Cloning and nucleotide sequencing of new glutaryl 7-ACA and cephalosporin C acylase genes from Pseudomonas strains

https://doi.org/10.1016/0922-338X(91)90155-AGet rights and content

Abstract

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.

References (30)

  • Y Shibuya et al.

    Isolation and properties of 7β-(4-carboxybutanamido) cephalosporanic acid acylase-producing bacteria

    Agric. Biol. Chem.

    (1981)
  • S Ichikawa et al.

    Purification and properties of 7β-(4-carboxybutanamido)-cephalosporanic acid acylase produced by mutants derived from Pseudomonas

    Agric. Biol. Chem.

    (1981)
  • K Tsuzuki et al.

    Enzymatic synthesis of 7-amino cephalosporanic acid

    Nippon Nogeikagaku Kaishi

    (1989)
  • A Matsuda et al.

    Cloning and characterization of the genes for two distinct cephalosporin acylases from a Pseudomonas strain

    J. Bacteriol.

    (1987)
  • G Schumacher et al.

    Penicillin acylase from E. coli: unique gene-protein relation

    Nucleic Acids Res.

    (1986)
  • Cited by (74)

    • Characterization of D-succinylase from Cupriavidus sp. P4-10-C and its application in D-amino acid synthesis

      2018, Journal of Bioscience and Bioengineering
      Citation Excerpt :

      GK16 (25), Pseudomonas sp. A14, and P. diminuta N176 (26). The summary of the amino acid sequence alignment between DSA and these enzymes is shown in Table 3.

    • Determination of the second autoproteolytic cleavage site of cephalosporin C acylase and the effect of deleting its flanking residues in the α-C-terminal region

      2014, Journal of Biotechnology
      Citation Excerpt :

      Recently, the CPC acylase has garnered great interest because it can directly convert CPC into 7-ACA; therefore, it possesses great potential for producing cephalosporin antibodies via a simpler process with low cost, although its catalytic performance, including its activity and product inhibition resistance, is not yet as satisfactory as that of the GL-7-ACA acylase (Sonawane, 2006). Some studies focused on identifying more active enzymes from different microbial sources, such as Pseudomonas sp. 130 (Oh et al., 2003; Pollegioni et al., 2005; Zhang et al., 2005) and Pseudomonas diminuta N176 (Aramori et al., 1991; Ishii et al., 1995; Saito et al., 1996). Other scientists engineered the enzyme either rationally or randomly to improve the catalytic specificity toward the substrate CPC by either the CPC acylase or GL-7-ACA acylase (Binder et al., 1994; Oh et al., 2003; Pollegioni et al., 2005; Shin et al., 2005; Wang et al., 2012a).

    View all citing articles on Scopus
    View full text