Abstract
The characteristics and amino acid sequence of an enantiomer-selective amidase active on R-(−)-2-(3′-benzoyl phenyl)propionamide [ R-(−)-ketoprofen amide] purified previously from Comamonas acidovorans KPO-2771-4 were studied. On gel filtration, this amidase appeared to be a monomer with a molecular mass of 55 kDa. It had maximal activity at 35°C and at pH values from 8.5 to 10.0. Except for Cu 2+, Zn 2+, Pb 2+, and p-chloromercuribenzoate, it was not affected by chelating reagents, carbonyl reagents, reductants, most metal ions, or thiol reagents. The amidase had hydrolyzing activity against a broad range of aliphatic, aromatic, and amino acid amides, evidence that it is a wide-spectrum amidase. Oligonucleotide probes designed from limited peptide sequence information were used to clone the corresponding gene. The nucleotide-determined sequence indicated that the amidase consists of 473 amino acids ( M w 50,464 Da). Significant homologies were found at the amino acid level between the R-enantiomer-selective amidase of C. acidovorans KPO-2771-4 and the S-enantiomer-selective enzymes from Rhodococcus sp., Brevibacterium sp., and Pseudomonas sp. Only 39% homology was conserved in the consensus region of these amidases.
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