Skip to main content
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1997 Sep;63(9):3412–3418. doi: 10.1128/aem.63.9.3412-3418.1997

Molecular cloning and analysis of the gene encoding the thermostable penicillin G acylase from Alcaligenes faecalis.

R M Verhaert 1, A M Riemens 1, J M van der Laan 1, J van Duin 1, W J Quax 1
PMCID: PMC168649  PMID: 9292993

Abstract

Alcaligenes faecalis penicillin G acylase is more stable than the Escherichia coli enzyme. The activity of the A. faecalis enzyme was not affected by incubation at 50 degrees C for 20 min, whereas more than 50% of the E. coli enzyme was irreversibly inactivated by the same treatment. To study the molecular basis of this higher stability, the A. faecalis enzyme was isolated and its gene was cloned and sequenced. The gene encodes a polypeptide that is characteristic of periplasmic penicillin G acylase (signal peptide-alpha subunit-spacer-beta subunit). Purification, N-terminal amino acid analysis, and molecular mass determination of the penicillin G acylase showed that the alpha and beta subunits have molecular masses of 23.0 and 62.7 kDa, respectively. The length of the spacer is 37 amino acids. Amino acid sequence alignment demonstrated significant homology with the penicillin G acylase from E. coli A unique feature of the A. faecalis enzyme is the presence of two cysteines that form a disulfide bridge. The stability of the A. faecalis penicillin G acylase, but not that of the E. coli enzyme, which has no cysteines, was decreased by a reductant. Thus, the improved thermostability is attributed to the presence of the disulfide bridge.

Full Text

The Full Text of this article is available as a PDF (668.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Balasingham K., Warburton D., Dunnill P., Lilly M. D. The isolation and kinetics of penicillin amidase from Escherichia coli. Biochim Biophys Acta. 1972 Jul 13;276(1):250–256. doi: 10.1016/0005-2744(72)90027-7. [DOI] [PubMed] [Google Scholar]
  2. Barbero J. L., Buesa J. M., González de Buitrago G., Méndez E., Péz-Aranda A., García J. L. Complete nucleotide sequence of the penicillin acylase gene from Kluyvera citrophila. Gene. 1986;49(1):69–80. doi: 10.1016/0378-1119(86)90386-0. [DOI] [PubMed] [Google Scholar]
  3. Brandl E. Zur Kenntnis der Penicillinamidase. Hoppe Seylers Z Physiol Chem. 1965;342(1):86–92. [PubMed] [Google Scholar]
  4. Brannigan J. A., Dodson G., Duggleby H. J., Moody P. C., Smith J. L., Tomchick D. R., Murzin A. G. A protein catalytic framework with an N-terminal nucleophile is capable of self-activation. Nature. 1995 Nov 23;378(6555):416–419. doi: 10.1038/378416a0. [DOI] [PubMed] [Google Scholar]
  5. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chiang C., Bennett R. E. Purification and properties of penicillin amidase from Bacillus megaterium. J Bacteriol. 1967 Jan;93(1):302–308. doi: 10.1128/jb.93.1.302-308.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Daumy G. O., Danley D., McColl A. S. Role of protein subunits in Proteus rettgeri penicillin G acylase. J Bacteriol. 1985 Sep;163(3):1279–1281. doi: 10.1128/jb.163.3.1279-1281.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Daumy G. O., Williams J. A., McColl A. S., Zuzel T. J., Danley D. Expression and regulation of the penicillin G acylase gene from Proteus rettgeri cloned in Escherichia coli. J Bacteriol. 1986 Oct;168(1):431–433. doi: 10.1128/jb.168.1.431-433.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Duggleby H. J., Tolley S. P., Hill C. P., Dodson E. J., Dodson G., Moody P. C. Penicillin acylase has a single-amino-acid catalytic centre. Nature. 1995 Jan 19;373(6511):264–268. doi: 10.1038/373264a0. [DOI] [PubMed] [Google Scholar]
  10. Erarslan A., Koçer H. Thermal inactivation kinetics of penicillin G acylase obtained from a mutant derivative of Escherichia coli ATCC 11105. J Chem Technol Biotechnol. 1992;55(1):79–84. doi: 10.1002/jctb.280550113. [DOI] [PubMed] [Google Scholar]
  11. Gribskov M., Burgess R. R. Sigma factors from E. coli, B. subtilis, phage SP01, and phage T4 are homologous proteins. Nucleic Acids Res. 1986 Aug 26;14(16):6745–6763. doi: 10.1093/nar/14.16.6745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hamilton-Miller J. M. Penicillinacylase. Bacteriol Rev. 1966 Dec;30(4):761–771. doi: 10.1128/br.30.4.761-771.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kang J. H., Hwang Y., Yoo O. J. Expression of penicillin G acylase gene from Bacillus megaterium ATCC 14945 in Escherichia coli and Bacillus subtilis. J Biotechnol. 1991 Feb;17(2):99–108. doi: 10.1016/0168-1656(91)90001-c. [DOI] [PubMed] [Google Scholar]
  14. Klei H. E., Daumy G. O., Kelly J. A. Purification and preliminary crystallographic studies of penicillin G acylase from Providencia rettgeri. Protein Sci. 1995 Mar;4(3):433–441. doi: 10.1002/pro.5560040310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Konstantinović M., Marjanović N., Ljubijankić G., Glisin V. The penicillin amidase of Arthrobacter viscosus (ATCC 15294). Gene. 1994 May 27;143(1):79–83. doi: 10.1016/0378-1119(94)90608-4. [DOI] [PubMed] [Google Scholar]
  16. Kutzbach C., Rauenbusch E. Preparation and general properties of crystalline penicillin acylase from Escherichia coli ATCC 11 105. Hoppe Seylers Z Physiol Chem. 1974 Jan;355(1):45–53. doi: 10.1515/bchm2.1974.355.1.45. [DOI] [PubMed] [Google Scholar]
  17. Ljubijankić G., Konstantinović M., Glisin V. The primary structure of Providencia rettgeri penicillin G amidase gene and its relationship to other gram negative amidases. DNA Seq. 1992;3(3):195–200. doi: 10.3109/10425179209034017. [DOI] [PubMed] [Google Scholar]
  18. Margolin A. L., Svedas V. K., Berezin I. V. Substrate specificity of penicillin amidase from E. coli. Biochim Biophys Acta. 1980 Dec 4;616(2):283–289. doi: 10.1016/0005-2744(80)90145-x. [DOI] [PubMed] [Google Scholar]
  19. Martín L., Prieto M. A., Cortés E., García J. L. Cloning and sequencing of the pac gene encoding the penicillin G acylase of Bacillus megaterium ATCC 14945. FEMS Microbiol Lett. 1995 Jan 15;125(2-3):287–292. doi: 10.1016/0378-1097(94)00510-x. [DOI] [PubMed] [Google Scholar]
  20. Matagne A., Joris B., Frère J. M. Anomalous behaviour of a protein during SDS/PAGE corrected by chemical modification of carboxylic groups. Biochem J. 1991 Dec 1;280(Pt 2):553–556. doi: 10.1042/bj2800553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Needleman S. B., Wunsch C. D. A general method applicable to the search for similarities in the amino acid sequence of two proteins. J Mol Biol. 1970 Mar;48(3):443–453. doi: 10.1016/0022-2836(70)90057-4. [DOI] [PubMed] [Google Scholar]
  22. Niersbach H., Kühne A., Tischer W., Weber M., Wedekind F., Plapp R. Improvement of the catalytic properties of penicillin G acylase from Escherichia coli ATCC 11105 by selection of a new substrate specificity. Appl Microbiol Biotechnol. 1995 Aug-Sep;43(4):679–684. doi: 10.1007/BF00164773. [DOI] [PubMed] [Google Scholar]
  23. Ohashi H., Katsuta Y., Hashizume T., Abe S. N., Kajiura H., Hattori H., Kamei T., Yano M. Molecular cloning of the penicillin G acylase gene from Arthrobacter viscosus. Appl Environ Microbiol. 1988 Nov;54(11):2603–2607. doi: 10.1128/aem.54.11.2603-2607.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Prieto I., Martín J., Arche R., Fernández P., Pérez-Aranda A., Barbero J. L. Penicillin acylase mutants with altered site-directed activity from Kluyvera citrophila. Appl Microbiol Biotechnol. 1990 Aug;33(5):553–559. doi: 10.1007/BF00172550. [DOI] [PubMed] [Google Scholar]
  25. Prieto I., Rodríguez M. C., Márquez G., Pérez-Aranda A., Barbero J. L. Changing glycine 21 for glutamic acid in the beta-subunit of penicillin G acylase from Kluyvera citrophila prevents protein maturation. Appl Microbiol Biotechnol. 1992 Feb;36(5):659–662. doi: 10.1007/BF00183245. [DOI] [PubMed] [Google Scholar]
  26. Roa A., Garcia J. L., Salto F., Cortes E. Changing the substrate specificity of penicillin G acylase from Kluyvera citrophila through selective pressure. Biochem J. 1994 Nov 1;303(Pt 3):869–875. doi: 10.1042/bj3030869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Schumacher G., Sizmann D., Haug H., Buckel P., Böck A. Penicillin acylase from E. coli: unique gene-protein relation. Nucleic Acids Res. 1986 Jul 25;14(14):5713–5727. doi: 10.1093/nar/14.14.5713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Seemuller E., Lupas A., Baumeister W. Autocatalytic processing of the 20S proteasome. Nature. 1996 Aug 1;382(6590):468–471. doi: 10.1038/382468a0. [DOI] [PubMed] [Google Scholar]
  29. Self A., Kay G., Lilly M. D., Dunnill P. The conversion of benzyl penicillin to 6-aminopenicillanic acid using an insoluble derivative of penicillin amidase. Biotechnol Bioeng. 1969 May;11(3):337–348. doi: 10.1002/bit.260110306. [DOI] [PubMed] [Google Scholar]
  30. von Heijne G. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 1986 Jun 11;14(11):4683–4690. doi: 10.1093/nar/14.11.4683. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES