Potential Application of N-Carbamoyl- -Alanine Amidohydrolase from Agrobacterium tumefaciens C58 for -Amino Acid Production

An N-carbamoyl-alanine amidohydrolase of industrial interest from Agrobacterium tumefaciens C58 ( carAt) has been characterized. carAt is most active at 30°C and pH 8.0 with N-carbamoyl-alanine as a substrate. The purified enzyme is completely inactivated by the metal-chelating agent 8-hydroxyquinoline-5sulfonic acid (HQSA), and activity is restored by the addition of divalent metal ions, such as Mn , Ni , and Co . The native enzyme is a homodimer with a molecular mass of 90 kDa from pH 5.5 to 9.0. The enzyme has a broad substrate spectrum and hydrolyzes nonsubstituted N-carbamoyl-, -, -, and -amino acids, with the greatest catalytic efficiency for N-carbamoyl-alanine. carAt also recognizes substrate analogues substituted with sulfonic and phosphonic acid groups to produce the -amino acids taurine and ciliatine, respectively. carAt is able to produce monosubstituted and -amino acids, showing better catalytic efficiency (kcat/Km) for the production of the former. For both types of monosubstituted substrates, the enzyme hydrolyzes N-carbamoyl-amino acids with a short aliphatic side chain better than those with aromatic rings. These properties make carAt an outstanding candidate for application in the biotechnology industry.