TKU024，係以烏賊軟骨粉末為唯一碳/氮源，篩選自台灣中部土壤之一株幾丁聚醣酶生產菌，經鑑定為Acinetobacter calcoaceticus。TKU024 幾丁聚醣酶較適生產條件為1.5% 烏賊軟骨粉末、0.1% K2HPO4、0.05% MgSO4．7H2O之液態培養基(pH 8)於37℃振盪培養4 天。將發酵所得上清液離心，經硫酸銨沉澱、DEAE-Sepharose、Mcaro-Prap DEAE 及Phenyl Sepharose 管柱層析後，可純化出兩個幾丁聚醣酶(C1/C2)，經SDS-PAGE 測得分子量分別為27 kDa及66 kDa。經胜肽質譜鑑定，C1 分別與Cellvibrio japonicus Ueda107 之幾丁質酶(GenBank 編號為gi1192361966)、Pseudomonas aeruginosa PA01 之琥珀脫氫酶鐵硫亞基(GenBank 編號為gi115596781)相似，相似度分別為4%及10%；C2與 Arthrobacter sp. 之幾丁質酶(GenBank 編號為gi127065204)相似，相似度為7%。於最適反應pH、最適反應溫度、pH 安定性、熱安定性方面，C1為pH 6、50℃、pH 8-9 及＜70℃，C2 為pH 7、60℃、pH 6 及＜60℃。C1 活性受5 mM Cu2+、Zn2+、Mn2+及2% (v/v) SDS 抑制，且於4~5 mM Mn2+完全抑制；C2 活性受5 mM Ca2+、Urea、EDTA、Zn2+、Mn2+抑制，但在1 mM Mn2+則有少許促進效果，於2% (v/v) SDS、Triton X-100、Tween 20 和0.5% (v/v)Tween 20 有輕微抑制效果。

Strain TKU024 was isolated from the soil in the middle Taiwan by using squid pen powder(SPP) as the sole carbon/nitrogen source and identified as Acinetobacter calcoaceticus. The optimized culture condition for chitosanase production was composed of 1.5% squid pen power(SPP), 0.1% K2HPO4 and 0.05% MgSO4．7H2O at pH 8 and incubated in 50 mL of liquid media in shaking flasks at 37℃ for 4 days.
Two chitosanases(C1/C2) were purified from the culture supernatant by using ammonium sulfate precipitation, chromatography procedures of DEAE-Sepharose, Macro-Prep DEAE, and Phenyl Sepharose. The molecular mass of C1 and C2 determined by SDS-PAGE were approximately 27 kDa and 66 kDa, respectively. The results of peptide mass mapping
indicated that C1 matched to chitinase from Cellvibrio japonicus Ueda107(GenBank accession number gi1192361966) with 4% sequence coverage and Pseudomonas aeruginosa PA01(GenBank accession number gi115596781) with 10% sequence coverage, and C2 matched to chitinase from Arthrobacter sp.(GenBank accession number gi127065204) with 7% sequence coverage. The optimum temperature, optimum pH, thermal stability, and pH stability of TKU024 chitosanase C1 and C2 were 50℃, pH 6,＜70℃, pH 8~9 and 60℃, pH 7,＜60℃, pH 6, respectively. C1 was inhibited by 5 mM Cu2+, Zn2+, Mn2+ and 2% (v/v) SDS, and was inhibited completely by 4~5 mM Mn2+ ; C2 was inhibited by 5 mM Ca2+, Urea, EDTA, Zn2+, Mn2+, 2% (v/v) SDS, Triton X-100, Tween 20 and 0.5% (v/v) Tween 20, but activated by 1 mM Mn2+.

目錄
頁次
封面內頁
授權書
簽名頁
中文摘要 ............................................................................................... Ⅰ
英文摘要 ............................................................................................... Ⅱ
謝誌…..…………………………………………………………………Ⅲ
目錄 ....................................................................................................... Ⅳ
圖目錄 ................................................................................................... Ⅷ
表目錄 ................................................................................................... Ⅸ
第一章 緒言 ........................................................................................... 1
第二章 文獻回顧 ................................................................................... 2
2.1 不動桿菌(Acinetobacter)簡介 ............................................. 2
2.2 幾丁質、幾丁聚醣之結構與來源 ....................................... 2
2.2.1 幾丁質 ......................................................................... 2
2.2.2 幾丁聚醣 ..................................................................... 3
2.3 幾丁質酶與幾丁聚醣酶 ...................................................... 4
2.3.1 幾丁質酶 ..................................................................... 4
V
2.3.2 幾丁聚醣酶 ................................................................. 5
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 幾丁聚醣酶之活性測定 ................................................... 11
3.6 幾丁聚醣酶較適生產條件探討 ........................................ 12
3.7 酵素之分離純化 ............................................................... 12
3.7.1 粗酵素液之製備 ...................................................... 12
3.7.2 陰離子交換層析 ....................................................... 13
3.7.3 疏水性層析 ............................................................... 13
3.8 蛋白質電泳分析 ................................................................ 14
3.9 酵素之特性分析 ................................................................ 14
3.9.1 幾丁聚醣酶最適反應溫度 ........................................ 14
3.9.2 幾丁聚醣酶熱安定性 ............................................... 15
3.9.3 幾丁聚醣酶最適反應pH .......................................... 15
3.9.4 幾丁聚醣酶pH 安定性............................................. 15
3.9.5 金屬離子及化學藥品對幾丁聚醣酶活性之影響 ..... 16
VI
3.9.6 界面活性劑對幾丁聚醣酶活性之影響 .................... 16
3.9.7 幾丁聚醣酶之基質特異性 ........................................ 17
第四章 結果與討論 ............................................................................. 18
4.1 幾丁聚醣酶生產菌株之篩選 ............................................ 18
4.2 幾丁聚醣酶較適生長條件探討......................................... 18
4.3 幾丁聚醣酶之純化分離 .................................................... 19
4.3.1 粗酵素液製備 ........................................................... 19
4.3.2 陰離子交換層析法 ................................................... 20
4.3.3 疏水性層析法 ........................................................... 20
4.3.4 綜合結果 ................................................................... 21
4.4 幾丁聚醣酶之分子量測定 ................................................ 22
4.4.1 SDS-PAGE ................................................................ 22
4.4.2 幾丁聚醣酶胜肽質譜鑑定 ........................................ 22
4.5 幾丁聚醣酶之特性分析 .................................................... 23
4.5.1 幾丁聚醣酶最適反應溫度及熱安定性 .................... 23
4.5.2 幾丁聚醣酶最適反應pH 及pH 安定性 .................. 24
4.5.3 金屬離子及化學藥品對幾丁聚醣酶活性之影響 ..... 24
4.5.4 界面活性劑對幾丁聚醣酶活性之影響 .................... 25
4.5.5 幾丁聚醣酶之基質特異性 ........................................ 25
第五章 結論 ......................................................................................... 48
VII
參考文獻 ............................................................................................... 49
VIII
圖目錄
頁次
圖2.1 幾丁聚醣、纖維素、幾丁質之化學結構圖 ................................ 6
圖2.2 幾丁質水解酵素水解途徑 .......................................................... 8
圖4.1 菌種鑑定與分析 ........................................................................ 26
圖4.2 SPP 濃度對TKU024 幾丁聚醣酶生產之影響 .......................... 27
圖4.3 培養溫度對TKU024 幾丁聚醣酶生產之影響 ......................... 28
圖4.4 培養液體積對TKU024 幾丁聚醣酶生產之影響 ..................... 29
圖4.5 培養液pH 對TKU024 幾丁聚醣酶生產之影響 ...................... 30
圖4.6 Acinetobacter calcoaceticusTKU024 培養於SPP 培養基所生產幾
丁聚醣酶之生長曲線圖 ........................................................................ 31
圖4.7 Acinetobacter calcoaceticus TKU024 所生產幾丁聚醣酶之純化
分離流程圖 ........................................................................................... 32
圖4.8 TKU024 幾丁聚醣酶之DEAE-Sepharose CL-6B 層析圖譜 ..... 33
圖4.9 TKU024 幾丁聚醣酶之Macro-Prep DEAE Cartridge 層析圖譜 ...
............................................................................................................... 34
圖4.10 TKU024 幾丁聚醣酶之Phenyl Sepharose 層析圖譜............... 35
圖4.11 幾丁聚醣酶(C1, C2)於SDS-PAGE 之分子量分析 ............... 37
圖4.12 幾丁聚醣酶C1 與C2 之(A)最適反應溫度; (B)熱安定性 ..... 43
圖4.13 幾丁聚醣酶C1 與C2 之(A)最適反應pH ; (B)pH 安定性..... 44
IX
表目錄
頁次
表2.1 Acinetobacter sp.其他方面的研究 ............................................... 7
表4.1 Acinetobacter calcoaceticus TKU024 幾丁聚醣酶之純化總表 . 36
表4.2 TKU024 幾丁聚醣酶胜肽質譜鑑定結果 .................................. 38
表4.3 微生物來源之幾丁質酶特性比較 ............................................ 39
表4.4 不動桿菌屬之幾丁聚醣酶特性比較......................................... 41
表4.5 微生物來源之幾丁聚醣酶特性比較......................................... 42
表4.6 金屬離子及化學藥品對幾丁聚醣酶活性之影響 ..................... 45
表4.7 界面活性劑對幾丁聚醣酶活性之影響 ..................................... 46
表4.8 TKU024 幾丁聚醣酶之基質特異性 .......................................... 47

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