β-Galactosidase from DSM 20075: Biochemical Characterization and Recombinant Expression for Applications in Dairy Industry.

β-Galactosidase encoding genes from DSM 20075 were cloned and successfully overexpressed in and using different expression systems. The highest recombinant β-galactosidase activity of ∼26 kU per L of medium was obtained when using an expression system based on the T7 RNA polymerase promoter in , which is more than 1000-fold or 28-fold higher than the production of native β-galactosidase from DSM 20075 when grown on glucose or lactose, respectively. The overexpression in using lactobacillal food-grade gene expression system resulted in ∼2.3 kU per L of medium, which is approximately 10-fold lower compared to the expression in . The recombinant β-galactosidase from overexpressed in was purified to apparent homogeneity and subsequently characterized. The and values for lactose and -nitrophenyl-β-d-galactopyranoside (NPG) were 15.7 ± 1.3 mM, 11.1 ± 0.2 µmol D-glucose released per min per mg protein, and 1.4 ± 0.3 mM, 476 ± 66 µmol -nitrophenol released per min per mg protein, respectively. The enzyme was inhibited by high concentrations of NPG with = 3.6 ± 0.8 mM. The optimum pH for hydrolysis of both substrates, lactose and NPG, is pH 6.5 and optimum temperatures for these reactions are 60 and 55 °C, respectively. The formation of galacto-oligosaccharides (GOS) in discontinuous mode using both crude recombinant enzyme from and purified recombinant enzyme from revealed high transgalactosylation activity of β-galactosidases from ; hence, this enzyme is an interesting candidate for applications in lactose conversion and GOS formation processes.