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Francisco J Plou - One of the best experts on this subject based on the ideXlab platform.
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selective synthesis of galactooligosaccharides containing β 1 3 linkages with β galactosidase from bifidobacterium bifidum saphera
Journal of Agricultural and Food Chemistry, 2020Co-Authors: Vera Fureder, Barbara Rodriguezcolinas, Lucia Fernandezarrojo, Antonio Ballesteros, Ana Poveda, Jesus Jimenezbarbero, Fadia V Cervantes, Francisco J PlouAbstract:The transglycosylation activity of a novel commercial β-galactosidase from Bifidobacterium bifidum (Saphera) was evaluated. The optimal conditions for the operation of this enzyme, measured with o-nitrophenyl-β-d-galactopyranoside, were 40 °C and pH around 6.0. Although at low lactose concentrations the property of this enzyme was basically hydrolytic, an increase of lactose concentration to 400 g/L resulted in a significant formation (107.2 g/L, 27% yield) of prebiotic galactooligosaccharides (GOS). The maximum amount of GOS was obtained at a lactose conversion of approximately 90%, which contrasts with other β-galactosidases, for which the highest GOS yield is achieved at 40-50% lactose conversion. Using high-performance anion-exchange chromatography with pulsed amperometric detection, semipreparative high-performance liquid chromatography-hydrophilic interaction liquid chromatography, mass spectrometry, and 1D and 2D NMR, we determined the structure of most of the GOS synthesized by this enzyme. The main identified products were Gal-β(1→3)-Gal-β(1→4)-Glc (3'-O-β-galactosyl-lactose), Gal-β(1→6)-Glc (Allolactose), Gal-β(1→3)-Glc (3-galactosyl-glucose), Gal-β(1→3)-Gal (3-galactobiose), and the tetrasaccharide Gal-β(1→3)-Gal-β(1→3)-Gal-β(1→4)-Glc. In general, B. bifidum β-galactosidase showed a tendency to form β(1→3) linkages followed by β(1→6) and more scarcely β(1→4).
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Galactooligosaccharide Production from Pantoea anthophila Strains Isolated from “Tejuino”, a Mexican Traditional Fermented Beverage
MDPI AG, 2017Co-Authors: Claudia V. Yañez-Ñeco, Francisco J Plou, Lorena Amaya-delgado, Barbara Rodriguez-colinas, Antonio O. Ballesteros, Anne Gschaedler, Javier ArrizonAbstract:Two Pantoea anthophila bacterial strains were isolated from “tejuino”, a traditional Mexican beverage, and studied as β-galactosidase producers for galactooligosaccharides synthesis. Using 400 g/L of lactose, 50 °C, and 15 U/mL of β-galactosidase activity with ethanol-permeabilized cells, the maximum galactooligosaccharides (GOS) yield determined by High performance anion exchange chromatography with pulse amperometric detection (HPAEC-PAD) was 136 g/L (34% w/w of total sugars) at 96% of lactose conversion for Bac 55.2 and 145 g/L (36% w/w of total sugars) at 94% of lactose conversion for Bac 69.1. The main synthesized products were the disaccharides Allolactose [Gal-β(1 → 6)-Glc] and 6-galactobiose [Gal-β(1 → 6)-Gal], as well as the trisaccharides 3′-galactosyl-lactose [Gal-β(1 → 3)-Gal-β(1 → 4)-Glc], 6-galactotriose [Gal-β(1 → 6)-Gal-β(1 → 6)-Gal], 3′-galactosyl-Allolactose [Gal-β(1 → 3)-Gal-β(1 → 6)-Glc], and 6′-galactosyl-lactose [Gal-β(1 → 6)-Gal-β(1 → 4)-Glc]. The β-galactosidases present in both strains showed a high transgalactosylation activity and formed principally β(1 → 3) and β(1 → 6) linkages. Considering the stability and bifidogenic properties of GOS containing such types of bonds, P. anthophila strains Bac 55.2 and Bac 69.1 possess a high potential as novel biocatalysts for prebiotic industrial production
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low lactose prebiotic enriched milk
Probiotics Prebiotics and Synbiotics#R##N#Bioactive Foods in Health Promotion, 2016Co-Authors: Francisco J Plou, Barbara Rodriguezcolinas, Lucia Fernandezarrojo, Antonio BallesterosAbstract:To mimic the multiple benefits of human milk oligosaccharides (HMOs), infant dairy products are usually supplemented with prebiotic galacto-oligosaccharides (GOS) and/or fructo-oligosaccharides. Instead of adding GOS to infant formula, an attractive alternative could be to form such oligosaccharides in situ during the typical treatment of milk with β-galactosidases to eliminate lactose. The objective of this work was to obtain milk with a significant presence of GOS and, at the same time, a low content of lactose. The formation of GOS in skim milk was studied with the β-galactosidases from Bacillus circulans and Kluyveromyces lactis , at 4 and 40 °C. With B . circulans β-galactosidase, the maximum GOS concentration was obtained when 50% of the initial lactose had disappeared. In contrast, the maximum GOS yield with K . lactis enzyme was achieved at 95% of lactose depletion. Using an enzyme dosage of 0.1% (v/v), GOS concentration with K . lactis β-galactosidase reached 7.0 g/L—the HMOs concentration in breast milk is between 5 and 15 g/L—with only 2.1 g/L of residual lactose (initially 45 g/L). The major GOS synthesized by this enzyme were 6-galactobiose, Allolactose, and 6′- O -β- galactosyl-lactose. Thermal inactivation after enzymatic treatment with β-galactosidase from K . lactis could provide low-lactose milk with the extra benefit of a significant content of prebiotic GOS
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galactooligosaccharides formation during enzymatic hydrolysis of lactose towards a prebiotic enriched milk
Food Chemistry, 2014Co-Authors: Barbara Rodriguezcolinas, Lucia Fernandezarrojo, Antonio Ballesteros, Francisco J PlouAbstract:The formation of galactooligosaccharides (GOS) in skim milk during treatment with several commercial β-galactosidases (Bacillus circulans, Kluyveromyces lactis and Aspergillus oryzae) was analysed in detail, at 4 and 40°C. The maximum GOS concentration was obtained at a lactose conversion of approximately 40-50% with B. circulans and A. oryzae β-galactosidases, and at 95% lactose depletion for K. lactis β-galactosidase. Using an enzyme dosage of 0.1% (v/v), the maximum GOS concentration with K. lactis β-galactosidase was achieved in 1 and 5h at 40 and 4 °C, respectively. With this enzyme, it was possible to obtain a treated milk with 7.0 g/L GOS - the human milk oligosaccharides (HMOs) concentration is between 5 and 15 g/L--and with a low content of residual lactose (2.1g/L, compared with 44-46 g/L in the initial milk sample). The major GOS synthesised by this enzyme were 6-galactobiose [Gal-β(1 → 6)-Gal], Allolactose [Gal-β(1 → 6)-Glc] and 6'-O-β-galactosyl-lactose [Gal-β(1 → 6)-Gal-β(1 → 4)-Glc].
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detailed analysis of galactooligosaccharides synthesis with β galactosidase from aspergillus oryzae
Journal of Agricultural and Food Chemistry, 2013Co-Authors: Paulina Urrutia, Barbara Rodriguezcolinas, Lucia Fernandezarrojo, Antonio Ballesteros, Lorena Wilson, Andres Illanes, Francisco J PlouAbstract:The synthesis of galactooligosaccharides (GOS) catalyzed by β-galactosidase from Aspergillus oryzae (Enzeco) was studied. Using 400 g/L of lactose and 15 U/mL, maximum GOS yield, measured by HPAEC-PAD, was 26.8% w/w of total carbohydrates, obtained at approximately 70% lactose conversion. No less than 17 carbohydrates were identified; the major transgalactosylation product was 6′-O-β-galactosyl-lactose, representing nearly one-third (in weight) of total GOS. In contrast with previous reports, the presence of at least five disaccharides was detected, which accounted for 40% of the total GOS at the point of maximum GOS concentration (Allolactose and 6-galactobiose were the major products). A. oryzae β-galactosidase showed a preference to form β(1→6) bonds, followed by β(1→3) and β(1→4) linkages. Results were compared with those obtained with β-galactosidases from Kluyveromyces lactis and Bacillus circulans. The highest GOS yield and specific productivity were achieved with B. circulans β-galactosidase. The ...
Barbara Rodriguezcolinas - One of the best experts on this subject based on the ideXlab platform.
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selective synthesis of galactooligosaccharides containing β 1 3 linkages with β galactosidase from bifidobacterium bifidum saphera
Journal of Agricultural and Food Chemistry, 2020Co-Authors: Vera Fureder, Barbara Rodriguezcolinas, Lucia Fernandezarrojo, Antonio Ballesteros, Ana Poveda, Jesus Jimenezbarbero, Fadia V Cervantes, Francisco J PlouAbstract:The transglycosylation activity of a novel commercial β-galactosidase from Bifidobacterium bifidum (Saphera) was evaluated. The optimal conditions for the operation of this enzyme, measured with o-nitrophenyl-β-d-galactopyranoside, were 40 °C and pH around 6.0. Although at low lactose concentrations the property of this enzyme was basically hydrolytic, an increase of lactose concentration to 400 g/L resulted in a significant formation (107.2 g/L, 27% yield) of prebiotic galactooligosaccharides (GOS). The maximum amount of GOS was obtained at a lactose conversion of approximately 90%, which contrasts with other β-galactosidases, for which the highest GOS yield is achieved at 40-50% lactose conversion. Using high-performance anion-exchange chromatography with pulsed amperometric detection, semipreparative high-performance liquid chromatography-hydrophilic interaction liquid chromatography, mass spectrometry, and 1D and 2D NMR, we determined the structure of most of the GOS synthesized by this enzyme. The main identified products were Gal-β(1→3)-Gal-β(1→4)-Glc (3'-O-β-galactosyl-lactose), Gal-β(1→6)-Glc (Allolactose), Gal-β(1→3)-Glc (3-galactosyl-glucose), Gal-β(1→3)-Gal (3-galactobiose), and the tetrasaccharide Gal-β(1→3)-Gal-β(1→3)-Gal-β(1→4)-Glc. In general, B. bifidum β-galactosidase showed a tendency to form β(1→3) linkages followed by β(1→6) and more scarcely β(1→4).
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low lactose prebiotic enriched milk
Probiotics Prebiotics and Synbiotics#R##N#Bioactive Foods in Health Promotion, 2016Co-Authors: Francisco J Plou, Barbara Rodriguezcolinas, Lucia Fernandezarrojo, Antonio BallesterosAbstract:To mimic the multiple benefits of human milk oligosaccharides (HMOs), infant dairy products are usually supplemented with prebiotic galacto-oligosaccharides (GOS) and/or fructo-oligosaccharides. Instead of adding GOS to infant formula, an attractive alternative could be to form such oligosaccharides in situ during the typical treatment of milk with β-galactosidases to eliminate lactose. The objective of this work was to obtain milk with a significant presence of GOS and, at the same time, a low content of lactose. The formation of GOS in skim milk was studied with the β-galactosidases from Bacillus circulans and Kluyveromyces lactis , at 4 and 40 °C. With B . circulans β-galactosidase, the maximum GOS concentration was obtained when 50% of the initial lactose had disappeared. In contrast, the maximum GOS yield with K . lactis enzyme was achieved at 95% of lactose depletion. Using an enzyme dosage of 0.1% (v/v), GOS concentration with K . lactis β-galactosidase reached 7.0 g/L—the HMOs concentration in breast milk is between 5 and 15 g/L—with only 2.1 g/L of residual lactose (initially 45 g/L). The major GOS synthesized by this enzyme were 6-galactobiose, Allolactose, and 6′- O -β- galactosyl-lactose. Thermal inactivation after enzymatic treatment with β-galactosidase from K . lactis could provide low-lactose milk with the extra benefit of a significant content of prebiotic GOS
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galactooligosaccharides formation during enzymatic hydrolysis of lactose towards a prebiotic enriched milk
Food Chemistry, 2014Co-Authors: Barbara Rodriguezcolinas, Lucia Fernandezarrojo, Antonio Ballesteros, Francisco J PlouAbstract:The formation of galactooligosaccharides (GOS) in skim milk during treatment with several commercial β-galactosidases (Bacillus circulans, Kluyveromyces lactis and Aspergillus oryzae) was analysed in detail, at 4 and 40°C. The maximum GOS concentration was obtained at a lactose conversion of approximately 40-50% with B. circulans and A. oryzae β-galactosidases, and at 95% lactose depletion for K. lactis β-galactosidase. Using an enzyme dosage of 0.1% (v/v), the maximum GOS concentration with K. lactis β-galactosidase was achieved in 1 and 5h at 40 and 4 °C, respectively. With this enzyme, it was possible to obtain a treated milk with 7.0 g/L GOS - the human milk oligosaccharides (HMOs) concentration is between 5 and 15 g/L--and with a low content of residual lactose (2.1g/L, compared with 44-46 g/L in the initial milk sample). The major GOS synthesised by this enzyme were 6-galactobiose [Gal-β(1 → 6)-Gal], Allolactose [Gal-β(1 → 6)-Glc] and 6'-O-β-galactosyl-lactose [Gal-β(1 → 6)-Gal-β(1 → 4)-Glc].
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detailed analysis of galactooligosaccharides synthesis with β galactosidase from aspergillus oryzae
Journal of Agricultural and Food Chemistry, 2013Co-Authors: Paulina Urrutia, Barbara Rodriguezcolinas, Lucia Fernandezarrojo, Antonio Ballesteros, Lorena Wilson, Andres Illanes, Francisco J PlouAbstract:The synthesis of galactooligosaccharides (GOS) catalyzed by β-galactosidase from Aspergillus oryzae (Enzeco) was studied. Using 400 g/L of lactose and 15 U/mL, maximum GOS yield, measured by HPAEC-PAD, was 26.8% w/w of total carbohydrates, obtained at approximately 70% lactose conversion. No less than 17 carbohydrates were identified; the major transgalactosylation product was 6′-O-β-galactosyl-lactose, representing nearly one-third (in weight) of total GOS. In contrast with previous reports, the presence of at least five disaccharides was detected, which accounted for 40% of the total GOS at the point of maximum GOS concentration (Allolactose and 6-galactobiose were the major products). A. oryzae β-galactosidase showed a preference to form β(1→6) bonds, followed by β(1→3) and β(1→4) linkages. Results were compared with those obtained with β-galactosidases from Kluyveromyces lactis and Bacillus circulans. The highest GOS yield and specific productivity were achieved with B. circulans β-galactosidase. The ...
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production of galacto oligosaccharides by the β galactosidase from kluyveromyces lactis comparative analysis of permeabilized cells versus soluble enzyme
Journal of Agricultural and Food Chemistry, 2011Co-Authors: Barbara Rodriguezcolinas, Lucia Fernandezarrojo, Dietmar Haltrich, Miguel De Abreu, Roseri De Beer, Ana Poveda, Jesus Jimenezbarbero, Antonio Ballesteros O Olmo, Maria Fernandezlobato, Francisco J PlouAbstract:The transgalactosylation activity of Kluyveromyces lactis cells was studied in detail. Cells were permeabilized with ethanol and further lyophilized to facilitate the transit of substrates and products. The resulting biocatalyst was assayed for the synthesis of galacto-oligosaccharides (GOS) and compared with two soluble β-galactosidases from K. lactis (Lactozym 3000 L HP G and Maxilact LGX 5000). Using 400 g/L lactose, the maximum GOS yield, measured by HPAEC-PAD analysis, was 177 g/L (44% w/w of total carbohydrates). The major products synthesized were the disaccharides 6-galactobiose [Gal-β(1→6)-Gal] and Allolactose [Gal-β(1→6)-Glc], as well as the trisaccharide 6-galactosyl-lactose [Gal-β(1→6)-Gal-β(1→4)-Glc], which was characterized by MS and 2D NMR. Structural characterization of another synthesized disaccharide, Gal-β(1→3)-Glc, was carried out. GOS yield obtained with soluble β-galactosidases was slightly lower (160 g/L for Lactozym 3000 L HP G and 154 g/L for Maxilact LGX 5000); however, the typic...
Reuben E. Huber - One of the best experts on this subject based on the ideXlab platform.
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structural explanation for Allolactose lac operon inducer synthesis by lacz β galactosidase and the evolutionary relationship between Allolactose synthesis and the lac repressor
Journal of Biological Chemistry, 2013Co-Authors: R W Wheatley, Summie Lo, L J Jancewicz, Megan L Dugdale, Reuben E. HuberAbstract:β-Galactosidase (lacZ) has bifunctional activity. It hydrolyzes lactose to galactose and glucose and catalyzes the intramolecular isomerization of lactose to Allolactose, the lac operon inducer. β-Galactosidase promotes the isomerization by means of an acceptor site that binds glucose after its cleavage from lactose and thus delays its exit from the site. However, because of its relatively low affinity for glucose, details of this site have remained elusive. We present structural data mapping the glucose site based on a substituted enzyme (G794A-β-galactosidase) that traps Allolactose. Various lines of evidence indicate that the glucose of the trapped Allolactose is in the acceptor position. The evidence includes structures with Bis-Tris (2,2-bis(hydroxymethyl)-2,2′,2″-nitrilotriethanol) and l-ribose in the site and kinetic binding studies with substituted β-galactosidases. The site is composed of Asn-102, His-418, Lys-517, Ser-796, Glu-797, and Trp-999. Ser-796 and Glu-797 are part of a loop (residues 795–803) that closes over the active site. This loop appears essential for the bifunctional nature of the enzyme because it helps form the glucose binding site. In addition, because the loop is mobile, glucose binding is transient, allowing the release of some glucose. Bioinformatics studies showed that the residues important for interacting with glucose are only conserved in a subset of related enzymes. Thus, intramolecular isomerization is not a universal feature of β-galactosidases. Genomic analyses indicated that lac repressors were co-selected only within the conserved subset. This shows that the glucose binding site of β-galactosidase played an important role in lac operon evolution.
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LacZ β-galactosidase: Structure and function of an enzyme of historical and molecular biological importance
Protein Science, 2012Co-Authors: Douglas H Juers, Reuben E. HuberAbstract:This review provides an overview of the structure, function, and catalytic mechanism of lacZ β-galactosidase. The protein played a central role in Jacob and Monod's development of the operon model for the regulation of gene expression. Determination of the crystal structure made it possible to understand why deletion of certain residues toward the amino-terminus not only caused the full enzyme tetramer to dissociate into dimers but also abolished activity. It was also possible to rationalize α-complementation, in which addition to the inactive dimers of peptides containing the "missing" N-terminal residues restored catalytic activity. The enzyme is well known to signal its presence by hydrolyzing X-gal to produce a blue product. That this reaction takes place in crystals of the protein confirms that the X-ray structure represents an active conformation. Individual tetramers of β-galactosidase have been measured to catalyze 38,500 ± 900 reactions per minute. Extensive kinetic, biochemical, mutagenic, and crystallographic analyses have made it possible to develop a presumed mechanism of action. Substrate initially binds near the top of the active site but then moves deeper for reaction. The first catalytic step (called galactosylation) is a nucleophilic displacement by Glu537 to form a covalent bond with galactose. This is initiated by proton donation by Glu461. The second displacement (degalactosylation) by water or an acceptor is initiated by proton abstraction by Glu461. Both of these displacements occur via planar oxocarbenium ion-like transition states. The acceptor reaction with glucose is important for the formation of Allolactose, the natural inducer of the lac operon.
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structural comparisons of tim barrel proteins suggest functional and evolutionary relationships between beta galactosidase and other glycohydrolases
Protein Science, 2008Co-Authors: Douglas H Juers, Reuben E. Huber, Brian W MatthewsAbstract:Beta-galactosidase (lacZ) from Escherichia coli is a 464 kDa homotetramer. Each subunit consists of five domains, the third being an alpha/beta barrel that contains most of the active site residues. A comparison is made between each of the domains and a large set of proteins representative of all structures from the protein data bank. Many structures include an alpha/beta barrel. Those that are most similar to the alpha/beta barrel of E. coli beta-galactosidase have similar catalytic residues and belong to the so-called "4/7 superfamily" of glycosyl hydrolases. The structure comparison suggests that beta-amylase should also be included in this family. Of three structure comparison methods tested, the "ProSup" procedure of Zu-Kang and Sippl and the "Superimpose" procedure of Diederichs were slightly superior in discriminating the members of this superfamily, although all procedures were very powerful in identifying related protein structures. Domains 1, 2, and 4 of E. coli beta-galactosidase have topologies related to "jelly-roll barrels" and "immunoglobulin constant" domains. This fold also occurs in the cellulose binding domains (CBDs) of a number of glycosyl hydrolases. The fold of domain 1 of E. coli beta-galactosidase is closely related to some CBDs, and the domain contributes to substrate binding, but in a manner unrelated to cellulose binding by the CBDs. This is typical of domains 1, 2, 4, and 5, which appear to have been recruited to play roles in beta-galactosidase that are unrelated to the functions that such domains provide in other contexts. It is proposed that beta-galactosidase arose from a prototypical single domain alpha/beta barrel with an extended active site cleft. The subsequent incorporation of elements from other domains could then have reduced the size of the active site from a cleft to a pocket to better hydrolyze the disaccharide lactose and, at the same time, to facilitate the production of inducer, Allolactose.
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trp 999 of β galactosidase escherichia coli is a key residue for binding catalysis and synthesis of Allolactose the natural lac operon inducer
Biochemistry, 2003Co-Authors: Reuben E. Huber, Shamina Hakda, Calvino Cheng, Claire G Cupples, Robert EdwardsAbstract:Trp-999 is a key residue for the action of β-galactosidases (Escherichia coli). Several site specific substitutions (Phe, Gly, Tyr, Leu) for Trp-999 were made. Each substitution caused greatly decreased affinities for substrates and inhibitors that bind in the “shallow” mode, while the affinities of inhibitors that bind in the “deep” mode were not decreased nearly as much. This shows that Trp-999 is important for binding in the shallow mode. The residue is also very important for binding glucose to galactosyl-β-galactosidase (as a transgalactosidic acceptor). Substitution greatly diminished the affinity for glucose. Substitutions also changed the activation thermodynamics and, subsequently, the rates of the catalytic reactions. The enthalpies of activation of the glycolytic bond cleavage step (galactosylation, k2) became less favorable while the entropies of activation of that step became more favorable as a result of the substitutions. Differing magnitudes of these enthalpic and entropic effects with ONP...
Lucia Fernandezarrojo - One of the best experts on this subject based on the ideXlab platform.
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selective synthesis of galactooligosaccharides containing β 1 3 linkages with β galactosidase from bifidobacterium bifidum saphera
Journal of Agricultural and Food Chemistry, 2020Co-Authors: Vera Fureder, Barbara Rodriguezcolinas, Lucia Fernandezarrojo, Antonio Ballesteros, Ana Poveda, Jesus Jimenezbarbero, Fadia V Cervantes, Francisco J PlouAbstract:The transglycosylation activity of a novel commercial β-galactosidase from Bifidobacterium bifidum (Saphera) was evaluated. The optimal conditions for the operation of this enzyme, measured with o-nitrophenyl-β-d-galactopyranoside, were 40 °C and pH around 6.0. Although at low lactose concentrations the property of this enzyme was basically hydrolytic, an increase of lactose concentration to 400 g/L resulted in a significant formation (107.2 g/L, 27% yield) of prebiotic galactooligosaccharides (GOS). The maximum amount of GOS was obtained at a lactose conversion of approximately 90%, which contrasts with other β-galactosidases, for which the highest GOS yield is achieved at 40-50% lactose conversion. Using high-performance anion-exchange chromatography with pulsed amperometric detection, semipreparative high-performance liquid chromatography-hydrophilic interaction liquid chromatography, mass spectrometry, and 1D and 2D NMR, we determined the structure of most of the GOS synthesized by this enzyme. The main identified products were Gal-β(1→3)-Gal-β(1→4)-Glc (3'-O-β-galactosyl-lactose), Gal-β(1→6)-Glc (Allolactose), Gal-β(1→3)-Glc (3-galactosyl-glucose), Gal-β(1→3)-Gal (3-galactobiose), and the tetrasaccharide Gal-β(1→3)-Gal-β(1→3)-Gal-β(1→4)-Glc. In general, B. bifidum β-galactosidase showed a tendency to form β(1→3) linkages followed by β(1→6) and more scarcely β(1→4).
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low lactose prebiotic enriched milk
Probiotics Prebiotics and Synbiotics#R##N#Bioactive Foods in Health Promotion, 2016Co-Authors: Francisco J Plou, Barbara Rodriguezcolinas, Lucia Fernandezarrojo, Antonio BallesterosAbstract:To mimic the multiple benefits of human milk oligosaccharides (HMOs), infant dairy products are usually supplemented with prebiotic galacto-oligosaccharides (GOS) and/or fructo-oligosaccharides. Instead of adding GOS to infant formula, an attractive alternative could be to form such oligosaccharides in situ during the typical treatment of milk with β-galactosidases to eliminate lactose. The objective of this work was to obtain milk with a significant presence of GOS and, at the same time, a low content of lactose. The formation of GOS in skim milk was studied with the β-galactosidases from Bacillus circulans and Kluyveromyces lactis , at 4 and 40 °C. With B . circulans β-galactosidase, the maximum GOS concentration was obtained when 50% of the initial lactose had disappeared. In contrast, the maximum GOS yield with K . lactis enzyme was achieved at 95% of lactose depletion. Using an enzyme dosage of 0.1% (v/v), GOS concentration with K . lactis β-galactosidase reached 7.0 g/L—the HMOs concentration in breast milk is between 5 and 15 g/L—with only 2.1 g/L of residual lactose (initially 45 g/L). The major GOS synthesized by this enzyme were 6-galactobiose, Allolactose, and 6′- O -β- galactosyl-lactose. Thermal inactivation after enzymatic treatment with β-galactosidase from K . lactis could provide low-lactose milk with the extra benefit of a significant content of prebiotic GOS
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galactooligosaccharides formation during enzymatic hydrolysis of lactose towards a prebiotic enriched milk
Food Chemistry, 2014Co-Authors: Barbara Rodriguezcolinas, Lucia Fernandezarrojo, Antonio Ballesteros, Francisco J PlouAbstract:The formation of galactooligosaccharides (GOS) in skim milk during treatment with several commercial β-galactosidases (Bacillus circulans, Kluyveromyces lactis and Aspergillus oryzae) was analysed in detail, at 4 and 40°C. The maximum GOS concentration was obtained at a lactose conversion of approximately 40-50% with B. circulans and A. oryzae β-galactosidases, and at 95% lactose depletion for K. lactis β-galactosidase. Using an enzyme dosage of 0.1% (v/v), the maximum GOS concentration with K. lactis β-galactosidase was achieved in 1 and 5h at 40 and 4 °C, respectively. With this enzyme, it was possible to obtain a treated milk with 7.0 g/L GOS - the human milk oligosaccharides (HMOs) concentration is between 5 and 15 g/L--and with a low content of residual lactose (2.1g/L, compared with 44-46 g/L in the initial milk sample). The major GOS synthesised by this enzyme were 6-galactobiose [Gal-β(1 → 6)-Gal], Allolactose [Gal-β(1 → 6)-Glc] and 6'-O-β-galactosyl-lactose [Gal-β(1 → 6)-Gal-β(1 → 4)-Glc].
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detailed analysis of galactooligosaccharides synthesis with β galactosidase from aspergillus oryzae
Journal of Agricultural and Food Chemistry, 2013Co-Authors: Paulina Urrutia, Barbara Rodriguezcolinas, Lucia Fernandezarrojo, Antonio Ballesteros, Lorena Wilson, Andres Illanes, Francisco J PlouAbstract:The synthesis of galactooligosaccharides (GOS) catalyzed by β-galactosidase from Aspergillus oryzae (Enzeco) was studied. Using 400 g/L of lactose and 15 U/mL, maximum GOS yield, measured by HPAEC-PAD, was 26.8% w/w of total carbohydrates, obtained at approximately 70% lactose conversion. No less than 17 carbohydrates were identified; the major transgalactosylation product was 6′-O-β-galactosyl-lactose, representing nearly one-third (in weight) of total GOS. In contrast with previous reports, the presence of at least five disaccharides was detected, which accounted for 40% of the total GOS at the point of maximum GOS concentration (Allolactose and 6-galactobiose were the major products). A. oryzae β-galactosidase showed a preference to form β(1→6) bonds, followed by β(1→3) and β(1→4) linkages. Results were compared with those obtained with β-galactosidases from Kluyveromyces lactis and Bacillus circulans. The highest GOS yield and specific productivity were achieved with B. circulans β-galactosidase. The ...
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production of galacto oligosaccharides by the β galactosidase from kluyveromyces lactis comparative analysis of permeabilized cells versus soluble enzyme
Journal of Agricultural and Food Chemistry, 2011Co-Authors: Barbara Rodriguezcolinas, Lucia Fernandezarrojo, Dietmar Haltrich, Miguel De Abreu, Roseri De Beer, Ana Poveda, Jesus Jimenezbarbero, Antonio Ballesteros O Olmo, Maria Fernandezlobato, Francisco J PlouAbstract:The transgalactosylation activity of Kluyveromyces lactis cells was studied in detail. Cells were permeabilized with ethanol and further lyophilized to facilitate the transit of substrates and products. The resulting biocatalyst was assayed for the synthesis of galacto-oligosaccharides (GOS) and compared with two soluble β-galactosidases from K. lactis (Lactozym 3000 L HP G and Maxilact LGX 5000). Using 400 g/L lactose, the maximum GOS yield, measured by HPAEC-PAD analysis, was 177 g/L (44% w/w of total carbohydrates). The major products synthesized were the disaccharides 6-galactobiose [Gal-β(1→6)-Gal] and Allolactose [Gal-β(1→6)-Glc], as well as the trisaccharide 6-galactosyl-lactose [Gal-β(1→6)-Gal-β(1→4)-Glc], which was characterized by MS and 2D NMR. Structural characterization of another synthesized disaccharide, Gal-β(1→3)-Glc, was carried out. GOS yield obtained with soluble β-galactosidases was slightly lower (160 g/L for Lactozym 3000 L HP G and 154 g/L for Maxilact LGX 5000); however, the typic...
Douglas H Juers - One of the best experts on this subject based on the ideXlab platform.
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LacZ β-galactosidase: Structure and function of an enzyme of historical and molecular biological importance
Protein Science, 2012Co-Authors: Douglas H Juers, Reuben E. HuberAbstract:This review provides an overview of the structure, function, and catalytic mechanism of lacZ β-galactosidase. The protein played a central role in Jacob and Monod's development of the operon model for the regulation of gene expression. Determination of the crystal structure made it possible to understand why deletion of certain residues toward the amino-terminus not only caused the full enzyme tetramer to dissociate into dimers but also abolished activity. It was also possible to rationalize α-complementation, in which addition to the inactive dimers of peptides containing the "missing" N-terminal residues restored catalytic activity. The enzyme is well known to signal its presence by hydrolyzing X-gal to produce a blue product. That this reaction takes place in crystals of the protein confirms that the X-ray structure represents an active conformation. Individual tetramers of β-galactosidase have been measured to catalyze 38,500 ± 900 reactions per minute. Extensive kinetic, biochemical, mutagenic, and crystallographic analyses have made it possible to develop a presumed mechanism of action. Substrate initially binds near the top of the active site but then moves deeper for reaction. The first catalytic step (called galactosylation) is a nucleophilic displacement by Glu537 to form a covalent bond with galactose. This is initiated by proton donation by Glu461. The second displacement (degalactosylation) by water or an acceptor is initiated by proton abstraction by Glu461. Both of these displacements occur via planar oxocarbenium ion-like transition states. The acceptor reaction with glucose is important for the formation of Allolactose, the natural inducer of the lac operon.
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structural comparisons of tim barrel proteins suggest functional and evolutionary relationships between beta galactosidase and other glycohydrolases
Protein Science, 2008Co-Authors: Douglas H Juers, Reuben E. Huber, Brian W MatthewsAbstract:Beta-galactosidase (lacZ) from Escherichia coli is a 464 kDa homotetramer. Each subunit consists of five domains, the third being an alpha/beta barrel that contains most of the active site residues. A comparison is made between each of the domains and a large set of proteins representative of all structures from the protein data bank. Many structures include an alpha/beta barrel. Those that are most similar to the alpha/beta barrel of E. coli beta-galactosidase have similar catalytic residues and belong to the so-called "4/7 superfamily" of glycosyl hydrolases. The structure comparison suggests that beta-amylase should also be included in this family. Of three structure comparison methods tested, the "ProSup" procedure of Zu-Kang and Sippl and the "Superimpose" procedure of Diederichs were slightly superior in discriminating the members of this superfamily, although all procedures were very powerful in identifying related protein structures. Domains 1, 2, and 4 of E. coli beta-galactosidase have topologies related to "jelly-roll barrels" and "immunoglobulin constant" domains. This fold also occurs in the cellulose binding domains (CBDs) of a number of glycosyl hydrolases. The fold of domain 1 of E. coli beta-galactosidase is closely related to some CBDs, and the domain contributes to substrate binding, but in a manner unrelated to cellulose binding by the CBDs. This is typical of domains 1, 2, 4, and 5, which appear to have been recruited to play roles in beta-galactosidase that are unrelated to the functions that such domains provide in other contexts. It is proposed that beta-galactosidase arose from a prototypical single domain alpha/beta barrel with an extended active site cleft. The subsequent incorporation of elements from other domains could then have reduced the size of the active site from a cleft to a pocket to better hydrolyze the disaccharide lactose and, at the same time, to facilitate the production of inducer, Allolactose.
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a structural view of the action of escherichia coli lacz beta galactosidase
Biochemistry, 2001Co-Authors: Douglas H Juers, Td Heightman, Andrea Vasella, John D Mccarter, Lloyd F Mackenzie, Stephen G Withers, Brian W MatthewsAbstract:The structures of a series of complexes designed to mimic intermediates along the reaction coordinate for β-galactosidase are presented. These complexes clarify and enhance previous proposals regarding the catalytic mechanism. The nucleophile, Glu537, is seen to covalently bind to the galactosyl moiety. Of the two potential acids, Mg2+ and Glu461, the latter is in better position to directly assist in leaving group departure, suggesting that the metal ion acts in a secondary role. A sodium ion plays a part in substrate binding by directly ligating the galactosyl 6-hydroxyl. The proposed reaction coordinate involves the movement of the galactosyl moiety deep into the active site pocket. For those ligands that do bind deeply there is an associated conformational change in which residues within loop 794−804 move up to 10 A closer to the site of binding. In some cases this can be inhibited by the binding of additional ligands. The resulting restricted access to the intermediate helps to explain why allolactos...
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A structural view of the action of Escherichia coli (lacZ) beta-galactosidase.
2001Co-Authors: Douglas H Juers, Td Heightman, Vasella A, Jd Mccarter, Mackenzie L, Sg Withers, Bw MatthewsAbstract:The structures of a series of complexes designed to mimic intermediates along the reaction coordinate for beta-galactosidase are presented. These complexes clarify and enhance previous proposals regarding the catalytic mechanism. The nucleophile, Glu537, is seen to covalently bind to the galactosyl moiety. Of the two potential acids, Mg(2+) and Glu461, the latter is in better position to directly assist in leaving group departure, suggesting that the metal ion acts in a secondary role. A sodium ion plays a part in substrate binding by directly ligating the galactosyl 6-hydroxyl. The proposed reaction coordinate involves the movement of the galactosyl moiety deep into the active site pocket. For those ligands that do bind deeply there is an associated conformational change in which residues within loop 794-804 move up to 10 A closer to the site of binding. In some cases this can be inhibited by the binding of additional ligands. The resulting restricted access to the intermediate helps to explain why Allolactose, the natural inducer for the lac operon, is the preferred product of transglycosylation