淡江大學覺生紀念圖書館 (TKU Library)
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系統識別號 U0002-2307201313332700
中文論文名稱 Bacillus cereus TKU033 所生產幾丁聚醣酶之純化及其應用
英文論文名稱 Purification and characterization of chitosanase from Bacillus cereus TKU033 and it’s application
校院名稱 淡江大學
系所名稱(中) 化學學系碩士班
系所名稱(英) Department of Chemistry
學年度 101
學期 2
出版年 102
研究生中文姓名 黃稚婷
研究生英文姓名 Chih-Ting Huang
學號 600180243
學位類別 碩士
語文別 中文
口試日期 2013-07-23
論文頁數 70頁
口試委員 指導教授-王三郎
委員-王全祿
委員-梁慈雯
委員-王三郎
中文關鍵字 Bacillus cereus  幾丁聚醣酶  烏賊軟骨 
英文關鍵字 Bacillus cereus  chitosanase  squid pen powder 
學科別分類 學科別自然科學化學
中文摘要 菌株 TKU033 係以烏賊軟骨為唯一碳/氮源,篩選至台灣三芝土壤經鑑定為 Bacillus cereus之幾丁聚醣酶生產菌。幾丁聚醣酶之較適培養條件為,以含有1.5% 烏賊軟骨粉、0.1%K2HPO4、0.05%MgSO4.7H2O之50mL液態培養基於 37℃搖瓶(150 rpm)培養2天,所得發酵上清液經硫酸銨沉澱、DEAE-Sepharose 及Macro-Prep DEAE Cartidge離子交換樹脂等步驟,純化出一種幾丁聚醣酶。此幾丁聚醣酶之分子量經SDS-PAGE測為43 kDa ,最適反應pH、最適溫度、pH安定性及熱安定性分別為 pH 5、50℃、pH5-7及<40℃,其活性會受 Cu2+ 、Mn2+ 和EDTA所抑制。
較適培養條件所製得B.cereus TKU033粗酵素液,幾丁聚醣酶水解水溶性幾丁聚醣,所得上清液添加於乳酸菌Lactobacillus paracasei 12193及 Lactobacillus kefri 14011的MRS液態培養基,可分別促進乳酸菌生長達141%及116% 之影響。
英文摘要 The chitosanase-producing strain TKU033 was isolated from soil in Taiwan, Sanzhi with squid pen powder as the sole carbon/nitrogen source and identified as Bacillus cereus. A chitosanase was purified from the culture supernatant by ammonium sulfate precipitation, DEAE-Sepharose and Macro-Prep DEAE Cartridge. The molecular mass of chitosanase determined by SDS-PAGE was approximately 43 kDa. The optimun pH , optimun temperature, pH stability, and thermal stability of chitosanase were pH 5,
50℃, pH5-7 and <40℃. The chitosanase activity was inhibited by Cu2+ , Mn2+ and EDTA.
The supernatant of the crude enzyme from B. cereus TKU033 hydrolyzed water soluble chitosan exhibited activity of enhancing growth for L. paracasei 12193 up to 141% and L.kefri 14011 up to 116%.
論文目次 目錄
頁次
中文摘要 I
英文摘要 II
目錄 III
圖目錄 VI
表目錄 VII

第一章 緒論 1
第二章 文獻回顧 2
2.1 Bacillus cereus 之簡介 2
2.2幾丁寡與幾丁聚醣 3
2.3幾丁寡醣 6
2.4 幾丁質酶與幾丁聚醣酶 7
2.5 蛋白酶 8
2.6 益生質(prebiotics) 8
2.7 幾丁類物質吸附色素 9
第三章 材料與方法 10
3.1 實驗菌株 10
3.2 實驗材料 10
3.3 實驗儀器 12
3.4 酵素生產菌株之篩選 12
3.5 幾丁聚醣酶活性測定 13
3.6 蛋白酶活性測定 13
3.7 幾丁質酶活性測定 14
3.8 較適培養條件探討 14
3.8.1 培養條件 14
3.8.2 碳/氮源濃度 14
3.9 酵素之分離純化 15
3.9.1 粗酵素液之製備 15
3.9.2 離子交換樹脂層析 15
3.10 蛋白質電泳分析 16
3.11 酵素之特性分析 16
3.11.1 酵素最適反應溫度 16
3.11.2 酵素熱安定性 16
3.11.3 酵素最適反應 pH 16
3.11.4 酵素 pH 安定性 17
3.11.5 金屬離子及化學藥品對酵素活性之影響 17
3.11.6 界面活性劑對酵素活性之影響 17
3.11.7 酵素之基質特異性 18
3.12 幾丁聚醣酶水解基質及寡醣分析 18
3.12.1 基質之水解 18
3.12.2 N-乙醯幾丁寡醣之製備 18
3.12.3 N-乙醯幾丁寡醣之組成分析 19
3.13 還原糖之測量 19
3.14 總糖量之測定 (H2SO4-Phenol 方法) 20
3.15 MALDI-TOF-MS 20
3.16 經 B. cereus TKU033粗酵素液水解基質促進乳酸菌生長 21
3.17 幾丁類物質吸附色素 21
第四章 結果與討論 22
4.1 幾丁聚醣酶與蛋白酶生產菌之篩選 22
4.1.1 菌株TKU033 之鑑定 22
4.2 酵素較適生產條件探討 22
4.2.1 培養液體基 23
4.2.2 不同碳/氮源濃度 23
4.2.3 較適培養條件探討結果 23
4.3 幾丁聚醣酶及蛋白酶之純化 25
4.3.1 粗酵素液之製備 25
4.3.2 離子交換樹脂層析 25
4.3.3 綜合結果 26
4.4 幾丁聚醣酶分子量之測定 27
4.4.1 SDS-PAGE 27
4.4.2 綜合結果 27
4.5 幾丁聚醣酶之特性分析 27
4.5.1 幾丁聚醣酶之最適反應溫度及熱安定性 27
4.5.2 幾丁聚醣酶之最適反應pH及pH安定性 28
4.5.3 金屬離子及化學藥品對幾丁聚醣酶之影響 28
4.5.4 界面活性劑對幾丁聚醣酶活性之影響 29
4.5.5 幾丁聚醣酶之基質特異性 30
4.6 水解基質之探討 30
4.6.1 還原糖與總糖含量之分析 30
4.6.2 利用 HPLC 分析幾丁寡醣組成 31
4.6.3 利用 MALDI-TOF-MS 進行幾丁寡醣組成分析 31
4.7 促進乳酸菌生長 32
4.8 幾丁類物質吸附色素 33
4.9 含硼化合物對三種不同幾丁聚醣酶活性之影響 33
第五章 結論 62
參考文獻 63

圖目錄
圖 2.1 幾丁質、幾丁聚醣及纖維素之結構............................................................... 4
圖 4.1 16SrDNA 部分鹼基序列分析及API試驗結果 ........................................... 34
圖 4.2 培養體積對 B. cereus TKU033生產幾丁聚醣酶及蛋白酶之影響 ........... 35
圖 4.3 不同碳/氮源對B. cereus TKU033生產幾丁聚醣酶之影響 ....................... 36
圖 4.4不同碳/氮源對B. cereus TKU033生產蛋白酶之影響 ................................ 37
圖 4.5 B. cereus TKU033 幾丁聚醣酶之 DEAE-Sepharose CL-6B 層析圖譜 ..... 38
圖 4.6 B. cereus TKU033 幾丁聚醣酶之Macro-Prep DEAE Cartridge 層析圖譜 38
圖 4.7 B. cereus TKU033 幾丁聚醣酶之SDS-PAGE 之分子量分析 ................... 39
圖 4.8 幾丁聚醣酶之 (A) 最適反應溫度 (B) 熱安定性 ...................................... 40
圖 4.9 幾丁聚醣酶之 (A) 最適反應 pH (B) pH 安定性 ...................................... 41
圖 4.10 銅離子對幾丁聚醣酶活性影響................................................................... 42
圖 4.11 水溶性幾丁聚醣 (WSC) 中添加B. cereus TKU033粗酵素液水解不同時
間所得總醣及還原糖含量.......................................................................................... 43
圖 4.12 WSC 經B. cereus TKU033 粗酵素液水解不同時間所得幾丁寡醣HPLC 分析圖(A) standard (1-6 醣) (B) 2 hr (C) 4hr (D) 6hr ............................................... 44
圖 4.13 WSC 經B. cereus TKU033 粗酵素液水解不同時間所得幾丁寡醣HPLC 分析圖 (A) standard (1-6 醣) (B) 12 hr (C) 24hr (D) 48hr ....................................... 45
圖 4.14 水溶性幾丁聚醣經 B. cereus TKU033 粗酵素液水解所得幾丁寡醣之 MALDI-TOF-MS分析圖 ........................................................................................... 46
圖4.15 水溶性幾丁寡醣經 B. cereus TKU033 粗酵素液水解所得幾丁寡醣添加於(A) L.paracasei 12193 (B) L.kefir 14011 生長之影響 .......................................... 49

表目錄
表2.1幾丁質與幾丁聚醣之應用................................................................................. 5
表 4.1篩選菌株之蛋白酶及幾丁聚醣活性表.......................................................... 50
表4.2 B. cereus TKU033 生產酵素之較適條件 ...................................................... 51
表4.3幾丁聚醣酶./蛋白酶生產菌酵素活性之比較 ................................................ 51
表4.4微生物來源之幾丁聚醣特性比較................................................................... 52
表4.5 B. cereus TKU033 幾丁聚醣酶純化總表 ...................................................... 54
表4.6金屬離子及化學藥品對幾丁聚醣酶活性之影響........................................... 55
表4.7界面活性劑對幾丁聚醣酶活性之影響........................................................... 56
表4.8 B. cereus TKU033 幾丁聚醣酶之基質特異性 .............................................. 57
表4.9水溶性幾丁聚醣經B. cereus TKU033粗酵素液水解不同時間之MALDI-TOF-MS 幾丁寡醣組成分析 ...................................................................... 58
表4.10 水溶性幾丁聚醣經B. cereus TKU033粗酵素液水解不同時間所得幾丁寡醣添加於乳酸菌 (L.paracasei 12193和L.kefir 14011) 生長之影響 ..................... 59
表4.11發酵及未發酵烏賊軟骨粉對色素之吸附率................................................. 60
表4.12含硼化合物對幾丁聚醣酶活性之影響......................................................... 61
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