[Objective] To increase the activity of the whole-cells, reduce the fermentation period and improve production efficiency, high molecular mass nitrile hydratase (H-NHase) from Rhodococcus rhodochrous J1 was over-expressed in Escherichia coli. [Methods] The native SD sequence upstream of the α-subunit gene nhhA and the activator gene nhhG was substituted by a stronger SD sequence, and the length of the intergenic region between the two genes was optimized. In addition, the rare codons within the gene were optimized. The modified nhhBAG was over-produced in recombinant E. coli BL21(DE3). After purification by ion-exchange chromatography, the molecular mass for the recombinant enzyme was determined by Size-exclusion chromatography. The catalytic conditions were optimized, and the process of biosynthesis of nicotinamide was simulated with continuous substrate-flow. [Results] H-NHase was successfully over-expressed in E. coli. The activity in cell-free extracts and the specific activity of the purified enzyme were 85.5±4.3 U/mg and 234.0±11.7 U/mg by using 3-Cyanopyridine as substrate, respectively. The molecular mass for recombinant H-NHase was 504.5±9.8 kD. The optimal catalysis pH, temperature, and substrate concentration using whole cells were 7.5, 25 °C, 400 mmol/L, respectively. The whole-cell activity was up to 256.0±10.4 U/mL. Under this condition, the transformation ratio by whole-cell was 99.9%. [Conclusion] The recombinant E. coli grows fast with a shorter fermentation period. The production efficiency of amides using the recombinant strain would be increased, which exhibits potential value for industrial production.