TKU015 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質酶及幾丁聚醣酶生產菌，經鑑定為Pseudomonas sp.。TKU015生產幾丁質酶及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之液態培養基(pH 8)在30℃搖瓶培養3天。所得發酵上清液，經硫酸銨沉澱、DEAE-Sepharose、Phenyl-Sepharose及Sephacryl S-100等層析步驟，純化出一種幾丁質酶及一種幾丁聚醣酶，經SDS-PAGE測其分子量分別為68 kDa及30 kDa。於最適反應pH、最適反應溫度、pH安定、熱安定性方面，幾丁質酶的為pH 5、50℃、pH 5-7及<60℃；幾丁聚醣酶的為pH 4、50℃、pH 3-9及<50℃。幾丁質酶活性受Mn2+、Fe2+所抑制，幾丁聚醣酶活性則受Mn2+、Cu2+、PMSF所抑制。
TKU015生產納豆激酶之較適生產條件為含有1% 蝦殼粉、0.1 %K2HPO4及0.05 %MgSO4．7H2O 之液態培養基 (pH 7) 於30℃搖瓶培養2天。所得發酵上清液，經硫酸銨沉澱、DEAE-Sepharose及Phenyl-Sepharose等層析分離步驟，純化出一種納豆激酶。經SDS-PAGE測其分子量為21 kDa，其最適反應pH、最適反應溫度、pH安定、熱安定性則分別為pH 7、50℃、pH 4-11及<37℃。納豆激酶活性受PMSF完全抑制，Fe2+則會提高其活性。

The chitinase and chitosanase producing strain, Pseudomonas sp. TKU015, was isolated from the soil in Taiwan. The optimized condition for chitinase and chitosanase production were found when the culture was shaken at 30℃for 3 days in 100mL of medium contain 0.5% shrimp shell powder (SSP), 0.1 % K2HPO4 and 0.05 % MgSO4．7H2O (pH8). One chitinase and one chitosanase were purified by chromatography procedures of DEAE-Sepharose, Phenyl-Sepharose, and Sephacryl S-100. The molecular mass of the chitinase and the chitosanase determined by SDS-PAGE was approximately 68 kDa and 30 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of chitinase were pH 5, 50℃, pH 4-6 and <60℃, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of chitosanase were pH 4, 50℃, pH 3-9 and <50℃, respectively. The chitinase was inactivated by Mn2+ and Fe2+；The chitosanase was inactivated by Mn2+, Cu2+ and PMSF.

One nattokinase was also purified from the second day culture supernatant of Pseudomonas sp. TKU015. The optimized condition for nattokinase production was found when the culture was shaken at 30℃for 2 days in 100mL of medium contain 1% SSP, 0.1 % K2HPO4 and 0.05 % MgSO4．7H2O (pH7). The molecular mass of the nattokinase determined by SDS-PAGE was approximately 21 kDa. The optimum pH, optimum temperature, pH stability, and thermal stability of nattokinase were pH 7, 50℃, pH 4-11 and <37℃, respectively. The nattokinase was inactivated by PMSF, and activated by Fe2+.

目錄	頁次

簽名頁
授權書
中文摘要	I
英文摘要	II
誌謝	V
目錄	VI
圖目錄	XII
表目錄	IXV

第一章 緒論	1
第二章 文獻回顧	3
     2.1 菌種之簡介	3
     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	4
     2.3 幾丁質酶與幾丁聚醣酶之應用	4
        2.3.1 幾丁質酶之應用	4
        2.3.2 幾丁聚醣酶之應用	5
     2.4 納豆激酶	                  5
     2.5 納豆激酶溶解血栓之功用	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 生產菌株之篩選	11
     3.5 懸浮態幾丁質之製備	11
     3.6 幾丁質酶之活性測定	12
     3.7 幾丁聚醣酶之活性測定	12
     3.8 納豆激酶之活性測定	         12
     3.9 酵素最適培養條件探討	13
        3.9.1 碳/氮源之選擇	         13
        3.9.2 碳/氮源濃度之影響	14
        3.9.3 培養基之酸鹼值	14
        3.9.4 培養溫度	         14
        3.9.5 培養體積(通氣量)	14
        3.9.6 培養時間	15
     3.10 酵素之分離純化	15
        3.10.1 粗酵素液之製備	15
        3.10.2 陰離子交換層析	15
        3.10.3 疏水性層析	16
        3.10.4 膠體過濾層析	16
     3.11 蛋白質電泳分析	16
     3.12 蛋白質定量分析	17
     3.13 酵素之特性分析	17
        3.13.1 酵素最適反應溫度	17
        3.13.2 酵素之熱安定性	17
        3.13.3 酵素最適反應pH	18
        3.13.4 酵素之pH安定性	18
        3.13.5 金屬離子與抑制劑對酵素活性之影響	18
        3.13.6 界面活性劑對酵素活性之影響	19
        3.13.7 有機溶劑對酵素活性之影響	19
     3.14 蛋白質轉印	20
第四章 結果與討論	                  21
     4.1 幾丁質酶生產菌之篩選	21
     4.2 菌株之兔特性	21
     4.3 碳/氮源之選擇	21
     4.4 幾丁質酶較適生長條件探討	         22
     4.5 幾丁質酶及幾丁聚醣酶之分離純化	23
        4.5.1 粗酵素液製備	23
        4.5.2 陰離子交換層析	24
        4.5.3 疏水性層析	24
        4.5.4 膠體過濾層析	24
        4.5.5 綜合結果	25
     4.6 幾丁質酶及幾丁聚醣酶之分子量測定	25
        4.6.1 SDS-PAGE	25
        4.6.2 綜合結果	26
     4.7 幾丁質酶與幾丁聚醣酶之特性分析	27
        4.7.1 幾丁質酶之最適反應溫度及熱安定性	27
        4.7.2 幾丁聚醣酶之最適反應溫度及熱安定性	27
        4.7.3 幾丁質酶之最適反應pH及pH安定性	28
        4.7.4 幾丁聚醣酶之最適反應pH及pH安定性	28
        4.7.5 金屬離子對幾丁質酶之影響	29
        4.7.6 金屬離子對幾丁聚醣酶之影響	29
        4.7.7 界面活性劑對幾丁質酶之影響	30
        4.7.8 界面活性劑對幾丁聚醣酶之影響	30
        4.7.9 有機溶劑對幾丁質酶之影響	31
        4.7.10 有機溶劑對幾丁聚醣酶之影響	31
     4.8 納豆激酶之活性測定	58
     4.9 碳/氮源之選擇	58
     4.10 納豆激酶較適生長條件探討	58
     4.11 納豆激酶之分離純化	59
        4.11.1 粗酵素液製備	         59
        4.11.2 陰離子交換層析	60
        4.11.3 疏水性層析	60
        4.11.4 綜合結果	60
     4.12 納豆激酶之分子量測定	61
        4.12.1 SDS-PAGE	61
        4.12.2 綜合結果	61
     4.13 納豆激酶之特性分析	62
        4.13.1 納豆激酶之最適反應溫度及熱安定性	62
        4.13.2 納豆激酶之最適反應pH及pH安定性	62
        4.13.3 金屬離子對納豆激酶之影響	63
        4.13.4 界面活性劑對納豆激酶之影響	63
        4.13.5 有機溶劑對納豆激酶之影響	64

第五章 結論	82
參考文獻	83
附錄	96
圖目錄                      	頁次

圖2.1 幾丁質及幾丁聚醣之構造	7
圖2.2 納豆激酶在血栓溶解系統中之直接與間接作用	8
圖4.1 Pseudomonas sp.TKU015之顯微鏡照片	32
圖4.2 16S rDNA部份核酸序列分析	33
圖4.3 脂肪酸組成分析鑑定系統分析結果	34
圖4.4 SSP濃度對TKU015幾丁質酶生產之影響	35
圖4.5 不同碳/氮源對TKU015幾丁質酶生產之影響	36
圖4.6 培養溫度對TKU015幾丁質酶生產之影響	37
圖4.7 培養pH對TKU015幾丁質酶生產之影響	38
圖4.8 培養液體積對TKU015幾丁質酶生產之影響	39
圖4.9 Pseudomonas sp. TKU015培養於SSP培養基所生產幾丁質酶、幾	丁聚醣酶及納豆激酶之生長曲線圖	40
圖4.10 Pseudomonas sp. TKU015所生產三種酵素(幾丁質酶、幾丁聚醣酶及納豆激酶)之分離純化流程圖	41
圖4.11 DEAE-Sepharose CL-6B之幾丁質酶、幾丁聚醣酶層析圖譜	                                    42
圖4.12 幾丁聚醣酶之Phenyl-Sepharose層析圖譜	43
圖4.13 幾丁質酶之Sephacryl S-100之層析圖譜	44
圖4.14 各酵素之SDS-PAGE電泳分析圖	47
圖4.15 幾丁質酶之最適pH及pH安定性	48
圖4.16 幾丁質酶之最適溫度及熱安定性		49
圖4.17 幾丁聚醣酶之最適pH及pH安定性	         50
圖4.18 幾丁聚醣酶之最適溫度及熱安定性	51
圖4.19 有機溶劑對幾丁質酶活性之影響	         54
圖4.20 有機溶劑對幾丁聚醣酶活性之影響	55
圖4.21 有機溶劑對幾丁質酶安定性之影響	56
圖4.22 有機溶劑對幾丁聚醣酶安定性之影響	57
圖4.23 SSP濃度對TKU015納豆激酶生產之影響	65
圖4.24 培養溫度對TKU015納豆激酶活性生產之影響	66
圖4.25 pH對TKU015納豆激酶活性生產之影響	67
圖4.26 培養液體積對TKU015納豆激酶活性生產之影響	68
圖4.27 納豆激酶之DEAE-Sepharose CL-6B層析圖譜	69
圖4.28 納豆激酶之Phenyl-Sepharose層析圖譜	70
圖4.29 納豆激酶之最適pH及pH安定性	72
圖4.30 納豆激酶之最適溫度及熱安定性	73
圖4.31 有機溶劑對納豆激酶活性之影響	76
圖4.32 有機溶劑對納豆激酶安定性之影響	77
表目錄                               	頁次

表4.1 Pseudomonas sp. TKU015幾丁質酶純化總表	45
表4.2 Pseudomonas sp. TKU015幾丁聚醣酶純化總表	46
表4.3 金屬離子對幾丁質酶與幾丁聚醣酶之影響	52
表4.4 界面活性劑對幾丁質酶與幾丁聚醣酶之影響	53
表4.5 Pseudomonas sp. TKU015納豆激酶純化總表	71
表4.6 金屬離子對納豆激酶活性之影響	74
表4.7 界面活性劑對納豆激酶活性之影響	75
表4.8 微生物來源之幾丁質酶特性比較	78
表4.9 微生物來源之幾丁聚醣酶特性比較	79
表4.10 微生物來源之納豆激酶特性比較	80
表4.11 Pseudomonas sp.TKU015所生產三種酵素(幾丁質酶、幾丁聚醣酶及納豆激酶)之特性	81

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