4.10. Preparation of Cell-Free Extract

Cell-free extracts from yeast cells harvested on completion of the culture carried out at 20 °C in the liquid medium were prepared by using three different methods: (1) 2 g of the wet biomass was sonicated for 5 min at 0 °C, vibrations amplitude 30% (Vibra Cell 71408, Bioblock Scientific, USA) in 50 mM potassium phosphate buffer, pH 7.6, enriched with 100 mM MgCl₂, 1 mM PMSF, and 1 mM β-mercaptoethanol. The residual insoluble cell debris was discarded after centrifugation (5000× g, 4 °C, 30 min); (2) the enzymatic-chemical lysis of biomass was performed using the commercial YeastBuster Protein Extraction Reagent (Novagen, Madison, WI, USA; now supplied by Merck KgaA, Darmstadt, Germany) according to the manufacturer’s recommendations; (3) 200 mg of glass beads (ϕ 0.25–0.5 mm) were added to 200 mg of cell pellets and were suspended in 1 mL of lysis buffer (20 mM potassium phosphate buffer, pH 7.4, enriched with 0.1% Triton X-100, 100 mM KCl, 8 mM MgCl₂, 150 mM NaCl, and 1 mM PMSF). The mixture was incubated alternately on a shaker (13,000 rpm at room temperature) and on ice for 30 s each, repeating 12 times. The residual insoluble cell debris was discarded after centrifugation (1500× g, 10 °C, 5 min).