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標題: Streptomyces sioyaensis endo-1,3-β-葡萄聚糖水解的純化、結晶與抑制劑評估測試
Purification, crystallization and survey of potential inhibitors for endo-1,3-β-glucanase from Streptomyces sioyaensis
作者: 汪詩祥
Wang, Shih-Hsiang
關鍵字: 葡萄聚糖水解;glucanase;抑制劑;inhibitor
出版社: 生物化學研究所
β-1,3-葡萄聚糖水解會專一性水解β-1,3鍵結的葡萄聚糖,廣泛存在自然界中植物體內主要是用來防禦真菌入侵,真菌體內是作為攻擊植物的工具,細菌體內則是水解真菌細胞壁以獲得養分。依水解葡萄聚糖的方式,可分為endo-1,3-β-葡萄聚糖水解 (EC 及exo-1,3-β-葡萄聚糖水解 (EC 兩種類型。由Streptomyces sioyaensis菌株中得到之endo-1,3-β-葡萄聚糖水解 ,N-端為葡萄聚糖水解催化區,屬於糖基水解酵素第16家族 (Glycoside Hydrolase family 16, GHF 16) 而C-端為碳水化合物結合區,屬於碳水化合物結合區第6家族 (Carbohydrate-Binding Module family 6, CBM 6)。計畫所研究為endo-1,3-β-葡萄聚糖水解 之N-端水解催化區,全長272 a.a.、分子量為29.4 kDa。此家族另一成員endo-1,3-1,4-β-葡萄聚糖水解 (EC,同樣能水解葡萄聚糖,但是水解β-1,3-1,4-鍵結的葡萄聚糖中的β-1,4鍵結。所研究蛋白質與endo-1,3-1,4-β-葡萄聚糖水解 序列相似性並不高,但實驗室初步解出之β-1,3-葡萄聚糖水解催化區結構與PDB資料庫中一些已知結構的endo-1,3-1,4-β-葡萄聚糖水解 有相似的結構外型,都有著以β-sheet為主的jellyroll-sandwich構型。透過DNS呈色活性分析的方式,找出潛在的受質以作為抑制劑,供之後浸入晶體中解析結構,研究β-1,3-葡萄聚糖水解 專一性水解β-1,3-葡萄聚糖的原因。蛋白質經滲透壓休克法萃取及純化後由OD280測量吸光值換算得到濃度為16.9 mg/mL,此外也由Microseeding方式培養出繞射品質好的晶體。選用4-Nitrophenyl β-D-glucopyranoside, 4-Nitrophenyl α-D-glucopyranoside, 4-Nitrophenyl β-D-galactopyranoside, 4-Nitrophenyl β-D- xylopyranoside四種分子模擬受質當作抑制劑,以DNS呈色活性分析法做測試,發現隨著抑制劑使用量的增加吸光值也顯著上升,當葡萄糖直接與抑制劑混合做DNS呈色活性分析測試,發現吸光值上升幅度和蛋白質、受質及抑制劑相混合後的活性測試結果相近,因此所使用的四種分子並未有顯著抑制活性的能力。雖然並未找到可作為抑制劑的分子,但計畫所使用的DNS呈色活性分析法為一合適的分析方法。

β-1,3-glucanases specially hydrolyze β-1,3-D-glucosidic linkages of β-1,3-glucans. Based on the product and the hydrolysis reactions catalyzed by the glucanase, β-1,3-glucanases are classified into exo-1,3-β-glucanase (EC and endo-1,3-β-glucanase (EC In plants, these enzymes are thought to be a type of defense system against fungal pathogens. In fungi, β-1,3-glucanases involved in fungal pathogen-plant interactions during pathogen attack. In bacteria, it is related to the assimilation of fungal cell walls as a food source. In this project, the endo-1,3-β-glucanase N-termainal catalytic domain from Streptomyces sioyaensis shares sequence similarity with bacterial endo-1,3-β- glucanases which is classified as glycosyl hydrolase family 16 (GHF 16). The protein fragment is 273 a.a. in lengh and the molecular weight is 29.4 kDa. The other homologous GHF 16 endo-1,3-1,4-β-glucanase (EC catalyze the hydrolysis of β-1,4- D- glucosidic linkages in β-D-glucans with mixed 1,3- and 1,4-linkages. The overall structure of endo-1,3-β-glucanase N-terminal catalytic domain and endo-1,3-1,4-β-glucanase share a similar jellyroll β-sandwich fold. Yet, their substrate specificity is very different. Here, we aim to investigate the substrate binding specificity of endo-1,3-β-glucanase through structural determination of the β-1,3- glucan and substrate co-crystal. The catalytic domain of endo-1,3-β-glucanase from Streptomyces sioyaensis was purified in large quantity and reproduce good quality crystals by using the vapor diffusion and microseeding techniques. We examined four derivatized mono saccharide compounds, 4-Nitrophenyl β-D- glucopyranoside, 4-Nitrophenyl α-D-glucopyranoside, 4-Nitrophenyl β-D- galactopyranoside, 4-Nitrophenyl β-D-xylopyranoside of endo-1,3-β-glucanase for potential inhibitors. Although we found all of the result did not show promising candidate for endo-1,3-β-glucanase co-crystals, the developed method can be used to screen for possible inhibitor candidates.
Appears in Collections:生物化學研究所

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