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. 1980 Mar;17(3):355–358. doi: 10.1128/aac.17.3.355

Purification and properties of a new beta-lactamase from Pseudomonas cepacia.

K Hirai, S Iyobe, M Inoue, S Mitsuhashi
PMCID: PMC283790  PMID: 6968539

Abstract

An inducible beta-lactamase was purified from a beta-lactam antibiotic-resistant strain (GN11164) of Pseudomonas cepacia. The purified enzyme preparation gave a single protein band on polyacrylamide gel electrophoresis. The specific enzyme activity for the hydrolysis of cephalothin as a substrate was 314.5 U/mg of protein. The optimal pH was about 8.0, and the optimal temperature was 45 degrees C. The isoelectric point was 9.3, and the molecular weight was estimated to be about 22,000 to 24,000 from gel filtration on a Sephadex G-200 column and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme activity was inhibited by iodine, p-chloromercuribenzoate, clavulanic acid, CP 45899, and cloxacillin. The beta-lactamase showed a unique substrate profile by hydrolyzing most of the cephalosporins, including cefuroxime, cefotaxime (HR 756), ampicillin, and penicillin G, at a high rate.

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Selected References

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

  1. Andrews P. Estimation of the molecular weights of proteins by Sephadex gel-filtration. Biochem J. 1964 May;91(2):222–233. doi: 10.1042/bj0910222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Dale J. W. Characterization of the -lactamase specified by the resistance factor R-1818 in E. coli K12 and other Gram-negative bacteria. Biochem J. 1971 Jul;123(4):501–505. doi: 10.1042/bj1230501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Dale J. W., Smith J. T. Some relationships between R-factor and chromosomal -lactamase in Gram-negative bacteria. Biochem J. 1971 Jul;123(4):507–512. doi: 10.1042/bj1230507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dale J. W., Smith J. T. The dimeric nature of an R-factor mediated beta-lactamase. Biochem Biophys Res Commun. 1976 Feb 9;68(3):1000–1005. doi: 10.1016/0006-291x(76)91245-6. [DOI] [PubMed] [Google Scholar]
  5. Fu K. P., Neu H. C. beta-lactamase stability of HR 756, a novel cephalosporin, compared to that of cefuroxime and cefoxitin. Antimicrob Agents Chemother. 1978 Sep;14(3):322–326. doi: 10.1128/aac.14.3.322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fujii-Kuriyama Y., Yamamoto M., Sugawara S. Purification and properties of beta-lactamase from Proteus morganii. J Bacteriol. 1977 Sep;131(3):726–734. doi: 10.1128/jb.131.3.726-734.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gelbart S. M., Reinhardt G. F., Greenlee H. B. Pseudomonas cepacia strains isolated from water reservoirs of unheated nebulizers. J Clin Microbiol. 1976 Jan;3(1):62–66. doi: 10.1128/jcm.3.1.62-66.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  9. Letarte R., Devaud-Felix M., Pechere J. C., Allard-Leprohon D. Enzymatic and immunological characterization of a new cephalosporinase from Enterobacter aerogenes. Antimicrob Agents Chemother. 1977 Aug;12(2):201–205. doi: 10.1128/aac.12.2.201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lindqvist R. C., Nordström K. Resistance of Escherichia coli to penicillins. VII. Purification and characterization of a penicillinase mediated by the R factor R1. J Bacteriol. 1970 Jan;101(1):232–239. doi: 10.1128/jb.101.1.232-239.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Matsubara N., Minami S., Muraoka T., Saikawa I., Mitsuhashi S. In vitro antibacterial activity of cefoperazone (T-1551), a new semisynthetic cephalosporin. Antimicrob Agents Chemother. 1979 Dec;16(6):731–735. doi: 10.1128/aac.16.6.731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Matthew M., Harris A. M. Identification of beta-lactamases by analytical isoelectric focusing: correlation with bacterial taxonomy. J Gen Microbiol. 1976 May;94(1):55–67. doi: 10.1099/00221287-94-1-55. [DOI] [PubMed] [Google Scholar]
  13. NOVICK R. P. Micro-iodometric assay for penicillinase. Biochem J. 1962 May;83:236–240. doi: 10.1042/bj0830236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Neu H. C., Fu K. P. Cefuroxime, a beta-lactamase-resistant cephalosporin with a broad spectrum of gram-positive and -negative activity. Antimicrob Agents Chemother. 1978 Apr;13(4):657–664. doi: 10.1128/aac.13.4.657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Neu H. C., Fu K. P. Clavulanic acid, a novel inhibitor of beta-lactamases. Antimicrob Agents Chemother. 1978 Nov;14(5):650–655. doi: 10.1128/aac.14.5.650. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Phillips I., Eykyn S., Curtis M. A., Snell J. J. Pseudomonas cepacia (multivorans) septicaemia in an intensive-care unit. Lancet. 1971 Feb 20;1(7695):375–377. doi: 10.1016/s0140-6736(71)92212-4. [DOI] [PubMed] [Google Scholar]
  17. Ross G. W., Chanter K. V., Harris A. M., Kirby S. M., Marshall M. J., O'Callaghan C. H. Comparison of assay techniques for beta-lactamase activity. Anal Biochem. 1973 Jul;54(1):9–16. doi: 10.1016/0003-2697(73)90241-8. [DOI] [PubMed] [Google Scholar]
  18. Sawai T., Mitsuhashi S., Yamagishi S. Drug resistance of enteric bacteria. XIV. Comparison of beta-lactamases in gram-negative rod bacteria resistant to alpha-aminobenzylpenicillin. Jpn J Microbiol. 1968 Dec;12(4):423–434. doi: 10.1111/j.1348-0421.1968.tb00415.x. [DOI] [PubMed] [Google Scholar]
  19. Sawai T., Takahashi K., Yamagishi S., Mitsuhashi S. Variant of penicillinase mediated by an R factor in Escherichia coli. J Bacteriol. 1970 Nov;104(2):620–629. doi: 10.1128/jb.104.2.620-629.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Speller D. C., Stephens M. E., Viant A. C. Hospital infection by Pseudomonas cepacia. Lancet. 1971 Apr 17;1(7703):798–799. doi: 10.1016/s0140-6736(71)91236-0. [DOI] [PubMed] [Google Scholar]
  21. Sykes R. B., Matthew M. The beta-lactamases of gram-negative bacteria and their role in resistance to beta-lactam antibiotics. J Antimicrob Chemother. 1976 Jun;2(2):115–157. doi: 10.1093/jac/2.2.115. [DOI] [PubMed] [Google Scholar]
  22. Tsuchiya K., Kida M., Kondo M., Ono H., Takeuchi M., Nishi T. SCE-963, a new broad-spectrum cephalosporin: in vitro and in vivo antibacterial activities. Antimicrob Agents Chemother. 1978 Oct;14(4):557–568. doi: 10.1128/aac.14.4.557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Tsuchiya K., Kondo M., Nagatomo H. SCE-129, antipseudomonal cephalosporin: in vitro and in vivo antibacterial activities. Antimicrob Agents Chemother. 1978 Feb;13(2):137–145. doi: 10.1128/aac.13.2.137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Waley S. G. A spectrophotometric assay of beta-lactamase action on penicillins. Biochem J. 1974 Jun;139(3):789–790. doi: 10.1042/bj1390789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]
  26. Yabuuchi E., Miyajima N., Hotta H., Oyama A., Tanaka N. Pseudomonas cepacia from blood of a burn patient. Med J Osaka Univ. 1970 Dec;21(1):1–6. [PubMed] [Google Scholar]
  27. Yaginuma S., Sawai T., Ono H., Yamagishi S., Mitsuhashi S. Biochemical properties of a cephalosporin beta-lactamase from Pseudomonas aeruginosa. Jpn J Microbiol. 1973 Mar;17(2):141–149. doi: 10.1111/j.1348-0421.1973.tb00718.x. [DOI] [PubMed] [Google Scholar]
  28. Yaginuma S., Sawai T., Yamagishi S., Mitsuhashi S. Beta-lactamase formation and resistance of Proteus morganii to various penicillins and cephalosporins. Jpn J Microbiol. 1974 Mar;18(2):113–118. doi: 10.1111/j.1348-0421.1974.tb00798.x. [DOI] [PubMed] [Google Scholar]

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