Homodimeric β-galactosidase from Lactobacillus delbrueckii subsp. bulgaricus DSM 20081: expression in Lactobacillus plantarum and biochemical characterization

Abstract

The lacZ gene from Lactobacillus delbrueckii subsp. bulgaricus DSM 20081, encoding a β-galactosidase of the glycoside hydrolase family GH2, was cloned into different inducible lactobacillal expression vectors for overexpression in the host strain Lactobacillus plantarum WCFS1. High expression levels were obtained in laboratory cultivations with yields of approximately 53000 U of β-galactosidase activity per liter of medium, which corresponds to ~170 mg of recombinant protein per liter and β-galactosidase levels amounting to 63% of the total intracellular protein of the host organism. The wild-type (nontagged) and histidine-tagged recombinant enzymes were purified to electrophoretic homogeneity and further characterized. β-Galactosidase from L. bulgaricus was used for lactose conversion and showed very high transgalactosylation activity. The maximum yield of galacto-oligosaccharides (GalOS) was approximately 50% when using an initial concentration of 600 mM lactose, indicating that the enzyme can be of interest for the production of GalOS.

Figure 1

Schematic overview of the pTH plasmids developed in this study. The structural gene lacZ (with or without a hexa-histidine tag) is controlled by the inducible promoters P_sppA (pSIP403 derivatives) or P_sppQ (pSIP409 derivatives). P_sppIP controls the structural genes of the two-component regulatory system, sppK, a histidine kinase, and sppR, a response regulator. Ery indicates the erythromycin resistance marker, and transcriptional terminators are marked by lollypop structures.

Figure 2

SDS-PAGE analysis of cell-free extracts of noninduced (A) and induced cells (B) of L. plantarum WCFS1 harboring pTH101 (lanes 1A, 1B), pTH103 (lanes 2A, 2B), pTH104 (lanes 3A, 3B), and pTH102 (lanes 5A, 5B). Lane 4 shows the Precision Plus Protein standard (Bio-Rad). The gel was stained with Coomassie blue.

Figure 3

Electrophoretic analysis of purified recombinant β-galactosidase from L. bulgaricus: (A) SDS-PAGE (lanes: 1, Precision plus Protein standard ladder (Bio-Rad); 2, purified recombinant enzyme); (B) native-PAGE (lanes: 3, activity staining of β-galactosidase using 4-methylumbelliferyl β-d-galactoside as substrate; 4, purified β-galactosidase; 5, high molecular mass protein ladder (GE Healthcare)).

Figure 4

Temperature and pH optima of the activity of recombinant β-galactosidase from L. bulgaricus: (○) lactose as substrate; (●) oNPG as substrate. Relative activities are given in comparison with the maximum activities measured under optimal conditions (100%), which were 412 and 237 U/mL with oNPG and lactose as the substrate, respectively, when determining the temperature optimum (A) and 680 and 106 U/mL with oNPG and lactose as the substrate, respectively, for the pH dependence of activity (B).

Figure 5

Composition of the sugar mixture during lactose conversion by recombinant β-galactosidase from L. bulgaricus. The reaction was carried out at 30 °C with an initial concentration of 600 mM lactose in 50 mM sodium phosphate buffer, pH 6.5, in the presence of 10 mM MgCl₂ using ∼1.5 U_lactose/mL of enzyme. (A) Time course of the conversion: (+), lactose; (●), glucose; (○), galactose; (▼) total galacto-oligosaccharides (GalOS). (B) Composition of the sugar mixture and individual GalOS components at different degrees of lactose conversion: (●), glucose; (○), galactose; (▼) total (GalOS); (⧫), β-d-Galp-(1→3)-d-Glc; (■), β-d-Galp-(1→3)-d-Gal; (◇), β-d-Galp-(1→3)-Lac; (△), β-d-Galp-(1→6)-d-Glc; (□), β-d-Galp-(1→6)-Lac; (◆), unidentified GalOS. Monosaccharides were measured enzymatically, lactose and GalOS were quantified by HPAEC-PAD and CE. Individual sugars are given as the percentage of total sugars (205 g/L) in the mixture.