系統識別號 | U0002-0507201715005700 |
---|---|
DOI | 10.6846/TKU.2017.00153 |
論文名稱(中文) | Bacillus cereus TKU044 所生產幾丁聚醣酶之純化及定性 |
論文名稱(英文) | Purification and Characterization of Chitosanases from Bacillus cereus |
第三語言論文名稱 | |
校院名稱 | 淡江大學 |
系所名稱(中文) | 化學學系碩士班 |
系所名稱(英文) | Department of Chemistry |
外國學位學校名稱 | |
外國學位學院名稱 | |
外國學位研究所名稱 | |
學年度 | 105 |
學期 | 2 |
出版年 | 106 |
研究生(中文) | 余浩廷 |
研究生(英文) | Hao-Ting Yu |
學號 | 604180066 |
學位類別 | 碩士 |
語言別 | 繁體中文 |
第二語言別 | |
口試日期 | 2017-06-21 |
論文頁數 | 58頁 |
口試委員 |
指導教授
-
王三郎
委員 - 陳佑汲 委員 - 王全祿 |
關鍵字(中) |
Bacillus cereus 幾丁聚醣酶 烏賊軟骨 |
關鍵字(英) |
Bacillus cereus chitosanase squid pens |
第三語言關鍵字 | |
學科別分類 | |
中文摘要 |
Bacillus cereus TKU044 係篩選自花蓮土壤之幾丁聚醣酶生產菌,以含有1%烏賊軟骨粉、0.1% K2HPO4、0.05% MgSO4.7H2O之100 mL液態培養基,於37℃進行搖瓶 (150 rpm)培養2天後,可得較高幾丁聚醣酶活性 (2.66U/mL)。 培養所得發酵上清液經硫酸銨沉澱、DEAE-Sepharose 及 Macro-Prep® DEAE Cartridge 陰離子交換層析等步驟,以285.9倍之純化度以及16%之回收率,純化出經 SDS-PAGE電泳測得分子量約為43 KDa 之一種幾丁聚醣酶。此幾丁聚醣酶的最適反應pH為7,pH安定性為6~7,最適反應溫度為40℃,而熱安定性則是低於50℃,其活性會受到Na+、Fe2+、Ba2+、Cu2+、Zn2+及EDTA所抑制。TKU044粗酵素液水解水溶性幾丁聚醣所得寡糖,具有抗氧化活性。 |
英文摘要 |
The chitosanase-producing strain TKU044 was isolated from the soils of Hualien with squid pen power (SPP) as the sole carbon/nitrogen source and identified as Bacillus cereus. A chitosanase was purified from the culture supernatant by ammonium sulfate precipitation, DEAE-Sepharose and Macro-Prep® DEAE Cartridge. The molecular mass of chitosanase determined by SDS-PAGE was approximately 43 kDa. The optimum pH, optimum temperature, pH stability, and thermal stability of the TKU044 chitosanase were pH 7, 40℃, pH 6~7 and under 50℃, respectively. The chitosanase activity was inhibited by Na+, Fe2+, Ba2+, Cu2+, Zn2+ and EDTA. The oligosaccharides prepared by hydrolyzing soluble chitosan with the crude chitosanase showed antioxidant activity. |
第三語言摘要 | |
論文目次 |
目錄 中文摘要 I 英文摘要 II 目錄 III 表目錄 VIII 圖目錄 IX 縮寫表 XI 第一章 緒論 1 第二章 文獻回顧 2 2.1臘狀芽孢桿菌(Bacillus cereus)之簡介 2 2.2 幾丁質 (chitin)與幾丁聚醣 (chitosan) 3 2.3 幾丁聚醣酶 (chitosanase) 6 2.4 N-乙醯幾丁寡醣和幾丁寡醣 8 2.5 抗氧化能力 8 2.6 DPPH 自由基清除率測定 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 11 3.4 酵素生產菌株之篩選 12 3.5 革蘭氏染色 12 3.6 API (Analytical Profile Index)之鑑定 13 3.7 幾丁聚醣酶活性測定 13 3.8 蛋白酶活性測定 14 3.9 幾丁聚醣酶較適生產條件探討 14 3.9.1碳/氮源種類 14 3.9.2不同碳源濃度 15 3.9.3不同培養體積 15 3.9.4不同溫度 15 3.10 幾丁聚醣酶之分離與純化 16 3.10.1粗酵素液的製備 16 3.10.2陰離子交換層析 (一) 16 3.10.3陰離子交換層析 (二) 16 3.11 蛋白質電泳分析 17 3.12 酵素之特性分析 18 3.12.1酵素最適反應溫度 18 3.12.2酵素熱安定性 18 3.12.3酵素最適反應pH 18 3.12.4酵素pH安定性 19 3.12.5金屬離子及化學藥品對酵素活性之影響 19 3.12.6界面活性劑對酵素活性之影響 20 3.12.7酵素之基質特異性 20 3.13 幾丁聚醣酶水解基質 20 3.13.1基質水解 20 3.13.2還原糖量測定 21 3.13.3總糖含量測定 21 3.13.4幾丁寡醣之製備 22 3.14 DPPH自由基清除能力之測定 22 第四章 結果與討論 23 4.1幾丁聚醣酶生產菌種之鑑定 23 4.2 幾丁聚醣酶生產條件之探討 25 4.2.1不同碳/氮源之探討 25 4.2.2不同碳源濃度探討 26 4.2.3培養體積之探討 27 4.2.4培養溫度之探討 27 4.2.5 B. cereus TKU044生產幾丁聚醣酶與蛋白酶之探討 27 4.2.6較適培養條件結果與比較 30 4.3幾丁聚醣酶純化與分離 32 4.3.1 粗酵素液的製備 33 4.3.2離子交換樹脂層析 (一) 33 4.3.3離子交換樹脂層析 (二) 35 4.3.4分離與純化綜合結果 37 4.3.5幾丁聚醣酶分子量之測定 38 4.4 幾丁聚醣酶之特性分析 40 4.4.1 酵素最適反應溫度及熱安定性 40 4.4.2 幾丁聚醣酶之最適反應pH及pH安定性 41 4.4.3 金屬離子及化學藥品對幾丁聚醣酶活性之影響 42 4.4.4 界面活性劑對幾丁聚醣酶活性之影響 43 4.4.5 幾丁聚醣酶之基質特異性 45 4.5 幾丁聚醣酶水解基質 46 4.5.1 還原糖和總糖含量分析 46 4.6抗氧化活性測定 47 4.6.1 DPPH自由基清除能力之測定 47 第五章 結論 51 參考文獻 52 表目錄 表 2.1 B. cereus 於真菌抑制之應用 2 表 2.2幾丁質與幾丁聚醣酶應用 5 表 3.1 緩衝溶液種類 19 表 3.2 DNS試劑之組成 21 表 4.1 TKU044之16S rDNA 部分基因序列 24 表 4.2 API鑑定結果 24 表 4.3 B. cereus TKU044所生產酵素之較適培養條件 30 表 4.4 以幾丁質水產廢棄物作唯一碳/氮源的幾丁聚醣酶/蛋白酶生產菌之較適培養條件比較 31 表 4.5 B. cereus TKU044幾丁聚醣酶之純化總表 37 表 4.6金屬離子和化學藥品對B. cereus TKU044所產生的幾丁聚醣 酶之影響 42 表 4.7界面活性劑對 B. cereus TKU044所生產之幾丁聚醣酶之影 響 44 表 4.8 B. cereus TKU044所生產幾丁聚醣酶基質特異性 45 表 4.9幾丁寡醣之DPPH自由基清除率之ED50 48 圖目錄 圖 2.1 纖維素、幾丁質和幾丁聚醣之結構 4 圖 2.2 幾丁聚醣的種類和作用位置 7 圖 2.3 幾丁聚醣的作用模式 7 圖 2.4 DPPH 結構 9 圖 2.5 抗氧化物清除 DPPH 自由基之反應 9 圖 4.1 Bacillus cereus TKU044之顯微鏡圖 23 圖 4.2碳/氮源對B. cereus TKU044生產幾丁聚醣酶之影響 25 圖 4.3 SPP濃度對B. cereus TKU044生產幾丁聚醣酶之影響 26 圖 4.4培養體積對B. cereus TKU044生產幾丁聚醣酶之影響 27 圖 4.5培養溫度對B. cereus TKU044幾丁聚醣酶生產之影響 28 圖 4.6 B. cereus TKU044生產幾丁聚醣與蛋白酶之影響 29 圖 4.7幾丁聚醣酶之純化流程圖 32 圖 4.8 B. cereus TKU044 幾丁聚醣酶之DEAE-Sepharose CL-6B 層析圖譜 34 圖 4.9B. cereus TKU044幾丁聚醣酶之Macro-Prep® DEAE Cartridge層析圖譜 36 圖 4.10 B. cereus TKU044幾丁聚醣酶之純化SDS-PAGE圖 39 圖 4.11幾丁聚醣酶之最適反應溫度及熱安定性 40 圖 4.12幾丁聚醣酶之最適反應pH及pH安定性 41 圖 4.13水溶性幾丁聚醣經B. cereus TKU044粗酵素液水解不同時間所得總糖及還原糖含量 43 |
參考文獻 |
Aranaz I, Mengibar M, Harris R, Panos I, Miralles B, Acosta N, Galed G, Heras A (2009) Functional characterization of chitin and chitosan. Current Chemical Biology, 3:203-230 Arslan S, Eyi A, Küçüksarı R (2014) Toxigenic genes, spoilage potential, and antimicrobial resistance of Bacillus cereus group strains from ice cream. Anaerobe, 25:42-46 Azuma K, Izumi R, Osaki T, Ifuku S, Morimoto M, Saimoto H, Minami S, Okamoto Y (2015) Chitin, chitosan, and its derivatives for wound healing: old and new materials. Journal of Functional Biomaterials, 6:104-142 Barikani M, Oliaei E, Seddiqi H, Honarkar H (2014) Preparation and application of chitin and its derivatives: a review. Iranian Polymer Journal, 23:307-326 Bernfeld P (1955) Amylase, α and β. Methods of Enzymology, 1:149-158 Cabib E, Bowers B, Sburlati A, Silverman SJ (1988) Fungal cell wall synthesis: The construction of a biological structure. Microbiological Science, 5:370-37 Chang WT, Chen YC, Jao CL (2007) Antifungal activity and enhancement of plant growth by Bacillus cereus grown on shellfish chitin wastes. Bioresource Technology, 98:1224-1230 Cortizoa MS, Berghoff CF, Alessandrini JL (2008) Characterization of chitin from Illex argentinus squid pen. Carbohydrate Polymers, 74:10-15 Franca EF, Lins RD, Freitas LC, Straatsma TP (2008) Characterization of chitin and chitosan molecular structure in aqueous solution. Journal of Chemical Theory and Computation, 4:2141-2149 Gao XA, Ju WT, Jung WJ, Park RD (2008) Purification and characterization of chitosanase from Bacillus cereus D-11. Carbohydrate Polymers, 72:513-520 Ge L, Zhang H, Chen K, Ma L, Xu Z (2010) Effect of chitin on the antagonistic activity of Rhodotorula glutinis against Botrytis cinerea in strawberries and the possible mechanisms involved. Food Chemistry, 120:490-495 Harish Prashanth KV, Tharanathan R (2007) Chitin/chitosan: modifications and their unlimited application potential-an overview. Trends in Food Science & Technology, 18:117-131 Jiang X, Chen D, Chen L, Yang G, Zou S (2012) Purification, characterization, and action mode of a chitosanase from Streptomyces roseolus induced by chitin. Carbohydrate Research, 355:40-44 Kong CS, Kim JA, Ahn B, Byun HG, Kim SK (2010) Carboxymethylations of chitosan and chitin inhibit MMP expression and ROS scavenging in human fibrosarcoma cells. Process Biochemistry, 45:179-186 Kong M, Chen XG, Xing K, Park HJ (2010) Antimicrobial properties of chitosan and mode of action: a state of the art review. International Journal of Food Microbiology, 144:51-63 Kofuji K, Qian CJ, Nishimura M, Sugiyama I, Murata Y, Kawashima S (2005) Relationship between physicochemical characteristics and functional properties of chitosan. European Polymer Journal, 41:2784-2791 Kumar R, Majeti NV (2000) A review of chitin and chitosan applications. Reactive and Functional Polymer, 46:1-27 Larson RA (1988) The antioxidants of higher plants. Phytochemistry, 27:969-978 Larson RA (1988) The antioxidants of higher plants. Phytochemistry, 27:969-978 Li TC, Sun XJ, Bi Y, Ge YH, Wang Y (2009) Antifungal activity of chitosan on Fusarium sulphureum in relation to dry rot of potato tuber. Agricultural Sciences in China, 8:597-604 Liang TW, Chen WT, Lin ZH, Kuo YH, Nguyen AD, Pan PS, Wang SL (2016) An amphiprotic novel chitosanase from Bacillus mycoides and its application in the production of chitooligomers with their antioxidant and anti-inflammatory evaluation. International Journal of Molecular Sciences, 17:1302 Liang TW, Lo BC, Wang SL (2015) Chitinolytic bacteria-assisted conversion of squid pen and its effect on dyes and pigments adsorption. Marine Drugs, 13:4576-4593 Liang TW, Chen Y, Pan PS, Wang SL (2014) Purification of chitinase/chitosanase from Bacillus cereus and discovery of an enzyme inhibitor. International Journal of Biological Macromolecules, 63:8-14 Liu CL, Lan CY, Fu CC, Juang RS (2014) Production of hexaoligochitin from colloidal chitin using a chitinase from Aeromonas schubertii. International Journal of Biological Macromolecules, 69:59-63 Liu Y, Ren X, Jie Liang J (2015) Antibacterial modification of cellulosic materials. Bioresource Technology, 10:1964-1985 Liu YL, Jiang S, Ke ZM, Wu HS, Chi CW, Guo ZY (2009) Recombinant expression of a chitosanase and its application in chitosan oligosaccharide production. Carbohydrate Research, 344:815-819 Mahdavi B, Rahimi A (2013) Seed priming with chitosan improves the germination and growth performance of ajowan under salt stress. Eurasian Journal of Biosciences, 7:69-76 Mazumder MAJ, Zahir MH, Zaman SF (2014) Advanced Materials for Gene Delivery. Advanced Materials Research, 995:29-47 Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31:426-428 Mishra K, Ojha H, Chaudhury NK (2012) Estimation of antiradical properties of antioxidants using DPPH assay: a critical review and results. Food Chemistry,130:1036-1043 Nagahama H, Kashiki T, New N, Jayakumar R, Furuike T, Tamura H (2008) Preparation of biodegradable chitin/gelatin membranes with GlcNAc for tissue engineering applications. Carbohydrate Polymers, 73:456-463 NavinChandran M, Iyapparaj P, Moovendhan S, Ramasubburayan R, Prakash S, Immanuel G, Palavesam A (2014) Influence of probiotic bacterium Bacillus cereus isolated from the gut of wild shrimp Penaeus monodon in turn as a potent growth promoter and immune enhancer in P. monodon. Fish & Shellfish Immunology, 36:38-45 Pacheco N, Garnika-Gonzalez M, Ramirez JY, Flores-Albino B, Gimeno M, Barzana E, Shirai K (2009) Effect of temperature on chitin and astaxanthin recoveries from shrimp waste using lactic acid bacteria. Bioresource Technology, 100:2849-2854 Pan YM, Wang K, Huang SQ, Wang HS, Mu XM, He CH, Ji XW, Zhang J, Huang FJ (2008) Antioxidant activity of microwave-assisted extract of longan (Dimocarpus Longan Lour.) peel, Food Chemistry, 106:1264-1270 Park HJ, Byun YJ, Kim YT, Whiteside WS, Bae HJ (2013) Processes and applications for edible coating and film materials from agropolymers. In J. H. Han (Ed.), Innovations in Food Packaging (pp.257-275) Patil NS, Waghmare SR, Jadhav JP (2013) Purification and characterization of an extracellular antifungal chitinase from Penicillium ochrochloron MTCC 517 and its application in protoplast formation. Process Biochemistry, 48:176-183 Poulsen PHB, Moller J, Magid J (2008) Determination of a relationship between chitinase activity and microbial diversity in chitin amended compost. Bioresource Technology, 99:4355-4359 Qi H, Zhang Q, Zhao T, Chen R, Zhang H, Niu X (2005) Antoxidation activity of different sulfate content derivatives of polysaccharide extracted from Ulva pertusa (Cholrophyta) in vitro. International Journal of Biological Macromolecules, 37:195-199 Raafat D, Von Bargen K, Haas A, Sahl HG (2008) Insights into the mode of action of chitosan as an antibacterial compound. Applied and Environmental Microbiology, 74:3764-3773 Sajomsang W, Gonil P (2010) Preparation and characterization of α-chitin from cicada sloughs. Materials Science and Engineering C, 30:357-363 Schallmey M, Singh A, Ward OP (2004) Developments in the use of Bacillus species for industrial production. Canadian Journal of Microbiology, 50:1-17 Sethulekshmi C (2014) Chitin and its benefits. International Journal of Advanced Research in Biological Sciences, 1:171-175 Sinha S, Chand S, Tripathi P (2014) Microbial degradation of chitin waste for production of chitosanase and food related bioactive compounds. Applied Biochemistry and Microbiology, 50:125-133 Su C, Wang D, Yao L, Yu Z (2006) Purification, characterization, and gene cloning of a chitosanase from Bacillus species strain S65. Journal of Agricultural and Food Chemistry, 54:4208-4214 Trimukhe KD, Varma AJ (2008) Complexation of heavy metals by crosslinked chitin and its deacetylated derivatives. Carbohydrate Polymers, 71:66-73 Thadathil N, Velappan SP (2014) Recent developments in chitosanase research and its biotechnological applications: A review. Food Chemistry, 150:392-399 Velioglu YS, Mazza G, Gao L, Oomah BD (1998) Antioxidant activity and total phenolics in selected fruits, vegetables and grain products. Journal of Agricultural and Food Chemistry, 46:4113-4117 Wang CL, Su JW, Liang TW, Nguyen AD, Wang SL (2014) Production, purification and characterisation of a chitosanase from Bacillus cereus. Research on Chemical Intermediates, 40:2237-2248 Wang SL, Liang TW (2017) Microbial reclamation of squid pens and shrimp shells. Research on Chemical Intermediates, 43:3445-3462 Wang SL, Li HT, Zhang LJ, Lin ZH, Kuo YH (2016) Conversion of squid pen to homogentisic acid via Paenibacillus sp. TKU036 and the antioxidant and anti-inflammatory activities of homogentisic acid. Marine Drugs, 14:183 Wang SL, Yen YH, Tzeng GC, Chienyan H (2005) Production of antifungal materials by bioconversion of shellfish chitin wastes fermented by Pseudomonas fluorescens K-188. Enzyme and Microbial Technology, 36:49-56 Wu SJ, Pan SK, Wang HB, Wu JH (2013) Preparation of chitooligosaccharides from cicada slough and their antibacterial activity.International Journal of Biological Macromolecules, 62:348-351 Wu Y, Wang Y, Luo G, Dai Y (2009) In situ preparation of magnetic Fe3O4-chitosan nanopraticles for lipase immobilization by cross-linking and oxidation in aqueous solution. Bioresource Technology, 100: 3459-3464 Wysokowski M, Petrenko I, Stelling AL, Stawski D, Jesionowski T, Ehrlich H (2015) Poriferan chitin as a versatile template for extreme biomimetics. Polymers, 7:235-265 Younes I, Rinaudo M (2015) Chitin and chitosan preparation from marine source. Structure, properties and applications. Marine Drugs, 13:1133-1174 Zhang CM, Yu SH, Zhang LS, Zhao ZY, Dong LL (2014) Effects of several acetylated chitooligosaccharides on antioxidation, antiglycation and NO generation in erythrocyte. Bioorganic & Medicinal Chemistry Letters, 24:4053-4057 Zhang H, Li R, Liu W (2011) Effects of chitin and its derivative chitosan on postharvest decay of fruits: a review. International Journal of Molecular Sciences, 12:917-934 |
論文全文使用權限 |
如有問題,歡迎洽詢!
圖書館數位資訊組 (02)2621-5656 轉 2487 或 來信