Abstract:Ethyl carbamate(EC, or urethane), a by-product of fermented food, has been shown to be possible carcinogenic and genotoxic to human. Enzymatic removal of EC from fermented foods and beverages is an efficient and safe method. Lysinibacillus fusiformis, a strain isolated from the gastrointestinal tracts of mice, showed the activity of hydrolyzing urethane. The protein with urethanase activity was purified to electrophoretic homogeneity by ammonium sulfate precipitation, ion exchange chromatography, hydrophobic interaction chromatography and gelfiltration chromatography. The monomeric molecular weight of this urethanase was detected to be approximate 50 kDa by SDS-PAGE. Enzymatic properties analysis demonstrated that the purified enzyme was stable at temperature range 10~45 ℃, the optimal reaction conditions of it was determined to be at 35 ℃ and at pH 7.0. Using urethane as the substrate, the kinetic constants of Km and Kcat were detected to be 37.2 mmol/L and 1 176.4 s-1, respectively. The activity was inhibited by metal ions while EDTA had no effect on enzyme activity. The results showed that the urethanase was not an ion-associated enzyme. In addition, the enzyme showed tolerance to low concentration of ethanol and NaCl, indicating its potential applications in removing urethane from fermented products(rice wine and soy sauce).
