Purification and characterization of vanadium bromoperoxidase from a marine macroalga, Boodlea composita.

• Main Authors: Chun-Hui Chang, 常竣揮
• Other Authors: Hsi-Pin Lin
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/s7c44a

碩士 === 元培科技大學 === 食品科學研究所 === 99 === 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. There enzymes are responsible for theemission of volatile bromomethanes such as bromoform and dibromomethane in the marine environment. There enzymes also produce small amounts of bromophenol, halogenated terpene, bromo-topsentins, diazonamides and jasplakinolides, which often have important biological activity, such as antimicrobial properties, feeding deterrents and pharmacological properties, such as anti-inflammatory and anti-cancer activities. 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 activities with modified thymol blue colorimetric assay method. We also isolated this enzyme with ammonium sulfate precipitation, DEAE-support ion-exchange chromatography, Q- sepharose FF chromatography and Superdex 200 10/300 gel chromatography. Then the characterization (such as Km, optimum pH, optimum temperature and metal ion dependence) of this enzyme was determined. The activity of vanadium bromoperoxidase(V-BrPO) is stable between 15－45℃, and the optimal reaction temperature is 45℃.The pH optimum for V-BrPO was pH 8.0.The enzyme activity was inhibited by metal ions of Na+, K+, Mg2+, Ca2+, Zn2+, Fe2+, Fe3+, Cu2+, Co2+ and EDTA, but cannot be affected by Mn2+ and NaF. However, V5+ ions can raise the activity of vanadium bromoperoxidase remarkably. The relative molecular masses were 25 kDa for V-BrPO, as determined by gel filtration. SDS-PAGE shows that V-BrPO was 22 kDa. The following kinetic parameters have been determined from initial velocities of thymol blue (TB) halogenations: KmBr=5.6 mM, KmI=10.7 mM, KmCl=461.8 mM.