*4.2. Isolation of Cell Envelope Fraction*

Microbial cells were harvested in the late exponential growth phase (after 48 h) that was determined by the turbidity of the cell suspension. Cells were collected by centrifugation at 7000 rpm for 20 min, washed once with a solution of 5% NaCl, and suspended in buffer containing 50 mM Na2HPO4, 100 mM NaCl, sucrose (5%, *w*/*v*), 1.5 mg of DNAase, and sodium azide (0.02%, *w*/*v*).The cells were disrupted by sonication (UZDN-2T insonator, Sumy, Ukraine) at 44 kHz (10 × 1 min cycles on ice). The unbroken cells were centrifuged at 10,000× *g* for 15 min at 4 ◦C, and the supernatant was centrifuged at 25,000× *g* (Heraeus Biofuge stratus, Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at 4 ◦C. The pellet was then washed with distilled water and used as cell envelope preparation (crude membrane fraction). It contained the total fraction of cytoplasmic and OM proteins.
