A highly thermostable ethyl carbamate-degrading urethanase from Thermoflavimicrobium dichotomicum

The carcinogen ethyl carbamate (EC) in food is a potential threat to health. Available urethanases cannot efficiently degrade EC because of their instability or low activity under acidic conditions. Here, a novel thermostable urethanase was identified in Thermoflavimicrobium dichotomicum using a database-mining approach. The enzyme displayed exceptional thermotolerance, with an optimum temperature of 75 °C, and exhibited 58.6 % of its maximum activity at 90 °C. After incubation at temperatures below 70 °C for 30 min, 100 % activity was maintained. Following treatment at 4 °C for 6 h, it retained 59-87 % of its activity at pH 4.0-5.0, demonstrating the highest acid stability reported so far. This enzyme showed good ethanol tolerance. 80.4 % of its activity was retained after incubation in 10 % (v/v) ethanol solution at 37 °C for 1 h. The enzyme exhibited the highest EC affinity (K_m, 3.545 mM), and catalytic efficiency (k_cat/K_m, 46.75 ± 2.34 s^-1·mM^-1) at pH 4.5. After reacting with 200 U/L purified enzyme at 30 °C for 5 h, 62.4 % and 9.7 % of EC were degraded from rice wine samples with pH 6.0 and 4.5, respectively. Furthermore, the enzyme exhibited significant hydrolytic activity against the 2A carcinogen acrylamide. These findings suggest that this urethanase is a promising industrial enzyme.