本篇研究目的在工業和廢棄處理當中來生產新穎幾丁聚醣酶。將幾丁殼素之廢棄物轉換成生物活性幾丁寡醣 (COS) ，幾丁聚醣酶是最具有潛力的應用之一。
    幾丁聚醣酶生產菌株篩選於台灣土壤，以0.5%烏賊軟骨粉為唯一碳/氮源，經鑑定為Bacillus mycoides。酵素從培養上清液經管住層析等步驟分離。胞外幾丁聚醣酶純化倍率為130倍與活性回收率35 %，測得分子量約為48 kDa。純化之幾丁聚醣酶最適反應溫度為50 °C，pH為6、10，熱安定性為30-50 °C，pH安定性為4-10。幾丁聚醣酶活性受到Cu2+, Ba2+, Zn2+, Fe2+, Mn2+, EDTA 及PMSF所抑制，其Km 及 Vmax分別為0.098 mg/mL 及 1.336 U/mL。
    HPLC及MALDI-TOF MS之組合結果分析，酵素水解水溶性幾丁聚醣所獲得之幾丁寡醣，其TKU038包括具有多種聚合度 (DP) ，範圍3-9。TKU038之發酵上清液與幾丁寡醣混合物具有DPPH·自由基清除活性。高聚合度之幾丁寡醣 DPPH·自由基清除活性較低聚合度之幾丁寡醣強。而幾丁寡醣也有抗發炎之活性與促進L. paracasei TKU010 及L. paracasei TKU012之生長。TKU038對於幾丁寡醣之生產在SPP廢棄處理與工業上有潛在的應用。

The objectives of this investigation were to produce a novel chitosanase for application in industries and waste treatment. The conversion of chitinous biowaste into bioactive chitooligomers (COS) is one of the most promising applications of chitosanase. 
A chitosanase-producing strain was isolated form Taiwan soil and identified as Bacillus mycoides. The chitosanase was produced using 0.5% (w/v) squid pen powder (SPP) as the sole carbon/nitrogen source, and these enzymes was purified from the culture supernatant by column chromatography. Extracellular chitosanase was purified to 130-fold with a 35% yield, and its molecular mass was approximately 48 kDa. The purified chitosanase exhibited optimum activity at 50 °C, pH 6, 10 and was stable at 30-50 °C, pH 4-10. The chitosanase was significantly inhibited by Cu2+, Ba2+, Zn2+, Fe2+, Mn2+, EDTA and PMSF. The value of Km and Vmax for chitosanase were 0.098 mg/mL and 1.336 U/mL, respectively. 
A combination of the HPLC and MALDI-TOF MS results showed that the chitosan oligosaccharides obtained from the hydrolysis of water-soluble chitosan by TKU038 comprise oligomers with various degrees of polymerization (DP), ranging from 3 to 9. The TKU038 culture supernatant and COS mixture exhibited 2,2-diphenyl-1-picrylhydrazyl (DPPH·) scavenging activities. The COS with high DP exhibited enhanced DPPH· radical scavenging compared with COS with low DP. The COS also had anti-inflammatory activity and enhanced the growth of L. paracasei TKU010 and L. paracasei TKU012. TKU038 has potential applications in SPP waste treatment and industries for COS production as a medical prebiotic.

目錄

授權書
簽名頁
誌謝
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........................... 3
  2.1 Bacillus mycoides之簡介........................... 3
  2.2幾丁質 (Chitin) 與幾丁聚醣 (Chitosan)........................... 3
    2.2.1幾丁質與幾丁聚醣之結構...........................	3
    2.2.2幾丁質與幾丁聚醣之製備...........................	6
  2.3幾丁質與幾丁聚醣之應用........................... 8
  2.4幾丁聚醣酶 (Chitosanase)........................... 10
    2.4.1 還原醣測定法........................... 12
  2.5 N-乙醯幾丁寡醣及幾丁寡醣...........................	12
  2.6 抗氧化........................... 16
    2.6.1 DPPH自由基清除能力........................... 18
第三章 材料與方法........................... 19
  3.1實驗菌株........................... 19
  3.2實驗材料........................... 19
  3.3實驗儀器........................... 20
  3.4菌株之篩選、分離、保存........................... 20
  3.5菌株之鑑定........................... 21
    3.5.1革蘭氏染色 (Gram staining)........................... 21
    3.5.2革蘭氏陽性菌DNA之萃取........................... 22
    3.5.3 16S rDNA 之定序分析........................... 23
    3.5.4 API (Analytical Profile Index) 之鑑定........................... 24
  3.6幾丁聚醣酶活性測定........................... 24
  3.7幾丁聚醣酶最適條件探討........................... 25
3.7.1 碳/氮源之選擇........................... 25
    3.7.2不同碳源濃度...........................	25
    3.7.3不同培養體積...........................	26
    3.7.4通氣量之探討...........................	26
    3.7.5不同溫度........................... 26
  3.8 幾丁聚醣酶之分離與純化........................... 27
    3.8.1粗酵素液之製備........................... 27
    3.8.2陰離子交換樹脂層析(一)........................... 27
    3.8.3陰離子交換樹脂層析(二)...........................  28
  3.9 幾丁聚醣酶分子量測定與鑑定........................... 28
    3.9.1蛋白質電泳分析 (SDS-PAGE)........................... 28
    3.9.2幾丁聚醣酶胜肽譜鑑定........................... 29
  3.10 幾丁聚醣酶之特性分析........................... 29
    3.10.1酵素最適反應溫度........................... 29
    3.10.2酵素熱安定性........................... 30
    3.10.3酵素最適pH值........................... 30
    3.10.4酵素pH安定性........................... 30
    3.10.5金屬離子與化學藥品對酵素活性作用之影響........................... 31
    3.10.6界面活性劑對酵素活性作用之影響........................... 31
    3.10.7酵素之基質特異性...........................	32
    3.10.8酵素動力學 (Enzyme kinetic)........................... 32
  3.11 幾丁聚醣酶水解基質及幾丁寡醣分析........................... 33
    3.11.1基質水解........................... 33
    3.11.2還原醣量之測定........................... 33
    3.11.3總醣量之測定........................... 34
    3.11.4幾丁寡醣之製備........................... 34
    3.11.5 HPLC進行幾丁寡醣組成分析........................... 34
    3.11.6 MALDI-TOF-MS進行幾丁寡醣組成分析........................... 35
  3.12抗氧化活性測定...........................	35
    3.12.1 DPPH自由基清除能力之測定........................... 35
  3.13抗菌之測試........................... 36
  3.14抗黴菌之測試........................... 37
  3.15促進乳酸菌之生長........................... 37
第四章 結果與討論........................... 39
  4.1幾丁聚醣酶生產菌株之篩選與鑑定........................... 39
  4.2幾丁聚醣酶較適生產條件探討........................... 43
4.2.1碳/氮源之選擇........................... 43
    4.2.2不同碳源濃度........................... 44
    4.2.3不同培養體積...........................	45
    4.2.4通氣量之探討...........................	46
    4.2.5不同溫度........................... 47
    4.2.6較適培養條件綜合結果與其他文獻比較........................... 48
4.3 幾丁聚醣酶之分離純化........................... 53
    4.3.1 粗酵素液之製備........................... 54
    4.3.2 陰離子交換樹脂層析 (一)...........................54
    4.3.3 陰離子交換樹脂層析 (二)........................... 56
    4.3.4 純化與分離之綜合結果........................... 58
  4.4 幾丁聚醣酶分子量之測定與鑑定........................... 59
    4.4.1 蛋白質電泳分析 (SDS-PAGE)........................... 59
    4.4.2 幾丁聚醣酶胜肽質譜鑑定........................... 62
  4.5 幾丁聚醣酶之特性分析........................... 63
    4.5.1酵素最適反應溫度及熱安定性........................... 63
    4.5.2酵素最適pH及pH安定性........................... 65
    4.5.3金屬離子與化學藥品對酵素活性作用之影響........................... 67
    4.5.4界面活性劑對酵素活性作用之影響........................... 69
    4.5.5酵素之基質特異性........................... 71
    4.5.6酵素動力學 (Enzyme kinetic)........................... 74
  4.6 幾丁聚醣酶水解基質及幾丁寡醣分析........................... 79
    4.6.1還原醣及總醣含量之分析........................... 79
    4.6.2 HPLC分析幾丁寡醣........................... 82
    4.6.3 MALDI-TOF-MS進行幾丁寡醣組成分析........................... 89
  4.7 抗氧化活性測定........................... 96
    4.7.1 DPPH自由基清除能力之測定...........................	96
  4.8抗腫瘤之測試........................... 100
  4.9抗發炎之測試........................... 104
  4.10抗菌之測試........................... 106
  4.11抗黴菌之測試........................... 109
  4.12促進乳酸菌之生長........................... 113
第五章 結論........................... 116
第六章 參考文獻........................... 117
附錄........................... 130
 
圖目錄

圖2.1纖維素、幾丁質與幾丁聚醣之結構...........................	5
圖2.2幾丁質製備之流程........................... 7
圖2.3幾丁聚醣酶的種類和切割位置...........................	11
圖2.4幾丁聚醣酶的作用模式...........................	12
圖2.6幾丁聚醣合成幾丁寡醣...........................	14
圖4.1 Bacillus mycoides TKU038之顯微鏡照片...........................	40
圖4.2 Bacillus mycoides TKU038所生產之DNA片段...........................	41
圖4.3 Bacillus mycoides TKU038之16S rDNA部分鹼基序列.......................	42
圖4.4不同碳/氮源對B. mycoides TKU038幾丁聚醣酶生產之影響.........................	43
圖4.5 SPP濃度對B. mycoides TKU038幾丁聚醣酶生產之影響...........................	44
圖4.6培養體積對B. mycoides TKU038幾丁聚醣酶生產之影響.........................	45
圖4.7通氣量對B. mycoides TKU038幾丁聚醣酶生產之影響...........................	46
圖4.8溫度對B. mycoides TKU038幾丁聚醣酶生產之影響...........................	47
圖4.9幾丁聚醣酶之純化流程圖...........................	53
圖4.10 B.mycoides TKU038幾丁聚醣酶之DEAE-Sepharose CL-6B層析圖譜...........................	55
圖4.11 B.mycoides TKU038幾丁聚醣酶之Macro-PrepRDEAECartridge層析圖譜...........................	57
圖4.12 B. mycoides TKU038幾丁聚醣酶之純化SDS-PAGE之分子量分析...........................	61
圖4.13幾丁聚醣酶之最適反應溫度及熱安定性...........................	64
圖4.14幾丁聚醣酶之最適反應pH及pH安定性...........................	66
圖4.15 (A)幾丁聚醣酶水解反應之Michaelis-Menten圖型 (B)幾丁聚醣酶動力參數之Lineweaver-Burk圖型...........................	75
圖4.16水溶性幾丁聚醣經B.mycoides TKU038所生產之粗酵素液水解不同時間所得還原醣及總醣含量...........................	80
圖4.17蝦殼粉經B.mycoides TKU038所生產之粗酵素液水解不同時間所得還原醣及總醣含量...........................	81
圖4.18 WSC經B.mycoides TKU038粗酵素液水解不同時間所得幾丁寡醣之HPLC分析圖 (a) 標準品 (b) 水解8小時。...........................	84
圖4.19 WSC經B.mycoides TKU038粗酵素液水解不同時間所得幾丁寡醣之HPLC分析圖 (a) 水解1天 (b) 水解2天。...........................	85
圖4.20 WSC經B.mycoides TKU038粗酵素液水解不同時間所得幾丁寡醣之HPLC分析圖 (a) 水解3天 (b) 水解4天。...........................	86
圖4.21 WSC經B.mycoides TKU038粗酵素液水解不同時間所得幾丁寡醣之HPLC分析圖 (a) 水解5天 (b) 水解6天。...........................	87
圖4.22 WSC經B.mycoides TKU038粗酵素液水解不同時間所得不同聚合度之幾丁寡醣之HPLC分析圖 (a) DP<8 (b) 8<DP<16。...........................	88
圖4.23水溶性幾丁聚醣經B.mycoides TKU038粗酵素液水解8小時及1天所得之MALDI-TOF-MS之分析圖...........................	91
圖4.24水溶性幾丁聚醣經B.mycoides TKU038粗酵素液水解2天及3天所得之MALDI-TOF-MS之分析圖...........................	92
圖4.25水溶性幾丁聚醣經B.mycoides TKU038粗酵素液水解4天及5天所得之MALDI-TOF-MS之分析圖...........................	93
圖4.26水溶性幾丁聚醣經B.mycoides TKU038粗酵素液水解6天所得之MALDI-TOF-MS之分析圖...........................	94
圖4.27發酵上清液及水解上清液之DPPH自由基清除活性...........................	98
圖4.28幾丁寡醣之DPPH自由基清除活性...........................	99
圖4.29幾丁寡醣對RAW 264.7抗發炎之影響...........................	105
圖4.30上清液、DP<8、8< DP<16及WSC對Aspergillus fumigates生長影響.......................	111
圖4.31上清液、DP<8、8< DP<16及WSC對Fusarium oxysporum
生長影響...........................	112
圖4.32 B.mycoides TKU038所生產之幾丁寡醣對於Lactobacillus paracasei TKU010生長影響...........................	114
圖4.33 B.mycoides TKU038所生產之幾丁寡醣對於Lactobacillus paracasei TKU012生長影響...........................115

表目錄

表2.1幾丁質與幾丁聚醣之應用...........................	9
表2.2幾丁寡醣之製備...........................	15
表2.3幾丁寡醣之應用...........................	16
表3.1緩衝溶液之種類...........................	31
表3.2 DNS試劑之組成...........................	33
表4.1 API鑑定分析結果...........................	42
表4.2 B. mycoides TKU038所生產幾丁聚醣酶活性之較適條件.......................	48
表4.3 以含幾丁質水產廢棄物為唯一碳/氮源之幾丁聚醣酶生產菌之  較適培養條件比較...........................	49
表4.4 B. mycoides TKU038幾丁聚醣酶之純化總表...........................	58
表4.5 B. mycoides TKU038幾丁聚醣酶胜肽質譜鑑定結果...........................	62
表4.6金屬離子與化學藥品對B. mycoides TKU038所生產之幾丁聚醣酶之影響.......................	68
表4.7界面活性劑對B. mycoides TKU038所生產之幾丁聚醣酶之影響.......................	70
表4.8 B. mycoides TKU038所生產之幾丁聚醣酶基質特異性........................	72
表4.9基質特異性之比較...........................	73
表4.10微生物來源之幾丁聚醣酶之特性比較...........................	76
表4.11水溶性幾丁聚醣水解不同時間對幾丁寡醣含量之影響...........................	83
表4.12水溶性幾丁聚醣經B.mycoides TKU038粗酵素液水解不同時間之MALDI-TOF-MS幾丁寡醣組成分析...........................	95
表4.13利用MTT測試對Hep G2、HEp-2、WiDr 及 A549 細胞增生的影響.........................	102
表4.14 COS對不同腫瘤之生長比較...........................	103
表4.15幾丁寡醣對RAW 264.7抗發炎之影響...........................	105
表4.16上清液、DP<8、8< DP<16及WSC對Staphylococcus aureus、Bacillus subtilis TKU007、Escherichia coli 及 Pseudomonas aeruginosa K187生長影響...........................	108
表4.17上清液、DP<8、8< DP<16及WSC對Aspergillus fumigates及Fusarium oxysporum生長影響...........................	110

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