菌株TKU027 係以蝦頭粉為唯一碳/氮源，篩選自台灣彰化土壤之幾丁質酶、幾丁聚醣酶及蛋白酶生產菌，經鑑定為 Bacillus cereus。幾丁質酶與蛋白酶較適生產條件為，分別於含有 1% 蝦頭粉末、0.1% K2HPO4、0.05% MgSO4．7H2O 之50 mL和100 mL 液態培養基（pH 6），於 37℃ 搖瓶（150 rpm）培養 2 天。將所得發酵液經離心、硫酸銨沉澱，以及DEAE-Sepharose與Sephacryl S-100 層析等步驟，純化出幾丁質酶CHI與幾丁聚醣酶CHS，經SDS-PAGE分別測定分子量為 65 kDa 及 63 kDa。CHI及CHS之最適反應pH、最適反應溫度、pH安定性、熱安定性分別為: pH 6、50℃、pH 5 – 8、< 40℃ 和 pH 6、60℃、pH 3 – 10、< 50℃。CHI 活性會受 Mn2+ 及PMSF 所抑制；CHS 活性則會受 Cu2+、Mn2+ 及EDTA 所抑制。
利用B. cereus TKU027 較適培養條件，將所得發酵上清液添加，分別可促進L. paracasei TKU012生長達 175% 、L. paracasei 12193 生長達144% 與 L. kefir 14011 生長達 132% 之影響。另外，利用製備自上清液的粗酵素液，水解水溶性幾丁聚醣後所得之寡醣，亦可促進 L. paracasei 12193 生長達 324% 與 L. kefir 14011 生長達 174% 之影響。此外利用PCR-DGGE技術，探討添加 B. cereus TKU027 與蝦頭粉，於淡水紅樹林土壤之菌相變化與生物降解，並於培養 5 週有最高總糖量（3511 &micro;g/g soil）及還原糖量（1442 &micro;g/g soil），提高總生菌數至 6 × 107 CFU/g soil。

The chitinase, chitosanase and protease-producing strain TKU027 was isolated from soil in Changhua of Taiwan with shrimp head powder as the sole carbon/nitrogen source and identified as Bacillus cereus. The optimized culture conditions for chitinase and protease production were found to be shaken at 37℃ for 2 days in 50 mL and 100 mL of medium containing 1% shrimp head powder (SHP) ,  0.1% K2HPO4 and 0.05% MgSO4．7H2O (pH 6). A chitinase CHI and a chitosanase CHS were purified from the culture supernatant by ammonium sulfate precipitation, DEAE-Sepharose and Sephacryl S-100. The molecular masses of CHI and CHS determined by SDS-PAGE were approximately 65 and 63 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of CHI and CHS were pH 6, 50℃, pH 5–8, <40℃ and pH 6, 60℃, pH 3–10, <50℃, respectively. The chitinase activity was inhibited by Mn2+ and PMSF. The chitosanase activity was inhibited by Cu2+, Mn2+ and EDTA.
The culture supernatant obtained from B. cereus TKU027 in the optimized culture conditions enhanced the growth of L. paracasei TKU012 most obviously up to 175%, followed by L. paracasei 12193 up to 144% and L. kefir 14011 up to 132%. The crude enzyme from B. cereus TKU027 culture supernatant hydrolyzed water soluble chitosan to produce N-acetyl chitooligosaccharides. The N-acetyl chitooligosaccharides also exhibited activity of enhancing growth for L. paracasei 12193 up to 324% and L. kefir 14011 up to 174%. In addition, B. cereus TKU027 and SHP were added in soil respectively to investigate the biodegradation of SHP and change of bacteria flora by PCR-DGGE analysis. The highest reducing sugar（1442 &micro;g/g soil）, total sugar（3511 &micro;g/g soil）and total viable cell counts（6 × 107 CFU/g soil）were found at the 5th week incubation with B. cereus TKU027 and SHP in Tamsui mangrove river soil.

中文摘要………………………………………………………Ⅰ
英文摘要………………………………………………………Ⅱ	
目錄……………………………………………………………Ⅳ
圖目錄…………………………………………………………Ⅷ
表目錄………………………………………………………Ⅹ

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……………………………………………………2
2.3 N-乙醯幾丁寡醣及幾丁寡醣……………………………………………………4
2.4 幾丁質酶與幾丁聚醣酶……………………………………………………6
2.5 蛋白酶……………………………………………………7
2.6 益生質(prebiotics)……………………………………………………8
2.7 DGGE 在環境微生物菌相分析……………………………………………………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 幾丁質酶之活性測定……………………………………………………13
3.6 幾丁聚醣酶之活性測定……………………………………………………13
3.7 蛋白酶活性之測定……………………………………………………14
3.8 較適培養條件探討……………………………………………………14
3.8.1 碳/氮源濃度……………………………………………………14
3.8.2 培養體積……………………………………………………15
3.8.3 培養溫度……………………………………………………15
3.8.4 培養基 pH 值……………………………………………………15
3.8.5 培養時間……………………………………………………15
3.9 幾丁質酶及幾丁聚醣酶之分離純化……………………………………………………16
3.9.1 粗酵素液之製備……………………………………………………16
3.9.2 陰離子交換層析……………………………………………………16
3.9.3 膠體過濾層析	……………………………………………………17
3.10 蛋白質電泳分析……………………………………………………17
3.11 酵素之特性分析……………………………………………………17
3.11.1 酵素最適反應溫度……………………………………………………17
3.11.2 酵素熱安定性……………………………………………………18
3.11.3 酵素最適反應 pH……………………………………………………18
3.11.4 酵素 pH 安定性……………………………………………………18
3.11.5 金屬離子及化學藥品對酵素活性之影響……………………………………………………19
3.11.6 界面活性劑對酵素活性之影響……………………………………………………19
3.11.7 酵素之基質特異性……………………………………………………20
3.12 N-乙醯幾丁寡醣製備……………………………………………………20
3.13 N-乙醯幾丁寡醣之組成分析……………………………………………………20
3.14 還原糖量之測定……………………………………………………21
3.15 總糖量之測定(H2SO4-Phenol法)……………………………………………………21
3.16 MALDI-TOF-MS……………………………………………………22
3.17 總生菌計數法……………………………………………………22
3.18 促進乳酸菌生長……………………………………………………22
3.18.1 經 B. cereus TKU027 發酵蝦頭粉……………………………………………………22
3.18.2 經 B. cereus TKU027 粗酵素液水解基質……………………………………………………23
3.19 DGGE（Denaturing Gradient Gel Electrophoresis）分析……………………………………………………23
第四章 結果與討論……………………………………………………25
4.1幾丁質酶與蛋白酶生產菌之篩選……………………………………………………25
4.1.1菌株TKU027之鑑定……………………………………………………25
4.2 酵素較適產生條件探討……………………………………………………27
4.2.1 碳/氮源濃度……………………………………………………27
4.2.2 培養液體積……………………………………………………27
4.2.3 通氣量……………………………………………………28
4.2.4培養溫度……………………………………………………28
4.2.5培養pH值……………………………………………………29
4.2.6較適培養條件探討結果……………………………………………………29
4.3 幾丁質酶及幾丁聚醣酶之分離純化……………………………………………………39
4.3.1 粗酵素液之製備……………………………………………………39
4.3.2 離子交換樹脂層析……………………………………………………39
4.3.3 膠體過濾層析	……………………………………………………40
4.3.4 綜合結果……………………………………………………40
4.4 幾丁質酶與幾丁聚醣酶之分子量測定……………………………………………………41
4.4.1 SDS-PAGE……………………………………………………41
4.4.2 幾丁質酶與幾丁聚醣酶胜肽質譜鑑定……………………………………………………41
4.4.3 綜合結果……………………………………………………41
4.5 幾丁質酶與幾丁聚醣酶之特性分析……………………………………………………49
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……………………………………………………49
4.5.2 幾丁質酶與幾丁聚醣酶之最適反應pH及pH安定性……………………………………………………49
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……………………………………………………50
4.5.4 界面活性劑對幾丁質酶與幾丁聚醣酶活性之影響……………………………………………………51
4.5.5 幾丁質酶與幾丁聚醣酶之基質特異性……………………………………………………51
4.6 經 B. cereus TKU027 發酵蝦頭粉所得幾丁寡醣組成分析……………………………………………………59
4.7 水解基質之探討……………………………………………………59
4.7.1 還原糖與總糖含量之分析……………………………………………………59
4.7.2 利用HPLC 進行幾丁寡醣組成分析……………………………………………………60
4.7.3 利用 MALDI-TOF-MS 進行幾丁寡醣組成分析……………………………………………………60
4.8 促進乳酸菌生長……………………………………………………77
4.8.1 經 B. cereus TKU027 發酵蝦頭粉之探討……………………………………………………77
4.8.2 經 B. cereus TKU027 粗酵素液水解基質之探討……………………………………………………78
4.9 土壤中的生物降解和微生物菌相變化之探討……………………………………………………85
4.9.1 總生菌數測定……………………………………………………85
4.9.2 還原糖和總糖含量之探討……………………………………………………85
4.9.3 DGGE分析……………………………………………………86
第五章 結論……………………………………………………93
參考文獻……………………………………………………94

圖目錄
圖 2.1 幾丁質、幾丁聚醣及纖維素之結構……………………………………………………5
圖 4.1 Bacillus cereus TKU027 之顯微鏡照片……………………………………………………25
圖 4.2 16S rDNA 部分鹼基序列分析及API 試驗……………………………………………………26
圖 4.3 SHP濃度對 B. cereus TKU027幾丁質酶及蛋白酶生產之影響……………………………………………………31
圖 4.4 培養液體積對B. cereus TKU027幾丁質酶及蛋白酶生產之影響……………………………………………………32
圖 4.5 通氣量對 B. cereus TKU027幾丁質酶及蛋白酶生產之影響……………………………………………………33
圖 4.6 培養溫度對B. cereus TKU027 幾丁質酶及蛋白酶生產之影響……………………………………………………34
圖 4.7 培養液 pH 對第2天 B. cereus TKU027幾丁質酶及蛋白酶生產之影響……………………………………………………35
圖 4.8 B. cereus TKU027 於含SHP培養基生產酵素之生長曲線圖……………………………………………………36
圖 4.9 B. cereus TKU027 所生產酵素之純化分離流程圖……………………………………………………43
圖 4.10 B. cereus TKU027 幾丁質酶與幾丁聚醣酶 DEAE-Sepharose CL-6B層析圖譜……………………………………………………44
圖 4.11 B. cereus TKU027 幾丁質酶之Sephacryl S-100層析圖譜……………………………………………………45
圖 4.12 B. cereus TKU027 幾丁聚醣酶之Sephacryl S-100 層析圖譜……………………………………………………45
圖 4.13 B. cereus TKU027 幾丁質酶與幾丁聚醣酶 SDS-PAGE 之分子量分析……………………………………………………47
圖 4.14 幾丁質酶與幾丁聚醣酶之 (A) 最適反應溫度 (B) 熱安定性……………………………………………………52
圖 4.15 幾丁質酶和幾丁聚醣酶之 (A) 最適反應 pH (B) pH安定性……………………………………………………53
圖 4.16利用HPLC進行幾丁寡醣組成分析 (A) 標準品 (B) B. cereus TKU027發酵含 1% SHP…………………………………………62
圖 4.17 (A) chitin β (B) 水溶性幾丁聚醣中添加 B. cereus TKU027粗酵素液於水解不同時間的回收率、還原糖及總糖含量之變化……………………………………………………63
圖 4.18 (A) chitin β (B) 水溶性幾丁聚醣經 B. cereus TKU027 粗酵素液於水解不同時間所剩的基質粉末……………………………………………………64
圖4.19 利用HPLC進行幾丁寡醣組成分析經 B. cereus TKU027 粗酵素液水解不同時間之 chitin β，(A) 2 hr ; (B) 4 hr ; (C) 6 hr ; (D) 12 hr……………………………………………………64
圖4.20 利用HPLC進行幾丁寡醣組成分析經 B. cereus TKU027 粗酵素液水解不同時間之 chitin β，(A) 24 hr；(B) 48 hr；(C) 72 hr……………………………………………………66
圖4.21 利用HPLC進行幾丁寡醣組成分析經 B. cereus TKU027 粗酵素液     水解不同時間之水溶性幾丁聚醣 (A) 2 h ; (B) 4 h; (C) 6 h ; (D) 12 h……………………………………………………67
圖4.22 利用HPLC進行幾丁寡醣組成分析經 B. cereus TKU027 粗酵素液水解不同時間之水溶性幾丁聚醣 (A) 24 h ; (B) 48 h ; (C) 72 h ……………………………………………………68
圖 4.23 chitin β 經 B. cereus TKU027 粗酵素液水解所得幾丁寡 MALDI-TOF-MS 分析圖……………………………………………………69
圖 4.24 水溶性幾丁聚醣經 B. cereus TKU027 粗酵素液水解所得幾丁寡      醣之MALDI-TOF-MS 分析圖……………………………………………………72
圖 4.25 B. cereus TKU027 發酵蝦頭粉末所得上清液及幾丁寡醣對於 (A)……………………………………………………L. paracasei TKU010 (B) L. paracasei TKU012 生長之影響……………………………………………………80
圖 4.26 B. cereus TKU027 發酵蝦頭粉末所得上清液及幾丁寡醣對於 (A) L. paracasei 12193 (B) L. kefir 14011 生長之影響……………………………………………………81
圖 4.27 (A) CHI β (B) WSC經 B. cereus TKU027 粗酵素液水解不同時間所得幾丁寡醣添加於 L. paracasei 12193 生長之影響……………………………………………………82
圖 4.28 (A) CHI β (B) WSC經 B. cereus TKU027 粗酵素液水解不同時間所得幾丁寡醣添加於 L. kefir 14011 生長之影響……………………………………………………83
圖 4.29 四種樣品於八週培養之總生菌數……………………………………………………88
圖 4.30 四種樣品於八週培養之總糖含量變化……………………………………………………89
圖 4.31 四種樣品於八週培養之還原糖含量變化……………………………………………………89
圖 4.32 16S rDNA 片段聚合酶鏈鎖反應-變性梯度凝膠電泳圖……………………………………………………90

表目錄
表 2.1 幾丁質與幾丁聚醣之應用……………………………………………………5
表 2.2 土壤中幾丁質分解酵素生產菌之碳/氮源……………………………………………………7
表 3.1 DNS 試劑組成……………………………………………………21
表 4.1 B. cereus TKU027 生產酵素之較適條件……………………………………………………37
表4.2 以含幾丁質水產廢棄物為幾丁質酶/蛋白酶生產菌唯一碳/氮源之較適生產條件比較……………………………………………………38
表 4.3 B. cereus TKU027 幾丁質酶之純化總表……………………………………………………46
表 4.4 B. cereus TKU027 幾丁聚醣酶之純化總表……………………………………………………46
表 4.5 B. cereus TKU027 幾丁質酶和幾丁聚醣酶胜肽質譜鑑定結果……………………………………………………48
表 4.6 金屬離子及化學藥品對幾丁質酶及幾丁聚醣酶活性之影響……………………………………………………54
表 4.7 界面活性劑對幾丁質酶及幾丁聚醣酶活性之影響……………………………………………………54
表 4.8 B. cereus TKU027 幾丁質酶及幾丁聚醣酶之基質特異性……………………………………………………55
表 4.9 微生物來源之幾丁質酶與幾丁聚醣酶特性比較……………………………………………………56
表 4.10 不同發酵上清液所得幾丁寡醣之組成……………………………………………………61
表 4.11 chitin β 經 B. cereus TKU027 粗酵素液水解不同時間之MALDI-  TOF-MS 幾丁寡醣組成分析……………………………………………………75
表 4.12 水溶性幾丁聚醣經 B. cereus TKU027 粗酵素液水解不同時間之 MALDI-TOF-MS 幾丁寡醣組成分析……………………………………………………76
表 4.13 B. cereus TKU027 發酵蝦頭粉末所得上清液及幾丁寡醣對於乳酸菌生長（12 與 48小時）之影響……………………………………………………79
表 4.14 CHI β和WSC經 B. cereus TKU027 粗酵素液水解不同時間所得幾丁寡醣添加於乳酸菌生長（12小時）之影響……………………………………………………84
表 4.15 CHI β和WSC經 B. cereus TKU027 粗酵素液水解不同時間所得幾丁寡醣添加於乳酸菌生長（48小時）之影響……………………………………………………84
表 4.16 四種樣品於八週培養之總生菌數……… …………………………88

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