台灣沿海海藻中偏釩溴過氧化酵素活性調查與純化

The Screening and Purification of Vanadium Bromoperoxidase from Taiwanese Marine Macroalga

很多種類的海藻皆含有多種鹵化次級代謝產物，其中的鹵化過氧化酵素（haloperoxidase），被認為在鹵化物以及過氧化氫的鹵化合成上扮演關鍵性的角色，這些酵素在海洋中具有排放揮發性溴甲烷，例如三溴甲烷（bromoform）和二溴甲烷（dibromomethane）的能力，並具有重要的生物活性。偏釩溴過氧化酵素（Vanadium bromoperoxidase）也具有上述特性，其主要來源為海藻。本研究中篩選台灣東北角、墾丁以及澎湖沿海的十五種海藻，以等量的0.1 M磷酸緩衝液（pH 8.0）粗萃取其酵素，再以改良式百里酚藍（Thymol blue）色度分析法測定偏釩溴過氧化酵素（V-BrPO）活性，發現以布氏藻（Boodlea composita） 的酵素活性最高。進一步以硫酸銨劃分、DEAE -sepharose離子交換層析、Q-sepharose離子交換層析以及Superdex 200 凝膠層析等方法，將偏釩溴過氧化酵素從布氏藻中分離出來並加以純化，可有效的將酵素純度提高300倍，經由Superdex 200 凝膠層析以及SDS-PAGE沖提後計算相對分子量約22 kDa。本研究證明台灣沿海海藻中偏釩溴過氧化酵素因種類而有不同活性，且本文所採用的方法可以有效分離純化酵素。

Many species of marine macroalgae contain a variety of halogenated secondary metabolites. Haloperoxidase, a halogenating enzyme, is considered to play a key role in their syntheses in the presence of halides and hydrogen peroxide. These enzymes are responsible for theemission of volatile bromomethanes such as bromoform and dibromomethane in the marine environment. Haloperoxidaseproduce small amounts of bromophenol, halogenated terpene, bromo-topsentins, diazonamides and jasplakinolides, which often have important biological activity. In this study, we collected 15 species of marine algae from Taiwan's northeast, Kenting and Penghu sea area for screening the vanadium-haloperoxidase enzyme (V-BrPO)activities with modified thymol blue colorimetric assay method.It was showed that Boodlea compositapossessed the vanadium-haloperoxidase enzyme activity.We also purifiedthis enzyme from B.composita with ammonium sulfate precipitation, DEAE-cellulose ion-exchange chromatography, Q-sepharose FF chromatography and Superdex 200 10/300 gel chromatography. The relative molecular masses of V-BrPO was22kDa separated fromgel filtrationand SDS-PAGE.