Evaluation of substrate promiscuity of an L ‐carbamoyl amino acid amidohydrolase from Geobacillus stearothermophilus CECT43

N‐carbamoyl‐amino‐acid amidohydrolase (also known as N‐carbamoylase) is the stereospecific enzyme responsible for the chirality of the D ‐ or L ‐amino acid obtained in the “Hydantoinase Process.” This process is based on the dynamic kinetic resolution of D , L ‐5‐monosubstituted hydantoins. In this work, we have demonstrated the capability of a recombinant L ‐N‐carbamoylase from the thermophilic bacterium Geobacillus stearothermophilus CECT43 (BsLcar) to hydrolyze N‐acetyl and N‐formyl‐ L ‐amino acids as well as the known N‐carbamoyl‐ L ‐amino acids, thus proving its substrate promiscuity. BsLcar showed faster hydrolysis for N‐formyl‐ L ‐amino acids than for N‐carbamoyl and N‐acetyl‐ L ‐derivatives, with a catalytic efficiency (k cat /K m ) of 8.58 × 10 5 , 1.83 × 10 4 , and 1.78 × 10 3 (s −1 M −1 ), respectively, for the three precursors of L ‐methionine. Optimum reaction conditions for BsLcar, using the three N‐substituted‐ L ‐methionine substrates, were 65°C and pH 7.5. In all three cases, the metal ions Co 2+ , Mn 2+ , and Ni 2+ greatly enhanced BsLcar activity, whereas metal‐chelating agents inhibited it, showing that BsLcar is a metalloenzyme. The Co 2+ ‐dependent activity profile of the enzyme showed no detectable inhibition at high metal ion concentrations. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010