点带石斑鱼幽门垂胰蛋白酶的纯化与特性分析.DOC

點帶石斑魚幽門垂胰蛋白酶的純化與特性分析 Purification and characterization of trypsin from the pyloric caeca of orange-spotted grouper, Epinephelus coioides 研究生：徐雅麗 Shiu, Yali 指導教授：劉俊宏 Liu, Chun-Hung 【摘要】 胰蛋白酶在消化過程中扮演重要角色，主要存在於幽門垂和小腸。其除了參與蛋白質消化外，更與消化道中其他酶原 (如：胰凝乳蛋白酶原和彈性蛋白酶原)的活化作用有關。因此，胰蛋白酶的活性常被做為魚類早期發育時，消化系統分化的指標。 研究目的為藉由硫酸銨沉澱 (飽和濃度 40-60%)、陰離子交換樹脂管柱層析和benzamidine sepharose管柱，純化點帶石斑魚幽門垂的胰蛋白酶。純化後的胰蛋白酶在SDS上顯示為單一蛋白質條帶，分子量為24kDa、純度161.85倍，回收率為4%。以Να-benzoyl-L-arginine ethyl ester作為基質，胰蛋白酶在pH 8和50℃具有最高活性；溫度20-50℃和 pH 6.0-8.0時，胰蛋白酶活性呈現穩定；NaCl (0-0.6 M)濃度增加會造成胰蛋白酶活性下降。大豆胰蛋白酶抑制劑和N-ρ-tosyl-L-lysine-chloromethyl ketone對純化後的胰蛋白酶具有明顯的抑制效果，而iodoacetic acid、ethylenediaminetetraacetic acid、1-(L-trans-epoxysuccinyl-leuclamino)-4- guanidinobutane和pepstatin A則有輕微的抑制效果。定序結果顯示兩個蛋白酶序列LGEHNI和 NLDNDIML與其他魚種的胰蛋白酶具有高相似性，證實此純化蛋白為胰蛋白酶。在不同組織胰蛋白酶活性檢測顯示，幽門垂活性最高，之後依序為前腸、中腸、後腸和脾臟，而在其他組織中亦存在活性，但活性非常低。胰蛋白酶在幽門垂、前腸、中腸和後腸四個 關鍵字：點帶石斑魚、胰蛋白酶純化、特性分析、幽門垂 【Abstract】 Trypsin plays an important role in digestive process, and it exists in the pyloric caeca and intestine of fish. It not only involves in protein digestion, but also involves with the activation of the other enzyme original in the alimentary canal. Therefore, the activity of trypsin is often used as an indicator of digestive system differentiation at larval early development. In this study, trypsin was isolated from the pyloric caeca of orange-spotted grouper, Epinephelus coioides by using ammonium sulphate precipitation (40-60% saturation), two DEAE-cellulose and benzamidine sepharose chromatography. After the final step, the enzyme was purified 161.85 fold and 4% of recovery. Purified trypsin was molecular weight of 24 kDa according to SDSanalysis, and it optimal activity was observed at pH 8.0 and 50 oC with Να-benzoyl-L-arginine ethyl ester as the substrate. It was stable within the temperatures range of 20-50 oC and pH range of 6.0-8.0. Trypsin activity decreased with NaCl concentration (0-0.6 M) increasing. The activity of purified trypsin was effectively inhib