Two-step purification of D(-)-specific carbamoylase from Agrobacterium tumefaciens AM 10

A simple, economical and rapid affinity chromatography procedure with red dye as a ligand has been described for the two-step purification of a relatively thermostable D(-)-carbamoylase from the cell-free extract of Agrobacterium tumefaciens AM 10. The enzyme was purified 232-fold to homogeneity with a recovery of 30% in the presence of 2 mM dithiothreitol. The specific activity of the enzyme was 7.88 U/mg protein. The enzyme is a dimer with a native molecular mass of 67 kDa and a subunit relative molecular mass of 38 kDa. The isoelectric point of the enzyme was found to be 5.83. The K-m values for N-carbamoyl-DL-methionine and N-carbamoyl-D-phenylglycine were 3.84 and 5.0 mM, respectively. (C) 2001 Academic Press.