Synthesis Of Galacto-oligosaccharides Research Articles

Improvement of chitosan derivatization for the immobilization of bacillus circulans β-galactosidase and its further application in galacto-oligosaccharide synthesis.

Chitosan was derivatized by two methodologies to design a robust biocatalyst of immobilized Bacillus circulans β-galactosidase from a low-cost support for its further application in the synthesis of galacto-oligosaccharides (GOS). In the first one, chitosan was derivatized by cross-linking with glutaraldehyde and activated with epichlorohydrin; in the second one, cross-linking and activation were done with epichlorohydrin in a two-step process, favoring first support cross-linking and then support functionalization (C-EPI-EPI). Epoxy groups were hydrolyzed and oxidized, obtaining two supports activated with different aldehyde concentrations (100-250 μmol/g). The expressed activity and stability of the immobilized biocatalysts varied according to the derivatization methodology, showing that both the cross-linking agent and the activation degree are key parameters in the final biocatalyst performance. The best compromise between expressed activity and thermal stability was obtained using C-EPI-EPI with 200 μmol of aldehyde groups per gram of support. The immobilization conditions were optimized, obtaining a biocatalyst with 280 IU/g, immobilization yields in terms of activity and protein of 17.3 ± 0.4 and 61.5 ± 3.9%, respectively, and a high thermal stability, with a half-life of 449 times the value of the soluble enzyme. The biocatalyst was applied to the synthesis of GOS in repeated batch operation without affecting the product composition. Four successive batches were required for obtaining a cumulative specific productivity higher than the one obtained with the soluble enzyme.

Biochemical and structural characterization of a thermostable β-glucosidase from Halothermothrix orenii for galacto-oligosaccharide synthesis.

Lactose is a major disaccharide by-product from the dairy industries, and production of whey alone amounts to about 200 million tons globally each year. Thus, it is of particular interest to identify improved enzymatic processes for lactose utilization. Microbial β-glucosidases (BGL) with significant β-galactosidase (BGAL) activity can be used to convert lactose to glucose (Glc) and galactose (Gal), and most retaining BGLs also synthesize more complex sugars from the monosaccharides by transglycosylation, such as galacto-oligosaccharides (GOS), which are prebiotic compounds that stimulate growth of beneficial gut bacteria. In this work, a BGL from the thermophilic and halophilic bacterium Halothermothrix orenii, HoBGLA, was characterized biochemically and structurally. It is an unspecific β-glucosidase with mixed activities for different substrates and prominent activity with various galactosidases such as lactose. We show that HoBGLA is an attractive candidate for industrial lactose conversion based on its high activity and stability within a broad pH range (4.5–7.5), with maximal β-galactosidase activity at pH 6.0. The temperature optimum is in the range of 65–70 °C, and HoBGLA also shows excellent thermostability at this temperature range. The main GOS products from HoBGLA transgalactosylation are β-d-Galp-(1→6)-d-Lac (6GALA) and β-d-Galp-(1→3)-d-Lac (3GALA), indicating that d-lactose is a better galactosyl acceptor than either of the monosaccharides. To evaluate ligand binding and guide GOS modeling, crystal structures of HoBGLA were determined in complex with thiocellobiose, 2-deoxy-2-fluoro-d-glucose and glucose. The two major GOS products, 3GALA and 6GALA, were modeled in the substrate-binding cleft of wild-type HoBGLA and shown to be favorably accommodated.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-014-6015-x) contains supplementary material, which is available to authorized users.

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

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 k cat/K m 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 with total GOS yields of 33% and 44% of total sugars obtained with β-gal I and β-gal II, respectively. The predominant transgalactosylation products are β-D-Galp-(1→6)-D-Glc (allolactose) and β-D-Galp-(1→3)-D-Lac, accounting together for more than 75% and 65% of the GOS formed by transgalactosylation by β-gal I and β-gal II, respectively, indicating that both enzymes have a propensity to synthesize β-(1→6) and β-(1→3)-linked GOS. The resulting GOS mixtures contained relatively high fractions of allolactose, which results from the fact that glucose is a far better acceptor for galactosyl transfer than galactose and lactose, and intramolecular transgalactosylation contributes significantly to the formation of this disaccharide.

Assessing the volumetric productivity of an ultrafiltration membrane bioreactor during the synthesis of galacto-oligosaccharides

Assessing the volumetric productivity of an ultrafiltration membrane bioreactor during the synthesis of galacto-oligosaccharides

Assessment of repetitive batch-wise synthesis of galacto-oligosaccharides from lactose slurry using immobilised β-galactosidase from Bacillus circulans

The synthesis of galacto-oligosaccharides (GOS) using covalently immobilised β-galactosidase from Bacillus circulans was carried out in a lactose slurry rather than in solution. Repeated batch-wise synthesis at 58 °C and 55% (w/w) lactose could be carried out for at least 15 successive runs. All 15 runs were completed within 4 h of reaction time. The reduction of heat exposure due to these short incubation times contributed to the retention of 60% of the initial activity. The product properties of the GOS mixture obtained with the immobilised biocatalyst were compared with the free enzyme product. Oligosaccharide yields and product pattern were highly similar as was the degree of polymerisation. The productivity of the immobilised enzyme system was compared to the free enzyme. The enzymatic productivity (i.e., g GOS g−1 enzyme) after 15 consecutive runs was 165% higher for the immobilised enzyme than for the free enzyme.

Enzyme, β-galactosidase immobilized on membrane surface for galacto-oligosaccharides formation from lactose: Kinetic study with feed flow under recirculation loop

The work focuses on producing galacto-oligosaccharides (GOS) through an enzymatic reaction with lactose under a partial recirculation loop by utilizing membrane-immobilized β-galactosidase. Cross-linking through covalent bonding, using gluteraldehyde, was employed to immobilize enzyme on a microporous polyvinylidene fluoride membrane. GOS synthesis was carried out in a laboratory fabricated reaction cell, whereby three immobilized membranes were housed in series. The reaction was conducted at varying initial lactose concentrations (ILCs) and feed flow rates at pH 6 and 40°C. A maximum GOS of 30% (dry basis) was obtained after 60h of reaction time, 50g/L ILC, 241U of enzyme (specific loading of 600U/g-membrane), and 0.5mL/min of feed flow rate at 56% lactose conversion. The GOS yield increased with increased ILC and decreased feed flow rate. The selectivity of GOS formation increased by increasing both the ILC and the feed flow rate, whereas the reverse was true for mono-saccharides. The immobilized enzyme retained ∼50% of its initial activity after 30 days of storage at 20°C, while the native enzyme lost 100% of its activity within 21 days. Furthermore, a five-step, nine-parameter model was developed, and simulated results showed excellent agreement with the experimental data.

Analysis, structural characterization, and bioactivity of oligosaccharides derived from lactose

The increasing interest for prebiotic carbohydrates as functional food ingredients has promoted the synthesis of galactooligosaccharides and new lactose derivatives. This review provides a comprehensive overview on the chromatographic analysis, structural characterization, and bioactivity studies of lactose-derived oligosaccharides. The most common chromatographic techniques used for the separation and structural characterization of this type of oligosaccharides, including GC and HPLC in different operational modes, coupled to various detectors are discussed. Insights on oligosaccharide MS fragmentation patterns, using different ionization sources and mass analyzers, as well as data on structural analysis by NMR spectroscopy are also described. Finally, this article deals with the bioactive effects of galacto oligosaccharides and oligosaccharides derived from lactulose on the gastrointestinal and immune systems, which support their consumption to provide significant health benefits.

Synthesis of prebiotic galactooligosaccharides from lactose using bifidobacterial β-galactosidase (BbgIV) immobilised on DEAE-Cellulose, Q-Sepharose and amino-ethyl agarose

The bifidobacterial β-galactosidase BbgIV was immobilised on DEAE-Cellulose and Q-Sepharose via ionic binding and on amino-ethyl- and glyoxal-agarose via covalent attachment, and was then used to catalyse the synthesis of galactooligosaccharides (GOS). The immobilisation yield exceeded 90% using ionic binding, while it was low using amino-ethyl agarose (25–28%) and very low using glyoxal agarose (<3%). This was due to the mild conditions and absence of chemical reagents in ionic binding, compared to covalent attachment. The maximum GOS yield obtained using DEAE-Cellulose and Q-Sepharose was similar to that obtained using free BbgIV (49–53%), indicating the absence of diffusion limitation and mass transfer issues. For amino-ethyl agarose, however, the GOS yield obtained was lower (42–44%) compared to that obtained using free BbgIV. All the supports tried significantly (P<0.05) increased the BbgIV operational stability and the GOS synthesis productivity up to 55°C. Besides, six successive GOS synthesis batches were performed using BbgIV immobilised on Q-Sepharose; all resulted in similar GOS yields, indicating the possibility of developing a robust synthesis process. Overall, the GOS synthesis operation performance using BbgIV was improved by immobilising the enzyme onto solid supports, in particular on Q-Sepharose.

Fed‐batch synthesis of galacto‐oligosaccharides with Aspergillus oryzae β‐galactosidase using optimal control strategy

Fed-batch synthesis of galacto-oligosaccharides (GOS) from lactose with β-galactosidase from Aspergillus oryzae was evaluated experimentally and reaction yield was maximized via optimal control technique. The optimal lactose and enzyme feed flow rate profiles were determined using a model for GOS synthesis previously reported by the authors. Experimentally it was found that fed-batch synthesis allowed an increase on the maximum total GOS concentration from 115 (batch synthesis) to 218 g L(-1) as consequence of the increase in total sugars concentration from 40 to 58% w/w. Such high concentration of total sugars was not attainable in batch operation because of the low solubility of lactose at the reaction temperature (40°C). Simulations predicted a GOS yield of 32.5 g g(-1) in fed-batch synthesis under optimal conditions, while experimentally the same yield as in batch synthesis was obtained (28 g g(-1) ). Besides, an enrichment of total oligosaccharides in GOS with a high polymerization degree (GOS-5 and GOS-6) was observed in the fed-batch synthesis. Experimental profiles for all sugars were similar to the ones predicted by simulation, which supports the use of this methodology for the optimization of GOS synthesis.

Synthesis of Galactooligosaccharides in the Cheese Whey-based Medium by a Lactase from Lactobacillus paracasei YSM0308

An enzyme <TEX>${\beta}$</TEX>-galactosidase or <TEX>${\beta}$</TEX>-galactohydrolase [EC3.2.1.23], commonly called lactase, mediates galacto-oligosaccharide (GOS) synthesis under conditions of high substrate concentrations. Also, lactase hydrolyzes <TEX>${\beta}$</TEX>(<TEX>$1{\rightarrow}4$</TEX>) lactose into glucose and galactose, the latter is successively transferred to free lactose to make various oligosaccharides via transgalactosylation. GOS is non-digestible to human digestive enzymes and has been used as a functional prebiotics. Among the 24 lactic acid bacteria (LAB) strains used, Lactobacillus paracasei YSM0308 was selected based on its exhibition of the highest <TEX>${\beta}$</TEX>-galactoside hydrolysis activity, and the crude lactase was prepared for examination of reaction conditions to affect the GOS synthesis. Lactase activity was measured with a spectrophotometer using ONPG (o-nitropheyl <TEX>${\beta}$</TEX>-D-galactopyranoside) method. Lactase activity was not detected in the culture supernatant and was mostly present in the cell pellet after centrifugation. Activity of the crude lactase preparation ranges from102 to 1,053 units/mL, with the highest activity determined for L. paracasei YSM0308. Optimal conditions for GOS synthesis are as follows: concentration of whey powder, pH, temperature, and time were 30%, pH 6.5-7.0, <TEX>$30^{\circ}C$</TEX>, and 4 h, respectively. The final GOS concentration was 19.41% (w/v) by the crude YSM0308 lactase, which was obtained from strain YSM0308 grown in the 10% (w/v) reconstituted whey-based medium.

Permeabilized probiotic Lactobacillus plantarum as a source of β-galactosidase for the synthesis of prebiotic galactooligosaccharides

Permeabilized probiotic Lactobacillus plantarum was used as a source of β-galactosidase for the synthesis of galactooligosaccharides (GOS) from lactose. β-galactosidase activity was highest when galactose (1,724 Miller Units) was used as a carbon source compared to lactose, sucrose or glucose at 37 °C, 18 h. Permeabilized cells had the highest transgalactosylation activity resulting in 34 % (w/w) GOS synthesis from 40 % (w/v) lactose at 50 °C over 12 h. HPLC revealed that the GOS were composed of 13 % disaccharides (non-lactose), 17 % trisaccharides and 4 % tetrasaccharides that were further confirmed by ESI–MS.

β-galactosidase stability at high substrate concentrations

Enzymatic synthesis of galacto-oligosaccharides is usually performed at high initial substrate concentrations since higher yields are obtained. We report here on the stability of β-galactosidase from Bacillus circulans at 25, 40, and 60°C in buffer, and in systems with initially 5.0 and 30% (w/w) lactose. In buffer, the half-life time was 220 h and 13 h at 25 and 40°C, respectively, whereas the enzyme was completely inactivated after two hours at 60°C. In systems with 5.0 and 30% (w/w) lactose, a mechanistic model was used to correct the o NPG converting activity for the presence of lactose, glucose, galactose, and oligosaccharides in the activity assay. Without correction, the stability at 5.0% (w/w) lactose was overestimated, while the stability at 30% (w/w) lactose was underestimated. The inactivation constant kd was strongly dependent on temperature in buffer, whereas only a slight increase in kd was found with temperature at high substrate concentrations. The enzyme stability was found to increase strongly with the initial substrate concentrations. The inactivation energy Ea appeared to be lower at high initial substrate concentrations.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-2-402) contains supplementary material, which is available to authorized users.

Effects of Carbohydrates on the oNPG Converting Activity of β-Galactosidases

The effects of high concentrations of carbohydrates on the o-nitrophenyl β-d-galactopyranoside (oNPG) converting activity of β-galactosidase from Bacillus circulans are studied to get a better understanding of the enzyme behavior in concentrated and complicated systems in which enzymatic synthesis of galacto-oligosaccharides is usually performed. The components that were tested were glucose, galactose, lactose, sucrose, trehalose, raffinose, Vivinal GOS, dextran-6000, dextran-70,000, and sarcosine. Small carbohydrates act as acceptors in the reaction. This speeds up the limiting step, which is binding of the galactose residue with the acceptor and release of the product. Simultaneously, both inert and reacting additives seem to cause some molecular crowding, which results in a higher enzyme affinity for the substrate. The effect of molecular crowding on the enzyme activity is small compared to the effect of carbohydrates acting in the reactions as acceptors. The effects of reactants on β-galactosidases from B. circulans, A. oryzae, and K. lactis are compared.

Immobilization of Bacillus circulans β-galactosidase and its application in the synthesis of galacto-oligosaccharides under repeated-batch operation

A highly active and stable derivate of immobilized Bacillus circulans β-galactosidase was prepared for the synthesis of galacto-oligosaccharides (GOS) under repeated-batch operation. B. circulans β-galactosidase was immobilized on monofunctional glyoxyl agarose and three heterofunctional supports: amino-, carboxy-, and chelate-glyoxyl agarose. Glyoxyl agarose was the support with highest immobilization yield and stability being selected for the optimization of immobilization conditions and application in GOS synthesis. A central composite rotatable design was conducted to optimize contacted protein and immobilization time, using maximum catalytic potential as the objective function. Optimal conditions of immobilization were 28.9mg/g and 36.4h of contact, resulting in a biocatalyst with 595IU/g and a half-life 89-fold higher than soluble enzyme. Immobilization process did not alter the synthetic capacity of β-galactosidase, obtaining the same GOS yield and product profile than the free enzyme. GOS yield and productivity remained unchanged along 10 repeated batches, with values of 39% (w/w) and 5.7g GOS/g of biocatalyst·batch. Total product obtained after 10 batches of reaction was 56.5g GOS/g of biocatalyst (1956g GOS/g protein). Cumulative productivity in terms of mass of contacted protein was higher for the immobilized enzyme than for its soluble counterpart from the second batch of synthesis onwards.

Characterization of β-Galactosidase Isoforms from Bacillus circulans and Their Contribution to GOS Production

A β-galactosidase preparation from Bacillus circulans consists of four isoforms called β-gal-A, β-gal-B, β-gal-C, and β-gal-D. These isoforms differ in lactose hydrolysis and galacto-oligosaccharide (GOS) synthesis at low substrate concentrations. For this reason, using a selection of the isoforms may be relevant for GOS production, which is typically done at high substrate concentrations. At initial lactose concentrations in between 0.44% and 0.68% (w/w), β-gal-A showed the least oligosaccharide formation, followed by β-gal-B and β-gal-C; most oligosaccharides were formed by β-gal-D. The differences in behavior were confirmed by studying the thermodynamics of lactose conversion with isothermal titration calorimetry since especially β-gal-A showed a different profile than the other isoforms. Also during the conversion of allolactose and 4-galactosyllactose at 0.44% and 0.61% (w/w), respectively, β-gal-A and β-gal-D showed clear differences. In contrast to above findings, the selectivity of the isoforms did hardly differ at an initial lactose concentration of 30% (w/w), except for a slightly higher production of galactose with β-gal-A. These differences were hypothesized to be related to the different accessibility of the active sites of the isoforms for different-sized reactants. The initial GOS formation rates of the isoforms indicate that β-gal-A and β-gal-B are the best isoforms for GOS production at high lactose concentrations.

Lactobacillus plantarum 70810 from Chinese paocai as a potential source of β-galactosidase for prebiotic galactooligosaccharides synthesis

A novel food-grade strain Lactobacillus plantarum 70810 producing β-galactosidase with high transgalactosylation activity was isolated from Chinese paocai. The galactooligosaccharides (GOS) were synthesized by using this enzyme with a maximum yield of 44.3 % (w/w) from 400 g/L lactose at 45 °C for 10 h. The β-galactosidase from this strain was purified to homogeneity by ammonium sulfate precipitation, anion exchange chromatography and gel filtration chromatography. It was a heterodimer arrangement of approximately 105 kDa composed of two subunits of 35 and 72 kDa. The optimal pH of the purified β-galactosidase was 8.0 for both o-nitrophenyl-β-d-galactopyranoside (oNPG) and lactose hydrolysis, and optimal temperature was 60 °C and 55 °C, respectively. Its K m and V max values for oNPG and lactose were 0.89 ± 0.05 mM, 194 ± 3.0 μmoL/min/mg protein, and 9.88 ± 0.16 mM, 15.88 ± 0.21 μmoL/min/mg protein, respectively. This enzyme was slightly inhibited by the hydrolysis products, that is, glucose and galactose. Since the β-galactosidase from L. plantarum 70810 exhibited higher transgalactosylation activity, strong affinity for lactose and low end-product inhibition, it was suggested to be a potential candidate for the synthesis of prebiotic GOS.

High stability of immobilized β-d-galactosidase for lactose hydrolysis and galactooligosaccharides synthesis

β-d-Galactosidase from Kluyveromyces lactis was immobilized on glutaraldehyde-activated chitosan and used in a packed-bed reactor for the continuous hydrolysis of lactose and the synthesis of galactooligosaccharides (GOS). The biocatalyst was tested for its optima pH and temperature, thermal stability in the presence of substrate and products, and operational stability. Immobilization increased the range of operational pH and temperature, and the enzyme thermal stability was sharply increased in the presence of lactose. Almost complete lactose hydrolysis was achieved for both milk whey and lactose solution at 37°C at flow rates up to 2.6mLmin−1. Maximal GOS concentration of 26gL−1 was obtained at a flow rate of 3.1mLmin−1, with a productivity of 186gL−1h−1. Steady-state operation for 15 days showed the reactor stability concerning lactose hydrolysis.

Synthesis and separation of galacto-oligosaccharides using membrane bioreactor

In the present investigation, a dead-end membrane module, equipped with β-galactosidase immobilized flat-sheet ultrafiltration (UF) membrane, has been considered as a membrane bioreactor (MBR) for galacto-oligosaccharide (GOS) synthesis and separation. β-galactosidase was immobilized on the membrane surface by the combination of a series of mechanisms, such as adsorption of polyethyleneimine (PEI) on membrane matrix, formation of PEI and β-galactosidase aggregates, and finally cross-linking of PEI and β-galactosidase complex on the membrane surface by glutaraldehyde. The optimum pH for PEI-enzyme aggregation was found to be 8.2 and the optimum pH for crosslinking of PEI-enzyme aggregates on the membrane surface was found as 7.0 when the ratio of PEI to β-galactosidase was 1:25. Stable immobilization could be achieved over broad temperature range (4°C to 65°C). It was found that at optimum condition β-galactosidase loading on the membrane surface was 85% at multilayer form. GOS was synthesized by MBR with retentate recirculation, using simulated lactose solution, as well as de-proteinated whey as a substrate. Optimum operating conditions for the GOS synthesis were found at 0.045M initial substrate concentration, 25mL·h−1 feed flow rate, 45°C temperature, 3.0kgfcm−2 trans-membrane pressure (TMP), 80 r.p.m. stirrer speed.

High Yield Production of a Soluble Bifidobacterial β-Galactosidase (BbgIV) in E. coli DH5α with Improved Catalytic Efficiency for the Synthesis of Prebiotic Galactooligosaccharides

The bifidobacterial β-galactosidase (BbgIV) was produced in E. coli DH5α at 37 and 30 °C in a 5 L bioreactor under varied conditions of dissolved oxygen (dO2) and pH. The yield of soluble BbgIV was significantly (P < 0.05) increased once the dO2 dropped to 0-2% and remained at such low values during the exponential phase. Limited dO2 significantly (P < 0.05) increased the plasmid copy number and decreased the cells growth rate. Consequently, the BbgIV yield increased to its maximum (71-75 mg per g dry cell weight), which represented 20-25% of the total soluble proteins in the cells. In addition, the specific activity and catalytic efficiency of BbgIV were significantly (P < 0.05) enhanced under limited dO2 conditions. This was concomitant with a change in the enzyme secondary structure, suggesting a link between the enzyme structure and function. The knowledge generated from this work is very important for producing BbgIV as a biocatalyst for the development of a cost-effective process for the synthesis of prebiotic galactooligosaccharides from lactose.

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.