Molecular and biochemical characterization of the recombinant amidase fromhyperthermophilic archaeon Sulfolobus solfataricus

We have cloned, sequenced, and overexpressed in Escherichia coli the amidas e gene from the hyperthermophilic archaeon Sulfolobus solfataricus (strain MT4). The recombinant thermophilic protein was expressed as a fusion protei n with an N-terminus six-histidine-residue affinity tag. The enzyme, the fi rst characterized archaeal amidase, is a monomer of 55,784 daltons, enantio selective, and active on 2- to 6-carbon aliphatic amides and on many aromat ic amides, over the pH range 4-9 and at temperatures from 60 degrees to 95 degreesC. The S. solfataricus amidase belongs to the class of amidases that share a characteristic signature, GGSS(S/G)GS, located in the central regi on of the protein, and which show remarkable variability in their individua l substrate specificities, can hydrolyze aliphatic or aromatic substrates, and share a large invariance of their primary structure.