Nitrile hydratase (NHase) is an important industrial enzyme used for acrylamide production from acrylonitrile hydration. The reaction kinetics of NHases in free resting cells of Rhodococcus sp. or Nocardia sp. was presented based on a bi-steady state assumption. Effects of substrate concentration

product concentration and hydration temperature were investigated experimentally and correlated by Levenberg-Marquardt method and global optimal method. The kinetics model showed that reaction would be inhibited when the concentration of product was high. When the mean concentration of substrate was lower than 10 g·L-1

the extrinsic hydration reaction rate showed a changeless base-value with respect to the altered substrate concentration

due to an instantaneous partial high concentration effect of substrate when being added into the reaction system. Effect of hydration temperature to reaction rate was rather significant. The nitrile hydratase activity at 28℃ was 3.3-fold of that at 15℃ under the same concentrations of substrate and product.