ABSTRACT
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.
Subject(s)
Agrobacterium tumefaciens/enzymology , Amidohydrolases/isolation & purification , Amidohydrolases/metabolism , Agrobacterium tumefaciens/growth & development , Amidohydrolases/chemistry , Chromatography, Affinity/methods , Chromatography, Ion Exchange/methods , Dithiothreitol , Electrophoresis, Polyacrylamide Gel/methods , Isoelectric Focusing , Kinetics , Molecular Weight , Substrate SpecificityABSTRACT
A new strain Bacillus coagulans BK07 was isolated from decomposed wood-bark, based on its ability to grow on ferulic acid as a sole carbon source. This strain rapidly decarboxylated ferulic acid to 4-vinylguaiacol, which was immediately converted to vanillin and then oxidized to vanillic acid. Vanillic acid was further demethylated to protocatechuic acid. Above 95% substrate degradation was obtained within 7 h of growth on ferulic acid medium, which is the shortest period of time reported to date. The major degradation products, was isolated and identified by thin-layer chromatography, high performance liquid chromatography and 1H-nuclear magnetic resonance spectroscopy were 4-vinylguaiacol, vanillin, vanillic acid and protocatechuic acid.
