本研究主要由台灣北部土壤篩選出一株蛋白酶、幾丁質酶及幾丁聚醣酶生產菌TKU020 ，以烏賊軟骨粉為唯一碳/氮源，經鑑定為Serratia sp.。TKU020生產蛋白酶、幾丁質酶及幾丁聚醣酶之較適培養條件為0.5% 烏賊軟骨粉、0.1 % K2HPO4及0.05 %MgSO4．7H2O，在25℃、pH 9之100 mL液態培養基搖瓶培養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，純化出一種蛋白酶、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55.4 kDa、64.5 kDa及55.4 kDa。其最適反應溫度、熱安定性、最適反應pH、pH安定方面，蛋白酶的分別為50℃、40℃、pH 10及pH 4-11；幾丁質酶為40℃、30℃、pH 4-8及pH 4-8；幾丁聚醣酶為40℃、40℃、pH 5及pH 7。蛋白酶活性不受金屬離子、EDTA、PMSF所抑制，幾丁質酶和幾丁聚醣酶皆受Mn2+、SDS所抑制。
    利用TKU020這株菌，分別用不同碳/氮源 (0~2%的蝦殼粉和烏賊軟骨粉)培養在25℃、pH 9之100 mL液態培養基分別培養0~5天，所得發酵上清液去探討其還原糖量和抗氧化之能力。

Strain TKU020 was isolated from the soil in the northern Taiwan by using squid pen powder (SPP) as the sole carbon/nitrogen source and identified as Serratia sp. .The optimized culture condition for protease, chitinase and chitosanase was composed of 0.5% squid pen powder (SPP), 0.1 % K2HPO4 and 0.05 % MgSO4．7H2O at pH 9 and incubate in 100 mL of liquid media in shaking flasks at 25℃ for 3 days.  
  One protease, chitinase and chitosanase were purified by chromatography procedures of DEAE-Sepharose, Phenyl-Sepharose, and Sephacryl S-100. The molecular mass of protease, chitinase and chitosanase determined by SDS-PAGE was approximately 49 kDa, 55.4 kDa and 65.4 kDa, respectively. The optimum temperature, thermal stability, optimum pH and pH stability of protease were 50℃, 40℃, pH 10 and pH 4-11, respectively. The optimum temperature, thermal stability, optimum pH and pH stability of chitinase were 40℃, 30℃, pH 4-8 and pH 4-8, respectively. The optimum temperature, thermal stability, optimum pH and pH stability of chitosanase were 40℃, 40℃, pH 5 and pH 7, respectively. The protease activity was not inhibited by tested metal ions, EDTA and PMSF；The chitinase and chitosanase were both inactivated by Mn2+ and SDS.
  In the second part , Serratia sp. TKU020 was cultured using different carbon source (0~2% shrimp shell powder and squid pen powder)and incubated in 100 mL of liquid media (pH 9) in shaking flasks at 25℃ for 0~5days , and the quantity of DPPH scavenging activity and reducing sugar in culture supernatant was studied.

目錄
	頁次
授權書
簽名頁
誌謝
中文摘要	I
英文摘要	III
目錄	V
圖目錄	X
表目錄	XII

第一章 緒論	1
第二章 文獻回顧	2
     2.1 沙雷氏菌屬之特性	2
     2.2 幾丁質與幾丁聚醣	2
     2.3 幾丁質酶與幾丁聚醣酶	3
     2.4 蛋白酶	3
     2.5 抗氧化活性	4

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 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	9
     3.8 蛋白酶之活性測定	10
     3.9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	11
        3.9.3 培養基之酸鹼值和溫度	11
        3.9.4 培養體積 (通氣量)	11
        3.9.5 較適培養時間	11
     3.10 酵素之分離純化	12
        3.10.1 粗酵素液之製備	12
        3.10.2 陰離子交換樹脂層析法	12
        3.10.3 疏水性層析	13
        3.10.4 膠體過濾層析	13
        3.10.5 蛋白質電泳分析	13
3.10.6 胜肽質譜鑑定	13
3.10.7 蛋白酶活性電泳	14
3.10.8 蛋白質定量分析	14
     3.11酵素生化特性分析	14
        3.11.1 酵素最適反應溫度	14
        3.11.2 酵素熱安定性	15
        3.11.3 酵素最適反應pH	15
        3.11.4 酵素pH安定性	15
        3.11.5 金屬離子與抑制劑對酵素活性之影響	16
        3.11.6 界面活性劑對酵素活性之影響	16
        3.11.7 幾丁質酶與幾丁聚醣酶之基質特異性	17
3.11.8蛋白酶之基質特異性	17
3.12 DPPH自由基清除能力之測定	18
     3.13 薄層色層分析	18
3.14 還原糖量之測定	19

第四章 結果與討論	20
     4.1 幾丁質酶/幾丁聚醣酶與蛋白酶生產菌之篩選	20
     4.2 菌株之特性	20
     4.3 碳/氮源之選擇	20
4.4 碳/氮源濃度之影響	21
4.5 培養基之酸鹼值和溫度	21
4.6 培養體積 (通氣量)	25
4.7 較適培養時間	25
4.8 酵素的較適生長條件	25
     4.9 幾丁質酶、幾丁聚醣酶與蛋白酶之純化分離	26
        4.9.1 粗酵素液製備	26
        4.9.2 陰離子交換層析	26
        4.9.3 疏水性層析	27
        4.9.4 膠體過濾層析	27
        4.9.5 綜合結果	27
     4.10 幾丁質酶、幾丁聚醣酶及蛋白酶之分子量判定	28
4.10.1 幾丁質酶、幾丁聚醣酶SDS-PAGE (銀染法)	28
4.10.2 蛋白酶SDS-PAGE (CBR染色法)	28
4.10.3 蛋白酶活性染色	28
        4.10.4 蛋白酶胜肽質譜鑑定	29
        4.10.5 綜合結果	29
     4.11 純化後幾丁質酶、幾丁聚醣酶與蛋白酶之生化特性分析	30
        4.11.1酵素之最適反應溫度及熱安定性	30
        4.11.2酵素之最適反應pH及pH安定性	31
        4.11.3 金屬離子與抑制劑對酵素酵素活性之影響	31
        4.11.4 界面活性劑對酵素活性之影響	32
        4.11.5 幾丁質酶與幾丁聚醣酶之基質特異性	32
        4.11.6 蛋白酶之基質特異性	33
     4.12 DPPH自由基清除能力之測定	33
     4.13薄層色層分析	34
     4.14還原糖量之測定	35
    
第五章 結論	80
參考文獻	81
附錄	90

圖目錄

圖4.1 Serratia sp.TKU020之顯微鏡照片	22
圖4.2 16S rDNA部份核酸序列分析	23
圖4.3生理生化試驗	24
圖4.4不同碳/氮源對於TKU020產生蛋白酶活性之影響	36
圖4.5不同碳/氮源對於TKU020產生幾丁質酶活性之影響	37
圖4.6不同碳/氮源對於TKU020產生幾丁聚醣酶活性之影響	38
圖4.7烏賊軟骨粉添加對TKU020產生蛋白酶活性之影響	39
圖4.8烏賊軟骨粉添加對TKU020產生幾丁質酶活性之影響	40
圖4.9烏賊軟骨粉添加對TKU020產生幾丁聚醣酶活性之影響	41
圖4.10溫度及pH對TKU020產生蛋白酶活性之影響	42
圖4.11溫度及pH對TKU020產生幾丁質酶活性之影響	43
圖4.12溫度及pH對TKU020產生幾丁聚醣酶活性之影響	44
圖4.13通氣量對TKU020產生蛋白酶活性之影響	45
圖4.14通氣量對TKU020產生幾丁質酶活性之影響	46
圖4.15通氣量對TKU020產生幾丁聚醣酶活性之影響	47
圖4.16 TKU020培養於烏賊培養基所產蛋白酶、幾丁質酶及幾丁聚醣酶變化情形	48
圖4.17 TKU020所生產酵素之純化分離流程圖	49
圖4.18 Serratia sp.之DEAE-Sepharose CL-6B層析圖譜	50
圖4.19 幾丁質酶和幾丁聚醣酶之Phenyl-Sepharose層析圖譜	51
圖4.20 Serratia sp.之DEAE-Sepharose CL-6B層析圖譜	52
圖4.21 蛋白酶之Sephacryl S-100之層析圖譜	53
圖4.22 幾丁質酶於SDS-PAGE之分子量分析	57
圖4.23幾丁聚醣酶於SDS-PAGE之分子量分析	58
圖4.24蛋白酶於SDS-PAGE之分子量分析	59
圖4.25蛋白酶活性染色	60
圖4.26 TKU020幾丁質酶之最適溫度及熱安定性	61
圖4.27 TKU020幾丁聚醣酶之最適溫度及熱安定性	62
圖4.28 TKU020蛋白酶之最適溫度及熱安定性	63
圖4.29 TKU020幾丁質酶之最適pH及pH安定性	64
圖4.30 TKU020幾丁聚醣酶之最適pH及pH安定性	65
圖4.31 TKU020蛋白酶之最適pH及pH安定性	66
圖4.32 碳/氮源濃度及發酵時間對DPPH自由基清除能力之影響	75
圖4.33 薄層色層分析TKU020發酵上清液之抗氧化物質	76
圖4.34 碳/氮源濃度及發酵時間對還原糖之影響	77
圖4.35 TKU020發酵上清液幾丁質酶、幾丁聚醣酶與還原醣之變化情形  78

表目錄

表4.1 Serratia sp. TKU020幾丁質酶純化總表	54
表4.2 Serratia sp. TKU020幾丁聚醣酶純化總表	55
表4.3 Serratia sp. TKU020蛋白酶純化總表	56
表4.4各種金屬離子與抑制劑對酵素之影響	67
表4.5各種界面活性劑對酵素之影響	68
表4.6幾丁質酶和幾丁聚醣酶之基質特異性	69
表4.7蛋白酶之基質特異性	70
表4.8 微生物來源之幾丁質酶和幾丁聚醣酶特性比較	71
表4.9 微生物來源之蛋白酶特性比較	73
表4.10TKU020蛋白酶胜肽質譜鑑定結果	79

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