由台灣北部土壤篩選到Bacillus cereus TKU006這株以蝦殼粉（shrimp shell powder；簡稱SSP）為主要碳源之蛋白酶和幾丁質酶生產菌。B. cereus TKU006生產蛋白酶和幾丁質酶之較適培養條件為2 %SSP、0.1 %K2HPO4、0.05 %MgSO4．7H2O，在25℃、pH 7之25 mL液體培養基，於250mL的三角錐型瓶中震盪培養2天，得最大之蛋白酶和幾丁質酶活性。B. cereus TKU006發酵上清液經硫酸銨沉澱、DEAE Sepharose、CM Sepharose、Phenyl-Sepharose、Sephacryl S-200和Sephacryl S-100可分離純化出蛋白酶和幾丁質酶。蛋白酶之回收率與比活性為0.07%與0.03 U/mg，而幾丁質酶的為11 %與0.62 U/mg。蛋白酶於經Sephacryl S-100純化步驟，活性幾已消失殆盡。蛋白質酶最適反應溫度與pH為50℃、9；熱穩定性及pH安定性分別為＜50℃與pH 3~11。幾丁質酶最適反應溫度與pH為40℃、5；熱穩定性及pH安定性分別為＜60℃與pH 3~11。幾丁質酶活性受Fe2+和Mn2+所抑制；蛋白酶活性受EDTA所抑制。幾丁質酶在丙酮、DMF或異戊醇存在下完全失活；蛋白酶在乙醇、正丁醇、二甲基甲醯胺(DMF)、異丙醇或乙酸乙酯存在下，只剩約20 %的殘餘活性。利用SDS-PAGE和膠體過濾層析法測得蛋白酶及幾丁質酶的分子量分別為39 kDa及35 kDa。

The protease-chitinase-producing by Bacillus cereus TKU006 was isolated from soil of northern of Taiwan. Shrimp shell powder (SSP) was used to be the carbon souse. The optimized culture condition for protease and chitinase production was composed of 2% SSP, 0.1 % K2HPO4 , 0.05 % MgSO4．7H2O, there have the maximum activated shaken in 250 mL Erlenmyer flask containing 25mL of above liquid medium at 25 ℃ and pH 7 for two days. The protease and chitinase of Bacillus cereus TKU006 were purified by ammonium sulfate precipitation, ionic exchange of DEAE-sepharose, CM-Sepharose, Phenyl-Sepharose, and sephadex S-200 and sephadex S-100 gel filtration . The overall activity yield of the purification protease were 0.07％,with specific protease activities of 0.03(U/mg). And, the overall activity yield of the purification chitinase were 11％,with specific chitinase activities of 0.62(U/mg). After purified by sephadex S-100, the protease was almost lost activity. The optimum pH, optimum temperature, pH stability, and thermal stability of protease were pH 9, 50℃, pH 3~11 and ＜50℃. The optimum pH, optimum temperature, pH stability, and thermal stability of chitinase were pH 5, 40℃, pH 3~11 and ＜60℃.The chitinase was inactivated by Fe2+ and Mn2+；the protease was inhibited completely by EDTA. The chitinase was inactivated by acetone, DMF and isoamyl alcohol； the protease in the presence of water-insoluble organic solvent such as ethyl alcohol, n-butyl alcohol, DMF, isopropyl alcohol and ethyl acetate, it retained only 20% of its activity. The molecular weight of the protease and the chitinase determined by SDS-PAGE and gel chromatography was approximately 39 kDa and 35 kDa, respectively.

封面內頁
簽名頁
授權書
誌謝………………………………………………………………………i
中文摘要………………………………………………………………ii
英文摘要………………………………………………………………iii
目錄………………………………………………………………………v
圖目錄……………………………………………………………………x
表目錄…………………………………………………………………xii

第一章 緒言……………………………………………………………1
第二章 文獻回顧………………………………………………………2
2.1 幾丁質與幾丁聚醣…………………………………………………2
2.1.1 幾丁質……………………………………………………………2
2.1.2 幾丁聚醣…………………………………………………………3
2.1.3 幾丁質與幾丁聚醣之應用………………………………………3
2.2 幾丁質酶……………………………………………………………8
2.3 蛋白酶……………………………………………………………10
2.4 蛋白酶於工業上之應用…………………………………………11
2.5  Bacillus cereus之特性………………………………………12
2.6 酵素純化…………………………………………………………13
2.6.1 硫酸銨沉澱……………………………………………………13
2.6.2 離子交換層析法………………………………………………13
2.6.3 疏水性層析法…………………………………………………13
2.6.4 膠體過濾層析法………………………………………………14
2.7 酵素之生化特性…………………………………………………15
2.8 酵素在有機溶劑中的應用………………………………………15
第三章 材料與方法……………………………………………………17
3.1 菌株………………………………………………………………17
3.2 培養基（液）……………………………………………………17
3.3 化學材料…………………………………………………………17
3.4 儀器………………………………………………………………18
3.5 菌株之篩選與分離純化…………………………………………19
3.6 菌種鑑定…………………………………………………………19
3.7 酵素最適培養條件探討…………………………………………20
3.7.1 不同碳源的影響………………………………………………20
3.7.2 碳源濃度的影響………………………………………………20
3.7.3 培養液pH………………………………………………………20
3.7.4 培養溫度………………………………………………………20
3.7.5 培養體積………………………………………………………21
3.7.6 培養時間………………………………………………………21
3.8 蛋白質定量分析…………………………………………………21
3.9 蛋白酶活性之測定………………………………………………22
3.10 幾丁質酶活性之測定……………………………………………22
3.11 蛋白酶酵素與幾丁質酶酵素之純化分離………………………23
3.11.1 大量培養………………………………………………………23
3.11.2 硫酸銨沉澱……………………………………………………23
3.11.3 離子交換樹脂層析法…………………………………………24
3.11.3.1 DEAE-Sepharose……………………………………………24
3.11.3.2 CM-Sepharose………………………………………………24
3.11.4 疏水性膠層析法………………………………………………25
3.11.5 膠體過濾層析法………………………………………………25
3.12 蛋白質電泳分析…………………………………………………26
3.13 蛋白酶酵素與幾丁質酶酵素之特性分析………………………28
3.13.1 最適反應溫度測定……………………………………………28
3.13.2 酵素熱安定的測定……………………………………………28
3.13.3 最適反應pH的測定……………………………………………28
3.13.4  pH安定性的測定……………………………………………29
3.13.5 金屬離子與抑制劑對酵素活性的影響………………………29
3.13.6 酵素之失活與復性……………………………………………30
3.13.7 有機溶劑對酵素之活性及安定性的影響……………………30
3.13.8 界面活性劑對酵素活性的影響………………………………30
3.14 基質特異性………………………………………………………31
第四章 結果與討論……………………………………………………32
4.1 生產蛋白酶與幾丁質酶之菌的篩選……………………………32
4.2 蛋白酶與幾丁質酶生產菌之鑑定………………………………32
4.3 較適酵素生產條件探討…………………………………………39
4.3.1 培養體積………………………………………………………39
4.3.2 碳源選擇………………………………………………………40
4.3.3 碳源濃度………………………………………………………40
4.3.4 培養pH…………………………………………………………40
4.3.5 培養溫度………………………………………………………41
4.3.6 培養時間………………………………………………………41
4.3.7 總結……………………………………………………………42
4.4 酵素之純化分離…………………………………………………51
4.4.1 大量粗酵素液之製備…………………………………………51
4.4.2 離子交換樹脂層析法…………………………………………51
4.4.3 疏水性層析法…………………………………………………53
4.4.4 膠體過濾層析法………………………………………………54
4.4.5 結果整合………………………………………………………54
4.5 酵素之生化特性分析……………………………………………64
4.5.1 酵素之最適反應溫度…………………………………………64
4.5.2 酵素之熱安定性………………………………………………64
4.5.3 酵素之最適反應pH……………………………………………64
4.5.4 酵素之pH安定性………………………………………………65
4.5.5 金屬離子及抑制劑對酵素之影響……………………………65
4.5.6 蛋白酶之復性…………………………………………………66
4.5.7 有機溶劑對酵素活性及安定性之影響………………………66
4.5.8 界面活性劑對酵素活性之影響………………………………67
4.6 純化後酵素之儲藏穩定性………………………………………81
4.7 酵素之基質特異性………………………………………………84
4.8 酵素之分子量判定………………………………………………86
第五章 結論……………………………………………………………91
參考文獻………………………………………………………………93
附錄……………………………………………………………………100

圖 目 錄
圖2.1幾丁質結構之三種型態	6
圖2.2幾丁質與幾丁聚醣之構造	7
圖4.1  Bacillus cereus TKU006之菌體內生孢子顯微照片………..34
圖4.2  Bacillus cereus TKU006之脂肪酸組成分析鑑定系統分析結                    
      果….………………..…………….…………………………...36
圖4.3 培養體積對酵素活性之影響………………………………...43
圖4.4 溶氧量對酵素活性之影響（50 rpm）……………………….44
圖4.5 溶氧量對酵素活性之影響（150 rpm）……………………..45
圖4.6 不同來源之碳源對酵素活性的影響………………………...46
圖4.7 碳源濃度對酵素活性之影響…………………………….…..47
圖4.8  pH對酵素活性之影響…………………………………...…..48
圖4.9 溫度對酵素活性之影響……………………………………...49
圖4.10 培養時間對酵素活性之影響…..…………………………...50
圖4.11 Bacillus cereus TKU006所生產酵素之純化分離流程圖…..56
圖  4.12       DEAE-Sepharose CL-6B之酵素層析圖譜………………....57
圖4.13           CM-Sepharose之酵素層析圖譜…………………………….58
圖4.14            Phenyl-Sepharose之酵素層析圖譜…………………………59
圖4.15           Sephacryl S-200之酵素層析圖譜……...…………………..60
圖4.16            Sephacryl S-100之酵素層析圖譜…………………………..61
圖4.17 酵素之最適反應溫度………………………….……………68
圖4.18 酵素之熱安定性…………………………………………….69
圖4.19 酵素之最適反應pH………………………………………...70
圖4.20 酵素之pH安定性…………………………………………..71
圖4.21 不同之有機溶劑（最終濃度20%，v/v）對蛋白酶活性之影        
       響……………………………………………………………77
圖4.22 不同之有機溶劑（最終濃度20%，v/v）對幾丁質酶活性之  
       影響…………………………………………………………78
圖4.23 不同之有機溶劑（最終濃度20%，v/v）對蛋白酶安定性之
       影響…………………………………………………………79
圖4.24 不同之有機溶劑（最終濃度20%，v/v）對幾丁質酶安定性
       之影響………………………………………………………80
圖4.25 酵素純化後在4℃下儲存安定性…………………………..82
圖4.26 酵素純化後在25℃下儲存安定性…………………………83
圖4.27 12.5%之SDS-PAGE檢測純化效果（DEAE-Sepharose後）        
       ………………………………………………………………88
圖4.28 12.5%之SDS-PAGE檢測純化效果（gel chromatography後）   
       ………………………………………………………………89
圖4.29 12.5%之SDS-PAGE檢測純化效果（不同通氣量與碳源濃度 
       之粗酵素液）………………………………………………..90

表 目 錄
表3.1 製備SDS-PAGE膠體溶液配方……………………………...27
表4.1  Bacillus cereus TKU006之16S rDNA部分鹼基序列………35
表4.2  Bacillus cereus TKU006之脂肪酸組成分析鑑定系統分析結
       果...............................................................................................37
表4.3  Bacillus cereus TKU006之VITEK鑑定套組分析系統…….38
表4.4 較適酵素生產條件…………………………………………...42
表4.5  Bacillus cereus TKU006蛋白酶純化概要…………………..62
表4.6  Bacillus cereus TKU006幾丁質酶純化概要………………..63
表4.7 各種物質對蛋白酶活性之影響……………………………...72
表4.8 各種物質對幾丁質酶活性之影響…………………………...73
表4.9 蛋白質酶之復性……………………………………………...74
表4.10 不同界面活性劑對蛋白酶活性之影響…………………….75
表4.11 不同界面活性劑對幾丁質酶活性之影響………………….76
表4.12 各種基質對蛋白酶活性之影響…………………………….85

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