Skip to main content
Log in

Improvement of the catalytic properties of penicillin G acylase from Escherichia coli ATCC 11105 by selection of a new substrate specificity

  • Original Paper
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Cloned penicillin G acylase (PGA) from Escherichia coli ATCC 11105 was mutagenized in vivo using N-methyl-N′-nitrosoguanidine. Mutants of PGA were selected by their ability to allow growth of the host strain E. coli M8820 with the new substrates phenylacetyl-β-alanyl-l-proline (PhAc-βAla-Pro) phthalyl-l-leucine (Pht-Leu) or phthalylglycyl-l-proline (Pht-Gly-Pro) as sole source of proline and leucine respectively. PGA mutants were purified and immobilized onto spherical methacrylate (G-gel). The immobilized form of mutant PGA selected with (PhAc-gbAla-Pro) hydrolyzed 95% of 9 mmol penicillin G 30% faster than wild-type PGA using the same specific activities. The specific activity of the soluble enzyme was 2.7-fold, and inhibition by phenylacetic acid was halved. Immobilized PGA mutant selected with Pht-Gly-Pro hydrolyzed penicillin G 20% faster than wild-type PGA. The K m of the soluble enzyme was increased 1.7-fold. Furthermore, the latter two mutants were also 3.6-fold more stable at 45° C than wild-type PGA. The specific activity of the mutant selected with Pht-Leu was 6.3-fold lower, and inhibition by phenylacetic acid was increased 13-fold.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Adelberg EA, Mandel M, Chen GCC (1965) Optimal conditions for mutagenesis by N-methyl-N′-nitro-N-nitrosoguanidinen in Escherichia coli K12. Biochem Biophys Res Commun 18:788–795

    Google Scholar 

  • Böck A, Wirth R, Schmid G, Schumacher G, Lang G (1983) The penicillin acylase from Escherichia coli ATCC 11105 consists of two dissimilar subunits. FEMS Microbiol Lett 20:135–139

    Google Scholar 

  • Burtscher H, Schumacher G (1992) Reconstitution in vivo of penicillin G acylase activity from separately expressed subunits. Eur J Biochem 205:77–83

    Google Scholar 

  • Casabadan MJ (1975) Fusion of the E. coli lac genes to the ara promoter: a general technique using bacteriophage Mu-1 insertions. Proc Natl Acad Sci USA 72:809–813

    Google Scholar 

  • Cole M (1969a) Deacylation of acylamino compounds other than penicillins by the cell-bound penicillin acylase of Escherichia coli. Biochem J 115:741–745

    Google Scholar 

  • Cole M (1969b) Factors affecting the synthesis of ampicillin and hydroxypenicillins by the cell-bound penicillin acylase of Escherichia coli. Biochem J 115:757–764

    Google Scholar 

  • Cole M (1969c) Penicillins and other acylamino compounds synthesized by the cell-bound penicillin acylase of Escherichia coli. Biochem J 115:747–756

    Google Scholar 

  • Cole M (1969d) Hydrolysis of penicillins and related compounds by the cell-bound penicillin acylase of Escherichia coli. Biochem J 115:733–739

    Google Scholar 

  • Cole M, Savidge T, Vanderhaeghe H (1975) Penicillin acylase (assay). Methods Enzymol 43:698–705

    Google Scholar 

  • Daumy GO, Danley D, McColl AS, Apostolakos D, Vinick FJ (1985) Experimental evolution of penicillin G acylases from Escherichia coli and Proteus rettgeri. J Bacteriol 163:925–932

    Google Scholar 

  • Degnen GE, Cox EC (1974) Conditional mutator gene in Escherichia coli: isolation, mapping, and effector studies. J Bacteriol 117:477–487

    Google Scholar 

  • Dixon, M (1953) The determination of enzyme inhibitor contants. Biochem J 55:170–171

    Google Scholar 

  • Dürckheimer W, Blumbach J, Lattrell R, Scheunemann KH (1985) Neue Entwicklungen auf dem Gebiet der β-Lactam-Antibiotika. Angew Chem 97:183–205

    Google Scholar 

  • Erarslan A, Kocer H (1992) Thermal inactivation kinetics of penicillin G acylase obtained from a mutant derivative of Escherichia coli ATCC 11105. J Chem Technol Biotechnol 55:79–84

    Google Scholar 

  • Erarslan A, Terzi I, Güray A, Bermek E (1991) Purification and kinetics of penicillin G acylase from a mutant strain of Escherichia coli ATCC 11105. J Chem Technol Biotechnol 51:27–40

    Google Scholar 

  • Ferrero MA, Reglero A, Mart′inez-Blanco H, Fern′andez-Ververde M, Luengo JM (1991) In vitro synthesis of new penicillins containing keto acids as side chains. antimicrob Agents Chemother 35:1931–1932

    Google Scholar 

  • Forney LJ, Wong DCL, Ferber DM (1989) Selection of amidases with novel substrate specifities from penicillin amidase of Escherichia coli. Appl Environ Microbiol 55:2550–2555

    Google Scholar 

  • Kasche V, Haufler U, Markowsky D, Melnyk S, Zeich A, Galunsky B (1987) Penicillin amidase from E. coli-enzyme heterogenity and stability. Ann NY Acad Sci: 97–102

  • Kaufmann W (1964) The possible implication of a bacterial enzyme in the biochemical mode of action of penicillins on gram negative bacteria. Biochem Biophys Res Commun 14:458–462

    Google Scholar 

  • Kaufmann W, Bauer K (1964) Variety of substrates for a bacterial benzyl penicillin-splitting enzyme. Nature 203:520

    Google Scholar 

  • Konecny J, Schneider A, Sieber M (1983) Kinetics and mechanism of acyl transfer by penicillin acylases. Biotechnol Bioeng 25:451–467

    Google Scholar 

  • Kutzbach C, Rauenbusch E (1974) Preparation and general properties of crystalline penicillin acylase from Escherichia coli ATCC 11105. Hoppe-Seylers Z Physiol Chem 354:45–53

    Google Scholar 

  • Lowry OH, Rosebrough NJ, Farr AL, Randall RH (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    Google Scholar 

  • Lucente G, Romeo A, Rossi D (1964) Use of Escherichia coli ATCC 9637 for the asymmetric hydrolysis of amino acid derivatives. Experientia 21:317–318

    Google Scholar 

  • Mart′in J, Prieto I, Barbero JL, P′erez-Gil J, Macheño JM, Arche R (1990) Thermodynamic profiles of penicillin G hydrolysis catalyzed by wild-type and Met → Ala168 mutant penicillin acylases from Kluyevera citrophila. Biochim Biophys Acta 1037:133–139

    Google Scholar 

  • Plaskie A, Roets E, Vanderhaeghe H (1978) Substrate specificity of penicillin acylase of E. coli. J Antibiot (Tokyo) 21:783–788

    Google Scholar 

  • Prieto I, Martín J, Arche R, Fernández P, Pérez-Aranda A (1990) Penicillin acylase mutants with altered site-directed activity from Kluyvera citrophila. Appl Microbiol Biotechnol 33:553–559

    Google Scholar 

  • Schumacher G, Sizmann D, Haug H, Buckel P, Böck A (1986) Penicillin acylase from E. coli: unique gene-protein relation. Nucleic Acids Res 14:5713–5727

    Google Scholar 

  • Spector T (1978) Refinement of the Coomassie-blue method of protein quantitation. Anal Biochem 86:142–146

    Google Scholar 

  • Tischer W (1989) Enzyme an Oberflächen. Boehringer Mannheim GmbH

  • Urabe I, Nanjo H, Okada H (1973) Effect of acetylation of Bacillus subtilis α-amylase on the kinetics of heat inactivation. Biochim Biophys Acta 302:73–79

    Google Scholar 

  • Williams JA, Zuzel TJ (1985) Penicillin G acylase (EC 3.4.1.11) substrate specificity modification by in vitro mutagenesis. J Cell Biochem 9b[Suppl]:99

    Google Scholar 

  • Woolf B (1932) Cited in: Haldane JBS, Stern KG, Steinkopff (eds) Allegemeine Chemie der Enzyme. Dresden Leipzig

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Niersbach, H., Kühne, A., Tischer, W. et al. Improvement of the catalytic properties of penicillin G acylase from Escherichia coli ATCC 11105 by selection of a new substrate specificity. Appl Microbiol Biotechnol 43, 679–684 (1995). https://doi.org/10.1007/BF00164773

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00164773

Keywords

Navigation