菌株TKU028係以蝦殼廢棄物作為唯一碳/氮源，篩選自彰化土壤之一株幾丁質酶及蛋白酶生產菌，經鑑定為Bacillus cereus。
    本研究藉由B. cereus TKU028發酵蝦殼廢棄物生產幾丁質酶及蛋白酶，並探討其較適培養條件。酵素純化係利用酵素與基質專一性之特性進行。B. cereus TKU028所生產幾丁質酶及蛋白酶，經由SDS-PAGE測得分子量分別為40kDa及43kDa，而最適反應pH、最適反應溫度、pH安定性及熱安定性分別為：pH5-6, 60℃, pH5-8, ≦ 60℃；pH9, 50-60℃, pH5-9, ≦ 40℃。幾丁質酶除了受Mn2+及EDTA有約40%抑制外，皆不受金屬離子及界面活性劑所影響；蛋白酶則受到Cu2+、SDS及EDTA抑制，判定屬於金屬型蛋白酶。
    B. cereus TKU028之幾丁質酶水解幾丁質所得寡糖，藉由HPLC分析得知以 (GlcNAc) 6為主；而蛋白酶因耐鹼及界面活性劑特性具潛力應用於洗滌劑添加物及去毛劑；PCR-DGGE結果顯示，B. cereus TKU028可與蝦蟹殼廢棄物應用於環境之生物復育。

Bacillus cereus TKU028, a chitinase and protease-producing strain, was isolated from the soil in Taiwan, by using shrimp head powder (SHP) as the sole carbon/nitrogen source.  A chitinase (CHT) and a protease (PRO) were purified by chitin affinity binding and casein affinity binding, respectively. The molecular masses of CHT and PRO determined by SDS- PAGE were approximately 40 kDa and 43 kDa, respectively.
    The optimum pH, optimum temperature, pH stability, and thermal stability of CHT and PRO were pH 5-6, 60 ℃, pH 5-8, and ≦60 ℃, and pH 9,50-60 ℃, pH 5-9, and ≦ 40℃, respectively. CHT was little inhibited by Mn2+ and EDTA, and PRO was inhibited by Cu2+ , EDTA, and 2 mM SDS. 
    The chitinase-hydrolyzed products of subtract degradation were analyzed by HPLC at 1-48 h intervals. It was observed the major products are (GlcNAc)6.  In addition, B. cereus TKU028 has unlimited potential to enhance the biodegradtion of shrimp shells in the seawater containing mangrove river sediment.

目錄
頁次
封面內頁
簽名頁
授權書
致謝
中文摘要	I
英文摘要	II
目錄	III
圖目錄	IX
表目錄	XII


第一章 緒論	1
第二章 文獻回顧	2
2.1 幾丁質與幾丁聚醣	2
2.2 幾丁質水解酵素	5
2.3 N-乙醯幾丁寡醣及幾丁寡醣	5
2.4 蛋白質水解酵素	6
2.5 Bacillus cereus之簡介	9
2.6 酵素純化	9
第三章 材料與方法	11
3.1 實驗菌株	11
3.2 實驗材料	11
3.3 實驗儀器	13
3.4幾丁質酶生產菌之篩選	14
3.5幾丁質酶之活性測定	14
3.6 蛋白酶活性之測定	15
3.7 幾丁質酶及蛋白酶較適生產條件探討	15
3.7.1碳/氮源濃度	15
3.7.2培養溫度	16
3.7.3 培養體積	16
3.7.4培養基通氣量	16
3.7.5培養時間	17
3.8幾丁質酶及蛋白酶之分離純化	17
3.8.1粗酵素液之製備	17
3.8.2親合性吸附法	17
3.8.2.1幾丁質親合性吸附	18
3.8.2.2酪蛋白親合性吸附	18
3.8.3陰離子交換樹脂	18
3.8.4疏水性層析法	19
3.9酵素之分子量測定	19
3.9.1 蛋白質電泳分析	19
3.10幾丁質酶及蛋白酶之特性分析	20
3.10.1酵素最適反應溫度	20
3.10.2 酵素熱安定性	20
3.10.3 酵素最適反應 pH	21
3.10.4 酵素 pH 安定性	21
3.10.5 金屬離子及化學藥品對酵素活性之影響	22
3.10.6 界面活性劑對幾丁聚醣酶活性之影響	22
3.10.7 酵素之基質特異性	23
3.10.7.1幾丁質酶	23
3.10.7.2 蛋白酶	23
3.11 還原糖量之測定	24
3.11.1 Imoto and Yagishita測定法	24
3.11.2 DNS測定法	24
3.12 幾丁質酶水解基質及寡醣分析	25
3.12.1 基質之水解	25
3.12.2 N-乙醯幾丁寡醣製備	25
3.12.3 N-乙醯幾丁寡醣之組成分析	25
3.13 蝦殼廢棄物及B. cereus TKU028對紅樹林河流泥沙之生物降解及微生物族群變化	26
3.13.1 採樣及土壤處理	26
3.13.2 樣品總生菌數及糖含量之測定	26
3.13.3 土壤內微生物DNA 之萃取純化	27
3.13.4 聚合酶鏈連鎖反應(Polymerase Chain Reaction, PCR)	27
3.13.5 變性梯度凝膠電泳(Denaturing Gradient Gel Electrophoresis, DGGE)	30
3.13.6 DGGE圖譜的統計分析	32
3.13.7 DNA定序	32
第四章 結果與討論	33
4.1 幾丁質酶與蛋白酶生產菌之篩選	33
4.1.1菌種鑑定	33
4.2 幾丁質酶及蛋白酶較適生產條件探討	36
4.2.1 碳/氮源濃度	36
4.2.2 培養溫度	37
4.2.3培養體積	37
4.2.4通氣量	38
4.2.5較適培養條件探討結果	38
4.3 幾丁質酶及蛋白質酶之純化	50
4.3.1 粗酵素液之製備	50
4.3.2 幾丁質親合性吸附/酪蛋白親合性吸附	50
4.3.3 離子交換樹脂/疏水性層析	51
4.3.4 比較幾丁質親合性吸附與傳統純化方式之優劣	51
4.3.5 綜合結果	51
4.4 幾丁質酶與蛋白質酶之分子量測定	52
4.4.1 SDS-PAGE	52
4.4.2 綜合結果	53
4.4.3 幾丁質酶胜肽質譜鑑定(peptide mass mapping)	54
4.5 幾丁質酶與蛋白酶之特性分析	54
4.5.1 幾丁質酶與蛋白酶之最適反應溫度及熱安定性	54
4.5.2 幾丁質酶與蛋白酶之最適反應pH及pH安定性	56
4.5.3 金屬離子及化學藥品對幾丁質酶與蛋白酶之影響	56
4.5.4 界面活性劑對幾丁質酶與蛋白酶活性之影響	57
4.5.5 幾丁質酶與蛋白酶之基質特異性	58
4.5.5.1 幾丁質酶之基質特異性	58
4.5.5.2 蛋白酶之基質特異性	58
4.6 幾丁質酶水解寡糖及應用	77
4.6.1 幾丁質酶水解不同基質及HPLC分析水解產物	77
4.6.2 添加幾丁質廢棄物(蝦頭粉)於紅樹林河流泥沙中之菌相變化及生物降解	77
4.6.2.1 土壤培養8週後之總生菌數及含醣量	78
4.6.2.2聚合酶鏈反應-變性梯度凝膠電泳分析(PCR-DGGE)	78
第五章 結論	85
參考文獻	85



圖目錄
頁次
圖2.1 幾丁質、幾丁聚醣及纖維素之結構	3
圖2.2幾丁質與幾丁聚糖之應用	4
圖3.1 PCR反應溫度控制圖	29
圖4.1 Bacillus cereus TKU028之顯微照片	34
圖4.2 16S rDNA部分鹼基序列分析及API試驗	35
圖4.3 不同碳/氮源對B. cereus TKU028生產幾丁質酶之影響	41
圖4.4 不同碳/氮源對B. cereus TKU028生產蛋白酶之影響	42
圖4.5 培養溫度對B. cereus TKU028生產幾丁質酶之影響	43
圖4.6 培養溫度對B. cereus TKU028生產蛋白酶之影響	44
圖4.7 培養液體積對B. cereus TKU028生產幾丁質酶之影響	45
圖4.8 培養液體積對B. cereus TKU028生產蛋白酶之影響	46
圖4.9 通氣量對B. cereus TKU028生產幾丁質酶及蛋白酶之影響	47
圖4.10 B. cereus TKU028培養於SHP培養基所生產幾丁質酶與蛋白酶之生長曲線圖	48
圖4.11 B. cereus TKU028所生產幾丁質酶與蛋白酶之純化流程圖	59
圖4.12 B. cereus TKU028所生產幾丁質酶之DEAE-Sepharose CL-6B層析圖譜	60
圖4.13 B. cereus TKU028所生產蛋白酶之Butyl-S Sepharose層析圖譜	61
圖4.14 比較不同方法純化B. cereus TKU028所生產幾丁質酶之DEAE-Sepharose CL-6B層析圖譜	64
圖4.15 Bacillus cereus TKU028幾丁質酶及蛋白酶SDS-PAGE之分子量分析	66
圖4.16 幾丁質酶最適反應溫度及熱安定性	68
圖4.17 蛋白酶最適反應溫度及熱安定性	69
圖4.18 基質存在下對蛋白酶於60℃熱安定性影響	70
圖4.19 幾丁質酶之最適反應pH及pH安定性	71
圖4.20 蛋白酶之最適反應pH及pH安定性	72
圖4.21幾丁質酶水解不同基質之殘餘量照片	79
圖4.22 幾丁質酶水解不同基質之殘餘量	80
圖4.23 幾丁質酶水解不同基質之總醣及還原醣	81
圖4.24 HPCL進行水溶性幾丁質水解產物分析	82
圖4.25 HPCL進行β型幾丁質水解產物分析	83
圖4.26 HPCL進行懸浮態幾丁質水解產物分析	84
圖4.27 不同條件下培養8週之土壤總生菌數	85
圖4.28不同條件下培養8週之土壤總醣含量	85
圖4.29不同條件下培養8週之土壤還原醣含量	85
圖4.30 樣品PCR產物之電泳圖	85
圖4.31 16S rDNA之聚合酶鏈反應-變性梯度凝膠電泳分析(PCR-DGGE)	85



表目錄
頁次
表2.1 蛋白質水解酵素之分類	8
表2.2 以蝦蟹殼、烏賊軟骨為唯一碳/氮源自台灣土壤篩選出之菌株	10
表3.1 DNS試劑之組成	24
表3.2 PCR所使用之引子	28
表3.3 PCR反應試劑成分	28
表3.4 不同濃度之Acrylamide/Bis所能分離之核酸長度	30
表3.5  7%之Acrylamide/Bis變性凝膠成份	31
表3.6  10% Ammonium persulfate之成份	31
表4.1 Bacillus cereus TKU028 生產幾丁質酶及蛋白酶之較適條件	40
表4.2 B. cereus TKU028、B. cereus TKU006及Serratia sp. TKU017之幾丁質酶及蛋白酶較適生產條件比較	49
表4.3 Bacillus cereus TKU028 所生產幾丁質酶之純化總表	62
表4.4 Bacillus cereus TKU028 所生產蛋白酶之純化總表	63
表4.5 比較不同方法純化Bacillus cereus TKU028 所生產幾丁質酶之純化總表	65
表4.6 Bacillus cereus TKU028幾丁質酶胜肽質譜鑑定結果	67
表4.7 化學物質對幾丁質酶及蛋白酶之影響	73
表4.8 Bacillus cereus TKU028幾丁質酶及蛋白酶之基質特異性	74
表4.9 微生物生產之幾丁質酶及蛋白酶特性比較	75
表4.10 培養8週之總生菌數	85
表4.11不同條件下培養8週之土壤總醣含量	85
表4.12不同條件下培養8週之土壤還原醣含量	85

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