Skip to main content

A lysozyme and magnetic bead based method of separating intact bacteria

Abstract

As a response to environmental stress, bacterial cells can enter a physiological state called viable but noncultivable (VBNC). In this state, bacteria fail to grow on routine bacteriological media. Consequently, standard methods of contamination detection based on bacteria cultivation fail. Although they are not growing, the cells are still alive and are able to reactivate their metabolism. The VBNC state and low bacterial densities are big challenges for cultivation-based pathogen detection in drinking water and the food industry, for example. In this context, a new molecular-biological separation method for bacteria using point-mutated lysozymes immobilised on magnetic beads for separating bacteria is described. The immobilised mutated lysozymes on magnetic beads serve as bait for the specific capture of bacteria from complex matrices or water due to their remaining affinity for bacterial cell wall components. Beads with bacteria can be separated using magnetic racks. To avoid bacterial cell lysis by the lysozymes, the protein was mutated at amino acid position 35, leading to the exchange of the catalytic glutamate for alanine (LysE35A) and glutamine (LysE35Q). As proved by turbidity assay with reference bacteria, the muramidase activity was knocked out. The mutated constructs were expressed by the yeast Pichia pastoris and secreted into expression medium. Protein enrichment and purification were carried out by SO3-functionalised nanoscale cationic exchanger particles. For a proof of principle, the proteins were biotinylated and immobilised on streptavidin-functionalised, fluorescence dye-labelled magnetic beads. These constructs were used for the successful capture of Syto9-marked Microccocus luteus cells from cell suspension, as visualised by fluorescence microscopy, which confirmed the success of the strategy.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11
Fig. 12a–d

References

  1. Gunasekera TS, Sorensen A, Attfield PV, Sorensen SJ, Veal DA (2002) Inducible gene expression by nonculturable bacteria in milk after pasteurization. Appl Environ Microbiol 68:1988–1993

    Article  CAS  Google Scholar 

  2. Oliver JD, Dagher M, Linden K (2005) Induction of Escherichia coli and Salmonella typhimurium into the viable but nonculturable state following chlorination of wastewater. J Water Health 3:249–257

    Google Scholar 

  3. Wolf PW, Oliver JD (1992) Temperature effects on the viable but non-culturable state of Vibrio vulnificus. FEMS Microbiol Lett 101:33–39

    Google Scholar 

  4. Oliver JD (2005) The viable but not culturable state in bacteria. J Microbiol 43:93–100

    Google Scholar 

  5. Byrd JJ, Xu HS, Colwell RR (1991) Viable but nonculturable bacteria in drinking water. Appl Environ Microbiol 57:875–878

    CAS  Google Scholar 

  6. Besnard V, Federighi M, Cappelier JM (2000) Development of a direct viable count procedure for the investigation of VBNC state in Listeria monocytogenes. Lett Appl Microbiol 31:77–81

    Article  CAS  Google Scholar 

  7. Allen MJ, Edberg SC, Reasoner DJL (2004) Heterotrophic plate count bacteria—what is their significance in drinking water? Int J Food Microbiol 92:265–274

    Google Scholar 

  8. EU (2005) Implementation of procedures based on the HACCP principles in certain food businesses (European Union Guidance Document). EU, Brussels

  9. Food and Agriculture Organisation/World Health Organisation (2006) Guidance to governments on the application of HACCP in small and/or less-developed food businesses (FAO Food and Nutrition Paper 86). FAO/WHO, Rome/Geneva, ISSN 0254-4725

  10. Varela Villarreal J, Schwartz T, Obst U (2010) Culture-independent techniques applied to food industry water surveillance—a case study. Int J Food Microbiol 141:147–155

    Google Scholar 

  11. Jollès P, Jollès J (1984) What’s new in lysozyme research? Mol Cell Biochem 63:165–189

    Article  Google Scholar 

  12. Blake CCF, Johnson LN, Mair GA, North ACT, Phillips DC, Sarma VR (1967) Crystallographic studies of the activity of hen egg-white lysozyme. Proc R Soc Lond B Biol Sci 167:378–388

    Google Scholar 

  13. Malcolm BA, Rosenberg S, Corey MJ, Allen JS, de Baetselier A, Kirsch JF (1989) Site-directed mutagenesis of the catalytic residues Asp-52 and Glu-35 of chicken egg white lysozyme. Proc Nat Acad Sci USA 86:133–137

    Google Scholar 

  14. Kuroki R, Yamada H, Moriyama T, Imoto T (1986) Chemical mutations of the catalytic carboxyl groups in lysozyme to the corresponding amides. J Biol Chem 261:13571–13574

    CAS  Google Scholar 

  15. Rachel D, Milton TWH (2005) Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J Mol Recognit 18:119–138

    Article  Google Scholar 

  16. Ling MM, Robinson BH (1997) Approaches to DNA mutagenesis: an overview. Anal Biochem 254:157–178

    Google Scholar 

  17. Inoue H, Nojima H, Okayama H (1990) High efficiency transformation of Escherichia coli with plasmids. Gene 96:23–28

    Google Scholar 

  18. Shugar D (1952) The measurement of lysozyme activity and the ultraviolet inactivation of lysozyme. Biochim Biophys Acta 8:302–309

    Google Scholar 

  19. Repaske R (1958) Lysis of gram-negative organisms and the role of versene. Biochim Biophys Acta 30:225–232

    Article  CAS  Google Scholar 

  20. Enroth H, Engstrand L (1995) Immunomagnetic separation and PCR for detection of Helicobacter pylori in water and stool specimens. J Clin Microbiol 33(8):2162–2165

    CAS  Google Scholar 

  21. Tu SI, Patterson D, Uknalis J, Irwin P (2000) Detection of Escherichia coli O157:H7 using immunomagnetic capture and luciferin-luciferase ATP measurement. Food Res Int 33:375–380

    Google Scholar 

  22. Sun W, Khosravi F, Albrechtsen H, Brovko LY, Griffiths MW (2002) Comparison of ATP and in vivo bioluminescence for assessing the efficiency of immunomagnetic sorbents for live Escherichia coli O157:H7 cells. J Appl Microbiol 92:1021–1027

    Article  CAS  Google Scholar 

  23. Kaeppler TE, Hickstein B, Peuker UA, Posten C (2008) Characterization of magnetic ion-exchange composites for protein separation from biosuspensions. J Biosci Bioeng 105:579–585

    Article  CAS  Google Scholar 

  24. Wadud S, Leon-Velarde CG, Larson N, Odumeru JA (2010) Evaluation of immunomagnetic separation in combination with ALOA Listeria chromogenic agar for the isolation and identification of Listeria monocytogenes in ready-to-eat foods. J Microbiol Meth 81:153–159

    Google Scholar 

  25. Vinu A, Murugesan V, Hartmann M (2004) Adsorption of lysozyme over mesoporous molecular sieves MCM-41 and SBA-15: influence of pH and aluminum incorporation. J Phys Chem B 108:7323–7330

    Google Scholar 

  26. Makrides SC (1996) Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev 60:512–538

    CAS  Google Scholar 

  27. Monsalve RI, Lu G, King TP (1999) Expressions of recombinant venom allergen, antigen 5 of Yellowjacket (Vespula vulgaris) and Paper Wasp (Polistes annularis), in bacteria or yeast. Protein Expr Purif 16:410–416

    Google Scholar 

Download references

Acknowledgements

The authors are thankful to Merck KGaA (Karl Holschuh, Darmstadt, Germany) for kindly donating the MagPrep SO3 particles for protein enrichment and purification.

The Research Group of KS is financed by the “Concept for the Future” of the Karlsruhe Institute of Technology (KIT) within the German Excellence Initiative. Additional funding was provided by the KIT Competence Field “Applied Life Science”.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Thomas Schwartz.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Diler, E., Obst, U., Schmitz, K. et al. A lysozyme and magnetic bead based method of separating intact bacteria. Anal Bioanal Chem 401, 253–265 (2011). https://doi.org/10.1007/s00216-011-5065-5

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-011-5065-5

Keywords

  • Bacteria separation
  • Magnetic beads
  • Lysozyme mutation
  • Protein purification
  • Binding capacity