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系統識別號 U0002-1707201213103400
中文論文名稱 Bacillus cereus TKU030 所生產幾丁聚醣酶之純化定性及抑制劑探討
英文論文名稱 Studies on the purification and characterization of a Bacillus cereus TKU030 chitosanase and its inhibitor
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 100
學期 2
出版年 101
研究生中文姓名 陳岳吟
研究生英文姓名 Yue-Yin Chen
學號 699160205
學位類別 碩士
語文別 中文
口試日期 2012-06-29
論文頁數 76頁
口試委員 指導教授-王三郎
委員-陳佑汲
委員-王全祿
中文關鍵字 Bacillus cereus  幾丁聚醣酶  烏賊軟骨  抑制劑 
英文關鍵字 Bacillus cereus  chitosanase  squid pen  inhibitor 
學科別分類 學科別自然科學化學
中文摘要 Bacillus cereus TKU030 係以烏賊軟骨為唯一碳/氮源,篩選自淡江大學
校園土壤之幾丁聚醣酶、幾丁質酶及蛋白酶之生產菌,本研究探討B. cereus
TKU030 發酵烏賊軟骨生產幾丁聚醣酶之較適培養條件,所得發酵上清液經硫
酸銨沉澱、Sephacryl S-100 膠體層析、DEAE-Sepharose 離子交換層析以及Sephacryl S-100 再膠體層析等步驟,純化出一種幾丁聚醣酶 (CHS)。CHS
之分子量經SDS-PAGE 測為43 kDa;最適反應溫度、最適反應pH、熱安定
性以及pH 安定性分別為40oC、pH 4、30-40oC 以及pH 3-7。於抑制劑方面,
CHS 活性會受Mn2+、Cu2+、Fe2+ 及含硼化合物TKUPSP017 所抑制。

TKUPSP017 濃度於0.05% (w/v) 時的抑制率達100%,抑制效果會隨
CHS 與TKUPSP017 預反應時間的增長而有所提升。此外,抑制劑結構對於
CHS 酵素活性的影響,以及添加TKUPSP017 於培養基對於B. cereus
TKU030 生長之影響亦有探討。CHS 被抑制不會影響TKU030 的生長,隨著
TKUPSP017 濃度提高,TKU030 生長狀況反而越好。
英文摘要 The chitosanase, chitinase and proease-producing strain
TKU030 was isolated from soil in Tamkang University with squid pen powder as the sole carbon/nitrogen source and identified as Bacillus cereus. A chitosanase (CHS) was purified from the culture supernatant by ammonium sulfate precipitation, Sephacryl S-100 gel chromatography, DEAE- Sepharose column chromatography and Sephacryl S-100 rechromatography . The molecular mass of CHS determined by SDS-PAGE was approximately 43 kDa. The optimum temperature, optimum pH, thermal stability, and pH stability of CHS were 40oC, pH 4, 30-40oC and pH 3-7, respectively.The activity of CHS was inhibited by Mn2+, Cu2+, Fe2+ and a boron-containing compound of TKUPSP017.

The inhibition of CHS activity reached 100% at 0.05% (w/v) of TKUPSP017. The inhibitory activity increased with the
preincubating time of TKUPSP017 and CHS. The effects of
inhibitor structures on enzyme activity and growth of strain TKU030 in media supplemented with TKUPSP017 were also studied.Growth of strain TKU030 was not influenced by inhibiting CHS activity but enhanced by increasing TKUPSP017 concentration.
論文目次 中文摘要....................................................I
英文摘要...................................................II
目錄.....................................................…IV
圖目錄.....................................................IX
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 ..................................5
2.3 幾丁質酶與幾丁聚醣酶 .....................................5
2.4 蛋白酶 .................................................6
2.5 Bacillus cereus 之簡介 .................................7
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 酵素生產菌株之篩選 ......................................11
3.5 幾丁質酶活性測定 ........................................12
3.6 幾丁聚醣酶活性測定 ......................................12
3.7 蛋白酶活性測定 .........................................13
3.8 較適培養條件之探討 ......................................13
3.8.1 培養液體積 ..........................................13
3.8.2 碳/氮源濃度與培養時間 .................................14
3.9 酵素之分離純化 .........................................14
3.9.1 粗酵素液之製備 .......................................14
3.9.2 陰離子交換層析 .......................................15
3.9.3 膠體過濾層析 .........................................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 ......................................17
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 ............................18
3.11.7 酵素之基質特異性 ....................................18
3.12 還原糖量之測定 ........................................19
3.13 總糖量之測定 (H2SO4-Phenol 法) ........................19
3.14 幾丁聚醣酶水解基質及寡醣分析 .............................20
3.14.1 基質之水解 .........................................20
3.14.2 N-乙醯幾丁寡醣之製備 .................................20
3.14.3 N-乙醯幾丁寡醣之組成分析 ..............................21
第四章 結果與討論 ...........................................22
4.1 幾丁聚醣酶、幾丁質酶及蛋白酶生產菌之篩選與鑑定 ...............22
4.2 酵素較適生產條件探討 ....................................22
4.2.1 培養液體.............................................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 ..................................24
4.3 幾丁質酶及幾丁聚醣酶之純化 ...............................24
4.3.1 粗酵素液之製備 .......................................24
4.3.2 離子交換樹脂層析 ......................................25
4.3.3 膠體過濾層析 .........................................26
4.3.4 綜合結果 ............................................26
4.4 幾丁聚醣酶分子量測定 ....................................28
4.4.1 SDS-PAGE ..........................................28
4.4.2 幾丁聚醣酶生態質譜鑑定 (peptide mass mapping) ..........28
4.5 幾丁質酶與幾丁聚醣酶之特異性分析 ...........................29
4.5.1 幾丁質酶與幾丁聚醣酶之最適反應溫度及熱安定性 ...............29
4.5.2 幾丁質酶與幾丁聚醣酶之最適pH 值及酸鹼安定性 ...............29
4.5.3 金屬離子及化學藥品對幾丁質酶與幾丁聚醣酶活性之影響...........30
4.5.4 界面活性劑對幾丁質酶與幾丁聚醣酶活性之影響 ................31
4.6 B. cereus TKU030 發酵烏賊軟骨所得幾丁寡醣組成分析 ..........31
4.7 水解基質之探討 .........................................32
4.7.1 總糖與還原糖含量之分析 .................................32
4.7.2 利用HPLC 分析幾丁寡醣組成 .............................32
4.8 B. cereus TKU030 幾丁聚醣酶之抑制劑探討 ..................33

第五章 結論 ...............................................68
第六章 參考文獻 ............................................69

圖目錄
圖 2.1 纖維素、幾丁質及幾丁聚醣之結構 ......................... 3
圖 4.1 16S rDNA 部分鹼基序列分析及API 試驗結果 .............. 36
圖 4.2 培養液體積對B. cereus TKU030 生產幾丁質酶、幾丁聚醣
酶及蛋白酶之影響 ...........................................37
圖 4.3 不同碳/氮源對B. cereus TKU030 生產幾丁質酶之影響 .......38
圖 4.4 不同碳/氮源對B. cereus TKU030 生產幾丁聚醣酶之影響.......39
圖 4.5 不同碳/氮源對B. cereus TKU030 生產蛋白酶之影響 .........40
圖 4.6 B. cereus TKU030 幾丁質與幾丁聚醣酶之Sephacryl
S-100 層析圖譜 ............................................41
圖 4.7 B. cereus TKU030 幾丁質與幾丁聚醣酶DEAE-Sepharose
CL-6B 層析圖譜 ............................................41
圖 4.8 B. cereus TKU030 幾丁聚醣酶之Sephacryl S-100 層析圖
譜 .......................................................42
圖 4.9 B. cereus TKU030 幾丁聚醣酶SDS-PAGE 之分子量分析.......43
圖 4.10 溫度對B. cereus TKU030 幾丁質酶 (A) 及幾丁聚醣酶
(B)之討論 .................................................44
圖 4.11 酸鹼度對B. cereus TKU030 幾丁質酶 (A) 及幾丁聚醣酶
(B) 之討論 ................................................45
圖 4.12 銅離子對幾丁質酶活性的影響 ............................46
圖 4.13 幾丁寡糖之HPLC 組成分析圖 (A) 標準品 (B) B. cereus
TKU030 發酵1% SPP 所得上清液 ...............................47
圖 4.14 WSC 經B. cereus TKU030 粗酵素液水解不同時間所得總
糖及還原糖含量 .............................................48
圖 4.15 WSC 經B. cereus TKU030 粗酵素液水解不同時間所得
幾丁寡醣之HPLC 分析圖 ..................................... 49
圖 4.16 WSC 經B. cereus TKU030 粗酵素液水解不同時間所得幾
丁寡醣之HPLC 分析圖 ....................................... 50
圖 4.17 抑制劑與幾丁聚醣酶預反應時間對酵素活性抑制率之影響........ 51
圖 4.18 幾丁聚醣培養基加入抑制劑TKUPSP017 對B. cereus
TKU030 生長之影響 ........................................ 51
圖 4.19 添加0.05 %TKUPSP017 於培養基對B. cereus TKU030
生長之影響 ................................................52
圖 4.20 添加幾丁聚醣酶抑制劑對B. cereus TKU030 生長之影響.......53

表目錄
表 2.1 幾丁質與幾丁聚醣之應用 .................................4
表 4.1 B. cereus TKU030 生產酵素之較適培養條件 .............. 54
表 4.2 不同細菌源幾丁質酶/蛋白酶之酵素活性比較 ..................54
表 4.3 不同細菌源之幾丁質酶與幾丁聚醣酶特性比較 .................55
表 4.4 B. cereus TKU030 幾丁質酶純化總表 (I) ................58
表 4.5 B. cereus TKU030 幾丁質酶純化總表 (II) ...............58
表 4.6 B. cereus TKU030 幾丁聚醣酶純化總表 (I) ..............59
表 4.7 B. cereus TKU030 幾丁聚醣酶純化總表 (II) .............59
表 4.8 各種化學品對幾丁質酶、幾丁聚醣酶和蛋白酶活性之影響..........60
表 4.9 界面活性劑對幾丁質酶及幾丁聚醣酶之影響 ...................61
表 4.10 Bacillus sp. TKU030 幾丁質酶及幾丁聚醣酶之基質特異性....63
表 4.12 抑制劑對B. cereus TKU030 幾丁聚醣酶之抑制效果 .........64
表 4.13 含硼化合物對Bacillus cereus TKU030 幾丁聚醣酶之影響
(%) ......................................................64
表 4.14 抑制劑TKUPSP017, TKUPSP074 及TKUPSP114 對其
他細菌生產幾丁聚醣酶之抑制活性 ............................... 65
表 4.15 含硼化合物對B. cereus TKU030 幾丁聚醣酶活性之抑制率 ... 66
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