CLONING AND NUCLEOTIDE SEQUENCING OF NEW GLUTARYL 7-ACA AND CEPHALOSPORIN-C ACYLASE GENES FROM PSEUDOMONAS STRAINS

We cloned a gene for the glutaryl 7-ACA acylase from Pseudomonas sp. A14 and genes for the cephalosporin C acylases from Pseudomonas diminuta N176 and V22 in Escherichia coli and determined their nucleotide sequences. Single open reading frames were recognized, which were composed of 2457 base pairs for the A14 gene and 2322 base pairs for the N176 and V22 genes. We also purified and characterized these enzymes from recombinant E. coli strains. Each enzyme was shown to be composed of two non-identical subunits. Determination of N-terminal amino acid sequences revealed that both subunits were encoded within an open reading frame, suggesting that both subunits of these enzymes are produced from a common precursor peptide. Comparison of amino acid sequences among these enzymes and other known glutaryl 7-ACA acylase and penicillin G acylase showed that there was 10 to 25% homology distributed heterogeneously along the sequences.