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

Abstract

β-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 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 E. coli, which is more than 1000-fold or 28-fold higher than the production of native β-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 ∼2.3 kU per L of medium, which is approximately 10-fold lower compared to the expression in E. coli. The recombinant β-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-β-d-galactopyranoside (oNPG) 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 o-nitrophenol released per min per mg protein, respectively. The enzyme was inhibited by high concentrations of oNPG with K_i,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 °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 β-galactosidases from L. helveticus; hence, this enzyme is an interesting candidate for applications in lactose conversion and GOS formation processes.

Keywords: Lactobacillus helveticus; expression systems; recombinant enzyme; β-galactosidase.

Figure 1

SDS-PAGE analysis of cell-free extract of crude Escherichia coli BL21 Star (DE3) (A), E. coli T7 Express containing the plasmid pGRO7 (B), Lactobacillus plantarum WCFS1 (C) and L. plantarum TLG02 (D) overexpressing β-galactosidase from Lactobacillus helveticus DSM 20075. E. coli BL21 Star (DE3) and T7 Express GRO carrying the plasmid pET21lacLMLh were cultivated in 300 mL LB medium; whereas L. plantarum WCFS1 and L. plantarum TLG02 harboring the plasmids p409lacLMLh and p609lacLMLh, respectively, were cultivated in 500 mL MRS at 30 °C. The cultivation and induction conditions are as described in Materials and Methods and samples were taken at different time points during cultivations (non-induced cultures) and after induction during cultivations (induced cultures). The arrows indicate the LacL and LacM subunits of the recombinant β-galactosidase. M denotes the Precision protein ladder (Biorad, CA, USA).

Figure 1

SDS-PAGE analysis of cell-free extract of crude Escherichia coli BL21 Star (DE3) (A), E. coli T7 Express containing the plasmid pGRO7 (B), Lactobacillus plantarum WCFS1 (C) and L. plantarum TLG02 (D) overexpressing β-galactosidase from Lactobacillus helveticus DSM 20075. E. coli BL21 Star (DE3) and T7 Express GRO carrying the plasmid pET21lacLMLh were cultivated in 300 mL LB medium; whereas L. plantarum WCFS1 and L. plantarum TLG02 harboring the plasmids p409lacLMLh and p609lacLMLh, respectively, were cultivated in 500 mL MRS at 30 °C. The cultivation and induction conditions are as described in Materials and Methods and samples were taken at different time points during cultivations (non-induced cultures) and after induction during cultivations (induced cultures). The arrows indicate the LacL and LacM subunits of the recombinant β-galactosidase. M denotes the Precision protein ladder (Biorad, CA, USA).

Figure 2

Time course of the cultivations of (A) E. coli BL21 Star (DE3) and E. coli T7 Express containing the plasmid pGRO7 and (B) L. plantarum WCFS1 and L. plantarum TLG02 overexpressing β-galactosidase from L. helveticus DSM 20075, resulting in the recombinant β-galactosidases EcoliBL21Lhβ-gal, EcoliGROLhβ-gal, Lp409Lhβ-gal and Lp609Lhβ-gal, respectively. All data points represent the average value from two independent experiments, and the percentage error was always less than 5%.

Figure 3

SDS-PAGE analysis of crude and purified β-galactosidase (lacLM) from L. helveticus DSM 20075 overexpressed in E. coli BL21 Star (DE3) and L. plantarum WCFS1. Lane 1, whole-cell lysates of E. coli BL21 Star (DE3) carrying the plasmid pET21lacLMLh with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) induction at 25 °C for 18 h; lane 2, whole-cell lysates of L. plantarum WCFS1 containing p409lacLMLh with 25 ng/mL inducing peptide pheromone IP-673 at 30 °C for 8 h; lane 3, purified recombinant β-galactosidase EcoliBL21Lhβ-gal. The arrows indicate the LacL and LacM subunits of the recombinant β-galactosidase. M denotes the Precision protein ladder (Biorad, CA, USA).

Figure 4

pH optimum (A) and temperature optimum (B) of recombinant β-galactosidase EcoliBL21Lhβ-gal using oNPG and lactose as the substrates. Relative activities are given in comparison to the maximum activities measured under optimal conditions (100%). All data points represent the average value from two independent experiments, and the percentage error was always less than 5%.

Figure 4

pH optimum (A) and temperature optimum (B) of recombinant β-galactosidase EcoliBL21Lhβ-gal using oNPG and lactose as the substrates. Relative activities are given in comparison to the maximum activities measured under optimal conditions (100%). All data points represent the average value from two independent experiments, and the percentage error was always less than 5%.

Figure 5

Hydrolysis of lactose catalyzed by recombinant β-galactosidases from L. helveticus, (A) crude enzyme Lp609Lhβ-gal and (B) purified EcoliBL21Lhβ-gal, as analyzed by thin layer chromatography (TLC) on pre-activated silica plates (eluent n-butanol/n-propanol/ethanol/water mixture: 2/3/3/2). The reactions were carried out at 50 °C with an initial lactose concentration of 205 g L⁻¹ in 50 mM sodium phosphate buffer (pH 6.5) containing 1 mM MgCl₂ and 1.5 U_Lac mL⁻¹ of recombinant β-galactosidase. The samples were taken at regular time intervals during the reactions. A commercially available GOS preparation, Vivinal (Friesland Foods Domo), was added for comparison (indicated by V-GOS). A standard mixture of lactose (Lac), glucose (Glc) and galactose (Gal) was used as standards.

Figure 6

Expression vectors for lacLM from L. helveticus DSM 20075 in L. plantarum based on the erythromycin resistance gene (erm) [31] (A) and the alanine racemase gene (alr) [32] (B) as selection markers. sppK and sppR, denoting a histidine kinase and a response regulator, respectively, are regulated by P_sppIP. Overlapping genes lacLM from L. helveticus are controlled by the inducible promoter PsppQ (pSIP409 derivatives).