In the present investigation, the purification of beta-galactosidase synthesized from Bacillus safensis (JUCHE I), a new strain isolated from casein whey, has been considered. Broth harvested after 28 h (100.8 x 10(3) s) was first centrifuged, and the sedimented cell pellet was sonicated to isolate intracellular beta-galactosidase. After sonication, the intracellular fluid was subjected to microfiltration (MF) (0.2 x 10(-6) m) and ultrafiltration (UF) (300 kg mol(-1)) in series using a flat-sheet poly(ether sulfone) (PES) membrane in a novel rotating-disk membrane module. In the present investigation, four-stage discontinuous diafiltration (DD) at a constant volume concentration factor (VCF = 2) was carried out. Hydrodynamic studies were conducted to determine the appropriate stirrer speed, transmembrane pressure (TMP), and membrane rotation speed. Finally, beta-galactosidase was purified based on the principle of salting-out upon addition of 40% (w/v) ammonium sulfate, followed by dialysis using a 1 kg mol(-1) membrane. Subsequently, purified beta-galactosidase was characterized using ortho-nitrophenyl-beta-galactoside (ONPG) and lactose as substrates.