ISOLATION OF AGROBACTERIUM SP. STRAIN KNK712 THAT PRODUCES N-CARBAMYL-D-AMINO ACID AMIDOHYDROLASE, CLONING OF THE GENE FOR THIS ENZYME, ANDPROPERTIES OF THE ENZYME

Agrobacterium sp. strain KNK712, which produced N-carbamyl-D-amino aci d amidohydrolase (DCase) was isolated from soil. The bacterium had D-s pecific hydantoinase activity also. Both enzymes are suitable for use in the production of D-amino acids. The DCase gene from Agrobacterium sp. strain KNK712 was cloned into Escherichia coil. The cloned DNA fra gment contained one open reading frame, predicted to encode a peptide of 304 amino acids, with a calculated molecular weight of 34,285, The DCase gene was overexpressed under the control of the lac promoter, an d DCase accounted for 50% of the soluble protein in the cells. The enz yme was purified and some properties were investigated. Both the optim um pH and the pH that gave greatest stability were about pH 7.0. The o ptimum temperature was 65 degrees C, and the enzyme was stable at 55 d egrees C. The enzyme had strict specificity toward N-carbamyl-D-amino acids, and was inhibited by thiol reagents, Cu2+, Hg2+, Ag+, and ammon ia.