實驗菌株TKU039 係篩選自新北市林口山區紅土之幾丁聚醣酶生產菌，屬於格蘭氏陽性，蕈狀孢芽桿菌，經由16S rDNA 部分鹼基序列分析與NCBI/BLAST資料庫比對，得知此菌株最接近Bacillus 屬，經由API鑑定系統判定為Bacillus mycoides，鑑定率為88.5 %。以含有1%烏賊軟骨粉、0.1% K2HPO4、0.05% MgSO4 ·7H2O之150 mL液態培養基，於37°C中搖瓶(150 rpm)培養5天可得較高幾丁聚醣酶活性(0.65U)。
依較佳培養條件培養所得之發酵液，經離心、硫酸銨沉澱、DEAE-Sepharose及Macro-Prep® DEAE Cartridge陰離子交換層析等步驟，純化度(61.7倍)以及活性回收率(4.2%)，純化後經SDS PAGE電泳測得分子量約為41 kDa之幾丁聚醣酶。此幾丁聚醣酶的最適反應pH為6、pH安定性為pH4~pH10 、最適反應溫度為50°C、於50°C以下皆具熱安定性。
此幾丁聚醣酶水解WSC後之寡醣經過MALDI-TOF-MS分析得知其組成多為3~6醣，並以6醣含量最多。

Chitosan has been more popular in recent years due to its good bio-compatibility, low toxicity and rarely no allergic reaction to human body. Chitosan also has other bioactivity like reduce blood pressure, lower cholesterol, increase immunity, and its antibacterial property can be used in many clinical applications. For the best resource of Chitin/Chitosan we extracted from the seafood wastes (squid pens, shrimp or crab shells),which is abundant of polysaccharides, and glucosamine that we can use enzyme hydrolysis to achieve it. In traditional way, using a lot of chemical to receive chitosan and its oligomer, but in nowadays, we use the natural way, chitosanase bacteria strain TKU039 identified as Bacillus sp. from the soil in New Taipei City Linkou mountain. TKU039 was culture in medium with the squid pen powder (SPP) and shrimp powder (SP).The SPP medium was found most suitable with 1.0% SPP as a sole carbon/nitrogen source optimized under cultured at 37°C with 150 rpm for 5 days in 150 mL of medium containing 1% SPP, 0.1% K2HPO4 and 0.05 % MgSO4·7H2O.

目錄

中文摘要	Ⅰ
中文摘要	Ⅱ
目錄	Ⅲ
圖目錄	Ⅶ
表目錄	Ⅸ
第一章 緒論	1
第二章 文獻回顧	2
  2.1實驗菌種Bacillus mycoides之簡介	2
  2.2幾丁質及幾丁聚醣	2
  2.3幾丁質酶及幾丁聚醣酶	4
  2.4 N-乙醯幾丁寡醣及幾丁寡醣	6 
第三章 材料與方法	7
  3.1 實驗菌株	7
  3.2實驗材料	7
  3.3實驗儀器	8
  3.4實驗菌株之篩選	9  
  3.5幾丁聚醣酶之活性測定	9   
  3.6 幾丁聚醣酶較適生產條件探討	10
3.6.1碳/氮源種類	10
    3.6.2培養濃度	10
    3.6.3培養體積	10
    3.6.4培養溫度	10
  3.7 幾丁聚醣酶之分離純化	11
    3.7.1 粗酵素液之製備	11
    3.7.2 陰離子交換層析	11
  3.8 蛋白質電泳分析	12
    3.8.1 蛋白質電泳分析	12
    3.8.2 幾丁聚醣酶胜肽質譜鑑定	12
  3.9 酵素之特性分析	13
    3.9.1 酵素最適反應溫度	13
    3.9.2 酵素熱安定性	13
    3.9.3 酵素最適反應pH	13
    3.9.4 酵素pH安定性	14
    3.9.5 介面活性劑對酵素活性之影響	14
    3.9.6 酵素之基質特異性	14
  3.10 幾丁聚醣酶水解基質及寡醣分析	15
    3.10.1 基質水解	15
    3.10.2 還原糖量之測定	15
    3.10.3 總糖量之測定	16
    3.10.4 N-乙醯幾丁寡醣製備	16
    3.10.5幾丁寡醣之組成分析	16
    3.10.6 MALDI-TOF-MS分析寡醣	17 
第四章 結果與討論	18
  4.1幾丁聚醣酶生產菌之篩選與鑑定	18
  4.2酵素較適生產條件探討	21
    4.2.1 較適碳/氮源種類	21
    4.2.2 較適培養液濃度	21
    4.2.3 較適培養液體積	21
    4.2.4 較適培養液溫度	22
    4.2.5 較適培養條件結果與比較	24
  4.3 幾丁聚醣酶之分離純化	27
    4.3.1 粗酵素液之製備	28
    4.3.2離子交換樹脂層析(Ⅰ)	28
    4.3.3離子交換樹脂層析(Ⅱ)	29
    4.3.4分離純化之綜合結果	31
  4.4 幾丁聚醣酶分子量之測定與鑑定	32
    4.4.1 SDS-PAGE	32
    4.4.2 幾丁聚醣酶胜肽質譜鑑定	32
  4.5 幾丁聚醣酶之特性分析	35
    4.5.1 最適反應溫度及熱安定性	35
    4.5.2 最適pH及pH安定性	37
    4.5.3 介面活性劑對幾丁聚醣酶活性之影響	37
    4.5.4 幾丁聚醣酶之基質特異性	39
    4.5.5 基質特異性之比較	40
    4.5.6 綜合比較	40
  4.6 水解基質之探討	43
    4.6.1 還原糖及總糖含量之分析	43
    4.6.2 HPLC進行幾丁寡醣之組成分析	43 
    4.6.3 MALDI-TOF-MS進行幾丁寡醣之組成分析	49 
第五章 結論	54
第六章 參考文獻	55

圖目錄

圖2.1 幾丁質跟幾丁聚醣之結構	3
圖2.2 結構圖(a)幾丁聚醣 (b)幾丁質中GlcNAc和GlcN分布 (c)部分去乙醯化幾丁聚醣 (d)全部去乙醯化幾丁聚醣	4
圖4.1 Bacillus mycoides TKU039之顯微鏡照片	18
圖4.2 B. mycoides TKU039之16S rDNA部分鹼基序列及API試驗分析結果	19
圖4.3 不同碳/氮源對B. mycoides TKU039生產幾丁聚醣酶影響	21
圖4.4 不同濃度對B. mycoides TKU039生產幾丁聚醣酶影響	22
圖4.5 不同體積對B. mycoides TKU039生產幾丁聚醣酶影響	23
圖4.6 不同溫度對B. mycoides TKU039生產幾丁聚醣酶影響	24
圖4.7 B. mycoides TKU039所生產酵素之純化流程圖	27
圖4.8 B. mycoides TKU039 幾丁聚醣酶之DEAE-Sepharose CL-6B層析圖譜	29
圖4.9 B. mycoides TKU039 幾丁聚醣酶之Macro-Prep® DEAE Cartridge層析圖譜	30
圖4.10 B. mycoides TKU039幾丁聚醣酶之純化SDS-PAGE圖	33
圖4.11 幾丁聚醣酶之最適反應溫度及熱安定性	36
圖4.12 幾丁聚醣酶之最適反應pH值及pH安定性	37
圖4.13水溶性幾丁聚醣經B. mycoides TKU039 粗酵素液水解不同時間所得總糖及還原醣含量	43
圖4.14 WSC經B.mycoides TKU039粗酵素液水解不同時間所得幾丁聚醣酶之HPLC分析圖(a)標準品(b)水解8小時	45
圖4.15 WSC經B.mycoides TKU039粗酵素液水解不同時間所得幾丁聚醣酶之HPLC分析圖(a) 水解一天(b)水解兩天	46
圖4.16 WSC經B.mycoides TKU039粗酵素液水解不同時間所得幾丁聚醣酶之HPLC分析圖(a)水解三天(b)水解四天	47
圖4.17 WSC經B.mycoides TKU039粗酵素液水解不同時間所得幾丁聚醣酶之HPLC分析圖-水解五天	48
圖4.18水溶性幾丁聚醣經B. mycoides TKU039粗酵素液水解不同時間之MALDI-TOF-MS幾丁寡醣組成分析圖	50
圖4.19水溶性幾丁聚醣經B. mycoides TKU039粗酵素液水解不同時間之MALDI-TOF-MS幾丁寡醣組成分析圖	51
圖4.20水溶性幾丁聚醣經B. mycoides TKU039粗酵素液水解不同時間之MALDI-TOF-MS幾丁寡醣組成分析圖	52


表目錄

表2.1 不同微生物之幾丁聚醣特性5 
表3.1 DNS試劑組成15 
表4.1 API鑑定分析結果19 
表4.2 B. mycoides TKU038 (左) & B. mycoides TKU039 (右) API鑑定分析結果比較20 
表4.3 B. mycoides TKU039酵素較適生產條件25 
表4.4 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26 
表4.5 B. mycoides TKU039幾丁聚醣酶之純化總表31 
表4.6 B. mycoides TKU039幾丁聚醣酶胜肽質譜鑑定結果34 
表4.7界面活性劑對B. mycoides TKU039所產幾丁聚醣酶影響38 
表4.8 B. mycoides TKU039所產之幾丁聚醣酶之基質特異性39 
表4.9基質特異性之比較41
表4.10 微生物來源之幾丁聚醣酶之特性比較42 
表4.11 水溶性幾丁聚醣經B. mycoides TKU039粗酵素液水解不同時間之MALDI-TOF-MS 幾丁寡醣組成分析53

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