Although providing an attractive route for production of acrylamide from acrylonitrile, utilization of nitrile hydratase enzymes has been limited by the requirement for low temperature bioconversion conditions. This report summarizes a search for thermostable nitrile hydratases from aerobic moderate thermophiles screened for ability to grow on acrylonitrile at concentrations to 1% at elevated temperatures. A new isolate Bacillus sp. BR449 constitutively expresses a thermostable nitrile hydratase with properties including low substrate inhibition and broad temperature range with optimal activity at 55 degrees C. With prolonged exposure, BR449 nitrile hydratase exhibited temperature-dependent inactivation by acrylonitrile, which is attributed to alkylation of nucleophilic sites on the enzyme.