A novel 7-β-(4-carboxybutanamido)-cephalosporanic acid acylase isolated from Pseudomonas strain C427 and its high-level production in Escherichia coli

Yoshinori Ishii, Yoshimasa Saito, Takao Fujimura, Takao Isogai, Hitoshi Kojo, Mitsuo Yamashita, Mineo Niwa, Masanobu Kohsaka

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

We cloned the gene for 7-β-(4-carboxybutanamido)-cephalosporanic acid (GL-7ACA) acylase from Pseudomonas strain C427. The DNA sequence revealed an open reading frame of 2154 bp coding for 718 amino acid residues. The deduced amino acid sequence consists of 4 structural domains: (i) a signal peptide (positions 1-27), (ii) a small subunit of the acylase (positions 28-190), designated as α, (iii) a spacer peptide (positions 191-198), (iv) a large subunit (positions 199-718), designated as β. Plasmids were constructed to direct the synthesis of the acylase in Escherichia coli and the following results were obtained. The active acylase consists of two subunits which are processed from a single precursor protein, removing the spacer peptide during processing. A proportion of active acylase is secreted into the periplasm and the remainder is retained in the cytoplasm. The amount of precursor protein accumulated in the cytoplasm is greatly reduced when plasmids for the acylase lacking the signal sequence are expressed. Therefore, processing is independent of the translocation of the gene product through the cytoplasmic membrane, in contrast to the situation for penicillin G acylase. A high level of active enzyme production was achieved with a plasmid coding for an acylase in which the amino terminal sequence (positions 1-32) of native acylase is replaced by MFPTT.

Original languageEnglish
Pages (from-to)591-597
Number of pages7
JournalJournal of Fermentation and Bioengineering
Volume77
Issue number6
DOIs
Publication statusPublished - 1994
Externally publishedYes

Fingerprint

amidase
Escherichia coli
Peptides
Amino acids
Genes
Proteins
Acids
DNA sequences
Processing
Enzymes
Plasmids
Protein Precursors
Membranes
Protein Sorting Signals
Penicillin Amidase
Amino Acids
Cephalosporins
glutarylamidocephalosporanic acid acylase

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology
  • Bioengineering

Cite this

A novel 7-β-(4-carboxybutanamido)-cephalosporanic acid acylase isolated from Pseudomonas strain C427 and its high-level production in Escherichia coli. / Ishii, Yoshinori; Saito, Yoshimasa; Fujimura, Takao; Isogai, Takao; Kojo, Hitoshi; Yamashita, Mitsuo; Niwa, Mineo; Kohsaka, Masanobu.

In: Journal of Fermentation and Bioengineering, Vol. 77, No. 6, 1994, p. 591-597.

Research output: Contribution to journalArticle

Ishii, Yoshinori ; Saito, Yoshimasa ; Fujimura, Takao ; Isogai, Takao ; Kojo, Hitoshi ; Yamashita, Mitsuo ; Niwa, Mineo ; Kohsaka, Masanobu. / A novel 7-β-(4-carboxybutanamido)-cephalosporanic acid acylase isolated from Pseudomonas strain C427 and its high-level production in Escherichia coli. In: Journal of Fermentation and Bioengineering. 1994 ; Vol. 77, No. 6. pp. 591-597.
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abstract = "We cloned the gene for 7-β-(4-carboxybutanamido)-cephalosporanic acid (GL-7ACA) acylase from Pseudomonas strain C427. The DNA sequence revealed an open reading frame of 2154 bp coding for 718 amino acid residues. The deduced amino acid sequence consists of 4 structural domains: (i) a signal peptide (positions 1-27), (ii) a small subunit of the acylase (positions 28-190), designated as α, (iii) a spacer peptide (positions 191-198), (iv) a large subunit (positions 199-718), designated as β. Plasmids were constructed to direct the synthesis of the acylase in Escherichia coli and the following results were obtained. The active acylase consists of two subunits which are processed from a single precursor protein, removing the spacer peptide during processing. A proportion of active acylase is secreted into the periplasm and the remainder is retained in the cytoplasm. The amount of precursor protein accumulated in the cytoplasm is greatly reduced when plasmids for the acylase lacking the signal sequence are expressed. Therefore, processing is independent of the translocation of the gene product through the cytoplasmic membrane, in contrast to the situation for penicillin G acylase. A high level of active enzyme production was achieved with a plasmid coding for an acylase in which the amino terminal sequence (positions 1-32) of native acylase is replaced by MFPTT.",
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AU - Fujimura, Takao

AU - Isogai, Takao

AU - Kojo, Hitoshi

AU - Yamashita, Mitsuo

AU - Niwa, Mineo

AU - Kohsaka, Masanobu

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