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Gene cloning, nucleotide sequencing, and purification and characterization of the Dâstereospecific aminoâacid amidase from Ochrobactrum anthropi SV3
- Komeda, Hidenobu, Asano, Yasuhisa
- European journal of biochemistry 2000 v.267 no.7 pp. 2028-2035
- Bacillus cereus, DNA, Escherichia coli, Ochrobactrum anthropi, Streptomyces, active sites, amidase, amides, amino acid sequences, ammonium sulfate, beta-lactamase, electrophoresis, fractionation, gene overexpression, genes, hydrophobicity, liquid chromatography, molecular cloning, molecular weight, pH, proteins, sequence homology, start codon
- The gene encoding the dâstereospecific aminoâacid amidase from Ochrobactrum anthropi SV3 was cloned and sequenced. Analysis of 7.3âkb of genomic DNA revealed the presence of six ORFs, one of which (daaA) encodes the dâaminoâacid amidase. This enzyme, DaaA, is composed of 363 aminoâacid residues (molecular mass 40â082âDa), and the deduced aminoâacid sequence exhibits homology to alkaline dâpeptidase from Bacillus cereus DF4âB (32% identity), ddâpeptidase from Streptomyces R61 (29% identity), and other penicillinârecognizing proteins. The DaaA protein contains the typical SXXK, YXN, and H(K)XG activeâsite motifs identified in the penicillinâbinding proteins and Î²âlactamases. The daaA gene modified in the nucleotide sequence upstream from its start codon was overexpressed in Escherichia coli. The activity of the recombinant DaaA enzyme in cellâfree extracts of E.âcoli was 33.6âUÂ·mgâ1 with dâphenylalaninamide as substrate, which is about 350âfold higher than in extracts of O.âanthropi SV3. This enzyme was purified to electrophoretic homogeneity by ammonium sulfate fractionation and three column chromatography steps. On gelâfiltration chromatography, DaaA appeared to be a monomer with a molecular mass of 40âkDa. It had maximal activity at 45âÂ°C and pHâ9.0, and was completely inactivated in the presence of phenylmethanesulfonyl fluoride or Zn2+. DaaA had hydrolyzing activity toward dâaminoâacid amides with aromatic or hydrophobic side chains, but did not act on the substrates for the ddâpeptidase and Î²âlactamase, despite their sequence similarity to DaaA. The characteristics of the recombinant DaaA are similar to those found for the native enzyme partially purified from O.âanthropi SV3.