Purification of β-galactosidase from recombinant Pichia pastoris using aqueous two-phase separation technique

ABSTRACT

In the current investigation, aqueous two-phase methodology with polyethylene glycol (PEG) 6000/phosphate salt has been used for single-step purification of β-galactosidase from recombinant Pichia pastoris. Optimized parameters with 12% (w/w) salt concentration, 25% (w/w) polymer concentration, 0.6 (mg/ml) protein load and 0.1 M ionic concentration resulted in a maximum of 4.7 purification fold with a 97% yield. The enzyme kinetic study of purified protein revealed V_m and K_m of 97.087 (U/mg), 0.027(U/mg), 0.07 (U/mg) and 0.7 (mM), 11.13 (mM), 10.73 (mM) with O-nitrophenyl-β-D-galactopyranoside, lactose and milk substrate, respectively.

KEYWORDS:

• Aqueous two-phase separation (ATPS)
• polyethylene glycol (PEG)
• phosphate salt
• β-galactosidase
• Pichia pastoris

Acknowledgments

We are grateful to the Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India for providing facilities to carry out this research.

Conflict of interest

The authors have declared no conflict of interest.