Abstract
Solubilization of trimethylamine-N-oxide demethylase (TMAOase) from walleye pollack myofibrillar fraction was examined by various treatments using detergents, acetone, and acid solutions. Without detergent treatments, TMAOase could be extracted from the myofibrillar fraction with 1 M NaCl at pH 4.5, and partially purified by diethylaminoethyl (DEAE)-cellulose and gel filtration chromatography. The specific activity increased 13 000-fold and the yield was 13%, as compared with those in the starting myofibrillar fraction. The enzyme was isolated by means of native PAGE. The partially purified enzyme converted TMAO stoichiometrically to demethylamine (DMA) and formaldehyde (FA), and showed the optimum pH at 7.0. The Km of the enzyme for TMAO was approximately 30 mM. The activation energy was calculated as 38.4 kJ/mol per deg in the temperature range of 0-30°C. The partially purified TMAOase required Fe2+ alone for activity. Reducing agents, such as ascorbate, cysteine, and dithiothreitol, were required to maintain the iron in the active form, Fe2+. The molecular weight of the enzyme was estimated to be 400 000 by the gel filtration chromatography and to be 25 000 on sodium dodecyl sulfate (SDS)-PAGE. The TMAOase activity was stable in the presence of SDS.
