Purification and Characterization of β-Galactosidase Synthesized from Bacillus safensis (JUCHE 1)
2013
Nath, Arijit | Chakrabarty, Sudip | Sarkar, Shubhrajit | Bhattacharjee, Chiranjib | Drioli, E. | Chowdhury, Ranjana
In the present investigation, the purification of β-galactosidase synthesized from Bacillus safensis (JUCHE 1), a new strain isolated from casein whey, has been considered. Broth harvested after 28 h (100.8 × 10³ s) was first centrifuged, and the sedimented cell pellet was sonicated to isolate intracellular β-galactosidase. After sonication, the intracellular fluid was subjected to microfiltration (MF) (0.2 × 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, β-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 β-galactosidase was characterized using ortho-nitrophenyl-β-galactoside (ONPG) and lactose as substrates.
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