Genetic and biochemical characterization of an enantioselective amidase from Agrobacterium tumefaciens strain d3.

摘要: An enantioselective amidase was purified to homogeneity from Agrobacterium tumefaciens d3. The enzyme has a molecular mass of about 490000 Da and is composed identical subunits with 63000 Da. converted racemic 2-phenylpropionamide the corresponding S-acid an enantiomeric excess (ee) value >95% at almost 50% conversion amide. digested trypsin amino acid sequences N terminus different tryptic peptides determined. These were used clone encoding gene. Finally, 9330 bp DNA fragment sequenced gene identified. deduced sequence showed homology other amidases bacterial genera. No indications structural coupling genes for nitrile hydratase could be found on cloned fragment. encoded by approximately 500 kb circular plasmid in A. heterologously expressed Escherichia coli and, as well 2-phenylpropionamide, shown hydrolyse α-chloro- α-methoxyphenylacetamide 2-methyl-3-phenylpropionamide highly enantioselectively. Some acids within conserved region common amongst all known (‘amidase signature’) changed site-specific mutagenesis significant changes relative activities amides observed.