The nitrile hydratase (NHase, EC 4.2.1.84) genes (alpha and beta subunit) and the corresponding activator gene from Rhodococcus equi TG328-2 were cloned and sequenced. This Fe-type NHase consists of 209 amino acids (alpha subunit, M-r 23 kDa) and 218 amino acids (beta subunit, M-r 24 kDa) and the NHase activator of 413 amino acids (M-r 46 kDa). Various combinations of promoter, NHase and activator genes were constructed to produce active NHase enzyme recombinantly in E. coli. The maximum enzyme activity (844 U/mg crude cell extract towards methacrylonitrile) was achieved when the NHase activator gene was separately co-expressed with the NHase subunit genes in E. coli BL21 (DE3). The overproduced enzyme was purified with 61% yield after French press, His-tag affinity chromatography, ultrafiltration and lyophilization and showed typical Fe-type NHase characteristics: besides aromatic and heterocyclic nitriles, aliphatic ones were hydrated preferentially. The purified enzyme had a specific activity of 6,290 U/mg towards methacrylonitrile. Enantioselectivity was observed for aromatic compounds only with E values ranging 5-17. The enzyme displayed a broad pH optimum from 6 to 8.5, was most active at 30A degrees C and showed the highest stability at 4A degrees C in thermal inactivation studies between 4A degrees C and 50A degrees C.