β-Galactosidase from Lactobacillus brevis PLA28: Purification, Characterization and Synthesis of Galacto-oligosaccharides

Abstract

Objective: Purification and characterization of β-galactosidase from Lactobacillus brevis PLA28 were carried out and transgalactosylation activity was also studied for the synthesis of galacto-oligosaccharides (GOS). Methods: β-Galactosidase was purified by using ammonium sulphate precipitation method and hydrophobic interaction chromatography. Reaction conditions were optimized for the assay of this β-galactosidase. GOS synthesis was carried out under the optimized reaction conditions in batch mode and the product formed was detected by thin layered chromatography (TLC). Results: β-Galactosidase was purified to 6.6 fold with a yield of 6% and specific activity of 4 U/mg protein. Molecular weight of the purified β-galactosidase was found to be 45 and 60 kDa on SDS PAGE and 105 kDa on native PAGE. The temperature and pH optima of purified enzyme were 30°C and pH 6.5 respectively. The enzyme was found to be stable at 30°C for 6 h. V max and K M of the purified β-galactosidase were calculated to be 6.6 U/mg protein and 8.33 mM respectively. GOS synthesis was observed at pH 6.5, 30°C in 6-8 h of incubation by purified enzyme. Conclusion: Lactobacillus brevis PLA28 β-galactosidase has exhibited capability for transgalactosylation reaction with lactose conversion to synthesize GOS.