Lysozyme activity was determined by EnzChek® Lysozyme
Assay Kit of Molecular Probes Invitrogen. Activity was tested by
using a fluorescence-based assay. Micrococcus lysodeikticus is
labeled with fluorescein as a fluorophore in such a quantity, that
fluorescence is quenched. Lysozyme activity cause the hydrolysis of
M. lysodeikticus cell wall fragments, therefore dequenching occurs,
and can be detected as an increase of fluorescence (Helal and
Melzig, 2008).
To 40 ml of sample volume 10 ml of reaction buffer, containing
0.1 M sodium phosphate and 0.1 M NaCl, were added, to reach a
total volume of 50 ml, and incubated protected from light for 30 min
at 37 C. Fluorescence was measured at an excitationwavelength of
485 nmand an emissionwavelength of 535 nm (Tecan infinite 200).
Experiments were carried out in two independent series and
measured in triplicates.
To enable a comparison between different species, lysozyme
activity and total protein amount have to be in correlation, therefore
the specific lysozyme activity was generated