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

Abstract

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-5- sulfonic acid (HQSA), and activity is restored by the addition of divalent metal ions, such as Mn2 , Ni2 , and Co2 . 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 2- and 3-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.