Thu Ha Nguyen | Foreign Trade University (original) (raw)
Papers by Thu Ha Nguyen
Applied Microbiology and Biotechnology, 2011
The gene encoding homodimeric β-galactosidase (lacA) from Bacillus licheniformis DSM 13 was clone... more The gene encoding homodimeric β-galactosidase (lacA) from Bacillus licheniformis DSM 13 was cloned and overexpressed in Escherichia coli, and the resulting recombinant enzyme was characterized in detail. The optimum temperature and pH of the enzyme, for both o-nitrophenyl-β-d-galactoside (oNPG) and lactose hydrolysis, were 50°C and 6.5, respectively. The recombinant enzyme is stable in the range of pH 5 to 9 at 37°C and over a wide range of temperatures (4–42°C) at pH 6.5 for up to 1 month. The K m values of LacA for lactose and oNPG are 169 and 13.7 mM, respectively, and it is strongly inhibited by the hydrolysis products, i.e., glucose and galactose. The monovalent ions Na+ and K+ in the concentration range of 1–100 mM as well as the divalent metal cations Mg2+, Mn2+, and Ca2+ at a concentration of 1 mM slightly activate enzyme activity. This enzyme can be beneficial for application in lactose hydrolysis especially at elevated temperatures due to its pronounced temperature stability; however, the transgalactosylation potential of this enzyme for the production of galacto-oligosaccharides (GOS) from lactose was low, with only 12% GOS (w/w) of total sugars obtained when the initial lactose concentration was 200 g/L.
Journal of Agricultural and Food Chemistry, 2006
The -galactosidases ( -Gals) of Lactobacillus reuteri L103 and L461 proved to be suitable biocata... more 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.
Journal of Agricultural and Food Chemistry, 2006
An antimicrobial peptides-producing strain was isolated from soil and identified as Bacillus subt... more An antimicrobial peptides-producing strain was isolated from soil and identified as Bacillus subtilis JM4 according to biochemical tests and 16S rDNA sequence analysis. The corresponding antimicrobial peptides were purified to homogeneity by ammonium sulfate precipitation, sequential SP-Sepharose Fast Flow, Sephadex G-25 and C18 reverse-phase chromatography, and in the final purification step, two active fractions were harvested, designated as Subpeptin JM4-A and Subpeptin JM4-B. The molecular weights, determined by mass spectrometry, were 1422.71 Da for Subpeptin JM4-A and 1422.65 Da for Subpeptin JM4-B, respectively. Amino acid sequencing showed that they differed from each other only at the seventh amino acid except for three unidentified residues, and the two peptides had no significant sequence homology to the known peptides in the database, indicating that they are two novel antimicrobial peptides. In addition, characteristic measurements indicated that both peptides had a relatively broad inhibitory spectrum and remained active over a wide pH and temperature range.
Biotechnology Journal, 2007
Galacto-oligosaccharides (GOS) are formed from lactose in discontinuous mode of conversion using ... more Galacto-oligosaccharides (GOS) are formed from lactose in discontinuous mode of conversion using β-galactosidase from Lactobacillus sp. (β-gal). The discontinuous process was optimized for technical application with regard to GOS yield, enzyme preparation, reaction temperature and substrate source. It proved to be advantageous to directly apply the crude cell-free enzyme extract for the conversion, since similar GOS yields and composition were obtained as when using the pure enzyme preparation, but expensive purification could be avoided. Reaction temperature was lowered to 17°C to limit microbial contamination when using technical substrates. Thereby GOS yield decreased from 30% to 28% of total sugars and enzyme demand increased 2.7-fold. Whey permeate was compared to buffered lactose solution as a substrate source. The initial reaction rate was found to be 1.8 times higher for the whey permeate substrate; however, GOS yield was slightly lower (approximately 25% of total sugar at 17°C) mainly due to smaller amounts of allolactose[β-D-Galp-(1→6)-D-Glc] and the trisaccharide β-D-Galp-(1→6)-D-Lac formed.
Journal of Biotechnology, 2007
1
Journal of Agricultural and Food Chemistry, 2008
This work presents the cloning and expression of the genes encoding heterodimeric beta-galactosid... more This work presents the cloning and expression of the genes encoding heterodimeric beta-galactosidases from Lactobacillus reuteri L103, Lactobacillus acidophilus R22, Lactobacillus plantarum WCFS1, and Lactobacillus sakei Lb790. These enzymes consist of two subunits of approximately 73 and 35 kDa, which are encoded by two overlapping genes, lacL and lacM, respectively. We have cloned these genes into the lactobacillal expression vectors pSIP403 and pSIP409, which are based on the sakacin P operon of L. sakei ( Sørvig et al. Microbiology 2005, 151, 2439- 2449 ), and expressed them in the host strains L. plantarum WCFS1 and L. sakei Lb790. Results varied considerably, ranging from 2.23 to 61.1 U/mg of beta-galactosidase activity, depending on the origin of the lacLM genes, the host strain, and the expression vector used. Highest expression levels were obtained in a laboratory cultivation of L. plantarum WCFS1 harboring the plasmid pEH3R containing the lacLM gene from L. reuteri L103. These cultivations yielded approximately 23 000 U of beta-galactosidase activity per liter, corresponding to the formation of roughly 100 mg of recombinant protein per liter of fermentation medium, and beta-galactosidase levels amounted to 55% of the total intracellular protein of the host organism. To further verify the suitability of this expression system, recombinant beta-galactosidase from L. reuteri was purified to apparent homogeneity. The properties of the purified enzyme were essentially identical with the properties of purified native beta-galactosidase from L. reuteri L103. The presented results lead the way to efficient overproduction of beta-galactosidase in a food-grade expression system, which is of high interest for applications in food industry.
Fems Microbiology Letters, 2007
β-Galactosidase from the probiotic strain Lactobacillus acidophilus R22 was purified to apparent ... more β-Galactosidase from the probiotic strain Lactobacillus acidophilus R22 was purified to apparent homogeneity by ammonium sulphate fractionation, hydrophobic interaction, and affinity chromatography. The enzyme is a heterodimer consisting of two subunits of 35 and 72 kDa, as determined by gel electrophoresis. The optimum temperature of β-galactosidase activity was 55°C (10-min assay) and the range of pH 6.5–8, respectively, for both o-nitrophenyl-β-d-galactopyranoside (oNPG) and lactose hydrolysis. The Km and Vmax values for lactose and oNPG were 4.04±0.26 mM, 28.8±0.2 μmol d-glucose released per min per mg protein, and 0.73±0.07 mM, 361±12 μmol o-nitrophenol released per min per mg protein, respectively. The enzyme was inhibited by high concentrations of oNPG with Ki,s=31.7±3.5 mM. The enzyme showed no specific requirements for metal ions, with the exception of Mg2+, which enhanced both activity and stability. The genes encoding this heterodimeric enzyme, lacL and lacM, were cloned, and compared with other β-galactosidases from lactobacilli. β-Galactosidase from L. acidophilus was used for the synthesis of prebiotic galacto-oligosaccharides (GOS) from lactose, with the maximum GOS yield of 38.5% of total sugars at about 75% lactose conversion.
Carbohydrate Research, 2008
Galacto-oligosaccharides, complex mixtures of various sugars, are produced by transgalactosylatio... more Galacto-oligosaccharides, complex mixtures of various sugars, are produced by transgalactosylation from lactose using b-galactosidase and are of great interest for food and feed applications because of their prebiotic properties. Most galacto-oligosaccharide preparations currently available in the market contain a significant amount of monosaccharides and lactose. The mixture of galacto-oligosaccharides (GalOS) in this study produced from lactose using recombinant b-galactosidase from Lactobacillus reuteri contains 48% monosaccharides, 26.5% lactose and 25.5% GalOS. To remove efficiently both monosaccharides and lactose from this GalOS mixture containing significant amounts of prebiotic non-lactose disaccharides, a biocatalytic approach coupled with subsequent chromatographic steps was used. Lactose was first oxidised to lactobionic acid using fungal cellobiose dehydrogenases, and then lactobionic acid and monosaccharides were removed by ion-exchange and size-exclusion chromatography. Two different cellobiose dehydrogenases (CDH), originating from Sclerotium rolfsii and Myriococcum thermophilum, were compared with respect to their applicability for this process. CDH from S. rolfsii showed higher specificity for the substrate lactose, and only few other components of the GalOS mixture were oxidised during prolonged incubation. Since these sugars were only converted once lactose oxidation was almost complete, careful control of the CDH-catalysed reaction will significantly reduce the undesired oxidation, and hence subsequent removal, of any GalOS components. Removal of ions and monosaccharides by the chromatographic steps gave an essentially pure GalOS product, containing less than 0.3% lactose and monosaccharides, in a yield of 60.3%.
Biotechnology Journal, 2010
A novel heterodimeric β-galactosidase with a molecular mass of 105 kDa was purified from crude ce... more A novel heterodimeric β-galactosidase with a molecular mass of 105 kDa was purified from 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 specific activity of 97 UoNPG/mg protein. The Km, kcat and kcat/Km values for lactose and o-nitrophenyl-β-D-galactopyranoside (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. Mg2+ ions enhanced both activity and stability 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-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.
Journal of Agricultural and Food Chemistry, 2007
Galacto-oligosaccharide (GOS) formation from lactose in discontinuous and continuous modes of con... more Galacto-oligosaccharide (GOS) formation from lactose in discontinuous and continuous modes of conversion was investigated using beta-galactosidase (beta-gal) from Lactobacillus reuteri. A continuous stirred tank reactor (CSTR) with an external crossflow membrane was set up, and continuous GOS production was analyzed and compared to the batchwise formed GOS product. Marked differences were detected for the two reactor setups. Above 65% lactose conversion, the GOS yield was lower for the CSTR due to a lower content of tri- and tetrasaccharides in the reaction mixture. In the CSTR, beta-gal from L. reuteri showed up to 2-fold higher specificity toward the formation of beta-(1-->6)-linked GOS, with beta-D-Galp-(1-->6)-D-Glc and beta-D-Galp-(1-->6)-D-Gal being the main GOS components formed under these conditions. This could be used to synthesize more defined GOS products.
Carbohydrate Research, 2010
Recombinant β-galactosidase from Lactobacillus plantarum WCFS1, homologously overexpressed in L. ... more 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.
Journal of Biotechnology, 2007
1
Journal of Biotechnology, 2007
Applied Microbiology and Biotechnology, 2011
The gene encoding homodimeric β-galactosidase (lacA) from Bacillus licheniformis DSM 13 was clone... more The gene encoding homodimeric β-galactosidase (lacA) from Bacillus licheniformis DSM 13 was cloned and overexpressed in Escherichia coli, and the resulting recombinant enzyme was characterized in detail. The optimum temperature and pH of the enzyme, for both o-nitrophenyl-β-d-galactoside (oNPG) and lactose hydrolysis, were 50°C and 6.5, respectively. The recombinant enzyme is stable in the range of pH 5 to 9 at 37°C and over a wide range of temperatures (4–42°C) at pH 6.5 for up to 1 month. The K m values of LacA for lactose and oNPG are 169 and 13.7 mM, respectively, and it is strongly inhibited by the hydrolysis products, i.e., glucose and galactose. The monovalent ions Na+ and K+ in the concentration range of 1–100 mM as well as the divalent metal cations Mg2+, Mn2+, and Ca2+ at a concentration of 1 mM slightly activate enzyme activity. This enzyme can be beneficial for application in lactose hydrolysis especially at elevated temperatures due to its pronounced temperature stability; however, the transgalactosylation potential of this enzyme for the production of galacto-oligosaccharides (GOS) from lactose was low, with only 12% GOS (w/w) of total sugars obtained when the initial lactose concentration was 200 g/L.
Journal of Agricultural and Food Chemistry, 2006
The -galactosidases ( -Gals) of Lactobacillus reuteri L103 and L461 proved to be suitable biocata... more 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.
Journal of Agricultural and Food Chemistry, 2006
An antimicrobial peptides-producing strain was isolated from soil and identified as Bacillus subt... more An antimicrobial peptides-producing strain was isolated from soil and identified as Bacillus subtilis JM4 according to biochemical tests and 16S rDNA sequence analysis. The corresponding antimicrobial peptides were purified to homogeneity by ammonium sulfate precipitation, sequential SP-Sepharose Fast Flow, Sephadex G-25 and C18 reverse-phase chromatography, and in the final purification step, two active fractions were harvested, designated as Subpeptin JM4-A and Subpeptin JM4-B. The molecular weights, determined by mass spectrometry, were 1422.71 Da for Subpeptin JM4-A and 1422.65 Da for Subpeptin JM4-B, respectively. Amino acid sequencing showed that they differed from each other only at the seventh amino acid except for three unidentified residues, and the two peptides had no significant sequence homology to the known peptides in the database, indicating that they are two novel antimicrobial peptides. In addition, characteristic measurements indicated that both peptides had a relatively broad inhibitory spectrum and remained active over a wide pH and temperature range.
Biotechnology Journal, 2007
Galacto-oligosaccharides (GOS) are formed from lactose in discontinuous mode of conversion using ... more Galacto-oligosaccharides (GOS) are formed from lactose in discontinuous mode of conversion using β-galactosidase from Lactobacillus sp. (β-gal). The discontinuous process was optimized for technical application with regard to GOS yield, enzyme preparation, reaction temperature and substrate source. It proved to be advantageous to directly apply the crude cell-free enzyme extract for the conversion, since similar GOS yields and composition were obtained as when using the pure enzyme preparation, but expensive purification could be avoided. Reaction temperature was lowered to 17°C to limit microbial contamination when using technical substrates. Thereby GOS yield decreased from 30% to 28% of total sugars and enzyme demand increased 2.7-fold. Whey permeate was compared to buffered lactose solution as a substrate source. The initial reaction rate was found to be 1.8 times higher for the whey permeate substrate; however, GOS yield was slightly lower (approximately 25% of total sugar at 17°C) mainly due to smaller amounts of allolactose[β-D-Galp-(1→6)-D-Glc] and the trisaccharide β-D-Galp-(1→6)-D-Lac formed.
Journal of Biotechnology, 2007
1
Journal of Agricultural and Food Chemistry, 2008
This work presents the cloning and expression of the genes encoding heterodimeric beta-galactosid... more This work presents the cloning and expression of the genes encoding heterodimeric beta-galactosidases from Lactobacillus reuteri L103, Lactobacillus acidophilus R22, Lactobacillus plantarum WCFS1, and Lactobacillus sakei Lb790. These enzymes consist of two subunits of approximately 73 and 35 kDa, which are encoded by two overlapping genes, lacL and lacM, respectively. We have cloned these genes into the lactobacillal expression vectors pSIP403 and pSIP409, which are based on the sakacin P operon of L. sakei ( Sørvig et al. Microbiology 2005, 151, 2439- 2449 ), and expressed them in the host strains L. plantarum WCFS1 and L. sakei Lb790. Results varied considerably, ranging from 2.23 to 61.1 U/mg of beta-galactosidase activity, depending on the origin of the lacLM genes, the host strain, and the expression vector used. Highest expression levels were obtained in a laboratory cultivation of L. plantarum WCFS1 harboring the plasmid pEH3R containing the lacLM gene from L. reuteri L103. These cultivations yielded approximately 23 000 U of beta-galactosidase activity per liter, corresponding to the formation of roughly 100 mg of recombinant protein per liter of fermentation medium, and beta-galactosidase levels amounted to 55% of the total intracellular protein of the host organism. To further verify the suitability of this expression system, recombinant beta-galactosidase from L. reuteri was purified to apparent homogeneity. The properties of the purified enzyme were essentially identical with the properties of purified native beta-galactosidase from L. reuteri L103. The presented results lead the way to efficient overproduction of beta-galactosidase in a food-grade expression system, which is of high interest for applications in food industry.
Fems Microbiology Letters, 2007
β-Galactosidase from the probiotic strain Lactobacillus acidophilus R22 was purified to apparent ... more β-Galactosidase from the probiotic strain Lactobacillus acidophilus R22 was purified to apparent homogeneity by ammonium sulphate fractionation, hydrophobic interaction, and affinity chromatography. The enzyme is a heterodimer consisting of two subunits of 35 and 72 kDa, as determined by gel electrophoresis. The optimum temperature of β-galactosidase activity was 55°C (10-min assay) and the range of pH 6.5–8, respectively, for both o-nitrophenyl-β-d-galactopyranoside (oNPG) and lactose hydrolysis. The Km and Vmax values for lactose and oNPG were 4.04±0.26 mM, 28.8±0.2 μmol d-glucose released per min per mg protein, and 0.73±0.07 mM, 361±12 μmol o-nitrophenol released per min per mg protein, respectively. The enzyme was inhibited by high concentrations of oNPG with Ki,s=31.7±3.5 mM. The enzyme showed no specific requirements for metal ions, with the exception of Mg2+, which enhanced both activity and stability. The genes encoding this heterodimeric enzyme, lacL and lacM, were cloned, and compared with other β-galactosidases from lactobacilli. β-Galactosidase from L. acidophilus was used for the synthesis of prebiotic galacto-oligosaccharides (GOS) from lactose, with the maximum GOS yield of 38.5% of total sugars at about 75% lactose conversion.
Carbohydrate Research, 2008
Galacto-oligosaccharides, complex mixtures of various sugars, are produced by transgalactosylatio... more Galacto-oligosaccharides, complex mixtures of various sugars, are produced by transgalactosylation from lactose using b-galactosidase and are of great interest for food and feed applications because of their prebiotic properties. Most galacto-oligosaccharide preparations currently available in the market contain a significant amount of monosaccharides and lactose. The mixture of galacto-oligosaccharides (GalOS) in this study produced from lactose using recombinant b-galactosidase from Lactobacillus reuteri contains 48% monosaccharides, 26.5% lactose and 25.5% GalOS. To remove efficiently both monosaccharides and lactose from this GalOS mixture containing significant amounts of prebiotic non-lactose disaccharides, a biocatalytic approach coupled with subsequent chromatographic steps was used. Lactose was first oxidised to lactobionic acid using fungal cellobiose dehydrogenases, and then lactobionic acid and monosaccharides were removed by ion-exchange and size-exclusion chromatography. Two different cellobiose dehydrogenases (CDH), originating from Sclerotium rolfsii and Myriococcum thermophilum, were compared with respect to their applicability for this process. CDH from S. rolfsii showed higher specificity for the substrate lactose, and only few other components of the GalOS mixture were oxidised during prolonged incubation. Since these sugars were only converted once lactose oxidation was almost complete, careful control of the CDH-catalysed reaction will significantly reduce the undesired oxidation, and hence subsequent removal, of any GalOS components. Removal of ions and monosaccharides by the chromatographic steps gave an essentially pure GalOS product, containing less than 0.3% lactose and monosaccharides, in a yield of 60.3%.
Biotechnology Journal, 2010
A novel heterodimeric β-galactosidase with a molecular mass of 105 kDa was purified from crude ce... more A novel heterodimeric β-galactosidase with a molecular mass of 105 kDa was purified from 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 specific activity of 97 UoNPG/mg protein. The Km, kcat and kcat/Km values for lactose and o-nitrophenyl-β-D-galactopyranoside (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. Mg2+ ions enhanced both activity and stability 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-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.
Journal of Agricultural and Food Chemistry, 2007
Galacto-oligosaccharide (GOS) formation from lactose in discontinuous and continuous modes of con... more Galacto-oligosaccharide (GOS) formation from lactose in discontinuous and continuous modes of conversion was investigated using beta-galactosidase (beta-gal) from Lactobacillus reuteri. A continuous stirred tank reactor (CSTR) with an external crossflow membrane was set up, and continuous GOS production was analyzed and compared to the batchwise formed GOS product. Marked differences were detected for the two reactor setups. Above 65% lactose conversion, the GOS yield was lower for the CSTR due to a lower content of tri- and tetrasaccharides in the reaction mixture. In the CSTR, beta-gal from L. reuteri showed up to 2-fold higher specificity toward the formation of beta-(1-->6)-linked GOS, with beta-D-Galp-(1-->6)-D-Glc and beta-D-Galp-(1-->6)-D-Gal being the main GOS components formed under these conditions. This could be used to synthesize more defined GOS products.
Carbohydrate Research, 2010
Recombinant β-galactosidase from Lactobacillus plantarum WCFS1, homologously overexpressed in L. ... more 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.
Journal of Biotechnology, 2007
1
Journal of Biotechnology, 2007