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- Title
Evaluation of substrate promiscuity of an L-carbamoyl amino acid amidohydrolase from Geobacillus stearothermophilus CECT43.
- Authors
Pozo-Dengra, Joaquín; Martínez-Gómez, Ana Isabel; Martínez-Rodríguez, Sergio; Clemente-Jiménez, Josefa María; Rodríguez-Vico, Felipe; Las Heras-Vízquez, Francisco Javier
- 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
- Subjects
AMINO acids; ENZYMES; HYDANTOIN; METHIONINE; METAL ions; ORGANIC synthesis
- Publication
Biotechnology Progress, 2010, Vol 26, Issue 4, p954
- ISSN
8756-7938
- Publication type
Article
- DOI
10.1002/btpr.410