Purification and enzymatic properties of TMAOase from muscles of Sepia esculenta

In order to extract and purify TMAOase, acetate buffer made of NaCl (0.1 mol/L) and Tris-CH₃COOH (pH 7.0, 20 mmol/L) was used to extract from the muscles of cuttlefish.After the process of acid, alkali, dialysis and concentrated treatment, the extract was further purified by DEAE-52 Sephacel and Sephacryl S-300 chromatography. The enzymatic properties of TMAOase were also studied. The results revealed that the enzyme purified by Sephacryl S-300 was 209.54 times purer than the crude enzyme. The optimum temperature of the curde and purified enzyme was 55 and 50 °C, respectively. Moreover, their thermal stability decreased significantly over this temperature. As the temperature reached 80 °C, crude enzyme still retaimed 21.9% of its activity. Whereas purified enzyme lost its activity totally. The optimum pH of the purified enzyme was 7.0, which stayed well at this pH, but decreased in acidic and alkaline conditions. As the pH increased to 9.0,especially, the activity of crude and pure enzyme reduced to 60.7% and 20.5%, respectively. The K_m of TMAOase measured by Lineweaver-Burk double reciprocal mapping method was 22.8 mmol/L. The purity of the obtained TMAOase was proved by SDS-PAGE, with a molecular weight of 21.3 ku. Chemicals of citric acid and CaCl₂ remarkably promoted the activity of the enzyme , while H₂O₂ and Na₂S significantly inhibited TMAOase activity.