Isolation of soil strains producing new cephalosporin acylases

Aramori, Ichiro; Fukagawa, Masao; Tsumura, Mana; Iwami, Morita; Yokota, Yoshiko; Kojo, Hitoshi; Kohsaka, Masanobu; Ueda, Yoshio; Imanaka, Hiroshi

doi:10.1016/0922-338x(91)90154-9

Cited by 33 publications

(14 citation statements)

References 9 publications

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“…Oligodeoxyribonucleotides were synthesized by a 381A or a 392 DNA synthesizer (Applied Biosystems). pCCN176-3 coding for native N176 acylase (Aramori et al, 1991), pCLaHtrp3t carrying a synthetic E. coli tryptophan promoter (Saito et al, 1987), and pTQiPAdtrp carrying a duplicated form of fd phage central terminator were used as starting plasmids. All DNA manipulations were performed according to the method of Maniatis et al (1982).…”

Section: Methodsmentioning

confidence: 99%

High‐level Production, Chemical Modification and Site‐directed Mutagenesis of a Cephalosporin C Acylase from Pseudomonas Strain N176

Ishii

Saito

Fujimura

et al. 1995

European Journal of Biochemistry

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A cephalosporin acylase from Pseudomonas strain N176 hydrolyses both 7-p-(4-~arboxybutanamido)-cephalosporanic acid (glutarylcephalosporanic acid) and cephalosporin C to 7-amino-cephalosporanic acid. However, its productivity in the original host was low and its activity against cephalosporin C was not sufficient for direct large-scale production of 7-amino-cephalosporanic acid. In order to overcome these problems, we established a high-level expression system for the acylase in Escherichia coli. Tyr270 in the acylase is reported to play an important role in the interaction with glutarylcephalosporanic acid, as determined from the reaction with an affinity-label reagent, 7p-(6-bromohexanoylamido) cephalosporanic acid [Ishii, Y., Saito, Y., Sasaki, H., Uchiyama Ferment. Bioeng. 77,, From carbamoylation with potassium cyanate and site-directed point mutagenesis of the cephalosporin C acylase, we have deduced that Tyr270 exists at a position where it can interact with a residue (possibly Ser239) corresponding to inactivation by carbamoylation. We mutated Met269 and Ala271 of the acylase and found that mutation of Met269 to Tyr or Phe caused a 1.6-fold and 1.7-fold increase, respectively, of specific activity against cephalosporin C as compared to that of the wild-type enzyme. Kinetic studies of these mutants revealed that their k,,, values increased, although their Km values against cephalosporin C were not changed. These data indicate that the mutation of Met269 near Tyr270 induces a minor conformational change to increase the stability of the activated complex with the enzyme and cephalosporin C. In particular, a mutant in which Met269 was replaced by Tyr was 2.5-fold more efficient in converting cephalosporin C to 7-aminocephalosporanic acid than the wild-type enzyme under conditions similar to those in a bio-reactor system.Keywords. Cephalosporin C ; site-directed mutagenesis ; 7-amino-cephalosporanic acid ; carbamoylation ; bioreactor.Recently, enzymic production of chemicals has been preferred to that of the chemical method because plant investment costs are lowered and the ecological problem of organic solvent disposal is eliminated. For 7-amino-cephalosporanic acid, a key intermediate in the production of cephem antibiotics, a two-step enzymic method is attractive in which cephalosporin C is converted to 7-~-(4-carboxybutanamido)cephalosporanic acid (glutarylcephalosporanic acid) with D-aminO acid oxidase and hydrogen peroxide, followed by hydrolysis of glutarylcephalosporanic acid to 7-amino-cephalosporanic acid with glutarylcephalosporanic acid acylase. Recently, a cephalosporin C acylase which directly catalyzes the hydrolysis of cephalosporin C to 7-amino-cephalosporanic acid and a-amino-adipic acid was isolated from a strain of Pseudomonas sp. N176 (Aramori et al., 1991a). It appeared to be useful for the one-step enzymic production of 7-amino-cephalosporanic acid from cephalosporin C. This acylase, designated as N176 acylase, consists of two polypeptide chains (a and p chains) which are formed from ...

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Section: Methodsmentioning

confidence: 99%

High‐level Production, Chemical Modification and Site‐directed Mutagenesis of a Cephalosporin C Acylase from Pseudomonas Strain N176

Ishii

Saito

Fujimura

et al. 1995

European Journal of Biochemistry

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“…1). Enzymes for the second step have been reported (Shibuya et al, 1981;Matsuda and Komatsu, 1985;Zhang et al, 1991;Aramori et al, 1991;Binder et al, 1993). However, the few that have been reported are intracellular and the process of cell disruption and the consequent increase in protein and nucleic acid concentration add to additional downstream…”

Section: Introductionmentioning

confidence: 99%

Cephalosporin modification: An extracellular glutaryl-7-ACA acylase from Bacillus sp.

1996

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“…B. laterosporus Jl, a cephalosporin acylase producer, was isolated from a soil sample (1 Bouillon agar plates were suspended in saline, harvested by centrifugation at 10,000 x g for 20 min and resuspended in 0.1 M phosphate buffer (pH 7.0) containing 0.1% Triton X-100 at a concentration of 7 ml/g (wet weight) of cells. The suspension was stirred with a magnetic bar for 2 h at room temperature and centrifuged at 8,000 x g for 10 min.…”

Section: Methodsmentioning

confidence: 99%

“…In our screening, we obtained a new glutaryl 7-ACA acylase producer named Jl which was identified as Bacillus laterosporus (1). We can expect some advantages of the glutaryl 7-ACA acylase from Bacillus sp.…”

mentioning

confidence: 99%

Cloning and nucleotide sequencing of a novel 7 beta-(4-carboxybutanamido)cephalosporanic acid acylase gene of Bacillus laterosporus and its expression in Escherichia coli and Bacillus subtilis

et al. 1991

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A strain of Bacillus species which produced an enzyme named glutaryl 7-ACA acylase which converts 7j1-(4-carboxybutanamido)cephalosporanic acid (glutaryl 7-ACA) to 7-amino cephalosporanic acid (7-ACA) was isolated from soil. The gene for the glutaryl 7-ACA acylase was cloned with pHSG298 in Escherichia coli JM109, and the nucleotide sequence was determined by the M13 dideoxy chain termination method. The

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Isolation of soil strains producing new cephalosporin acylases

Cited by 33 publications

References 9 publications

High‐level Production, Chemical Modification and Site‐directed Mutagenesis of a Cephalosporin C Acylase from Pseudomonas Strain N176

High‐level Production, Chemical Modification and Site‐directed Mutagenesis of a Cephalosporin C Acylase from Pseudomonas Strain N176

Cephalosporin modification: An extracellular glutaryl-7-ACA acylase from Bacillus sp.

Cloning and nucleotide sequencing of a novel 7 beta-(4-carboxybutanamido)cephalosporanic acid acylase gene of Bacillus laterosporus and its expression in Escherichia coli and Bacillus subtilis

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