 หัวเรื่อง:ไม่มีชื่อไทย (ชื่ออังกฤษ : Characterization of Non-Heme Haloperoxidase from Marine Red Algae, Gracilaria sp.) ผู้เขียน: Jirasak Kongkiattikajorn, Sayampong Pongdam สื่อสิ่งพิมพ์:pdf AbstractIn this study, the haloperoxidase has been isolated from the marine red algae, Gracilaria sp. and has been characterized. Haloperoxidase requires vanadium for enzyme activity. The enzyme was purified by ion-exchange and size-exclusion chromatography. The relative molecular mass was 619 kDa, as determined by gel filtration. The UV spectrum of the peroxidase did not show absorbance in the Soret band indicating a non-heme protein, like vanadium-dependent haloperoxidases. Reconstitution experiments in the presence of several metal ions showed that only vanadium completely restored the enzyme activity. The enzyme was also moderately thermostable, keeping full activity up to 50?C for 30 min. It has a pH optimum for bromoperoxidase activity at 6.0 and exhibits activities over a broad pH range from 4.5 to 8.5 It was inhibited by ethylene diamine tetra acetic acid (EDTA). Brominating activity was noticed using monochlorodimedone (MCD) as a substrate. The preliminary steady-state kinetic study was performed and apparent Michaelis–Menten kinetic parameters were determined for the substrate monochlorodimedone. The kinetic parameter has been determined from a steady-state analysis of the bromination: Km MCD = 2.74 ? 10-4M. Enzyme was inactivated by diafiltration against 100 mM citrate-phosphate pH 3.8 buffer in the presence of 1 mM EDTA, followed by a second diafiltration with 20 mM Tris-HCl (pH 8.0). Reactivation studies were carried out with ferric chloride, manganese sulphate, manganese sulphate, potassium chloride, cobolt sulphate, nigel chloride, cupric chloride, zinc chloride and sodium vanadate in the later buffer. The thermostability of the enzyme was tested by heating the extract at 30-80?C for 30 min and checking the activity (bromide assay). The enzyme from the marine red alga, Gracilaria sp. was found to be a non-heme bromoperoxidase. |