Cloning and heterologous expression of an enantioselective amidase from Rhodococcus erythropolis strain MP50.
The gene for an enantioselective amidase was cloned from Rhodococcus erythropolis MP50, which utilizes various aromatic nitriles via a nitrile hydratase/amidase system as nitrogen sources. The gene encoded a protein of 525 amino acids which corresponded to a protein with a molecular mass of 55.5 kDa. The deduced complete amino acid sequence showed homology to other enantioselective amidases from different bacterial genera. The nucleotide sequence approximately 2.5 kb upstream and downstream of the amidase gene was determined, but no indications for a structural coupling of the amidase gene with the genes for a nitrile hydratase were found. The amidase gene was carried by an approximately 40-kb circular plasmid in R. erythropolis MP50. The amidase was heterologously expressed in Escherichia coli and shown to hydrolyze 2-phenylpropionamide, alpha-chlorophenylacetamide, and alpha-methoxyphenylacetamide with high enantioselectivity; mandeloamide and 2-methyl-3-phenylpropionamide were also converted, but only with reduced enantioselectivity. The recombinant E. coli strain which synthesized the amidase gene was shown to grow with organic amides as nitrogen sources. A comparison of the amidase activities observed with whole cells or cell extracts of the recombinant E. coli strain suggested that the transport of the amides into the cells becomes the rate-limiting step for amide hydrolysis in recombinant E. coli strains.
Trott
Sandra
S
Institut für Mikrobiologie, Universität Stuttgart, 70569 Stuttgart, Germany.
Bürger
Sibylle
S
Calaminus
Carsten
C
Stolz
Andreas
A
D000081:Acetamides / Q000378:metabolism; D000577:Amides / Q000378:metabolism; D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics*; D044482:Benzeneacetamides; D003001:Cloning, Molecular; D004926:Escherichia coli; D015870:Gene Expression; D008969:Molecular Sequence Data; D009584:Nitrogen / Q000378:metabolism; D012240:Rhodococcus / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein
D000081:Acetamides; D000577:Amides; D044482:Benzeneacetamides; C005293:2-phenylacetamide; D000581:Amidohydrolases; C019665:amidase; D009584:Nitrogen