Evaluation of substrate promiscuity of an L‐carbamoyl amino acid amidohydrolase from Geobacillus stearothermophilus CECT43 |
| |
Authors: | Joaquín Pozo‐Dengra Ana Isabel Martínez‐Gómez Sergio Martínez‐Rodríguez Josefa María Clemente‐Jiménez Felipe Rodríguez‐Vico Francisco Javier Las Heras‐Vázquez |
| |
Institution: | Dept. de Química‐Física, Bioquímica y Química Inorgánica. Edificio C.I.T.E. I., Universidad de Almería, La Ca?ada de San Urbano, Almería, Spain E‐04120 |
| |
Abstract: | 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 (kcat/Km) of 8.58 × 105, 1.83 × 104, and 1.78 × 103 (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 Co2+, Mn2+, and Ni2+ greatly enhanced BsLcar activity, whereas metal‐chelating agents inhibited it, showing that BsLcar is a metalloenzyme. The Co2+‐dependent activity profile of the enzyme showed no detectable inhibition at high metal ion concentrations. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 |
| |
Keywords: | L‐amino acid production L‐N‐carbamoylase substrate promiscuity acylase process hydantoinase process formylase process |
|
|