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.
Sareen
D
D
Institute of Microbial Technology, Sector 39A, Chandigarh 160036, India.
Sharma
R
R
Nandanwar
H S
HS
Vohra
R M
RM
D016960:Agrobacterium tumefaciens / Q000201:enzymology* / Q000254:growth & development; D000581:Amidohydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D004229:Dithiothreitol; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D007525:Isoelectric Focusing; D007700:Kinetics; D008970:Molecular Weight; D013379:Substrate Specificity
D000581:Amidohydrolases; C080477:N-carbamoyl-D-amino acid amidohydrolase; D004229:Dithiothreitol