TKU016 係以蝦殼粉為唯一碳/氮源，篩選自台灣北部土壤之一株幾丁聚醣酶及蛋白酶生產菌，經鑑定為Serratia sp.。TKU016生產幾丁聚醣酶及蛋白酶之較適培養條件為含有1% 蝦殼粉、0.1 % K2HPO4及0.05 %MgSO4．7H2O之液態培養基(pH 7)在30℃搖瓶培養3天。所得發酵上清液，經硫酸銨沉澱、DEAE-Sepharose及Sephacryl S-100等層析步驟，可分離出一種幾丁聚醣酶及一種蛋白酶，經SDS-PAGE測其分子量分別為 65 kDa及53 kDa。於最適反應pH、最適反應溫度、pH安定、熱安定性方面，幾丁質酶的條件為pH 7、50℃、pH 4-8及<80℃；蛋白酶的為pH 11、40℃、pH 5-11及<90℃。幾丁聚醣酶活性受Mn2+所抑制。蛋白酶對偶氮白蛋白、偶氮酪蛋白為基質時，具有較佳的活性，對於凝膠蛋白、纖維蛋白、彈性蛋白的活性不佳。

The chitosanase and protease producing strain, Serratia sp. TKU016, was isolated from the soil in Taiwan. The optimized condition for chitosanase and protease production were found when the culture was shaken at 30℃for 3 days in 100mL of medium contain 1% shrimp shell powder (SSP), 0.1 % K2HPO4 and 0.05 % MgSO4．7H2O (pH8). One chitosanase and one protease were purified by chromatography procedures of DEAE-Sepharose, and Sephacryl S-100. The molecular mass of the chitosanase and protease determined by SDS-PAGE was approximately 65 kDa and 53 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of chitosanase were pH 7、40℃、pH 4-8 and <80℃, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of protease were pH 11、40℃、pH 5-11 and <90℃,respectively. The chitosanase was inactivated by Mn2+. The protease showed good activity toward azocasein and azoalbumin as substrates, low activity with gelatin, fibrin, and elastin.

目錄
	頁次

授權書
簽名頁
誌謝
中文摘要	I
英文摘要	II
目錄	Ⅲ
圖目錄	Ⅷ
表目錄	Ⅹ

第一章 緒論	1
第二章 文獻回顧	2
     2.1 菌種之簡介	2
     2.2 幾丁質與幾丁聚醣之應用	3
        2.2.1 幾丁質之應用	3
        2.2.2 幾丁聚醣之應用	3
     2.3 幾丁質酶與幾丁聚醣酶之應用	3
        2.3.1 幾丁質酶	3
        2.3.2 幾丁聚醣酶	4
     2.4 蛋白酶	4
        2.4.1 蛋白酶分類	4
        2.4.2 蛋白酶應用	5
第三章 材料與方法	6
     3.1 實驗菌株	6
     3.2 實驗材料	6
     3.3 實驗儀器	7
     3.4 生產菌株之篩選	8
     3.5 懸浮態幾丁質之製備	8
     3.6 幾丁質酶之活性測定	9
     3.7 幾丁聚醣酶之活性測定	10
     3.8 蛋白酶之活性測定	10
     3.9 酵素最適培養條件探討	10
        3.9.1 碳/氮源之選擇	10
        3.9.2 碳/氮源濃度之影響	10
        3.9.3 培養基之酸鹼值	10
        3.9.4 培養溫度	11
        3.9.5 培養體積(通氣量)	11
        3.9.6 培養時間	10
     3.10 酵素之分離純化	11
        3.10.1 粗酵素液之製備	11
        3.10.2 陰離子交換層析	12
        3.10.3膠體過濾層析	12
     3.11 蛋白質電泳分析	12
     3.12 蛋白質定量分析	13
     3.13 酵素之特性分析	13
        3.13.1 酵素最適反應溫度	14
        3.13.2 酵素之熱安定性	14
        3.13.3 酵素最適反應pH	14
        3.13.4 酵素之pH安定性	14
        3.13.5 金屬離子與抑制劑對酵素活性之影響	15
        3.13.6 界面活性劑對酵素活性之影響	15
        3.13.7 蛋白酶基質特異性	15
        3.13.8 幾丁質酶基質特異性	16
第四章 結果與討論	17
     4.1 幾丁質酶生產菌之篩選	17
     4.2 菌株之特性	17
     4.3 碳/氮源之選擇	18
     4.4 幾丁質酶及蛋白酶較適生長條件探討	18
     4.5 幾丁質酶之分離純化	19
        4.5.1 粗酵素液製備	19
        4.5.2 陰離子交換層析	19
        4.5.3膠體過濾層析	20
        4.5.4綜合結果	20         4.6 幾丁質酶及蛋白酶分子量測定	21
        4.6.1 SDS-PAGE	21
        4.6.2 綜合結果	21
     4.7 幾丁質酶與蛋白酶之特性分析	21
        4.7.1幾丁質酶之最適反應pH及pH安定性	21
        4.7.2蛋白酶之最適反應pH及pH安定性性	22
        4.7.3幾丁質酶之最適反應溫度及熱安定性	22
        4.7.4蛋白酶之最適反應溫度及熱安定性	22
        4.7.5 金屬離子及抑制劑對幾丁質酶及蛋白酶之影響	22
        4.7.6界面活性劑對幾丁質酶及蛋白酶之影響	22
        4.7.7蛋白酶之基質特異性	23
        4.7.8幾丁質酶之基質特異性	23
        
     4.8 發酵上清液DPPH清除率試驗	23
     4.9 蛋白酶胜肽質普鑑定	24
第五章 結論	57
參考文獻	58
附錄	71















圖目錄
	頁次

圖2.1 幾丁質及幾丁聚醣之構造	5
圖4.1 Serratia sp.TKU016之顯微鏡照片	25
圖4.2 16S rDNA部份核酸序列分析	26
圖4.3 API鑑定系統分析結果	27
圖4.4 SSP濃度對TKU016幾丁質酶/幾丁聚醣酶生產之影響	29
圖4.5 SSP濃度對TKU016蛋白酶生產之影響	30
圖4.6 培養溫度對TKU016幾丁質酶/幾丁聚醣酶生產之影響	31
圖4.7培養溫度對TKU016蛋白酶生產之影響	32
圖4.8 培養pH對TKU016幾丁質酶/幾丁聚醣酶生產之影響	33
圖4.9培養pH對TKU016蛋白酶生產之影響	34
圖4.10培養液體積對TKU016幾丁質酶/幾丁聚醣酶生產之影響	35
圖4.11培養液體積對TKU016蛋白酶生產之影響	36
圖4.12 Serratia sp. TKU016所生產幾丁質酶/幾丁聚醣酶及蛋白酶之生長曲線………………………………………………………………………………3
圖4.13 Serratia sp. TKU016所生產幾丁質酶及蛋白酶之分離純化流程圖
	38
圖4.14 DEAE-Sepharose CL-6B之幾丁質酶/幾丁聚醣酶及蛋白酶層析圖譜	39
圖4.15幾丁質酶/幾丁聚醣酶之Sephacryl S-100之層析圖譜	40
圖4.16蛋白酶之Sephacryl S-100之層析圖譜		41
圖4.17 幾丁質酶/幾丁聚醣酶及蛋白酶之SDS-PAGE電泳分析圖	43
圖4.18 幾丁質酶/幾丁聚醣酶之最適pH及pH安定性	44
圖4.19蛋白酶之最適pH及pH安定性	45
圖4.20幾丁質酶/幾丁聚醣酶之最適溫度及熱安定性	46
圖4.21蛋白酶之最適溫度及熱安定性	47
圖4.22蝦殼粉及烏賊軟骨為培養基DPPH清除效果之薄層層析	54
圖4.24 蝦殼粉及烏賊軟骨為培養基之DPPH清除率	55
圖4.26 蛋白酶之胜肽質普鑑定結果	56














表目錄
	頁次

表4.1 各種碳源對TKU016幾丁質酶及蛋白酶活性之影響	28
表4.2 Serratia sp. TKU016幾丁質酶純化總表	42
表4.3 Serratia sp. TKU016蛋白酶純化總表	42
表4.4金屬離子對蛋白酶活性之影響	48
表4.5界面活性劑對幾丁質酶及蛋白酶之影響	49
表4.6蛋白酶基質特異性	50
表4.7 幾丁質酶基質特異性	51
表4.8 微生物來源之幾丁質酶特性比較	52
表4.9 微生物來源之蛋白酶特性比較	53

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