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

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