TKU018 係以蝦殼粉為唯一碳/氮源，篩選自台灣北部土壤之幾丁聚醣酶生產菌，經鑑定為Bacillus cereus。TKU018生產幾丁聚醣酶之較適培養條件為含有0.5% 蝦殼粉、0.1 % K2HPO4及0.05 %MgSO4．7H2O之100 mL液態培養基 (pH 9)於37℃下搖瓶培養3天。所得發酵上清液，經硫酸銨沉澱、DEAE-Sepharose、Phenyl-Sepharose等層析步驟，純化出二種幾丁聚醣酶(C1、C2) ，經SDS-PAGE測其C1、C2分子量分別為44 kDa及22 kDa。C1、C2之最適反應溫度、最適反應pH、熱安定性、pH安定性分別為 (60℃、pH 5、<40℃、pH 5~7)，(50℃、pH 7、<50℃、pH 4~7)。C1其活性受Zn&sup2;+所抑制，C2則不受影響，在SDS存在下會完全抑制C1，而 C1、C2在0.5% Tween 20存在下，分別還維持原活性之100%及105%。而用烏賊軟骨粉在不同濃度下發酵所得上清液，第三天具有較佳的DPPH自由基清除力。

關鍵字：Bacillus cereus；幾丁聚醣酶；蝦殼粉

The chitosanase producing strain, Bacillus cereus TKU018, was isolated from the soil in the northern Taiwan. The optimized condition for chitosanase production were found when the culture was shaken at 37℃for 3 days in 100mL of medium contain 0.5% shrimp shell powder (SSP), 0.1 % K2HPO4 and 0.05 % MgSO4．7H2O (pH9). Two chitosanases (C1、C2) were purified by chromatography procedures of DEAE-Sepharose, Phenyl-Sepharose. The molecular mass of the C1 and C2 determined by SDS-PAGE was approximately 44 kDa and 22 kDa, respectively. C1、C2 The optimum temperature, optimum pH, thermal stability of C1 and C2 were (60℃、pH 5、<40℃、pH 5~7), (50℃、pH 7、<50℃、pH 4~7), respectively. The C1 was inactivated by Zn&sup2;+ , while the C2 was not affected；In the presence of SDS, the C1 was inactivated. C1、C2 retained 100% and 105% of its original activity in the presence of 0.5% Tween 20, respectively.
When cultured on various different concentrations of squid pen powder (SPP),  the supernatant of third day have better DPPH radical scavenging activity.

目錄
	頁次
授權書
簽名頁
誌謝	
中文摘要	I
英文摘要	II
目錄	IⅤ
圖目錄	IX
表目錄	XI

第一章 緒論	1
第二章 文獻回顧	2
     2.1 Bacillus cereus之簡介 2
     2.2 幾丁質與幾丁聚醣	3
     2.3 幾丁質酶與幾丁聚醣酶 5
     2.4 抗氧化活性	6
第三章 材料與方法	9
     3.1 實驗菌株	9
     3.2 實驗材料	9
     3.3 實驗儀器	10
     3.4 菌株篩選	11
     3.5 菌種鑑定	11
     3.6幾丁聚醣酶之活性測定 11
     3.7 最適生產條件探討	12
        3.7.1 碳/氮源	12
        3.7.2培養基之酸鹼值	12
        3.7.3培養溫度	12
        3.7.4培養體積	13
        3.7.5培養時間	13
     3.8 幾丁聚醣酶之分離純化	13
        3.8.1 粗酵素液之製備	13
        3.8.2 離子交換樹脂層析	14
        3.8.3 疏水性層析	14
     3.9 電泳分析	15
     3.10 酵素之特性分析	15
        3.10.1 酵素最適反應溫度	15
        3.10.2 酵素之熱安定性	16
        3.10.3 酵素最適反應pH	16
        3.10.4 酵素之pH安定性	16
        3.10.5 各種化學藥品對酵素活性之影響	17
        3.10.6 界面活性劑對酵素活性之影響	17
        3.10.7 幾丁聚醣酶之基質特異性	18
3.11　DPPH自由基清除能力之測定	18
3.12 不同滅菌時間對酵素抗氧化之影響	19
3.13 還原糖之測定	19
3.14 薄層層析法	19
第四章 結果與討論	20
     4.1 幾丁聚醣酶生產菌之篩選 20
     4.2 菌株之特性 20
     4.3 酵素較適生長條件探討 20
4.3.1 碳/氮源之選擇 20
4.3.2 培養溫度	21
4.3.3 培養pH	21
4.3.4 培養體積	22
4.3.5 較適條件探討結果 22
4.3.6 總結 23
4.4 幾丁聚醣酶之分離純化 24
        4.4.1 粗酵素液之製備 24
        4.4.2 離子交換層析	24
        4.4.3 疏水性層析	25
        4.4.4 綜合結果	25
     4.5 幾丁聚醣酶之分子量測定	26
        4.5.1 SDS-PAGE (銀染法)	26
4.5.2 SDS-PAGE (CBR染色法)	26
        4.5.3 綜合結果	26
     4.6 幾丁聚醣酶之特性分析	27
        4.6.1 幾丁聚醣酶之最適反應溫度  27
        4.6.2 幾丁聚醣酶之熱安定性	27
        4.6.3 幾丁聚醣酶之最適反應pH	28
        4.6.4 幾丁聚醣酶之pH安定性	28
        4.6.5 各種化學藥品對幾丁聚醣酶之影響	29
        4.6.6 界面活性劑對幾丁聚醣酶之影響	29
        4.6.7 幾丁聚醣酶之基質特異性	30
        4.6.8 總結 30 
     4.7 DPPH自由基清除能力	 31
     4.8 不同滅菌時間對酵素抗氧化之影響  31
     4.9 還原糖之測定	32
     4.10 薄層層析法	32
    第五章 結論	57
參考文獻	58
圖目錄

圖2.1  幾丁質、幾丁聚醣及纖維素之構造	7
圖4.1  Bacillus cereus TKU018之顯微鏡照片	34
圖4.2  Bacillus cereus TKU018之16S rDNA部份核酸序列分析35
圖4.3  SSP濃度對TKU018幾丁聚醣酶生產之影響	36
圖4.4  培養溫度對TKU018幾丁聚醣酶生產之影響	37
圖4.5  培養pH對TKU018幾丁聚醣酶生產之影響	38
圖4.6  培養液體積對TKU018幾丁聚醣酶生產之影響	39
圖4.7  TKU018培養於SSP培養基所生產幾丁聚醣酶之生長曲線圖40
圖4.8  Bacillus cereus TKU018所生產酵素之分離純化流程圖41
圖4.9  DEAE-Sepharose CL-6B幾丁聚醣酶層析圖譜	42
圖4.10  幾丁聚醣酶(C1)之Phenyl-Sepharose層析圖譜	43
圖4.11  幾丁聚醣酶(C2)之DEAE-Sepharose CL-6B層析圖譜	44
圖4.12  幾丁聚醣酶於SDS-PAGE之分子量分析	46
圖4.13  幾丁聚醣酶之最適反應溫度(A)及熱安定性(B)	47
圖4.14  幾丁聚醣酶之最適pH(A)及pH安定性(B)	48
圖4.15  0 ~ 4天不同濃度的SSP (A)和SPP (B)發酵上清液之DPPH自由基清除能力  52
圖4.16  0 ~ 4天以不同滅菌時間及不同濃度的SSP (A)和SPP (B) 發酵上清液之DPPH自由基清除能力  53
圖4.17  0 ~ 4天以不同濃度的SSP (A)和SPP (B) 與水解時間對還原糖量的影響  54
圖4.18  0 ~ 4天之0.5%的SSP (A)與SPP (B)薄層層析  55

表目錄

表2.1幾丁質、幾丁聚醣及其衍生物在食品上的用 8
表4.1 TKU018之較適生產條件	23
表4.2 Bacillus cereus TKU018所生產幾丁聚醣酶之特性	30
表4.3 Bacillus cereus TKU018幾丁聚醣酶純化總表	45
表4.4各種化學藥品對幾丁聚醣酶之影響	49
表4.5界面活性劑對幾丁聚醣酶之影響	50
表4.6幾丁聚醣酶之基質特異性 	51
表4.7 Bacillus spp.幾丁聚醣酶特性比較  56

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