Micrococcus lysodeikticus lyophilized cell

Lyophilized lysozyme from chicken egg white, Micrococcus lysodeikticus lyophilized cells, was purchased from Sigma-Aldrich (St. Louis, MO, USA). Potassium phosphate monobasic (purity ≥ 99.0%) was purchased from Fluka (Charlotte, NC, USA) and Micro BCA™ and BCA™ Protein Assay Kit were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Milli-Q water (obtained from a Milli-Q Integral water purification system from Merck, Darmstadt, Germany) was used for the buffer preparation.
1-Ethyl-3-methylimidazolium perfluorobutanesulfonate, [C₂C₁Im][C₄F₉SO₃] (≥97% mass fraction purity), 1-ethyl-3-methylpyridinium perfluorobutanesulfonate, [C₂C₁py][C₄F₉SO₃] (≥99% mass fraction purity), and cholinium ((2-hydroxyethyl)trimethylammonium) perfluorobutanesulfonate, [N_1112(OH)][C₄F₉SO₃] (≥97% mass fraction purity) were supplied by IoLiTec GmbH (Salzstraße, Heilbronn, Germany). To reduce volatile chemicals and water contents, all FILs were dried under vacuum (4 Pa) with vigorous stirring at about 50 °C for at least 2 days, immediately prior to their use. No further purification was carried out and the purity of all FILs was checked by ¹H and ¹⁹F NMR. The chemical structures of the fluorinated ionic liquids used in this work are presented in Table 1.

Lysozyme activity in midgut juice samples was assayed with Micrococcus lysodeikticus lyophilized cells (Sigma-Aldrich), measuring the rate of lysis of bacterial cells. Different volumes of midgut juice were diluted to 20 μl with 66 mM potassium phosphate buffer, pH 6.2. Then, the diluted samples were incubated with 980 μl of a suspension of 0.02 % (w/v) M. lysodeikticus lyophilized cells in the same buffer. The mixture was subjected to a continuous absorbance reading at 450 nm at 45°C. One unit/ml (U/ml) of lysozyme activity was defined as the amount of enzyme that causes a decrease in absorbance by 1 unit per min per ml of midgut juice sample.

The lysozyme activity was determined using a turbidimetric assay [35 (link)]. In total, 25 µL of samples (serum, gut lavage, and skin mucus) was placed separately into the wells of a microplate. Then, 75 µL of substrate containing 0.2 mg of Micrococcus lysodeikticus lyophilized cell (Sigma-Aldrich, St. Louis, MO, USA)/mL PBS at pH 6.3 was added. The absorbance was measured at 450 nm after 1 min and 60 min incubation with gentle shaking at room temperature (25 ± 2 °C) using a Multiskan spectrum microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). A unit of lysozyme activity was defined as the amount of enzyme causing a decrease in absorbance of 0.001 per min, and expressed as a U/mg unit.