Protein and glucose 6-phosphate dehydrogenase releasing from baker's yeast cells disrupted by a vertical bead mill

Saccharomyces cerevisiae cells were disrupted at 5 degrees C in a 200-ml vertical bead mill using glass beads (diameter: 0.5 mm) as abrasive. The parameters varied were: agitation (1100; 1700; 2300 and 3100 rpm), glass beads-volume (50, 75 and 100 mi) and cell concentration (170, 250 and 325 g/l). Before disruption the cells were thoroughly rinsed in physiological buffer and suspended in 50 mM Tris-HCl (pH 7.5) containing 2 mM MgCl2, 2 mM aminocaproic acid, 2 mM dithiothreitol and 1 mM phenylmethylsulfonyl fluoride. After removing the cell debris (centrifugation at 10 000 x g/15 min), the total protein concentration and the activity of glucose 6-phosphate dehydrogenase (G6PDH) were measured. The high cell disruption rate (6 x 10(11) cells/min) occurred 3 min after the beginning of the procedure, carried out at 3100 rpm with 325 g/l of cells and 100 mi of glass beads. Moreover, a significant release of protein and G6PDH required 6 and 3 min of agitation, respectively. Thus G6PDH liberation and the cell disruption ale coupled events. Furthermore, this disruption procedure was highly reproductive. (C) 2000 Elsevier Science Ltd. All rights reserved.