Bifidobacterial β-Galactosidase-Mediated Production of Galacto-Oligosaccharides: Structural and Preliminary Functional Assessments (2025)

Structural Comparison of Different Galacto-oligosaccharide Mixtures Formed by β-Galactosidases from Lactic Acid Bacteria and Bifidobacteria

Journal of Agricultural and Food Chemistry

The LacLM-type β-galactosidase from Lactobacillus helveticus DSM 20075 expressed in both Escherichia coli (EcoliBL21Lhβ-gal) and Lactobacillus plantarum (Lp609Lhβ-gal) was tested for their potential to form galacto-oligosaccharides (GOS) from lactose. The Lh-GOS mixture formed by β-galactosidase from L. helveticus, together with three GOS mixtures produced using β-galactosidases of both the LacLM and the LacZ type from other lactic acid bacteria, namely, L. reuteri (Lr-GOS), L. bulgaricus (Lb-GOS), and Streptococcus thermophilus (St-GOS), as well as two GOS mixtures (Br-GOS1 and Br-GOS2) produced using β-galactosidases (β-gal I and β-gal II) from Bifidobacterium breve, was analyzed and structurally compared with commercial GOS mixtures analyzed in previous work (Vivinal GOS, GOS I, GOS III, and GOS V) using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), high-performance size-exclusion chromatography with a refractive index (RI) detector (HPSEC-RI), and one-dimensional 1 H NMR spectroscopy. β-Galactosidases from lactic acid bacteria and B. breve displayed a preference to form β-(1→6)and β-(1→3)-linked GOS. The GOS mixtures produced by these enzymes consisted of mainly DP2 and DP3 oligosaccharides, accounting for ∼90% of all GOS components. GOS mixtures obtained with βgalactosidases from lactic acid bacteria and B. breve were quite similar to the commercial GOS III mixture in terms of product spectrum and showed a broader product spectrum than the commercial GOS V mixture. These GOS mixtures also contained a number of GOS components that were absent in the commercial Vivinal GOS (V-GOS).

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

PloS one, 2014

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 wit...

Galactooligosaccharides formation during enzymatic hydrolysis of lactose: Towards a prebiotic-enriched milk

Food Chemistry, 2014

The formation of galacto-oligosaccharides (GOS) in skim milk during the treatment with 14 several commercial β-galactosidases (Bacillus circulans, Kluyveromyces lactis and 15 Aspergillus oryzae) was analyzed in detail, at 4°C and 40°C. The maximum GOS 16 concentration was obtained at a lactose conversion of approximately 40-50% with B. 17 circulans and A. oryzae β-galactosidases, and at 95% lactose depletion for K. lactis β-18 galactosidase. Using an enzyme dosage of 0.1% (v/v), the maximum GOS concentration with 19 K. lactis β-galactosidase was achieved in 1 h and 5 h at 40°C and 4°C, respectively. With this 20 enzyme, it was possible to obtain a treated milk with 7.0 g/L GOS −the human milk 21 oligosaccharides (HMOs) concentration is between 5 and 15 g/L−, and with a low content of 22 residual lactose (2.1 g/L, compared with 44-46 g/L in the initial milk sample). The major 23 GOS synthesized by this enzyme were 6-galactobiose [Gal-β(1→6)-Gal], allolactose [Gal-24 β(1→6)-Glc] and 6´-O-β-galactosyl-lactose [Gal-β(1→6)-Gal-β(1→4)-Glc]. 25 26 Lactose-free milk, Human milk oligosaccharides. 28 29 3

β-Galactosidase from Lactobacillus plantarum WCFS1: biochemical characterization and formation of prebiotic galacto-oligosaccharides

Carbohydrate Research, 2010

Recombinant β-galactosidase from Lactobacillus plantarum WCFS1, homologously overexpressed in L. plantarum, was purified to apparent homogeneity using p-aminobenzyl 1-thio-β-Dgalactopyranoside affinity chromatography and subsequently characterized. The enzyme is a heterodimer of the LacLMfamily type, consisting of a small subunit of 35 kDa and a large subunit of 72 kDa. The optimum pH for hydrolysis of its preferred substrates o-nitrophenyl-β-D-galactopyranoside (oNPG) and lactose is 7.5 and 7.0, and optimum temperature for these reactions is 55 and 60 °C, respectively. The enzyme is most stable in the pH range of 6.5-8.0. The Km, k cat and kcat/Km values for oNPG and lactose are 0.9 mM, 92 s-1, 130 mM-1 s-1 and 29 mM, 98 s-1, 3.3 mM-1 s-1, respectively. The L. plantarum β-galactosidase possesses a high transgalactosylation activity and was used for the synthesis of prebiotic galactooligosaccharides (GOS). The resulting GOS mixture was analyzed in detail, and major components were identified by using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) as well as capillary electrophoresis. The maximal GOS yield was 41% (w/w) of total sugars at 85% lactose conversion (600 mM initial lactose concentration). The enzyme showed a strong preference for the formation of β-(1→6) linkages in its transgalactosylation mode, while β-(1→3)-linked products were formed to a lesser extent, comprising 80% and 9%, respectively, of the newly formed glycosidic linkages in the oligosaccharide mixture at maximum GOS formation. The main individual products formed were β-DGalp-( 1→6)-D-Lac, accounting for 34% of total GOS, and β-D-Galp-(1→6)-D-Glc, making up 29% of total GOS.

Galactosidase from Lactobacillus plantarum WCFS1: biochemical characterization and formation of prebiotic galacto-oligosaccharides

Carbohydrate Research, 2010

Recombinant β-galactosidase from Lactobacillus plantarum WCFS1, homologously overexpressed in L. plantarum, was purified to apparent homogeneity using p-aminobenzyl 1-thio-β-Dgalactopyranoside affinity chromatography and subsequently characterized. The enzyme is a heterodimer of the LacLMfamily type, consisting of a small subunit of 35 kDa and a large subunit of 72 kDa. The optimum pH for hydrolysis of its preferred substrates o-nitrophenyl-β-D-galactopyranoside (oNPG) and lactose is 7.5 and 7.0, and optimum temperature for these reactions is 55 and 60 °C, respectively. The enzyme is most stable in the pH range of 6.5-8.0. The Km, k cat and kcat/Km values for oNPG and lactose are 0.9 mM, 92 s-1, 130 mM-1 s-1 and 29 mM, 98 s-1, 3.3 mM-1 s-1, respectively. The L. plantarum β-galactosidase possesses a high transgalactosylation activity and was used for the synthesis of prebiotic galactooligosaccharides (GOS). The resulting GOS mixture was analyzed in detail, and major components were identified by using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) as well as capillary electrophoresis. The maximal GOS yield was 41% (w/w) of total sugars at 85% lactose conversion (600 mM initial lactose concentration). The enzyme showed a strong preference for the formation of β-(1→6) linkages in its transgalactosylation mode, while β-(1→3)-linked products were formed to a lesser extent, comprising 80% and 9%, respectively, of the newly formed glycosidic linkages in the oligosaccharide mixture at maximum GOS formation. The main individual products formed were β-DGalp-( 1→6)-D-Lac, accounting for 34% of total GOS, and β-D-Galp-(1→6)-D-Glc, making up 29% of total GOS.

Galactooligosaccharides derived from lactose and lactulose: Influence of structure on Lactobacillus, Streptococcus and Bifidobacterium growth

International Journal of Food Microbiology, 2011

The effect of structure on the fermentative properties of potential prebiotic trisaccharides derived from lactulose like 6′-galactosyl-lactulose (β-D-galactopyranosyl-(1 → 6)-β-D-galactopyranosyl-(1 → 4)-β-D-fructopyranose), 4′-galactosyl-lactulose (β-D-galactopyranosyl-(1 → 4)-β-D-galactopyranosyl-(1 → 4)-β-D-fructopyranose), and 1-galactosyl-lactulose (β-D-galactopyranosyl-(1 → 4)-β-D-fructopyranosyl-(1 → 1)-β-D-galactopyranose); and from lactose like 4′-galactosyl-lactose (β-D-galactopyranosyl-(1→ 4)-β-D-galactopyranosyl-(1 → 4)-β-Dglucopyranose) and 6′-galactosyl-lactose (β-D-galactopyranosyl-(1 → 6)-β-D-galactopyranosyl-(1 → 4)-β-Dglucopyranose), has been assessed in vitro. Fermentations with twelve pure strains of Lactobacillus, Streptococcus and Bifidobacterium were carried out using the purified trisaccharides as the sole carbon source, and bacteria growth was evaluated at 600 nm by means of a microplate reader during 48 h. Maximum growth rates (μ max) and lag phase were calculated. In general, all the strains tested were able to utilize lactulose and pure trisaccharides derived from lactulose and lactose when they were used as sole carbon source. Nonetheless, glycosidic linkage and/or the monosaccharide composition of the trisaccharides affected the individual strains lag phase, cell densities and growth rates. A general preference towards β-galactosyl residues β(1-6) and β(1-1) linked over those β(1-4) linked was observed, and some strains showed higher cell densities and speed of growth on 6′-galactosyl-lactulose than on 6′-galactosyl-lactose. This is the first study of the effect of lactulose-derived oligosaccharides on pure culture growth which shows that transglycosylation of lactulose allows for obtaining galactooligosaccharides with new glycosidic structures and would open new routes to the synthesis of compounds with potential prebiotic effects.

Galacto-oligosaccharide Synthesis from Lactose Solution or Skim Milk Using the β-Galactosidase from Bacillus circulans

Journal of Agricultural and Food Chemistry, 2012

The synthesis of galactooligosaccharides (GOS) catalyzed by a novel commercial preparation 2 of β-galactosidase from Bacillus circulans (Biolactase TM ) was studied and the products 3 characterized by MS and NMR. Using 400 g/l lactose and 1.5 enzyme units per ml, the 4 maximum GOS yield, measured by HPAEC-PAD analysis, was 165 g/l (41 % w/w of total 5 carbohydrates in the mixture). The major transgalactosylation products were the trisaccharide Gal-β(1→4)-Gal-β(1→4)-Glc and the tetrasaccharide Gal-β(1→4)-Gal-β(1→4)-Gal-β(1→4)-7 Glc. The GOS yield increased to 198 g/l (49.4 % w/w of total carbohydrates) using a higher 8 enzyme concentration (15 U/ml), which minimized the enzyme inactivation under reaction 9 conditions. Using skim milk (with a lactose concentration of 46 g/l), the enzyme also 10 displayed transgalactosylation activity: maximum GOS yield accounted for 15.4% (7.1 g/l), which was obtained at 50% lactose conversion. 12 13 Keywords: glycosidase, galacto-oligosaccharides, prebiotics, transglycosylation, beta-14 galactosidase, oligosaccharides. 15 7 (GOS). 2,3 GOS are non-cariogenic, reduce the level of cholesterol in serum, prevent colon 8 cancer and exhibit prebiotic properties. In fact, GOS constitute the major part of 9 oligosaccharides in human milk. 4-6 The properties of GOS depend significantly on their 10 chemical composition, structure and degree of polymerization. 7 Depending on the origin of β-11 galactosidase, the yield and composition of GOS vary notably. 8-11 The most studied β-12 galactosidases are those from Kluyveromyces lactis, 12-14 Rhizopus oryzae, 15 Bifidobacterium 13 sp. 16 and Bacillus circulans. 17 14 Regarding the β-galactosidase from Bacillus circulans, different isoforms have been 15 reported in the commercial preparation Biolacta (Daiwa Kasei). At least three isoforms with 16 different behaviour in GOS production were characterized: β-galactosidase-1 showed very low 17 transglycosylation activity, 18 β-galactosidase-2 contributed most significantly to GOS 18 synthesis, 18,19 and β-galactosidase-3 was able to produce GOS with β(1→3) bonds. 20 More 19 recently, Song et al. 21 described four isoforms with different molecular size in Biolacta: β-gal-20 A (189 kDa), β-gal-B (154 kDa), β-gal-C (134 kDa) and β-gal-D (91 kDa). The transferase 21 activity of β-galactosidase from B. circulans has been applied to the synthesis of 22 lactosucrose, 22 N-acetyl-lactosamine 23 and other galactosylated derivatives. 24 Interestingly, the 23 enzyme is able to catalyze the galactosylation of different acceptors in the presence of organic 24 2. EXPERIMENTAL PROCEDURES 1 Materials 2 Biolactase TM (batch no. MB-878) is a liquid β-galactosidase preparation from Bacillus 3 circulans produced by Kerry Ingredients and Flavours (http://www.kerry.com) that was 4 supplied by Biocon (Spain). Glucose, galactose, lactose monohydrate and o-nitrophenyl-β-D-5 galactopyranoside (ONPG) were from Sigma-Aldrich. 3-Galactobiose, 4-galactobiose, 6-6 galactobiose, 6-O-β-galactosyl-glucose (allolactose) and 4-O-β-galactosyl-lactose were from 7 Carbosynth (Berkshire, UK). Skim milk "Hacendado" was purchased from Mercadona 8 supermarket (Spain). All other reagents and solvents were of the highest available purity and 9 used as purchased. 10 11 Activity assay 12 The enzymatic activity towards o-nitrophenyl-β-D-galactopyranoside (ONPG) was measured 13 at 40 ºC following o-nitrophenol release at 405 nm using a microplate reader (Versamax, 14 Molecular Devices). The reaction was started by adding 10 µl of the enzyme (conveniently 15 diluted) to 190 µl of 15 mM ONPG in 0.1 M sodium acetate buffer (pH 5.5). The increase of 16 absorbance at 405 nm was followed in continuous mode during 5 min. The extinction molar 17 coefficient of o-nitrophenol at pH 5.5 was determined (537 M -1 cm -1 ). One unit (U) of activity 18 was defined as that corresponding to the hydrolysis of 1 µmol of ONPG per min. 19 20 SDS-PAGE. SDS-PAGE was performed on 8% polyacrylamide gels and the proteins were 21 stained with colloidal Coomassie Blue (Protoblue Safe, National Diagnostics) diluted with 22 ethanol. HMW-SDS calibration quit (53-220 kDa) was from GE Healthcare Bio-Sciences. 23 LMW-SDS calibration quit (15-150 kDa) was from Novagen. 24 25 6 Thermostability of B. circulans β β β β-galactosidase 1

β-Galactosidase from Lactobacillus pentosus: Purification, characterization and formation of galacto-oligosaccharides

Biotechnology Journal, 2010

Abbreviations: β-Gal, β-galactosidase; CE, capillary electrophoresis; GOS, galactooligosaccharides; HPAEC-PAD, high-performance anion exchange chromatography with pulsed amperometric detection; MUGAbstract A novel heterodimeric β-galactosidase with a molecular mass of 105 kDa was purified from the crude cell extracts of the soil isolate Lactobacillus pentosus KUB-ST10-1 using ammonium sulphate fractionation followed by hydrophobic interaction and affinity chromatography. The electrophoretically homogenous enzyme has a 5 specific activity of 97 U oNPG /mg protein. The K m , k cat and k cat /K m values for lactose and oNPG were 38 mM, 20 s -1 , 530 M -1 ·s -1 and 1.67 mM, 540 s -1 , 325,000 M -1 ·s -1 , respectively. The temperature optimum of β-galactosidase activity was 60-65°C for a 10-min assay, which is considerably higher than the values reported for other lactobacillal β-galactosidases. Mg 2+ ions enhanced both activity and stability 10 significantly. L. pentosus β-galactosidase was used for the production of prebiotic galacto-oligosaccharides (GOS) from lactose. A maximum yield of 31% GOS of total sugars was obtained at 78% lactose conversion. The enzyme showed a strong preference for the formation of β-(1→3) and β-(1→6) linkages, and the main transgalactosylation products identified were the disaccharides β-D-Galp-(1→6)-D-15 Glc, β-D-Galp-(1→3)-D-Glc, β-D-Galp-(1→6)-D-Gal, β-D-Galp-(1→3)-D-Gal, and the trisaccharides β-D-Galp-(1→3)-D-Lac, β-D-Galp-(1→6)-D-Lac.

Synthesis of Galactooligosaccharides in Milk and Whey: A Review

Comprehensive Reviews in Food Science and Food Safety, 2018

Galactooligosaccharides (GOS) are synthesized by the enzyme β‐galactosidase during the hydrolysis of lactose. In this so‐called transgalactosylation reaction the galactosyl moiety is transferred to another sugar molecule instead of water resulting in oligosaccharides of different chain lengths and glycosidic linkages. Because their structures are similar to oligosaccharides present in human breast milk, they act as prebiotics, which has been shown for infants and adults to be alike. While so far most of the research to maximize GOS yield has been carried out using buffered lactose solution as a starting material, more and more work is now conducted with dairy by‐products such as whey and whey permeate, or even milk, for direct GOS synthesis in order to develop new GOS‐enriched dairy products. This review aims to summarize the results obtained with various dairy liquids, and it rates their suitabilities to act as raw material for GOS production. Most of the studies using whey or milk...

Production of Prebiotic Galacto-Oligosaccharides from Lactose Using β-Galactosidases from Lactobacillus reuteri

Journal of Agricultural and Food Chemistry, 2006

The -galactosidases ( -Gals) of Lactobacillus reuteri L103 and L461 proved to be suitable biocatalysts for the production of prebiotic galacto-oligosaccharides (GOS) from lactose. Maximum GOS yields were 38% when using an initial lactose concentration of 205 g/L and at ∼80% lactose conversion. The product mixtures were analyzed by capillary electrophoresis (CE) and high-performance anionexchange chromatography with pulsed amperometric detection (HPAEC-PAD). Disaccharides other than lactose and trisaccharides made up the vast majority of GOS formed. The main products were identified as -D-Galp-(1f6)-D-Glc (allolactose), -D-Galp-(1f6)-D-Gal, -D-Galp-(1f3)-D-Gal, -D-Galp-(1f6)-Lac, and -D-Galp-(1f3)-Lac. There were no major products with 1f4 linkages formed. Both intermolecular and intramolecular transgalactosylation were observed. D-Galactose proved to be a very efficient galactosyl acceptor; thus, a relatively large amount of galactobioses was formed. Monosaccharides could be conveniently separated from the mixture by chromatography using a strong cation-exchange resin.