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 k cat/K m 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.

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

“…Incubation of the enzyme samples in 50 mM NaPB (pH 6.5) with the presence of 1 mM MgCl 2 at 40 °C and 50 °C showed approximately 15-fold and nine-fold increase in the half-life time ( τ 1/2 ) of activity, respectively, compared to the samples incubated at the same conditions but without MgCl 2 (Table 2). This positive effect of Mg 2+ on bacterial β-galactosidases has been demonstrated for both LacLM-type [10,35] and LacZ-type β-galactosidases [12,25,36]. The previous studies on metal-binding sites in the structure of β-galactosidase from E. coli has revealed binding sites for Mg 2+ , which are located in the direct vicinity of the active site and might take a part in the catalytic mechanism and stabilization of the enzyme [36,37,38].…”

A multifunctional thermophilic glycoside hydrolase from Caldicellulosiruptor owensensis with potential applications in production of biofuels and biochemicals

“…The GalOS productivity by CoGH1A was more than 12 times higher than that of the β-galactosidases from Lactobacillus delbrueckii subsp. Bulgaricus (19.8 g L −1 h −1 ) [ 23 ], Bifidobacterium breve (14.7 g L −1 h −1 ) [ 51 ], marine metagenomic library (20.6 g L −1 h −1 ) [ 52 ], and Thermotoga maritima (18.20 g L −1 h −1 ) [ 39 ]. Time for reaching the maximum yield by the four β-galactosidases was 5–10 h (Table 6 ), which is at least fivefold longer than that by CoGH1A.…”

From by-product to valuable components: Efficient enzymatic conversion of lactose in whey using β-galactosidase from Streptococcus thermophilus

“…These LacZ β-galactosidases are more frequent in other LAB including S. salivarius and S. thermophilus [44] or bifidobacteria including Bifidobacterium bifidum [45], Bifidobacterium longum subsp. infantis [46], or Bifidobacterium breve [47]. When overexpressing β-galactosidases from L. reuteri and L. bulgaricus in the host L. plantarum WCFS1, the highest yields obtained under optimized fermentation conditions were ~35–40 kU and ~53 kU of β-galactosidase activity per liter of culture, respectively [34,48].…”