Immobilized mutant lysozyme as target for specific interaction and subsequent separation of bacteria from complex matrices by magnetic forces
Diler, E. / Schwartz, T. (2011)
VAAM 2011, Karlsruhe, Deutschland, 3.-6. April, 2011
Diler, E. / Schwartz, T. (2011): „Immobilized mutant lysozyme as target for specific interaction and subsequent separation of bacteria from complex matrices by magnetic forces“. In: VAAM 2011, Karlsruhe, Deutschland, 3.-6. April, 2011
Background: As a response to environmental stress, bacterial cells can enter a physiological state called viable but non cultivable where bacteria fail to grow on routine bacteriologic media. Cultivation based contamination detection in food industry and drinking water conditioning fail on these bacteria and can have severe effects on public health.
Objectives: The aim of the project is the development of a separation method for bacteria using catalytic inactivated lysozymes immobilized on magnetic beads for selective separation of intact bacterial cells for subsequent cultivation independent pathogen detection.
Methods: To diminish lytic activity of lysozyme catalytic amino acid glutamate 35 was exchanged with glutamine and alanin by site directed mutagenesis. The mutated proteins and the wildtype reference were expressed by Pichia pastoris and secreted into expression medium enabling direct protein enrichment and purification from expression broth by cationic exchanger particles. Turbidity assays were used to prove the properties of the proteins. The proteins got biotinylated and immobilized on streptavidin functionalized, fluorescence dye labelled magnetic beads. These constructs were used for capture of Syto9 marked Micrococcus luteus cells from cell suspension.
Conclusion: The recombinant proteins were successfully expressed and selectively enriched by using cationic magnetic beads. The proteins retained affinity for bacterial cell wall components but not the bacteriolytic activity. Immobilization on magnetic beads enabled separation of reference bacteria from cell suspension and delivered the first hint for the success of the strategy.