陳昭瑩臺灣大學：植物病理與微生物學研究所郭淑慧Guo, Shu-HueiShu-HueiGuo2007-11-272018-06-292007-11-272018-06-292006http://ntur.lib.ntu.edu.tw//handle/246246/58045Bacillus circulans WL-12為一幾丁質分解細菌，可產生至少六種不同的幾丁質分解酵素，其中以幾丁質分解酵素A1 (ChiA1)水解膠狀幾丁質(colloidal chitin)之能力最高。純化ChiA1研究其對真菌之作用，發現ChiA1對Botrytis elliptica並無明顯之抑菌活性。另一方面，在拮抗菌Bacillus cereus 28-9中發現，它可產生幾丁質分解酵素CW (ChiCW)和幾丁質分解酵素CH (ChiCH)，其中ChiCW能有效地抑制B. elliptica之孢子發芽。ChiA1與ChiCW具有三個重要的功能區，分別是催化區、連結區和基質結合區，其中基質結合區在酵素與受質結合時扮演著重要的角色。為探討基質結合區對ChiA1及ChiCW不同抑菌表現之影響，本研究利用ChiA1刪除突變及ChiA1-ChiCW重組蛋白質，以膠狀幾丁質為反應受質，測定ChiA1刪除突變及重組蛋白質粗抽液之酵素活性，發現ChiA1基質結合區及連結區缺失時，會降低酵素對膠狀幾丁質的水解活性；並且發現若在ChiA1刪除突變蛋白質C端接上ChiCW基質結合區，能略為提高酵素對膠狀幾丁質的水解活性。於研究中進一步純化ChiA1(C-2F)和ChiA1(C-2F)W(B)，以大分子幾丁質為受質時，缺少基質結合區的ChiA1(C-2F)對受質的水解活性皆較ChiA1降低；而重組蛋白質ChiA1(C-2F)W(B)對不同受質的水解能力轉為類似於ChiCW對受質的水解活性。在B. elliptica孢子發芽試驗中，觀察到ChiA1(C-2F)與ChiA1一樣對B. elliptica孢子發芽並無明顯之抑制能力；而ChiA1(C-2F)W(B)則有約40%的孢子發芽抑制率，此現象與之前推測ChiCW基質結合區能有效提供幾丁質分解酵素對B. elliptica孢子發芽之抑制作用相符，因此證實ChiCW的基質結合區確實會改變ChiA1對受質的水解能力以及對真菌的作用。Bacillus circulans WL-12 and Bacillus cereus 28-9 are chitinase-producing bacteria. B. circulans WL-12 excretes at least six different chitinases, and ChiA1 is the main chitinolytic enzyme. In our previous study, ChiA1 showed very weak or no inhibitory activity on conidial germination of Botrytis elliptica. B. cereus 28-9 produces two chitinase, ChiCW and ChiCH. An in vitro assay showed that ChiCW had inhibitory activity on conidial germination of B. elliptica. ChiA1 and ChiCW have three kinds of domains, catalytic domain, fibronectin type III-like domain and substrate-binding domain. The substrate-binding domains of chitinases have been identified to play an important role in binding activities. In this study, the effect of the substrate-binding domain with the activity of ChiA1 and ChiCW toward fungi were examined. Deletion and replacement of the chitin-binding domain of ChiA1 generated three deletion mutants and three recombinants in that the chitin-binding domain of ChiA1 was replaced by the chitin-binding domain of ChiCW. Chitin-binding domain (and FN3 domain) deleted ChiA1 showed low hydrolytic activity toward colloidal chitin substrate; however, the hydrolytic activity regained by a fusion with the chitin-binding domain of ChiCW. In order to understand the effect of chitin-binding domain, ChiA1(C-2F)W(B) and ChiA1(C-2F) were purified from E. coli harboring pNTU129 and pNTU124. ChiA1(C-2F) also had very weak inhibitory activity like ChiA1, but ChiA1(C-2F)W(B) had 40% inhibitory activity on conidial germination of B. elliptica. It revealed that the chitin-binding domain of ChiCW could provide the antifungal capability to a chitinase originally lack of antifungal activity, especially toward B. elliptica.目錄 壹、中文摘要 5 貳、英文摘要 6 参、前言 7 肆、前人研究 9 一、幾丁質與幾丁質分解酵素 9 二、幾丁質分解酵素功能區之作用及相關應用 10 三、Bacillus circulans WL-12 ChiA1 和Bacillus cereus 28-9 ChiCW 12 伍、材料方法 14 一、供試菌株與質體 14 二、以B. cereus ChiCW基質結合區置換B. circulans ChiA1基質結合區建構重組蛋白質 14 2-1. 引子 14 2-2.質體DNA抽取 15 2-3. 聚合酵素連鎖反應及產物檢查 15 2-4. DNA片段回收與純化 16 2-5. 大腸桿菌勝任細胞的製備 16 2-6. DNA黏接及大腸桿菌細胞轉形 16 2-7. 轉形大腸桿菌之篩選 17 2-8. 重組蛋白質ChiA1(C-2F)W(B)和ChiA1(C-F)W(B)表現質體的建構 17 三、大腸桿菌細胞周質蛋白質抽取 18 四、ChiA1(C-2F)W(B)蛋白質純化 18 五、蛋白質及酵素活性分析 19 5-1. 蛋白質定量 19 5-2. SDS-聚丙烯醯胺膠體電泳分析 (SDS-PAGE；sodium dodecyl sulfate-polyacry- lamide gel electrophoresis) 19 5-3. 膠體中幾丁質分解酵素的偵測 20 5-4. 4-MU-(GlcNAc)3基質螢光分析 20 5-5. 還原醣測定 21 5-6. Trypan blue-glycol chitin培養基之酵素活性分析 21 六、抑菌能力分析 21 6-1. B. elliptica孢子發芽試驗 21 6-2. B. elliptica菌絲生長試驗 22 七、蛋白質與基質的結合分析 22 陸、結果 23 一、質體的建構與蛋白質表現分析 23 二、ChiA1、ChiCW、ChiA1刪除蛋白質及重組蛋白質粗抽液之酵素活性分析 23 三、E. coli DH5a(pNTU129) 之生長曲線.................................................... 26 四、ChiA1(C-2F)W(B)蛋白質純化............................................................... 27 五、ChiA1(C-2F) 蛋白質純化........................................................................ 27 六、ChiA1⣺77;ChiA1(C-2F)⣺77;ChiA1(C-2F)W(B)及ChiCW之酵素分析....... 28 柒、討論............................................................................................................... 30 捌、參考文獻 ........................................................................................................ 354011185 bytesapplication/pdfen-USBacillus circulansBacillus cereusChiA1ChiCW幾丁質分解酵素chitinasechitin-binding domainotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/58045/1/ntu-95-R93633021-1.pdf