β-Galactosidase from a cold-adapted bacterium: purification, characterization and application for lactose hydrolysis

Fernandes, S.; Geueke, B.; Delgado, O.; Coleman, J.; Hatti-Kaul, R.
March 2002
Applied Microbiology & Biotechnology;Mar2002, Vol. 58 Issue 3, p313
Academic Journal
The enzyme β-galactosidase was purified from a cold-adapted organism isolated from Antarctica. The organism was identified as a psychrotrophic Pseudoalteromonas sp. The enzyme was purified with high yields by a rapid purification scheme involving extraction in an aqueous two-phase system followed by hydrophobic interaction chromatography and ultrafiltration. The β-galactosidase was optimally active at pH 9 and at 26 °C when assayed with o-nitrophenyl-β-D-galactopyranoside as substrate for 2 min. The enzyme activity was highly sensitive to temperature above 30 °C and was undetectable at 40 °C. The cations Na+, K+, Mg2+ and Mn2+ activated the enzyme while Ca2+, Hg2+, Cu2+ and Zn2+ inhibited activity. The shelf life of the pure enzyme at 4 °C was significantly enhanced in the presence of 0.1% (w/v) polyethyleneimine. The pure β-galactosidase was also evaluated for lactose hydrolysis. More than 50% lactose hydrolysis was achieved in 8 h in buffer at an enzyme concentration of 1 U/ml, and was increased to 70% in the presence of 0.1% (w/v) polyethyleneimine. The extent of lactose hydrolysis was 40–50% in milk. The enzyme could be immobilized to Sepharose via different chemistries with 60–70% retention of activity. The immobilized enzyme was more stable and its ability to hydrolyze lactose was similar to that of the soluble enzyme.


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