Bacillus cereus TKU022係以蝦頭粉為主要碳/氮源，篩選自台灣北部土壤之幾丁聚醣酶生產菌，發酵蝦頭粉所得離心上清液具有幾丁聚醣酶及蛋白酶活性。幾丁聚醣酶較適生產條件為 1.5% 蝦頭粉、0.1% K2HPO4、0.05% MgSO4．7H2O 之50mL液態培養基（pH5）於 37℃振盪培養 2天。發酵所得離心上清液經硫酸銨沉澱、DEAE-Sepharose、Phenyl Sepharose 、Macro-Prep DEAE及Sephacryl S-100等層析步驟後，可純化出一種幾丁聚醣酶及一種蛋白酶，經 SDS-PAGE測定分子量分別為 44 kDa及45kDa。其幾丁聚醣酶及蛋白酶之最適反應pH、最適反應溫度、pH 安定性、熱安定性分別為 pH 7, 60 ℃, pH 7-10, <40 ℃及pH 10, 50-60℃, pH 6-10, <50 ℃；幾丁聚醣酶活性會受Cu2+及Mn2+所抑制，而非離子型界面活性劑 Triton X-100、Tween 20及離子型界面活性劑SDS 則不具抑制效果；蛋白酶則會受到Mn2+、EDTA及SDS所抑制，在2 mM之存在下Triton X-100、Tween 20、Tween 40還維持其原活性97 %、105 %及94 %。
TKU022以較適生產幾丁聚醣酶培養基（1.5 % SHP, 37 ℃, pH 5）培養8天，所得發酵上清液於第5天有最高之還原醣(649 &micro;g/mL)產量；並0.05 %之還原醣添加至MRS broth中可提升Lactobacillus paracasei TKU010之生長速率。

The chitosanase -producing bacterium, Bacillus cereus TKU022, was isolated from soil in the north Taiwan by using shrimp head powder as the sole carbon/nitrogen source. The supernatant of the culture medium contains the chitosanase and the protease the activity. The optimized condition for chitosanase production was found when the culture was shaken at 37°C for two days in 50mL of medium containing 1.5% SHP, 0.1% K2HPO4, 0.05 % MgSO4．7H2O ( pH 5). The chitosanase and protease were purified from the culture supernatant by ammonium sulfate precipitation, DEAE-Sepharose, Phenyl Sepharose, Macro-Prep DEAE and Sephacryl S-100. The molecular masses of the chitosanase and protease determined by SDS-PAGE were approximately 44 and 45 kDa, respectively. The optimum pH, optimum temperature, pH stability and thermal stability of TKU022 chitosanase and protease were pH 7, 60 ℃, pH 7-10, <40 ℃and pH 10, 50-60 ℃, pH 6-10, <50 ℃, respectively. The chitosanase activity was inhibited by Cu2+ and Mn2+, but not by Tween 20, Triton X-100 (nonionic surfactant) and SDS (anionic surfactant).The protease activity was inhibited by Mn2+, EDTA and SDS, but retained 97 %, 105 % and 94% of its original activity in the presence of 2 % Triton X-100, 2 % Tween 20 and 2 % Tween 40, respectively.
B. cereus TKU022 was incubated for 8 days under the optimized culture conditions（1.5 % SHP, 37 ℃, pH 5）and analyzed the reducing sugar. TKU022 culture supernatant incubated for 5 days had the highest reducing sugar（649 &micro;g/mL）and enhanced the growth rate of Lactobacillu paracasei TKU010 in MRS broth.

中文摘要........................................Ⅰ
英文摘要........................................Ⅲ
目錄............................................Ⅴ
圖目錄..........................................ⅩI
表目錄..........................................ⅩⅢ

第一章 緒論......................................1
第二章 文獻回顧..................................2
2.1 Bacillus cereus之簡介........................2
2.2 幾丁質與幾丁聚醣.............................4
2.3 幾丁質及幾丁聚醣水解酵素.....................7
2.4 N-乙醯幾丁寡醣及幾丁寡醣.....................8
2.5 蛋白酶.......................................9
2.5.1 蛋白酶的分類與命名.........................9
2.5.2蛋白酶在工業上之應用........................11
2.6 抗氧化.......................................13
第三章 材料與方法................................14
3.1 實驗菌株.....................................14
3.2 實驗材料.....................................14
3.3 實驗儀器.....................................16
3.4幾丁聚醣酶生產菌之篩選........................17
3.5幾丁聚醣酶之活性測定..........................17
3.6 蛋白酶活性之測定.............................18
3.7 幾丁聚醣酶較適生產條件探討...................18
3.7.1碳/氮源濃度.................................18
3.7.2培養溫度....................................19
3.7.3 培養體積...................................19
3.7.4培養基pH值..................................19
3.7.5培養時間....................................19
3.8幾丁聚醣酶及蛋白酶之分離純化..................20
3.8.1粗酵素液之製備..............................20
3.8.2陰離子交換樹酯..............................20
3.8.3疏水性層析法................................21
3.8.4膠體過濾層析................................21
3.9酵素之分子量測定..............................22
3.9.1 蛋白質電泳分析.............................22
3.9.2  膠體過濾層析..............................22
3.10幾丁聚醣酶及蛋白酶之特性分析.................23
3.10.1酵素最適反應溫度...........................23
3.10.2 酵素熱安定性..............................23
3.10.3 酵素最適反應 pH...........................23
3.10.4 酵素 pH 安定性............................24
3.10.5 金屬離子及化學藥品對酵素活性之影響........24
3.10.6 界面活性劑對幾丁聚醣酶活性之影響..........25
3.10.7 酵素之基質特異性..........................25
3.10.7.1幾丁聚醣酶	...............................25
3.10.7.2 蛋白酶..................................25
3.11 粗酵素液之蛋白酶及幾丁聚醣酶活性關係........26
3.12 還原醣量之測定..............................26
3.13 抗氧化分析..................................27
3.13.1 DPPH 自由基清除能力之測定.................27
3.14 N-乙醯幾丁寡醣製備..........................27
3.15 促進乳酸菌生長..............................28
第四章 結果與討論................................29
4.1 幾丁聚醣酶生產菌之篩選.......................29
4.1.1幾丁聚醣酶與蛋白質酶生產菌株之鑑定..........29
4.2 較適生產條件探討.............................30
4.2.1 碳/氮源濃度................................31
4.2.2 培養溫度...................................31
4.2.3培養體積....................................31
4.2.4 培養基pH值.................................32
4.2.5較適培養條件探討結果........................32
4.2.6 TKU022發酵SHP之產物分析....................33
4.3 幾丁聚醣酶及蛋白質酶之分離純化...............41
4.3.1 粗酵素液之製備.............................41
4.3.2 離子交換樹脂/疏水性層析....................41
4.3.3 膠體過濾層析...............................42
4.3.4 綜合結果...................................43
4.4 幾丁聚醣酶與蛋白質酶之分子量測定.............43
4.4.1 SDS-PAGE...................................43
4.4.2膠體過濾層析................................43
4.4.3 綜合結果...................................44
4.4.4 幾丁聚醣酶胜肽質譜鑑定(peptide mass mapping).45
4.5 幾丁聚醣酶與蛋白酶之特性分析.................45
4.5.1  幾丁聚醣酶與蛋白酶之最適反應pH及pH安定性........45
4.5.2  幾丁聚醣酶與蛋白酶之最適反應溫度及熱安定性......46
4.5.3  金屬離子及化學藥品對幾丁聚醣酶與蛋白酶之影響....46
4.5.4  界面活性劑對幾丁聚醣酶與蛋白酶活性之影響........47
4.5.5 幾丁聚醣酶與蛋白酶之基質特異性	..............48
4.5.5.1 幾丁聚醣酶之基質特異性....................48
4.5.5.2 蛋白酶之基質特異性........................48
4.6還原醣與抗氧化分析.............................49
4.7促進乳酸菌生長.................................49
第五章 結論.......................................69
參考文獻..........................................70

圖目錄
                                                  
圖2.1 幾丁質、幾丁聚醣及纖維素之結構...............4
圖2.2  幾丁質水解酵素水解途徑......................7
圖4.1 16S rDNA部分鹼基序列分析及API試驗............30
圖4.2 SHP濃度對TKU022幾丁聚醣酶生產之影響..........35
圖4.3 培養溫度對TKU022幾丁聚醣酶生產之影響.........35
圖4.4 培養液體積對TKU022幾丁聚醣酶及蛋白酶生產之影響....36
圖4.5培養液pH對第2天TKU022幾丁聚醣酶與蛋白酶生產之影響..37
圖4.6 B. cereus TKU022培養於SHP培養基所生產幾丁聚醣酶與蛋白酶之生長曲線圖.....................................38
圖4.7 B. subtilis TKU007於SHP培養基所產蛋白酶及幾丁聚醣酶之生長曲線圖.........................................38
圖 4.8 TKU022 幾丁聚醣酶與蛋白酶活性關係...........40
圖4.9 B. cereus TKU022所生產幾丁聚醣酶與蛋白酶之純化分離流程圖.................................................51
圖4.10 TKU022幾丁聚醣酶與蛋白酶之DEAE-Sepharose CL-6B層析圖譜.................................................52
圖4.11 TKU022幾丁聚醣酶之Macro-Prep DEAE Cartridge層析圖譜.................................................52
圖4.12 TKU022蛋白酶之Phenyl Sepharose層析圖譜......53
圖4.13 TKU022幾丁聚醣酶之Sephacryl S-100層析圖譜...53
圖4.14 TKU022蛋白酶之Sephacryl S-100層析圖譜.......54
圖4.15 TKU022幾丁聚醣酶SDS-PAGE之分子量分析........56
圖 4.16 分子量標準品和蛋白酶於Sephacryl S-100之層析圖譜.................................................57
圖4.17 幾丁聚醣酶與蛋白酶之(A)最適反應pH (B)pH安定性...59
圖4.18 幾丁聚醣酶與蛋白酶之(A)最適反應溫度(B)熱安定性..60
圖4.19發酵上清液還原醣量、DPPH 自由基清除能力之影響與微生物生長之變化情形.........................................68
圖4.20 TKU022所生產幾丁寡醣對於L. paracasei TKU010生長影響.....................................................68

表目錄
                                         
表 2.1 Bacillus spp.酵素於工業上的應用..................3
表2.2 幾丁質與幾丁聚醣之應用............................6
表2.3 蛋白酶的分類......................................11
表2.4 蛋白酶的應用......................................12
表 4.1 B. cereus TKU022 生產幾丁聚醣酶之較適條件........33
表 4.2 B. cereus TKU022、B. subtilis TKU007、Bacillus sp. TKU004幾丁聚醣酶較適生產條件之比較......................39
表4.3 Bacillus sp. TKU022 幾丁聚醣酶之純化總表..........55
表4.4 Bacillus sp. TKU022 蛋白酶之純化總表..............55
表4.5 TKU022 幾丁聚醣酶胜肽質譜鑑定結果.................58
表4.6 金屬離子及化學藥品對幾丁聚醣酶及蛋白酶之影響......61
表4.7界面活性劑對幾丁聚醣酶及蛋白酶之影響...............62
表4.8 TKU022幾丁聚醣酶之基質特異性......................63
表4.9 TKU022蛋白酶之基質特異性..........................63
表4.10 Bacillus spp.幾丁聚醣酶特性比較..................64
表4.11 Bacillus spp.蛋白酶特性比較......................66

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