菌株Bacillus cereus TKU034係篩選自新北市三芝山區土壤之幾丁聚醣酶及蛋白酶生產菌，以含有1%烏賊軟骨粉、0.1% K2HPO4、0.05% MgSO4．7H2O之100mL液態培養基，於37°C中搖瓶(150 rpm)培養4天可得較高幾丁聚醣酶活性(0.68U)。
    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-PrepR DEAE Cartridge陰離子交換層析等步驟，以高純化度(441倍)以及高活性回收率(50.9%)，純化出經SDS PAGE電泳測得分子量約為43 kDa之一種幾丁聚醣酶。此幾丁聚醣酶的最適反應pH為7、pH安定性為5~7.5 、最適反應溫度為50°C、於50°C以下皆具熱安定性，其活性會受到Fe2+、Ca2+、Cu2+、Zn2+、Mn2+及EDTA所抑制。
    TKU034之發酵上清液或水解WSC之水解上清液具有DPPH自由基清除能力及亞鐵離子螯合能力，且其發酵上清液對格蘭氏陽性菌S. aureus及B. subtilis之生長具有抑制之效果。色素吸附方面，烏賊軟骨粉經過TKU034發酵後對於食用色素之吸附效果提高10%左右，對於分散染料則提高約40%，且吸附後之染料不易再被脫附之性質對於汙水處理方面有著潛力。

The chitosanase and protease producing strain, TKU034, was isolated from the soil in Sanzhi, Taiwan with squid pen powder (spp) as the sole carbon/nitrogen source and identified as Bacillus cereus.The optimized culture conditions for chitosanase production by B. cereus TKU034 was found when the organism was cultured at 37°C with 150 rpm for 4 days in 100 mL of medium containing 1% SPP、0.1% K2HPO4 and 0.05%  MgSO4．7H2O.
    The chitosanase (CHS) with 441-fold of purifying rate and 50.9% of activity yield was purified from the culture supernatant of B. cereus TKU034 by ammonium sulfate precipitation, DEAE-Sepharose and Macro-PrepR DEAE Cartridge chromatography. The molecular mass of CHS determined by SDS-PAGE was approximately to be 43 kDa. The optimum pH, pH stability , optimum temperature and thermal stability of CHS was pH 7, pH 5-7.5, 50°C, <50°C. The CHS was inhibited by Fe2+、Ca2+、Cu2+、Zn2+、Mn2+ and EDTA.
    The antioxidation activities of culture supernatant of B. cereus TKU034 and hydrolyzates of water soluble chitosan were measured with DPPH radical scavenging capacity and ferrous ion chelating ability.The culture supernatant has inhibitory effect against S. aureus and B. subtilis TKU007.The pigments adsorption capacity of fermented SPP was better than that of unfermented SPP.

目錄

中文摘要..................................................Ⅰ
中文摘要..................................................Ⅱ
目錄......................................................Ⅲ
圖目錄....................................................Ⅷ
表目錄....................................................Ⅺ

第一章 緒論................................................1
第二章 文獻回顧............................................3
  2.1實驗菌種Bacillus cereus之簡介.........................3
  2.2幾丁質及幾丁聚醣......................................4
  2.3幾丁質酶及幾丁聚醣酶..................................9
  2.4 N-乙醯幾丁寡醣及幾丁寡醣............................10
  2.5蛋白酶...............................................11
  2.6幾丁類物質吸附色素...................................11
第三章 材料與方法.........................................13
  3.1 實驗菌株............................................13
  3.2實驗材料.............................................13
  3.3實驗儀器.............................................15
  3.4實驗菌株之篩選.......................................16
  3.5實驗菌株之生長曲線測定...............................16
  3.6酵素活性之測定.......................................17
    3.6.1幾丁聚醣酶之活性測定	............................17
    3.6.2懸浮態幾丁質之製備...............................17
    3.6.3幾丁質酶之活性測定...............................18
    3.6.4幾丁藍之活性測定.................................18
    3.6.5蛋白酶之活性測定.................................19
  3.7 幾丁聚醣酶及蛋白酶較適生產條件探討..................19
3.7.1 培養體積............................................19
    3.7.2 碳/氮源種類及濃度...............................20
  3.8 幾丁聚醣酶之分離純化................................20
    3.8.1 粗酵素液之製備..................................20
    3.8.2 陰離子交換層析(一)..............................20
    3.8.3 陰離子交換層析(二) .............................21
  3.9 蛋白質電泳分析......................................21
  3.10 酵素之特性分析.....................................22
    3.10.1 酵素最適反應溫度...............................22
    3.10.2 酵素熱安定性...................................22
    3.10.3 酵素最適反應pH.................................23
    3.10.4 酵素pH安定性...................................23
    3.10.5 金屬離子及化學藥品對酵素活性之影響.............23
    3.10.6 界面活性劑對酵素活性之影響.....................24
    3.10.7 酵素之基質特異性...............................24
  3.11 幾丁聚醣酶水解基質及寡醣分析.......................25
    3.11.1 基質水解.......................................25
    3.11.2 還原糖量之測定.................................25
    3.11.3 總糖量之測定...................................26
    3.11.4 N-乙醯幾丁寡醣製備.............................26
    3.11.5幾丁寡醣之組成分析..............................26
    3.11.6 MALDI-TOF-MS分析寡醣...........................27
  3.12 抗氧化活性測定.....................................27
    3.12.1 DPPH自由基清除能力之測定.......................27
    3.12.2 還原力之測定...................................28
    3.12.3 酚類化合物之定量分析...........................29
    3.12.4鐵離子螯合能力之測定............................29
  3.13 抗菌試驗...........................................30
  3.14 幾丁類物質對色素之影響.............................30
    3.14.1 色素吸附.......................................30
    3.14.2 色素脫附.......................................31
第四章 結果與討論.........................................32
  4.1幾丁聚醣酶與蛋白酶生產菌之篩選與鑑定.................32
  4.2 實驗菌株之生長曲線測定..............................34
  4.3酵素較適生產條件探討.................................35
    4.3.1 較適培養液體積..................................35
    4.3.2 較適碳/氮源種類及濃度...........................35
    4.3.3較適培養條件結果與比較...........................38
  4.4 幾丁聚醣酶之分離純化................................42
    4.4.1 粗酵素液之製備..................................42
    4.4.2離子交換樹脂層析(一).............................44
    4.4.3離子交換樹脂層析(二).............................46
    4.4.4分離純化之綜合結果...............................48
  4.5 幾丁聚醣酶分子量之測定與鑑定........................53
    4.5.1 SDS-PAGE........................................53
    4.5.2 幾丁聚醣酶胜肽質譜鑑定..........................53
  4.6 幾丁聚醣酶之特性分析................................56
    4.6.1 最適反應溫度及熱安定性..........................56
    4.6.2 最適pH及pH安定性................................57
    4.6.3 金屬離子及化學藥品對幾丁聚醣酶活性之影響........59
    4.6.4 界面活性劑對幾丁聚醣酶活性之影響................59
    4.6.5 幾丁聚醣酶之基質特異性..........................62
    4.6.6基質特異性之比較.................................62
    4.6.7綜合比較.........................................62
  4.7 水解基質之探討......................................70
    4.7.1 還原糖及總糖含量之分析..........................70
    4.7.2 HPLC進行幾丁寡醣之組成分析......................72
    4.7.3 MALDI-TOF-MS進行幾丁寡醣之組成分析..............75
  4.8 抗氧化活性測定......................................81
    4.8.1 DPPH自由基清除能力之測定........................81
    4.8.2還原力之測定.....................................82
    4.8.3 酚類化合物之定量分析............................83
    4.8.4 鐵離子螯合能力之測定............................84
  4.9 抗菌試驗............................................85
  4.10幾丁類物質對色素之影響..............................87
    4.10.1 色素吸附.......................................87
    4.10.2 色素脫附.......................................90
第五章 結論...............................................93
第六章 參考文獻...........................................95


圖目錄

圖2.1 α-chitin與β-chitin 之結構..........................5
圖2.2 幾丁質、幾丁聚醣及纖維素之結構.......................7
圖4.1 Bacillus cereus TKU034之顯微鏡照片..................32
圖4.2 B. cereus TKU034之16S rDNA部分鹼基序列及API試驗分析結果........................................................33
圖4.3 B. cereus TKU034之生長曲線及幾丁聚醣酶活性..........34
圖4.4 培養液體積對B. cereus TKU034生產幾丁聚醣酶及蛋白酶之影響........................................................36
圖4.5 不同碳/氮源對B. cereus TKU034生產幾丁聚醣酶與蛋白酶之影響......................................................37
圖4.6 B. cereus TKU034所生產酵素之純化流程圖..............43
圖4.7 B. cereus TKU034 幾丁聚醣酶之DEAE-Sepharose CL-6B層析圖譜......................................................45
圖4.8 B. cereus TKU034 幾丁聚醣酶之Macro-PrepR DEAE Cartridge層析圖譜.........................................47
圖4.9 B. cereus TKU034幾丁聚醣酶之純化SDS-PAGE圖..........54
圖4.10 幾丁聚醣酶之最適反應溫度及熱安定性.................56
圖4.11 幾丁聚醣酶之最適pH值(A)及pH安定性(B)...............58
圖4.12水溶性幾丁聚醣經B. cereus TKU034 粗酵素液水解不同時間所得總糖及還原醣含量......................................71
圖4.13 WSC經B. cereus TKU034粗酵素液水解不同時間所得幾丁聚寡醣之HPLC分析圖............................................73
圖4.14 WSC經B. cereus TKU034粗酵素液水解不同時間所得幾丁聚寡醣之HPLC分析圖............................................74
圖4.15水溶性幾丁聚醣經B. cereus TKU034粗酵素液水解不同時間之MALDI-TOF-MS幾丁寡醣組成分析圖............................76
圖4.16 水溶性幾丁聚醣經B. cereus TKU034粗酵素液水解不同時間之MALDI-TOF-MS幾丁寡醣組成分析圖..........................77
圖4.17水溶性幾丁聚醣經B. cereus TKU034粗酵素液水解不同時間之MALDI-TOF-MS幾丁寡醣組成分析圖............................78
圖4.18 水溶性幾丁聚醣經B. cereus TKU034粗酵素液水解不同時間之MALDI-TOF-MS幾丁寡醣組成分析圖..........................79
圖4.19發酵上清液及水解上清液之DPPH自由基清除能力..........81
圖4.20發酵上清液及水解上清液之還原力測定..................82
圖4.21發酵上清液及水解上清液之酚類化合物定量分析..........83
圖4.22發酵上清液及水解上清液之鐵離子螯合能力..............84
圖4.23發酵及未發酵烏賊軟骨粉對於食用色素之吸附率..........88
圖4.24發酵及未發酵烏賊軟骨粉對於分散染料之吸附率..........89
圖4.25以不同溶液對SPP粉脫附食用色素R40效果之比較..........90
圖4.26發酵及未發酵烏賊軟骨粉對於分散染料之脫附率..........91
圖4.27發酵及未發酵烏賊軟骨粉對於分散染料之脫附率..........92

 
表目錄

表2.1蝦蟹殼與烏賊軟骨粉組成成份比較........................6
表2.2 幾丁質與幾丁聚醣之應用...............................8
表3.1 DNS試劑組成.........................................25
表4.1 B. cereus TKU034 酵素生產之較適培養條件.............38
表4.2以含幾丁質水產廢棄物為唯一碳/氮源之幾丁聚醣酶/幾丁質酶/蛋白酶酶生產菌之較適培養條件比較..........................39
表4.3上清液及硫酸銨沉澱後之幾丁聚醣酶及蛋白酶之活性比較...42
表4.4 B. cereus TKU034幾丁聚醣酶之純化總表................49
表4.5 B. cereus TKU034與其它幾丁聚醣酶生產菌純化總表之比較........................................................50
表4.6 B. cereus TKU034幾丁聚醣酶胜肽質譜鑑定結果..........55
表4.7金屬離子及化學品對B. cereus TKU034所產之幾丁聚醣酶之影響........................................................60
表4.8界面活性劑對B. cereus TKU034所產之幾丁聚醣酶之影響...61
表4.9 B. cereus TKU034所產之幾丁聚醣酶之基質特異性........63
表4.10基質特異性之比較....................................64
表4.11 微生物來源之幾丁聚醣酶、幾丁質酶及蛋白酶之特性比較.65
表4.12水溶性幾丁聚醣經B. cereus TKU034粗酵素液水解不同時間之MALDI-TOF-MS 幾丁寡醣組成分析.............................80
表4.13發酵上清液對Staphylococcus aureus、Bacillus subtilis TKU007、Escherichia. coli及Pseudomonas aeruginosa K187生長之影響......................................................86

黃稚婷 (2014) Bacillus cereus TKU033 所生產幾丁聚醣酶之純化及其應用。私立淡江大學化學所碩士論文。第51-62頁。

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