Thermophilic Nitrile-degrading Enzymes
Rui A.M. PEREIRA* and Don A. COWAN
Department of Biochemistry and Molecular Biology, Gower St., London WC1E 6BT, UK
Thermophilic bacteria capable of growing on aromatic and aliphatic nitriles as sole carbon and nitrogen sources were isolated from terrestrial hydrothermal sources. Aliphatic "nitrilase" activities were constitutive while distinct aromatic "nitrilase" activities were inducible by benzonitrile but not acetonitrile. Addition of benzonitrile completely and irreversibly inhibited "acetonitrilase" activity.
Chromatographic separation revealed the presence of two nitrile degrading enzyme systems; an aliphatic nitrile-specific hydratase/arnidase complex (RCN → RCONH2 → RCOOH) and an aromatic-specific nitrilase (RCN → RCOOH). The subunit composition of the nitrile hydrarases (α2β2) was similar to mesophilic homologues. N-terminal sequence analysis of theβ subunit showed 75% homology with the Rhodococcus nitrile hydrarase, while theα subunit showed no significant homology to any published sequence.
The stabilities of the various nitrile-degrading enzymes in cell-encapsulated, crude cell extract and purified forms were compatible with the thermal origins of the source organisms (e.g., t1/2 (50℃) = 2.5h; t1/2 (37℃) = 45h), very substantially greater than the mesophilic homologues.
The nitrile hydratases showed broad specificity for short and long chain mono- and dinitriles and cyclic aliphatic nitriles. Specific activities on acrylonitrile were comparable with published data on mesophilic enzymes. Immobilised whole cell preparations were capable of converting acrylonitrile to acrylic acid at substrate concentrations up to 750 mM. Addition of low concentrations of urea substantially inhibited intracellular amidase activity resulting in accumulation of acrylamide.
