Two β-galactosidases from the human isolate Bifidobacterium breve DSM 20213: molecular cloning and expression, biochemical characterization and synthesis of galacto-oligosaccharides

Abstract

Two β-galactosidases, β-gal I and β-gal II, from Bifidobacterium breve DSM 20213, which was isolated from the intestine of an infant, were overexpressed in Escherichia coli with co-expression of the chaperones GroEL/GroES, purified to electrophoretic homogeneity and biochemically characterized. Both β-gal I and β-gal II belong to glycoside hydrolase family 2 and are homodimers with native molecular masses of 220 and 211 kDa, respectively. The optimum pH and temperature for hydrolysis of the two substrates o-nitrophenyl-β-D-galactopyranoside (oNPG) and lactose were determined at pH 7.0 and 50°C for β-gal I, and at pH 6.5 and 55°C for β-gal II, respectively. The kcat/Km values for oNPG and lactose hydrolysis are 722 and 7.4 mM-1s-1 for β-gal I, and 543 and 25 mM-1s-1 for β-gal II. Both β-gal I and β-gal II are only moderately inhibited by their reaction products D-galactose and D-glucose. Both enzymes were found to be very well suited for the production of galacto-oligosaccharides with total GOS yields of 33% and 44% of total sugars obtained with β-gal I and β-gal II, respectively. The predominant transgalactosylation products are β-D-Galp-(1→6)-D-Glc (allolactose) and β-D-Galp-(1→3)-D-Lac, accounting together for more than 75% and 65% of the GOS formed by transgalactosylation by β-gal I and β-gal II, respectively, indicating that both enzymes have a propensity to synthesize β-(1→6) and β-(1→3)-linked GOS. The resulting GOS mixtures contained relatively high fractions of allolactose, which results from the fact that glucose is a far better acceptor for galactosyl transfer than galactose and lactose, and intramolecular transgalactosylation contributes significantly to the formation of this disaccharide.

Figure 1. pH (A and B) and temperature (C and D) optimum of β-galactosidase activity for B. breve β-gal I (•) and β-gal II (○) using oNPG (A and C) and lactose (B and D) as substrate.

Values are the mean of two independent experiments and the standard deviation was always less than 5%.

Figure 2. pH stability of the β-galactosidases from B. breve β-gal I (•) and β-gal II (○) incubated at 37°C in Britton-Robinson buffer over a pH range of pH 5.0–9.0 for 4 h (solid lines) and 10 h (dashed lines).

The residual activity was measured after 4(B) and oNPG was used as substrate for the enzyme assay. Values are the mean of two independent experiments and the standard deviation was always less than 5%.

Figure 3. Time course of GOS formation (A) and formation and degradation of GOS during lactose conversion (B) catalyzed by B. breve β-gal I (•) and β-gal II (○).

The reaction was performed at 30°C at an initial lactose concentration of 200 g L⁻¹ in sodium phosphate buffer (pH 6.5) and 1 mM MgCl₂ using 1.5 U_Lac mL⁻¹ _. Values are the mean of two independent experiments and the standard deviation was always less than 5%.

Figure 4. Formation and degradation of individual GOS formed by B. breve β-gal I (A) and β-gal II (B) during lactose conversion.

Reaction conditions: initial lactose concentration of 200 g L⁻¹ in 50 mM sodium phosphate buffer (pH 6.5) with 1 mM Mg²⁺ and 30°C and 1.0 U_Lac mL⁻¹ β-gal I or 2.5 U_Lac mL⁻¹ β-gal II. Symbols: (•) D-Galp-(1→6)-D-Glc; (▪) D-Galp-(1→6)-D-Gal; (▴) Galp-(1→3)-D-Gal; (Δ) D-Galp-(1→3)-D-Glc; (□) D-Galp-(1→3)-D-Lac; (○) D-Galp-(1→4)-Lac, (+) D-Galp-(1→6)-D-Lac.

Figure 5. D-Glucose/D-Galactose (solid lines) and GalGlc/GalGal (dashed lines) ratios (A); GalGlc/GalLac (solid lines) and GalGal/GalLac (dashed lines) ratios (B) during lactose conversion by B. breve β-gal I (close symbol) and β-gal II (open symbol).

The reactions were performed at 30°C at an initial lactose concentration of 200 g L⁻¹ in sodium phosphate buffer (pH 6.5) and 1 mM MgCl₂ with 1.0 U_Lac mL⁻¹ β-gal I or 2.5 U_Lac mL⁻¹ β-gal II.