Lens culinaris β-galactosidase (Lsbgal): Insights into its purification, biochemical characterization and trisaccharides synthesis

Present work describes the purification of an acidic beta-galactosidase from Lens culinaris (Lsbgal) to homogeneity via 857 fold with specific activity of 87 U/mg. The molecular mass of purified Lsbgal was estimated similar to 76 kDa by Size Exclusion Chromatography on Superdex-200 (AKTA purifier) and on SDS-PAGE, showed hetero-dimeric subunits i.e. 45 kDa and 30 kDa. The purified Lsbgal showed glycoproteinous nature when applied to Con-A Sepharose chromatography. Biochemical studies revealed that optimum condition for purified Lsbgal against o, nitophenyl beta-D-galactopyranoside (ONPG) as a substrate was pH 3.0, 58 degrees C with an activation energy (E-a) 8.1 kcal/mole and Q(10) 1.8. Lsbgal hydrolyses ONPG with K-m value 1.21 mM and V-max 90.90 mu moles/min/mg. Purified Lsbgal when incubated with high lactose concentration showed transgalactosylation activity which lead to the formation of trisaccharides as a major product of total GOS. Therefore, the purified Lsbgal could be used as potential alternative in food industry and would be further explicated for trisaccharides synthesis.