Nitrilase of Rhodococcus rhodochrous J1 - Conversion into the active form by subunit association

Nitrilase-containing resting cells of Rhodococcus rhodochrous J1 converted acrylonitrile and benzonitrile to the corresponding acids, but the purified nitrilase hydrolyzed only benzonitrile, and not acrylonitrile. The activit y of the purified enzyme towards acrylonitrile was recovered by preincubati on with 10 mm benzonitrile, but not by preincubation with aliphatic nitrile s such as acrylonitrile. It was shown by light-scattering experiments, that preincubation with benzonitrile led to the assembly of the inactive, purif ied and homodimeric 80-kDa enzyme to its active 410-kDa aggregate, which wa s proposed to be a decamer. Furthermore, the association concomitant with t he activation was reached after dialysis of the enzyme against various salt s and organic solvents, with the highest recovery reached at 10% saturated ammonium sulfate and 50% (v/v) glycerol, and by preincubation at increased temperatures or enzyme concentrations.