β-galactosidase assay

Colonies defined the growth profiles were grown in a 5 mL SD medium (without Trp/Leu or Trp) at 30 °C. The cells in 500 µL of saturated culture were recovered with centrifugation and re-suspended in 300 µL Z- buffer (0.1 M sodium phosphate pH 7.0, 10 mM KCl, 1 mM MgSO₄, and 0.35% β-mercaptoethanol). The cells in suspensions were disintegrated in a freeze-thaw procedure repeated five times in liquid nitrogen. Then, 60 µL o-nitrophenyl-β-d-galactopyranoside (ONPG) (4 mg/mL) substrate was added and incubated for 60 minutes at 37 °C. To stop the reactions 300 µL Na₂CO₃ (0.5 M) was added. The supernatants were recovered by centrifugation at 15000 rpm for 5 min, and then the absorbance of samples at 420 nm (OD₄₂₀) was measured. The β-galactosidase activities were calculated from a standard curve plotted with a recombinant β-galactosidase enzyme (Roche).