The BPE crude extract was centrifuged at 3100 (or 18,000)
×
g for 15 min
(Thermo Scientific IEC CL40R, WaltHam, USA) to remove cell debris and obtain a BPE clarified crude extract. The isoelectric precipitation from the clarified extract
was done according to a previous report [22]. Briefly, clarified extract was diluted
1:2 with 50 mM dibasic phosphate buffer in a stainless steel precipitation tank (25 L
maximum capacity, radius 20 cm and height 20 cm) equipped with a bottom release
valve. The mixture was acidified with hydrochloric acid (HCl 0.1 N) to adjust pH to 4.
Temperature was kept constant at 4 ◦C using a cooling jacket with water as coolant
fluid. The sample was mixed at 50 rpm using a Rushton impeller (diameter of 12 cm)
for 20 min and then allowed to settle for 15 h. The precipitate was collected through
the bottom release valve and centrifuged for 1 h at 4 ◦C at 3100 or (18,000)
×
g in
order to obtain a BPE enriched pellet.