Screening, Characterization, and Cloning of the Gene forN-Carbamyl-D-Amino acid Amidohydrolase from Thermotolerant Soil Bacteria

For the production of D-amino acids, thermotolerant bacteria producing N-carbamyl-D-amino acid amidohydrolase were isolated from soil by enrichment culture at 45 degrees C with N-carbamyl-D-amino acids as the sole nitrogen source. The enzyme activities and substrate specificities of these strains were examined by the resting cells reaction. One of the enzymes, produced by Pseudomonas sp. strain KNK003A, was purified and characterized, and the amino acids of its N-terminal region were sequenced. A DNA fragment containing the gene for a thermostable N-carbamyl-D-amino acid amidohydrolase was then cloned into Escherichia coli. The gene encoded a peptide of 312 amino acids, with a calculated molecular weight of 35,000. The similarity of the deduced amino acid sequences of this enzyme and a related enzyme from a mesophile, Agrobacterium sp. strain KNK712, was 60%. A database was searched for similar sequences.