Beta-galactosidase from a cold-adapted bacterium: purification, characterization and application for lactose hydrolysis.
The enzyme beta-galactosidase was purified from a cold-adapted organism isolated from Antarctica. The organism was identified as a psychotrophic 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 beta-galactosidase was optimally active at pH 9 and at 26 degrees C when assayed with o-nitrophenyl-beta-D-galactopyranoside as substrate for 2 min. The enzyme activity was highly sensitive to temperature above 30 degrees C and was undetectable at 40 degrees 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 degrees C was significantly enhanced in the presence of 0.1% (w/v) polyethyleneimine. The pure beta-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.
Fernandes
S
S
Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, Sweden. Sheryl.Fernandes@biotek.lu.se
Geueke
B
B
Delgado
O
O
Coleman
J
J
Hatti-Kaul
R
R
D000064:Acclimatization; D000864:Antarctic Regions; D003080:Cold Temperature; D004795:Enzyme Stability; D005227:Fatty Acids / Q000032:analysis; D005285:Fermentation; D005690:Galactose / Q000378:metabolism; D006358:Hot Temperature; D006868:Hydrolysis; D007785:Lactose / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000378:metabolism; D031161:Pseudoalteromonas / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D012623:Seawater / Q000382:microbiology; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D005227:Fatty Acids; D011092:Polyethylene Glycols; D001616:beta-Galactosidase; D007785:Lactose; D005690:Galactose