereus TKU044生產幾丁聚醣酶之影響	25
圖 4.3 SPP濃度對B. cereus TKU044生產幾丁聚醣酶之影響	26
圖 4.4培養體積對B. cereus TKU044生產幾丁聚醣酶之影響	27
圖 4.5培養溫度對B. cereus TKU044幾丁聚醣酶生產之影響	28
圖 4.6 B. cereus TKU044生產幾丁聚醣與蛋白酶之影響	29
圖 4.7幾丁聚醣酶之純化流程圖	32
圖 4.8 B. cereus TKU044 幾丁聚醣酶之DEAE-Sepharose CL-6B 層析圖譜	34
圖 4.9B. cereus TKU044幾丁聚醣酶之Macro-Prep® DEAE 
Cartridge層析圖譜	36
圖 4.10 B. cereus TKU044幾丁聚醣酶之純化SDS-PAGE圖	39

圖 4.11幾丁聚醣酶之最適反應溫度及熱安定性	40
圖 4.12幾丁聚醣酶之最適反應pH及pH安定性	41
圖 4.13水溶性幾丁聚醣經B. cereus TKU044粗酵素液水解不同時間所得總糖及還原糖含量	43
 

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