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系統識別號 U0002-1907201104421200
中文論文名稱 Bacillus cereus TKU028 所生產幾丁質酶及蛋白酶之純化、定性與應用
英文論文名稱 Purification and characterization of a chitinase and a protease from Bacillus cereus TKU028 and their applications
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 99
學期 2
出版年 100
研究生中文姓名 謝東諺
研究生英文姓名 Tung-Yen Hsieh
學號 698180055
學位類別 碩士
語文別 中文
口試日期 2011-07-12
論文頁數 96頁
口試委員 指導教授-王三郎
委員-王三郎
委員-王全祿
委員-梁慈雯
中文關鍵字 仙人掌桿菌  幾丁質酶  蛋白酶  蝦頭粉 
英文關鍵字 Bacillus cereus  chitinase  protease  shrimp head powder 
學科別分類 學科別自然科學化學
中文摘要 菌株TKU028係以蝦殼廢棄物作為唯一碳/氮源,篩選自彰化土壤之一株幾丁質酶及蛋白酶生產菌,經鑑定為Bacillus cereus。
本研究藉由B. cereus TKU028發酵蝦殼廢棄物生產幾丁質酶及蛋白酶,並探討其較適培養條件。酵素純化係利用酵素與基質專一性之特性進行。B. cereus TKU028所生產幾丁質酶及蛋白酶,經由SDS-PAGE測得分子量分別為40kDa及43kDa,而最適反應pH、最適反應溫度、pH安定性及熱安定性分別為:pH5-6, 60℃, pH5-8, ≦ 60℃;pH9, 50-60℃, pH5-9, ≦ 40℃。幾丁質酶除了受Mn2+及EDTA有約40%抑制外,皆不受金屬離子及界面活性劑所影響;蛋白酶則受到Cu2+、SDS及EDTA抑制,判定屬於金屬型蛋白酶。
B. cereus TKU028之幾丁質酶水解幾丁質所得寡糖,藉由HPLC分析得知以 (GlcNAc) 6為主;而蛋白酶因耐鹼及界面活性劑特性具潛力應用於洗滌劑添加物及去毛劑;PCR-DGGE結果顯示,B. cereus TKU028可與蝦蟹殼廢棄物應用於環境之生物復育。
英文摘要 Bacillus cereus TKU028, a chitinase and protease-producing strain, was isolated from the soil in Taiwan, by using shrimp head powder (SHP) as the sole carbon/nitrogen source. A chitinase (CHT) and a protease (PRO) were purified by chitin affinity binding and casein affinity binding, respectively. The molecular masses of CHT and PRO determined by SDS- PAGE were approximately 40 kDa and 43 kDa, respectively.
The optimum pH, optimum temperature, pH stability, and thermal stability of CHT and PRO were pH 5-6, 60 ℃, pH 5-8, and ≦60 ℃, and pH 9,50-60 ℃, pH 5-9, and ≦ 40℃, respectively. CHT was little inhibited by Mn2+ and EDTA, and PRO was inhibited by Cu2+ , EDTA, and 2 mM SDS.
The chitinase-hydrolyzed products of subtract degradation were analyzed by HPLC at 1-48 h intervals. It was observed the major products are (GlcNAc)6. In addition, B. cereus TKU028 has unlimited potential to enhance the biodegradtion of shrimp shells in the seawater containing mangrove river sediment.
論文目次 目錄
頁次
封面內頁
簽名頁
授權書
致謝
中文摘要 I
英文摘要 II
目錄 III
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 幾丁質水解酵素 5
2.3 N-乙醯幾丁寡醣及幾丁寡醣 5
2.4 蛋白質水解酵素 6
2.5 Bacillus cereus之簡介 9
2.6 酵素純化 9
第三章 材料與方法 11
3.1 實驗菌株 11
3.2 實驗材料 11
3.3 實驗儀器 13
3.4幾丁質酶生產菌之篩選 14
3.5幾丁質酶之活性測定 14
3.6 蛋白酶活性之測定 15
3.7 幾丁質酶及蛋白酶較適生產條件探討 15
3.7.1碳/氮源濃度 15
3.7.2培養溫度 16
3.7.3 培養體積 16
3.7.4培養基通氣量 16
3.7.5培養時間 17
3.8幾丁質酶及蛋白酶之分離純化 17
3.8.1粗酵素液之製備 17
3.8.2親合性吸附法 17
3.8.2.1幾丁質親合性吸附 18
3.8.2.2酪蛋白親合性吸附 18
3.8.3陰離子交換樹脂 18
3.8.4疏水性層析法 19
3.9酵素之分子量測定 19
3.9.1 蛋白質電泳分析 19
3.10幾丁質酶及蛋白酶之特性分析 20
3.10.1酵素最適反應溫度 20
3.10.2 酵素熱安定性 20
3.10.3 酵素最適反應 pH 21
3.10.4 酵素 pH 安定性 21
3.10.5 金屬離子及化學藥品對酵素活性之影響 22
3.10.6 界面活性劑對幾丁聚醣酶活性之影響 22
3.10.7 酵素之基質特異性 23
3.10.7.1幾丁質酶 23
3.10.7.2 蛋白酶 23
3.11 還原糖量之測定 24
3.11.1 Imoto and Yagishita測定法 24
3.11.2 DNS測定法 24
3.12 幾丁質酶水解基質及寡醣分析 25
3.12.1 基質之水解 25
3.12.2 N-乙醯幾丁寡醣製備 25
3.12.3 N-乙醯幾丁寡醣之組成分析 25
3.13 蝦殼廢棄物及B. cereus TKU028對紅樹林河流泥沙之生物降解及微生物族群變化 26
3.13.1 採樣及土壤處理 26
3.13.2 樣品總生菌數及糖含量之測定 26
3.13.3 土壤內微生物DNA 之萃取純化 27
3.13.4 聚合酶鏈連鎖反應(Polymerase Chain Reaction, PCR) 27
3.13.5 變性梯度凝膠電泳(Denaturing Gradient Gel Electrophoresis, DGGE) 30
3.13.6 DGGE圖譜的統計分析 32
3.13.7 DNA定序 32
第四章 結果與討論 33
4.1 幾丁質酶與蛋白酶生產菌之篩選 33
4.1.1菌種鑑定 33
4.2 幾丁質酶及蛋白酶較適生產條件探討 36
4.2.1 碳/氮源濃度 36
4.2.2 培養溫度 37
4.2.3培養體積 37
4.2.4通氣量 38
4.2.5較適培養條件探討結果 38
4.3 幾丁質酶及蛋白質酶之純化 50
4.3.1 粗酵素液之製備 50
4.3.2 幾丁質親合性吸附/酪蛋白親合性吸附 50
4.3.3 離子交換樹脂/疏水性層析 51
4.3.4 比較幾丁質親合性吸附與傳統純化方式之優劣 51
4.3.5 綜合結果 51
4.4 幾丁質酶與蛋白質酶之分子量測定 52
4.4.1 SDS-PAGE 52
4.4.2 綜合結果 53
4.4.3 幾丁質酶胜肽質譜鑑定(peptide mass mapping) 54
4.5 幾丁質酶與蛋白酶之特性分析 54
4.5.1 幾丁質酶與蛋白酶之最適反應溫度及熱安定性 54
4.5.2 幾丁質酶與蛋白酶之最適反應pH及pH安定性 56
4.5.3 金屬離子及化學藥品對幾丁質酶與蛋白酶之影響 56
4.5.4 界面活性劑對幾丁質酶與蛋白酶活性之影響 57
4.5.5 幾丁質酶與蛋白酶之基質特異性 58
4.5.5.1 幾丁質酶之基質特異性 58
4.5.5.2 蛋白酶之基質特異性 58
4.6 幾丁質酶水解寡糖及應用 77
4.6.1 幾丁質酶水解不同基質及HPLC分析水解產物 77
4.6.2 添加幾丁質廢棄物(蝦頭粉)於紅樹林河流泥沙中之菌相變化及生物降解 77
4.6.2.1 土壤培養8週後之總生菌數及含醣量 78
4.6.2.2聚合酶鏈反應-變性梯度凝膠電泳分析(PCR-DGGE) 78
第五章 結論 85
參考文獻 85



圖目錄
頁次
圖2.1 幾丁質、幾丁聚醣及纖維素之結構 3
圖2.2幾丁質與幾丁聚糖之應用 4
圖3.1 PCR反應溫度控制圖 29
圖4.1 Bacillus cereus TKU028之顯微照片 34
圖4.2 16S rDNA部分鹼基序列分析及API試驗 35
圖4.3 不同碳/氮源對B. cereus TKU028生產幾丁質酶之影響 41
圖4.4 不同碳/氮源對B. cereus TKU028生產蛋白酶之影響 42
圖4.5 培養溫度對B. cereus TKU028生產幾丁質酶之影響 43
圖4.6 培養溫度對B. cereus TKU028生產蛋白酶之影響 44
圖4.7 培養液體積對B. cereus TKU028生產幾丁質酶之影響 45
圖4.8 培養液體積對B. cereus TKU028生產蛋白酶之影響 46
圖4.9 通氣量對B. cereus TKU028生產幾丁質酶及蛋白酶之影響 47
圖4.10 B. cereus TKU028培養於SHP培養基所生產幾丁質酶與蛋白酶之生長曲線圖 48
圖4.11 B. cereus TKU028所生產幾丁質酶與蛋白酶之純化流程圖 59
圖4.12 B. cereus TKU028所生產幾丁質酶之DEAE-Sepharose CL-6B層析圖譜 60
圖4.13 B. cereus TKU028所生產蛋白酶之Butyl-S Sepharose層析圖譜 61
圖4.14 比較不同方法純化B. cereus TKU028所生產幾丁質酶之DEAE-Sepharose CL-6B層析圖譜 64
圖4.15 Bacillus cereus TKU028幾丁質酶及蛋白酶SDS-PAGE之分子量分析 66
圖4.16 幾丁質酶最適反應溫度及熱安定性 68
圖4.17 蛋白酶最適反應溫度及熱安定性 69
圖4.18 基質存在下對蛋白酶於60℃熱安定性影響 70
圖4.19 幾丁質酶之最適反應pH及pH安定性 71
圖4.20 蛋白酶之最適反應pH及pH安定性 72
圖4.21幾丁質酶水解不同基質之殘餘量照片 79
圖4.22 幾丁質酶水解不同基質之殘餘量 80
圖4.23 幾丁質酶水解不同基質之總醣及還原醣 81
圖4.24 HPCL進行水溶性幾丁質水解產物分析 82
圖4.25 HPCL進行β型幾丁質水解產物分析 83
圖4.26 HPCL進行懸浮態幾丁質水解產物分析 84
圖4.27 不同條件下培養8週之土壤總生菌數 85
圖4.28不同條件下培養8週之土壤總醣含量 85
圖4.29不同條件下培養8週之土壤還原醣含量 85
圖4.30 樣品PCR產物之電泳圖 85
圖4.31 16S rDNA之聚合酶鏈反應-變性梯度凝膠電泳分析(PCR-DGGE) 85



表目錄
頁次
表2.1 蛋白質水解酵素之分類 8
表2.2 以蝦蟹殼、烏賊軟骨為唯一碳/氮源自台灣土壤篩選出之菌株 10
表3.1 DNS試劑之組成 24
表3.2 PCR所使用之引子 28
表3.3 PCR反應試劑成分 28
表3.4 不同濃度之Acrylamide/Bis所能分離之核酸長度 30
表3.5 7%之Acrylamide/Bis變性凝膠成份 31
表3.6 10% Ammonium persulfate之成份 31
表4.1 Bacillus cereus TKU028 生產幾丁質酶及蛋白酶之較適條件 40
表4.2 B. cereus TKU028、B. cereus TKU006及Serratia sp. TKU017之幾丁質酶及蛋白酶較適生產條件比較 49
表4.3 Bacillus cereus TKU028 所生產幾丁質酶之純化總表 62
表4.4 Bacillus cereus TKU028 所生產蛋白酶之純化總表 63
表4.5 比較不同方法純化Bacillus cereus TKU028 所生產幾丁質酶之純化總表 65
表4.6 Bacillus cereus TKU028幾丁質酶胜肽質譜鑑定結果 67
表4.7 化學物質對幾丁質酶及蛋白酶之影響 73
表4.8 Bacillus cereus TKU028幾丁質酶及蛋白酶之基質特異性 74
表4.9 微生物生產之幾丁質酶及蛋白酶特性比較 75
表4.10 培養8週之總生菌數 85
表4.11不同條件下培養8週之土壤總醣含量 85
表4.12不同條件下培養8週之土壤還原醣含量 85

參考文獻 Adrangi S, Faramarzi MA, Shahverdi AR, and Sepehrizadeh Z (2010) Purification and characterization of two extracellular endochitinases from Massilia timonae. Carbohydrate Research, 345: 402-407.
Banik RM and Prakash M (2004) Laundry detergent compatibility of the alkaline protease from Bacillus cereus. Microbiological Research, 159: 135-140.
Chen H, Liu LJ, Zhu JJ, Xu B, and Li R (2010) Effect of soybean oligosaccharides on blood lipid, glucose levels and antioxidant enzymes activity in high fat rats. Food Chemistry, 119: 1633-1636.
Dahiya N, Tewari R, and Hoondal GS (2006) Biotechnological aspects of chitinolytic enzymes: a review. Applied Microbiology and Biotechnology, 71: 773–782.
Ghaouth AE, Arul J, Grenier J, and Asselin A (1992) Effect of chitosan and other polyions on chitin deacetylase in Rhizopus stolonifer. Experimental Mycology, 16: 173-177.
Ghorbel-Frikha B, Sellami-Kamoun A, Fakhfakh N, Haddar A, Manni L, and Nasri M (2005) Production and purification of a calcium-dependent protease from Bacillus cereus BG1. Journal of Industrial Microbiology and Biotechnology, 32: 186-194.
Han Y, Yang B, Zhang F, Miao X, and Li Z (2009) Characterization of antifungal chitinase from marine Streptomyces sp. DA11 associated with South China Sea sponge Craniella australiensis. Marine Biotechnology, 11:132-140.
Harish Prashanth KV and Tharanathan RN (2007) Chitin/chitosan: modifications and their unlimited application potential -- an overview. Trends in Food Science & Technology, 18: 117-131.
Hsieh YC, Wu YJ, Chiang TY, Kuo CY, Shrestha KL, Chao CF, Huang YC, Chuankhayan P, Wu WG, Li YK, and Chen CJ (2010) Crystal structures of Bacillus cereus NCTU2 chitinase complexes with chitooligomers reveal novel substrate binding for catalysis: a chitinase without chitin binding and insertion domains. The Journal of Biological Chemistry, 285: 31603-31615.
Ivanova N, Sorokin A, Anderson I, Galleron N, Candelon B, Kapatral V, Bhattacharyya A, Reznik G, Mikhailova N, Lapidus A, Chu L, Mazur M, Goltsman E, Larsen N, D'Souza M, Walunas T, Grechkin Y, Pusch G, Haselkorn R, Fonstein M, Ehrlich SD, Overbeek R, and Kyrpides N (2003) Genome sequence of Bacillus cereus and comparative analysis with Bacillus anthracis. Nature, 423: 87-91.
Jeon YJ, Park PJ, and Kim SK (2001) Antimicrobial effect of chitooligosaccharides produced by bioreactor. Carbohydrate Polymers, 44: 71-76.
Kumar MNVR (2000) A review of chitin and chitosan applications. Reactive and Functional Polymers, 46: 1-27.
Liang TW, Chen YJ, Yen YH, and Wang SL (2007) The antitumor activity of the hydrolysates of chitinous materials hydrolyzed by crude enzyme from Bacillus amyloliquefaciens V656. Process Biochemistry, 42: 527-534.
Liu D, Cai J, Xie CC, Liu C, and Chen YH (2010) Purification and partial characterization of a 36-kDa chitinase from Bacillus thuringiensis subsp. colmeri, and its biocontrol potential. Enzyme and Microbial Technology, 46: 252-256.
Ma C, Ni X, Chi Z, Ma L, and Gao L (2007) Purification and characterization of an alkaline protease from the marine yeast Aureobasidium pullulans for bioactive peptide production from different sources. Marine Biotechnology, 9: 343-351.
Neri DFM, Balcão VM, Costa RS, Rocha ICAP, Ferreira EMFC, and Torres DPM (2009) Galacto-oligosaccharides production during lactose hydrolysis by free Aspergillus oryzae β-galactosidase and immobilized on magnetic polysiloxane-polyvinyl alcohol. Food Chemistry, 115: 92-99.
Qiang X, YongLie C, and QianBing W (2009) Health benefit application of functional oligosaccharides. Carbohydrate Polymers, 77: 435-441.
Queiroz JA, Tomaz CT, and Cabral JMS (2001) Hydrophobic interaction chromatography of proteins. Journal of Biotechnology, 87: 143-159.
Schallmey M, Singh A, and Ward OP (2004) Developments in the use of Bacillus species for industrial production. Canadian Journal of Microbiology, 50: 1-17.
Shikha, Sharan A, and Darmwal NS (2007) Improved production of alkaline protease from a mutant of alkalophilic Bacillus pantotheneticus using molasses as a substrate. Bioresource Technology, 98: 881-885.
Shrinivas D and Naik GR (2011) Characterization of alkaline thermostable keratinolytic protease from thermoalkalophilic Bacillus halodurans JB 99 exhibiting dehairing activity. International Biodeterioration and Biodegradation, 65: 29-35.
Suetsuna K (2000) Antioxidant peptides from the protease digest of prawn (Penaeus japonicus) muscle. Marine Biotechnology, 2: 5-10.
Sundararajan S, Kannan CN, and Chittibabu S (2011) Alkaline protease from Bacillus cereus VITSN04: Potential application as a dehairing agent. Journal of Bioscience and Bioengineering, 111:128-133.
Tauc P, Cochet S, Algiman E, Callebaut I, Cartron JP, Brochon JC , and Bertrand O (1998) Ion-exchange chromatography of proteins: modulation of selectivity by addition of organic solvents to mobile phase-application to single-step purification of a proteinase inhibor form corn and study of the mechanism of selectivity modulation. Journal of Chromatography, 825: 17-27.
Toyokawa Y, Takahara H, Reungsang A, Fukuta M, Hachimine Y, and Tachibana S (2010) Purification and characterization of a halotolerant serine proteinase from thermotolerant Bacillus licheniformis RKK-04 isolated from Thai fish sauce. Applied Microbiology and Biotechnology, 86: 1867 - 1875.
Waghmare SR, Ghosh JS (2010) Chitobiose production by using a novel thermostable chitinase from Bacillus licheniformis strain JS isolatedfrom a mushroom bed. Carbohydrate Research, 345:2630-2635
Wang SL and Chio SH (1998) Deproteinization of shrimp and crab shell with the protease of Pseudomonas aeruginosa K-187. Enzyme and Microbial Technology, 22: 629-633.
Wang SL and Yeh PY (2006) Production of a surfactant- and solvent-stable alkaliphilic protease by bioconversion of shrimp shell wastes fermented by Bacillus subtilis TKU007. Process Biochemistry, 41: 1545-1552.
Wang SL, Kao TY, Wang CL, Yen YH, Chern MK, and Chen YH (2006a) A solvent stable metalloprotease produced by Bacillus sp. TKU004 and its application in the deproteinization of squid pen for β-chitin preparation. Enzyme and Microbial Technology, 39: 724-731.
Wang SL, Kao TY, Wang CL, Yen YH, Chern MK, and Chen YH (2006b) A solvent stable metalloprotease produced by Bacillus sp. TKU004 and its application in the deproteinization of squid pen for β-chitin preparation. Enzyme and Microbial Technology, 39: 724-731.
Wang SL and Yeh PY (2008) Purification and characterization of a chitosanase from a nattokinase producing strain Bacillus subtilis TKU007. Process Biochemistry, 43: 132-138.
Wang SL, Chen SJ, and Wang CL (2008a) Purification and characterization of chitinases and chitosanases from a new species strain Pseudomonas sp. TKU015 using shrimp shells as a substrate. Carbohydrate Research, 343: 1171-1179.
Wang SL, Lin HT, Liang TW, Chen YJ, Yen YH, and Guo SP (2008b) Reclamation of chitinous materials by bromelain for the preparation of antitumor and antifungal materials. Bioresource Technology, 99: 4386-4393.
Wang SL, Chao CH, Liang TW, and Chen CC (2009a) Purification and characterization of protease and chitinase from Bacillus cereus TKU006 and conversion of marine wastes by these enzymes. Marine Biotechnology, 11: 334-344.
Wang SL, Lin CL, Liang TW, Liu KC, and Kuo YH (2009b) Conversion of squid pen by Serratia ureilytica for the production of enzymes and antioxidants. Bioressource Technology, 100: 316-323.
Wang SL, Wu PC, and Liang TW (2009c) Utilization of squid pen for the efficient production of chitosanase and antioxidants through prolonged autoclave treatment. Carbohydrate Research, 344: 979-984.
Wang SL, Chang TJ, and Liang TW (2010a) Conversion and degradation of shellfish wastes by Serratia sp. TKU016 fermentation for the production of enzymes and bioactive materials. Biodegradation, 21: 321-333.
Wang SL, Li JY, and Liang TW (2010b) Conversion of shrimp shell by using Serratia sp. TKU017 fermentation for the production of enzymes and antioxidants. Journal of Microbiology and Biotechnology, 20: 117-126.
Wang SL, Liang TW, Lin BS, Wang CL, Wu PC, and Liu JR (2010c) Purification and characterization of chitinase from a new species strain Pseudomonas sp. TKU008. Journal of Microbiology and Biotechnology, 20: 1001-1005.
Wang SL, Liang TW, and Yen YH (2011) Bioconversion of chitin-containing wastes for the production of enzymes andbioactive materials. Carbohydrate Polymers, 84: 732-742.
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