Maximum Galacto-oligosaccharides Yield Research Articles

β-galactosidase GALA from Bacillus circulans with high transgalactosylation activity

ABSTRACT β-galactosidase catalyzes lactose hydrolysis and transfers reactions to produce prebiotics such as galacto-oligosaccharides (GOS) with potential applications in the food industry and pharmaceuticals. However, there is still a need for improved transgalactosylation activity of β-galactosidases and reaction conditions of GOS production in order to maximize GOS output and reduce production costs. In this study, a β-galactosidase gene, galA, from Bacillus circulans was expressed in Pichia pastoris, which not only hydrolyzed lactose but also had strong transgalactosylation activity to produce GOS. Response surface methodology was adopted to investigate the effects of temperature, enzyme concentration, pH, initial lactose concentration, and reaction time on the production of GOS and optimize the reaction conditions for GOS. The optimal pH for the enzyme was 6.0 and remained stable under neutral and basic conditions. Meanwhile, GALA showed most activity at 50°C and retained considerable activity at a lower temperature 30–40°C, indicating this enzyme could work under mild conditions. The enzyme concentration and temperature were found to be the critical parameters affecting the transgalactosylation activity. Response surface methodology showed that the optimal enzyme concentration, initial lactose concentration, temperature, pH, and reaction time were 3.03 U/mL, 500 g/L, 30°C, 5.08, and 4 h, respectively. Under such conditions, the maximum yield of GOS was 252.8 g/L, accounting for approximately 50.56% of the total sugar. This yield can be considered relatively high compared to those obtained from other sources of β-galactosidases, implying a great potential for GALA in the industrial production and application of GOS.

Synthesis of β(1 → 3) and β(1 → 6) galactooligosaccharides from lactose and whey using a recombinant β-galactosidase from Pantoea anthophila

Background: Milk whey, a byproduct of the dairy industry has a negative environmental impact, can be used as a raw material for added-value compounds such as galactooligosaccharides (GOS) synthesis by β-galactosidases. Results: B-gal42 from Pantoea anthophila strain isolated from tejuino belonging to the glycosyl hydrolase family GH42, was overexpressed in Escherichia coli and used for GOS synthesis from lactose or milk whey. Crude cell-free enzyme extracts exhibited high stability; they were employed for GOS synthesis reactions. In reactions with 400 g/L lactose, the maximum GOS yield was 40% (w/w) measured by HPAEC-PAD, corresponding to 86% of conversion. This enzyme had a strong predilection to form GOS with β(1 → 6) and β(1 → 3) galactosyl linkages. Comparing GOS synthesis between milk whey and pure lactose, both of them at 300 g/L, these two substrates gave rise to a yield of 38% (60% of lactose conversion) with the same product profile determined by HPAEC-PAD. Conclusions: B-gal42 can be used on whey (a cheap lactose source) to produce added value products such as galactooligosaccharides. Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0cm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:Calibri,sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Times New Roman; mso-bidi-theme-font:minor-bidi;}

Immobilization of β-galactosidase on chitosan-coated magnetic nanoparticles and its application for synthesis of lactulose-based galactooligosaccharides

Abstract β-Galactosidase was covalently immobilized on the chitosan-coated magnetic nanoparticles activated with glutaraldehyde. The effect of three factors including protein content, pH and time on the yield of immobilization was investigated, and the optimum values were 0.45 mg/ml, 4.9 and 4.9 h, respectively. The maximum yield of immobilization reached 98.8% in term of enzyme activity. The stability of β-galactosidase significantly was improved after immobilization from 2.5 days to over 6 days of half-life at 60 °C and was active in wider ranges of temperature (45–60 °C), and pH (4.0–5.5) compared to soluble form. The activity of immobilized enzyme remained 85% of origin after eight recycles (at 60 °C, pH 4.5 for 4 h). Different initial lactulose concentrations (0.58–2.34 M) were applied for synthesis of galactooligosaccharides. The maximum yield of galactooligosaccharides was 17% mol/mol at the initial lactulose concentration of 2.34 M at 36 h of reaction. It showed that both hydrolytic and trans-galactosylation activities of β-galactosidase remained well after immobilization. The kinetic model of immobilized enzyme was determined. These results revealed that immobilized enzyme on chitosan-coated magnetic nanoparticles has application potentials in the production of prebiotic lactulose-based galactooligosaccharides.

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

β-Galactosidase from Streptococcus thermophilus was overexpressed in a food-grade organism, Lactobacillus plantarum WCFS1. Laboratory cultivations yielded 11,000U of β-galactosidase activity per liter of culture corresponding to approximately 170mg of enzyme. Crude cell-free enzyme extracts obtained by cell disruption and subsequent removal of cell debris showed high stability and were used for conversion of lactose in whey permeate. The enzyme showed high transgalactosylation activity. When using an initial concentration of whey permeate corresponding to 205gL−1 lactose, the maximum yield of galacto-oligosaccharides (GOS) obtained at 50°C reached approximately 50% of total sugar at 90% lactose conversion, meaning that efficient valorization of the whey lactose was obtained. GOS are of great interest for both human and animal nutrition; thus, efficient conversion of lactose in whey into GOS using an enzymatic approach will not only decrease the environmental impact of whey disposal, but also create additional value.

Detailed Analysis of Galactooligosaccharides Synthesis with β-Galactosidase from Aspergillus oryzae

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 specificity of the linkages formed and distribution of di-, tri-, and higher GOS varied significantly among the three β-galactosidases.

Homodimeric β-Galactosidase from Lactobacillus delbrueckii subsp. bulgaricus DSM 20081: Expression in Lactobacillus plantarum and Biochemical Characterization

The lacZ gene from Lactobacillus delbrueckii subsp. bulgaricus DSM 20081, encoding a β-galactosidase of the glycoside hydrolase family GH2, was cloned into different inducible lactobacillal expression vectors for overexpression in the host strain Lactobacillus plantarum WCFS1. High expression levels were obtained in laboratory cultivations with yields of approximately 53000 U of β-galactosidase activity per liter of medium, which corresponds to ∼170 mg of recombinant protein per liter and β-galactosidase levels amounting to 63% of the total intracellular protein of the host organism. The wild-type (nontagged) and histidine-tagged recombinant enzymes were purified to electrophoretic homogeneity and further characterized. β-Galactosidase from L. bulgaricus was used for lactose conversion and showed very high transgalactosylation activity. The maximum yield of galacto-oligosaccharides (GalOS) was approximately 50% when using an initial concentration of 600 mM lactose, indicating that the enzyme can be of interest for the production of GalOS.

Production of Galacto-oligosaccharides by the β-Galactosidase from Kluyveromyces lactis: Comparative Analysis of Permeabilized Cells versus Soluble Enzyme

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 typical profile with a maximum GOS concentration followed by partial hydrolysis of the newly formed oligosaccharides was not observed with the soluble enzymes. Results were correlated with the higher stability of β-galactosidase when permeabilized whole cells were used.

Characterization of a Heterodimeric GH2 β-Galactosidase from Lactobacillus sakei Lb790 and Formation of Prebiotic Galacto-oligosaccharides

The lacLM genes from Lactobacillus sakei Lb790, encoding a heterodimeric β-galactosidase that belongs to glycoside hydrolase family GH2, were cloned and heterologously expressed in Escherichia coli . Subsequently, the recombinant β-galactosidase LacLM was purified to apparent homogeneity and characterized. The enzyme is a β-galactosidase with narrow substrate specificity because o-nitrophenyl-β-D-galactopyranoside (oNPG) was efficiently hydrolyzed, whereas various structurally related oNP analogues were not. The K(m) and k(cat) values for oNPG and lactose were 0.6 mM and 180 s(-1) and 20 mM and 43 s(-1), respectively. The enzyme is inhibited competitively by its two end-products D-galactose and D-glucose (K(i) values of 180 and 475 mM, respectively). As judged by the ratio of the inhibition constant to the Michaelis constant, K(i)/K(m), this inhibition is only very moderate and much less pronounced than for other microbial β-galactosidases. β-Galactosidase from L. sakei possesses high transgalactosylation activity and was used for the synthesis of galacto-oligosaccharides (GalOS), employing lactose at a concentration of 215 g/L. The maximum GalOS yield was 41% (w/w) of total sugars at 77% lactose conversion and contained mainly non-lactose disaccharides, trisaccharides, and tetrasaccharides with approximately 38, 57, and 5% of total GalOS formed, respectively. The enzyme showed a strong preference for the formation of β-(1→6)-linked transgalactosylation products, whereas β-(1→3)-linked compounds were formed to a lesser extent and β-(1→4)-linked reaction products could not be detected.

OPTIMIZATION OF GALACTOOLIGO-SACCHARIDE PRODUCTION FROM LACTOSE USING β-GLYCOSIDASES FROM HYPERTHERMOPHILES

The maximal production of galactooligosaccharides (GOS) from lactose by (β-glycosidases from the hyperthermophilic archaea, Sulfolobus solfataricus (LacS) (derived from lacS gene) and Pyrococcus furiosus (CelB) (derived from celB gene) was optimized. The performance of these enzymes under extreme reaction conditions, temperatures up to 95°C and lactose concentrations up to 90% (w/v), were studied. The highly thermostable enzymes were shown to be very well suited for oligosaccharide synthesis. For both LacS and CelB the maximum yield of GOS increased with increasing lactose concentration, up to 70% (w/v). The maximum yield of GOS also increased with increasing temperature, and the optimal pH for synthesis was different at different temperatures. The sum of tri- and tetrasaccharides yielded 37% (w/w) in an optimally designed reaction for LacS, and a maximum yield of 40% (w/w) was attained for CelB. Compared to aqueous solution, an increase of the tetrasaccharide/trisaccharide ratio was obtained in two-phase systems with heptane and nonane. These two enzymes from hyperthermophilic organisms were shown to give higher GOS yields at high substrate concentrations than a β-galactosidase from a thermophilic/mesophilic organism (Aspergillus oryzae).