Abstract:

The fermentation supernatant of Bacillus stearothermophilus XG24 received salting out with ammonium sulfate, separation on DEAE-Sepharose Fast Flow anion exchange column and purification on Sephadex G-75 gel filtration column to obtain high-purity thermostable β-galactosidase, which was subsequently subjected to enzymatic characterization with o-nitrophenyl- β-D-galactopyranoside (ONPG) as a substrate. After the above separation and purification procedures, the purity of this enzyme showed a 54.5-fold increase, with an activity recovery of 20.4%, and the specific activity of the purified enzyme was 32.7 U/mg protein. The optimal pH and temperature for the reaction of this enzyme were 6.5 and 65 ℃, respectively. It was stable at temperatures below 70 ℃ or in a pH range between 4.0 and 8.0. Its activity was notably promoted by Mg2+, Mn2+, Fe2+ and Co2+, whereas Cu2+, Ag+ and Hg2+ were almost able to entirely inhibit its activity. The Km towards ONPG was determined to be 4.32 mmol/L. The SDS-PAGE analysis revealed that this enzyme was a single-chain protein. Based on the results of Sephadex G-75 gel filtration chromatographic measurement, it was deduced that the apparent molecular weight of this enzyme was 64 kD. Therefore, Bacillus stearothermophilus XG24-derived thermostable β-galactosidase has high application potential in the dairy industry.

Key words: Bacillus stearothermophilus, β-galactosidase, purification, characterization