Abstract
The β-galactosidases from Lactobacillus reuteri L103 (Lreuβgal), Lactobacillus delbrueckii subsp. bulgaricus DSM 20081 (Lbulβgal), and Bifidobacterium breve DSM 20281 (Bbreβgal-I and Bbreβgal-II) were investigated in detail with respect to their propensity to transfer galactosyl moieties onto lactose, its hydrolysis products d-glucose and d-galactose, and certain sugar acceptors such as N-acetyl-d-glucosamine (GlcNAc), N-acetyl-d-galactosamine (GalNAc), and l-fucose (Fuc) under defined, initial velocity conditions. The rate constants or partitioning ratios (kNu/kwater) determined for these different acceptors (termed nucleophiles, Nu) were used as a measure for the ability of a certain substance to act as a galactosyl acceptor of these β-galactosidases. When using Lbulβgal or Bbreβgal-II, the galactosyl transfer to GlcNAc was 6 and 10 times higher than that to lactose, respectively. With lactose and GlcNAc used in equimolar substrate concentrations, Lbulβgal and Bbreβgal-II catalyzed the formation of N-acetyl-allolactosamine with the highest yields of 41 and 24%, respectively, as calculated from the initial GlcNAc concentration.
Highlights
Β-Galactosidases (β-D-galactoside galactohydrolase, EC 3.2.1.23; βgal) have long been known to catalyze the hydrolysis of lactose into glucose and galactose, as well as the transfer of a galactosyl moiety to suitable acceptors
If lactose is present in excess, βgal will use lactose, or its hydrolysis products, glucose and galactose, as alternative galactosyl acceptors to form galacto-oligosaccharides (GOS) (Scheme 1)
Part of the human milk oligosaccharide (HMO) core structure or structurally related compounds can be accessed, albeit not in pure form, through an approach based on β-galactosidase-catalyzed transglycosylation with lactose as donor and N-acetylglucosamine (GlcNAc) as acceptor
Summary
Β-Galactosidases (β-D-galactoside galactohydrolase, EC 3.2.1.23; βgal) have long been known to catalyze the hydrolysis of lactose into glucose and galactose, as well as the transfer of a galactosyl moiety to suitable acceptors. Hydrolysis and Galactosyl Transfer Reactions during the β-Galactosidase-Catalyzed Conversion of Lactosea aE, enzyme; Lac, lactose; Gal, galactose; Glc, glucose; Nu, nucleophile. Bulgaricus DSM 20081 (L. bulgaricus, Lbulβgal), L. reuteri L103 (Lreuβgal), and Bifidobacterium breve DSM 20281 βgal I (Bbreβgal-I) and βgal-II (Bbreβgal-II) to transfer galactosyl moiety to different acceptors such as lactose (Lac), glucose (D-Glc), galactose (D-Gal), L-fucose (Fuc), N-acetyl-D-glucosamine (GlcNAc), and N-acetyl-D-. For purification and identification of GlcNAc transfer products, a 10 mL discontinuous batch reaction using Bbreβgal-II (5 ULac/mL) was carried out at 30 °C using initial equimolar concentrations of lactose and GlcNAc (600 mM each) dissolved in 50 mM sodium phosphate buffer (pH 6.5) with 1 mM Mg2+.
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