菌株TKU031係以烏賊軟骨粉為唯一碳/氮源，篩選自台灣淡水三芝土壤，經鑑定為Bacillus cereus之幾丁聚醣酶及蛋白酶生產菌。幾丁聚醣酶較適生產條件為，以含有1％烏賊軟骨粉、0.1 % K2HPO4、0.05 % MgSO4．7H2O之100mL液態培養基( pH 7)，於37℃搖瓶 ( 150 rpm ) 培養2天。所得發酵液經離心、硫酸銨沉澱以及 DEAE–Sepharose與 Macro-PrepR DEAE Cartridge陰離子交換層析等步驟，純化出的一種經SDS–PAGE測定分子量為43 kDa幾丁聚醣酶CHS。CHS的最適反應pH、最適反應溫度、pH安定性以及熱安定性分別為pH 5、50℃、pH 5–9以及＜50℃。CHS的酵素活性活性會受1mM的 Zn2+、Cu2+、Fe2+及5mM 的Mn2+、EDTA所抑制。
    於較適培養，添加0.1%硼砂進行培養發現可促進B. cereus TKU031菌體生長達122%，若添加0.1%硼砂及0.05%硼酸則可促進B. cereus TKU031幾丁聚醣酶之生產，相對活性分別是未添加之培養基培養第3天177％及第2天的 195％。

Strain TKU031, a chitosanase– and protease– producing bacteria, was isolated from the soil of Sanjhih, Taiwan with squid pen powder (SPP) as the sole carbon/nitrogen source and identified as Bacillus cereus. Optimized culture condition for chitosanase production was found when the organism was cultured at 37℃ for two days in 100mL medium (pH 7) containing 1%SPP (w/v), 0.1 % K2HPO4、0.05 % MgSO4．7H2O.
One chitosanase (CHS) was purified from the culture supernatant of B. cereus TKU031 by ammonium sulfate precipitation, DEAE–Sepharose chromatography and Macro-PrepR DEAE Cartridge chromatography. The molecular weight of CHS was determined by SDS-PAGE approximately to be 43kDa. The optimum pH, optimum temperature, pH stability, and thermal stability of CHS was pH 5, 50℃, pH 5–9, <50℃. CHS was inhibited by EDTA and completely inactivated by 1mM Zn2+、Cu2+、Fe2+, and 5mM Mn2+.
In addition, the growth of B. cereus TKU031 was found to be promoted up to 1.22-fold by adding 0.1% sodium tetraborate in the culture medium. The productivities of chitosanase were also increased by adding 0.1% sodium tetraborate and 0.05% boric acid, which enhanced 1.77- and 1.95- folds, respectively.

目錄

中文摘要.......................................................................................................................Ⅰ
英文摘要.......................................................................................................................Ⅱ
目錄...............................................................................................................................Ⅳ
圖目錄...........................................................................................................................Ⅹ
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-乙醯幾丁寡醣及幾丁寡醣.................................................................................5

2.3 幾丁質酶及幾丁聚醣酶........................................................................................5

2.4 蛋白酶....................................................................................................................6

2.5益生質(prebiotics)...................................................................................................7

2.6 Bacillus cereus 之簡介...........................................................................................7

第三章  材料與方法.....................................................................................................8

3.1實驗菌株 ...............................................................................................................8

3.2 實驗材料................................................................................................................8

 3.3實驗儀器.................................................................................................................9

 3.4 酵素生產菌株之篩選............................................................................................9

 3.5幾丁聚醣酶之活性測定.......................................................................................10

 3.6蛋白酶活性測定...................................................................................................10

 3.7較適培養條件探討...............................................................................................11

 3.7.1碳/氮源濃度.......................................................................................................11

 3.7.2 培養體積...........................................................................................................11

 3.8酵素之分離純化...................................................................................................11

 3.8.1 粗酵素液製備...................................................................................................11

 3.8.2陰離子交換層析 (一)........................................................................................11

 3.8.3陰離子交換層析 (二)........................................................................................12

 3.9蛋白質電泳分析...................................................................................................12

 3.10 酵素性分析........................................................................................................12

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

 3.10.2酵素熱安定性..................................................................................................13

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

 3.10.4酵素pH安定性...............................................................................................13

 3.10.5 金屬離子及化學藥品對酵素活性之影響.....................................................14

 3.10.6 界面活性劑對酵素活性之影響.....................................................................14

 3.10.7 酵素基質特異性.............................................................................................14

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

 3.12 總醣量之測定....................................................................................................15

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

 3.13.1 基質水解.........................................................................................................16

 3.13.2 N-乙醯幾丁寡醣製備......................................................................................16

 3.13.3 N-乙醯幾丁寡醣之組成分析..........................................................................16

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

 4.1 幾丁聚醣酶與蛋白酶生產菌之篩選..................................................................17
.
 4.1.1 菌株TKU031 之鑑定......................................................................................17

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

 4.2.1碳/氮源濃度.......................................................................................................17

 4.2.2培養液體積........................................................................................................18

 4.3 幾丁聚醣酶之純化分離......................................................................................18

 4.3.1粗酵素液製備....................................................................................................19

 4.3.2 離子交換樹脂層析 (一)..................................................................................19

 4.3.2離子交換樹脂層析 (二)...................................................................................20

 4.3.3 綜合結果...........................................................................................................20

 4.4 幾丁聚醣酶之分子量測定..................................................................................21

 4.4.1 SDS-PAGE.........................................................................................................21

 4.5 幾丁聚醣酶之特性分析......................................................................................22

 4.5.1幾丁聚醣酶之最適反應溫度及熱安定性........................................................22

 4.5.2幾丁聚醣酶之最適反應pH及pH安定性......................................................22

 4.5.3 金屬離子及化學品對幾丁聚醣酶活性之影響...............................................23

 4.5.4 界面活性劑對幾丁聚醣之影響.......................................................................23

 4.5.5幾丁聚醣酶之基質特異性................................................................................24

 4.6 水解基質之探討..................................................................................................24

 4.6.1 還原糖及總糖含量之分析...............................................................................24

 4.6.2利用MALDI-TOF-MS進行幾丁寡醣分析.....................................................24

 4.7促進乳酸菌生長...................................................................................................25

 4.8 抗菌測試..............................................................................................................26

 4.9 硼化合物對B.cereus TKU031之影響...............................................................26

 4.9.1 菌體生長影響...................................................................................................26

 4.9.2 幾丁聚醣酶活性影響.......................................................................................27

 4.9.3 綜合結果...........................................................................................................28

第五章 結論.................................................................................................................30

第六章 參考文獻.........................................................................................................66






















圖目錄

圖2.1 幾丁質、幾丁聚醣及纖維素之結構................................................................3

圖4.1 B. cereus TKU031之16S rDNA 部分鹼基序列分析....................................31

圖4.2 SPP濃度對B.cereus TKU031 所產生幾丁聚醣酶及蛋白酶之影響
......................................................................................................................................32

圖4.3培養液體積B.cereus TKU031 所產生幾丁聚醣酶及蛋白酶之影響
......................................................................................................................................33

圖4.4 B.cereus TKU031 幾丁聚醣酶DEAE-Sepharose CL-6B層析圖譜
......................................................................................................................................34

圖4.5 B.cereus TKU031 幾丁聚醣酶Macro-PrepR DEAE Cartridge層析圖譜
......................................................................................................................................35

圖4.6 B.cereus TKU031 幾丁聚醣酶之 SDS-PAGE 圖.........................................36

圖4.7 幾丁聚醣酶之最適反應溫度及熱安定性......................................................37

圖4.8 幾丁聚醣酶之最適反應pH及pH安定性....................................................38

圖4.9 水溶性幾丁聚醣經 B.cereus TKU031粗酵素液水解不同時間所得總醣及還原醣含量......................................................................................................................49

圖4.10水溶性幾丁聚醣經B.cereus TKU031粗酵素液水解不同時間之MALDI-TOF-MS幾丁寡醣組成分析圖.....................................................................40

圖4.11 WSC 經 B.cereus TKU031 粗酵素液水解所得上清液添加於乳酸菌生長之影響......................................................................................................................... 43

圖4.12 WSC 經B.cereus TKU031粗酵素液水解所得上清液添加於 (A) S. aureus (B) P. aeruginosa K-187 生長之影響.........................................................................44

圖4.13 硼酸添加於 Bacillus cereus 生長之影響....................................................45

圖4.14 硼砂添加於 Bacillus cereus生長之影響.....................................................46

圖4.15硼酸添加於培養基對 Bacillus cereus 所生產之幾丁聚醣酶之影響.........47

圖4.16硼砂添加於培養基對 Bacillus cereus 所生產之幾丁聚醣酶之影響.........48











表目錄

表2.1 幾丁質與幾丁聚醣之應用.................................................................................4

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

表4.1 API試驗.............................................................................................................49

表4.2 B. cereus TKU031及以含幾丁質水產廢棄物為幾丁聚醣酶/幾丁質酶/蛋白酶生產菌較適培養條件之比較.......................................................................................50

表4.3蛋白酶生產較適條件上清液及硫銨沉澱之幾丁聚醣酶及蛋白酶活性........51
.
表4.4 B.cereus TKU031 幾丁聚醣酶純化總表.........................................................52

表4.5 金屬離子及化學品對幾丁聚醣酶之影響.......................................................53

表4.6 不同濃度金屬離子對幾丁聚醣酶之影響.......................................................54

表4.7 界面活性劑對幾丁聚醣酶CHS活性之影響.................................................55

表4.8 B.cereus TKU031幾丁聚醣酶之基質特異性..................................................56

表4.9 微生物來源之幾丁聚醣酶、幾丁質酶特與蛋白酶特性比較.......................57

表4.10 WSC 經B.cereus TKU031粗酵素液水解不同時間之水解率......................61

表4.11 水溶性幾丁聚醣經B.cereus TKU031粗酵素液水解不同時間之MALDI-TOF-MS幾丁寡醣組成分析.........................................................................62

表4.12 B.cereus TKU031 水解水溶性幾丁聚醣所得上清液對乳酸菌生長(24與48小時)之影響..................................................................................................................63

表4.13硼酸添加於培養基對 Bacillus cereus 所生產之幾丁聚醣酶之影響。
.......................................................................................................................................64

表4.14硼砂添加於培養基對 Bacillus cereus 所生產之幾丁聚醣酶之影響。
.......................................................................................................................................64

表4.15硼化合物對B.cereus幾丁聚醣酶之影響......................................................65

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