title: 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 SO(3)-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.
authors.affiliation: Microbiology of Natural and Technical Interfaces Department, Institute of Functional Interfaces (IFG), Karlsruhe Institute of Technology (KIT)-Northern Campus, Eggenstein-Leopoldshafen, Germany.
authors.forename: Ebru
authors.initials: E
authors.lastname: Diler
authors.forename: Ursula
authors.initials: U
authors.lastname: Obst
authors.forename: Katja
authors.initials: K
authors.lastname: Schmitz
authors.forename: Thomas
authors.initials: T
authors.lastname: Schwartz
mesh_terms: D001419:Bacteria / Q000166:cytology / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D004800:Enzymes, Immobilized / Q000235:genetics / Q000378:metabolism; D008280:Magnetics / Q000379:methods*; D016982:Micrococcus luteus / Q000166:cytology / Q000302:isolation & purification / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D009113:Muramidase / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000235:genetics; D017354:Point Mutation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic
chemical_list: D004800:Enzymes, Immobilized; D011994:Recombinant Proteins; D009113:Muramidase