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系統識別號 U0002-2607201315344700
中文論文名稱 Penicillium janthinellum D4 所生產 幾丁聚醣酶之純化與定性
英文論文名稱 Purification and characterization of chitosanase from Penicillium janthinellum D4
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 黃淳靖
研究生英文姓名 Chun-Ching Huang
學號 698160537
學位類別 碩士
語文別 中文
口試日期 2013-07-04
論文頁數 73頁
口試委員 指導教授-王三郎
委員-王全祿
委員-梁慈雯
中文關鍵字 幾丁聚醣酶  烏賊軟骨  抑制劑 
英文關鍵字 Penicillium 
學科別分類 學科別自然科學化學
中文摘要 Penicillium janthinellum D4株篩選自越南Tay Nguyen大學之幾丁聚醣酶生

產菌。本研究探討P. janthinellum D4發酵烏賊軟骨生產幾丁聚醣酶之較適條

件,所得發酵上清液經硫酸銨沉澱、DEAE-Sepharose離子交換層析、

Macro-prep DEAE Cartridge等步驟,純化出兩種幾丁聚醣酶F1及F2。幾丁聚

醣酶F2之分子量經SDS-PAGE測量為49kDa,其最適反應溫度、最適反應

pH、熱安定性以及pH安定性分別為60℃、pH 7~9、25~40℃以及pH 10。於

抑制劑方面,幾丁聚醣酶F2的活性會受Fe2+、Cu2+、Mn2+所抑制。

英文摘要 The chitosanase producing strain Penicillium janthinellum D4, was

isolated from soil in Tay Nguyen University in Vietnam. Two

chitosanases F1 F2 were purified from the culture supernatant by

ammonium sulfate precipitation, DEAE-Sepharose column

chromatography and Macro-prep DEAE Cartridge. The molecular mass

of CHS was estimated to be 49kDa by SDS-PAGE. The optimum

temperature, optimum pH, thermal stability, and pH stability of CHS F2

were 60℃, pH 7~9, 25~40℃, and pH 10, respectively. The activity of CHS F2 was

inhibited by Fe2+,Cu2+,and Mn2+.

論文目次 目錄

中文摘要.............................................I

英文摘要............................................ II

目錄.................................................III

圖目錄...............................................VIII

表目錄...............................................XII

第一章 緒論........................................1

第二章 文獻回顧......................................2

2.1 幾丁質與幾丁聚醣.................................2

2.2 N-乙醯幾丁寡醣與幾丁寡醣.........................3

2.3 幾丁質酶與幾丁聚醣酶.............................4

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 幾丁質酶活性測定.................................9

3.5 幾丁聚醣酶活性測定...............................9

3.6 較適培養條件之探討...............................10

3.6.1 培養液體積.....................................10

3.6.2 碳/氮源濃度與培養時間..........................10

3.7 酵素之分離純化...................................11

3.7.1 粗酵素液之製備.................................11

3.7.2 陰離子交換層析 (一) ...........................11

3.7.3 陰離子交換層析 (二) ...........................11

3.7.4 陽離子交換層析.................................12



3.7.5 膠體過濾層析...................................12

3.8 酵素之特性分析...................................13

3.8.1 酵素最適反應溫度...............................13

3.8.2 酵素熱安定性...................................13

3.8.3 酵素最適反應 pH................................13

3.8.4 酵素 pH安定性..................................14

3.8.5 金屬離子、化學藥品及抑制劑對酵素活性之影響.....14

3.9 還原醣量之測定...................................15

3.10 總醣量之測定 (H2SO4-Phenol 法) .................15

3.11 幾丁聚醣酶水解基質及寡醣分析....................16

3.11.1 基質之水解....................................16

3.11.2 N-乙醯幾丁寡醣之製備..........................16

3.12 MALDI-TOF-MS....................................16

第四章 結果與討論....................................17

4.1 幾丁質酶、幾丁聚醣酶生產菌之篩選.................17

4.2 酵素較適生產條件探討.............................17

4.2.1 培養液體積.....................................18

4.2.2 不同碳/氮源濃度................................18

4.3 幾丁聚醣酶之純化.................................21

4.3.1 粗酵素液之製備.................................21

4.3.2 陰離子交換樹脂層析(一).........................21

4.3.3 陰離子交換樹脂層析(二).........................22

4.3.4 陽離子交換樹脂層析.............................22

4.3.5 綜合結果.......................................23

4.4 幾丁聚醣酶分子量測定.............................24



4.4.1 SDS-PAGE.......................................24

4.5 幾丁聚醣酶之特異性分析...........................24

4.5.1 幾丁聚醣酶之最適反應溫度及熱安定性.............24

4.5.2 幾丁聚醣酶之最適pH值與酸鹼安定性...............25

4.5.3 金屬離子及化學藥品對幾丁聚醣酶活性之影響.......25

4.6 P. janthinellum D4幾丁聚醣酶之抑制劑探討.........26

4.7 還原醣與總醣含量之分析...........................28

4.7.1 利用MALDI-TOF-MS進行幾丁寡醣組成分析...........28

第五章 結論..........................................66

第六章 文獻回顧......................................67

圖目錄

圖 2.1 幾丁質、幾丁聚醣、部分乙醯化幾丁聚醣的化學結構....3

圖 4.1 (A) 培養體積對P. janthinellum D4生產幾丁聚醣酶的影響........................................................29

圖 4.1 (B) 培養體積對P. janthinellum D4生產幾丁聚醣酶的影響........................................................30

圖 4.2 烏賊軟骨粉(SPP)添加濃度對P. janthinellum D4生產幾丁聚醣酶的影響........................................................31

圖 4.3 烏賊軟骨粉(SPP)和葡萄醣(Glucose)添加濃度對P. janthinellum D4生產幾丁聚醣酶的影響.......................32

圖 4.4 烏賊軟骨粉(SPP)添加濃度對Paecilomyces lilacinus D31生產幾丁聚醣酶的影響......................................33

圖 4.5 烏賊軟骨粉(SPP)和葡萄醣(Glucose)添加濃度對Paecilomyces lilacinus D31生產幾丁聚醣酶的影響............34


圖 4.6 烏賊軟骨粉(SPP)添加濃度對Paecilomyces lilacinus D31生產幾丁質酶的影響........................................35

圖 4.7 烏賊軟骨粉(SPP)和葡萄醣(Glucose)添加濃度對Paecilomyces lilacinus D31生產幾丁質酶的影響..............36

圖 4.8 Czapek 培養基和SPP添加濃度及添加懸浮態幾丁質對Paecilomyces lilacinus D31生產幾丁聚醣酶的影響............37

圖 4.9 Czapek 培養基和SPP添加濃度及添加懸浮態幾丁質對Paecilomyces lilacinus D31生產幾丁質酶的影響..............38

圖 4.10 Czapek 培養基和烏賊軟骨粉(SPP)、蝦頭殼粉(SHP)添加濃度對Paecilomyces lilacinus D31生產幾丁聚醣酶的影響......39

圖 4.11 Czapek 培養基和烏賊軟骨粉(SPP)、蝦頭殼粉(SHP)添加濃度對Paecilomyces lilacinus D31生產幾丁質酶的影響........40

圖 4.12 Penicillium janthinellum D4幾丁聚醣酶F2之DEAE-Sepharose CL-6B層析圖譜...................................41




圖 4.13 Penicillium janthinellum D4幾丁聚醣酶F2之Macro-Prep DEAE Cartridge 層析圖................................42

圖 4.14 Penicillium janthinellum D4幾丁聚醣酶F1之CM 層析........................................................43

圖 4.15 Penicillium janthinellum D4幾丁聚醣酶F1之CM 層析........................................................44

圖 4.16 P. janthinellum D4幾丁聚醣酶F1之S-100 層析......45

圖 4.17 Penicillium janthinellum D4幾丁聚醣酶F1之CM 層析........................................................46

圖 4.18 P. janthinellum D4幾丁聚醣酶F2 SDS-PAGE之分子量分析........................................................47

圖 4.19 Penicillium janthinellum D4幾丁聚醣酶F1 SDS-PAGE之分子量分析................................................48

圖 4.20 溫度對P. janthinellum D4幾丁聚醣酶F2之影響......49

圖 4.21 酸鹼度對P. janthinellum D4幾丁聚醣酶F2之影響....50

圖 4.22 WSC經P. janthinellum D4粗酵素液F2水解不同時間所得總醣及還原醣含量..........................................51

圖 4.23 水溶性幾丁聚醣經P. janthinellum D4粗酵素液F2水解所得幾丁寡醣之MALDI-TOF-MS分析圖............................52


表目錄

表2.1 微生物來源之幾丁聚醣酶特性比較.........................................6

表3.1 DNS試劑組成...............................................................................15

表4.1 P. janthinellum D4幾丁聚醣酶F2純化總表............................56

表4.2 Penicillium janthinellum D4幾丁聚醣酶F1純化總表。...........57

表4.3 各種化學品對幾丁聚醣酶F2活性之影響...............................58

表 4.4 各種化學品對幾丁聚醣酶F2活性之影響..............................59

表 4.5 含硼化合物對P. janthinellum D4幾丁聚醣酶F2活性之抑制率..........................................................................................................60

表 4.6 含硼化合物001對P. janthinellum D4幾丁聚醣酶活性之抑制率..........................................................................................................62

表 4.7 P. janthinellum D4生產酵素之較適條件...................................63

表 4.8水溶性幾丁聚醣經Penicillium janthinellum D4粗酵素液水解不同時間之MALDI-TOF-MS幾丁寡醣組成分析..............................64,65
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