Yield Of Oligosaccharides Research Articles

Enzymatic synthesis of oligosaccharides by recombinant <i>Eschericia coli</i> - expressed <i>Candida molichiana</i> &#946-glucosidase II

In the present study, the biosynthetic activity of recombinant β-glucosidase from E. coli was described. The purified β-glucosidase from the recombinant E. coli – expressed C. molichiana (E. coli pBG22: JM109) was obtained at the end of the fermentation process (24 h) with an enzyme activity of 180 IU/ml and the enzyme retained 93% of its initial activity in 20% (V/V) DMSO (dimethyl sulphoxide). The initial substrate concentration, the incubation time and temperature and the type of substrate was reported to influence the biosynthesis of oligosaccharides. The efficiency of the biosynthetic reaction increased to about 2-fold in the presence of DMSO. The yield of oligosaccharides was higher in the presence of sophorose than gentiobiose and cellobiose with DMSO.Egyptian Journal of Biotechnology Vol. 25 (1) 2007: pp. 45-59

Production of chitosan oligosaccharides using chitosanase immobilized on amylose-coated magnetic nanoparticles

We developed chitosanase immobilized on amylose-coated magnetic nanoparticles and utilized it to produce chitosan oligosaccharides. The magnetic nanoparticles used consisted of magnetite (Fe 3O 4) and their surface was coated with amylose. Hydroxyl groups of amylose deposited on the nanoparticles were activated chemically and then used to immobilize the chitosanase via multipoint covalent bonds. Compared to the conventional physical-adsorption method, 1.4–2.0 times the amount of chitosanase was immobilized via multipoint covalent bonding under the same experimental conditions. The immobilized chitosanase was used to produce pentamers and hexamers of chitosan oligosaccharides, which possess beneficial biological activities. In batch hydrolysis of chitosan using the immobilized chitosanase, a high yield of target oligosaccharides (40% of the used chitosan, based on weight) was successfully obtained. The immobilized chitosanase could be recovered and reused repeatedly.

Hydrothermal degradation of polysaccharides in a semi-batch reactor: product distribution as a function of severity parameter

The naturally occurring polysaccharides, starch and pectic acid, were hydrothermally degraded in a semi-batch reactor coupled with a plug-flow reactor (PFR) to produce valuable chemicals including mono- and oligosaccharides over a temperature range from 160 to 240 °C at 10 MPa. The solid polysaccharide samples were solubilized in a semi-batch reactor, and the water-soluble (WS) components instantly entered the PFR in which further degradation took place. The reaction temperature and/or residence time in the PFR significantly affected the yields of oligosaccharides, monosaccharides and secondary decomposition products, and also the average molecular weight of WS components. The product distributions were expressed with severity, which combines the effects of temperature and time on the progress of the reaction, as a single reaction parameter. Using this parameter, it was found that similar product distributions were obtained at equal levels of severity. The severity parameter enabled easy interpretation of the change in product distribution for these reactions.

High yield production of monomer-free chitosan oligosaccharides by pepsin catalyzed hydrolysis of a high deacetylation degree chitosan

The high molecular weight of chitosan, which results in a poor solubility at neutral pH values and high viscosity aqueous solutions, limits its potential uses in the fields of food, health and agriculture. However, most of these limitations are overcome by chitosan oligosaccharides obtained by enzymatic hydrolysis of the polymer. Several commercial enzymes with different original specificities were assayed for their ability to hydrolyze a 93% deacetylation degree chitosan and compared with a chitosanase. According to the patterns of viscosity decrease and reducing end formation, three enzymes—cellulase, pepsin and lipase A—were found to be particularly suitable for hydrolyzing chitosan at a level comparable to that achieved by chitosanase. Unlike the appreciable levels of both 2-amino-2-deoxy- d-glucose and 2-acetamido-2-deoxy- d-glucose monomers released from chitosan by the other enzymes after a 20 h-hydrolysis (4.6–9.1% of the total product weight), no monomer could be detected following pepsin cleavage. As a result, pepsin produced a higher yield of chitosan oligosaccharides than the other enzymes: 52% versus as much as 46%, respectively. Low molecular weight chitosans accounted for the remaining 48% of hydrolysis products. The calculated average polymerization degree of the products released by pepsin was around 16 units after 20 h of hydrolysis. This product pattern and yield are proposed to be related to the bond cleavage specificity of pepsin and the high deacetylation degree of chitosan used as substrate. The optimal reaction conditions for hydrolysis of chitosan by pepsin were 40 °C and pH 4.5, and an enzyme/substrate ratio of 1:100 (w/w) for reactions longer than 1 h.

Transformation of maltose into prebiotic isomaltooligosaccharides by a novel α-glucosidase from Xantophyllomyces dendrorhous

The transglycosylation activity of a novel α-glucosidase from the basidiomycetous yeast Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) was studied using maltose as glucosyl donor. The enzyme synthesized oligosaccharides with α-(1 → 2), α-(1 → 4) and α-(1 → 6) bonds. Using 200 g/l maltose, the yield of oligosaccharides was 53.8 g/l, with prebiotic oligosaccharides containing at least one α-(1 → 6) linkage (panose, 6- O-α-glucosyl-maltotriose and 6- O-α-isomaltosyl-maltose) being the major products (47.1 g/l). The transglycosylatying yield was 3.6 times higher than the observed with the α-glucosidase from Saccharomyces cerevisiae (53.8 vs. 14.7 g/l). Moreover, when increasing the maltose concentration up to 525 g/l, the maximum production of tri- and tetrasaccharides reached 167.1 g/l, without altering the percentage of oligosaccharides in the mixture. Compared with other microbial α-glucosidases in which the main transglycosylation product is a disaccharide, the enzyme from X. dendrorhous yields a final product enriched in trisaccharides and tetrasaccharides.

Optimization of extraction methods for determination of the raffinose family oligosaccharides in leguminous vine peas ( Pisum sativum L.) and effects of blanching

The extraction procedure for analysis of the raffinose family oligosaccharides (RFOs) in blanched leguminous vine peas was optimized. For this purpose, 25 pea samples from different cultivars and of different maturity were chosen at random. The effect of blanching was also studied in four cultivars. The yield of oligosaccharides was almost double on average when extracted with 50% ethanol as compared with 80% ethanol. This was generally valid for all three oligosaccharides, with some exceptions for verbascose, where 80% ethanol was shown to be as effective as 50% ethanol. Extraction at room temperature and at boiling temperature gave similar yields, while 15 min of extraction gave lower yields than 30 and 60 min of extraction, which were similar. The loss of oligosaccharides following blanching was 32% on average, with verbascose showing the highest loss (56.4%), followed by raffinose (28.4%) and stachyose (16.3%). It can be concluded that the procedure used for extraction is of great importance when analysing the RFOs in immature peas, and that there is considerable loss of these sugars during blanching, which is the process commonly used to prepare the frozen commercial product.

Noncatalytic hydrolysis of guar gum under hydrothermal conditions

Guar gum, a naturally occurring heteropolysaccharide made of mannose and galactose, was hydrolytically degraded without a catalyst in a batch reactor to produce water-soluble (WS) saccharides including mono- and oligosaccharides. The degradation was carried out under hydrothermal conditions over ranges of temperature from 180 to 240 °C and of reaction time from 3 to 60 min. Guar gum was readily dissolved and hydrolyzed, and the major products identified in the WS components were oligosaccharides with degrees of polymerization up to about 20, monosaccharides containing mannose and galactose, and 5-hydroxymethyl-2-furaldehyde (5-HMF). At 200 °C, the oligosaccharide yield, obtained from the difference between the yields of the total WS saccharides and monosaccharides, showed the highest value of 94.4% at 7 min among all conditions studied, on the basis of the saccharide content in the initial sample. The oligosaccharide yield decreased with reaction time, and the yield of monosaccharides correspondingly increased, and reached the highest value of 34.5% (mannose 22.8%, galactose 11.7%) at 60 min. The monosaccharides produced were further decomposed to secondary products such as 5-HMF. The maximum yield of 5-HMF obtained was 26.3% at 220 °C and 30 min. The production and the decomposition of galactose somewhat preceded those of mannose.

Purification and characterisation of an intracellular enzyme with β-glucosidase and β-galactosidase activity from the thermophilic fungus Talaromyces thermophilus CBS 236.58

An intracellular beta-glycoside hydrolase with beta-glucosidase and beta-galactosidase activity, designated beta-glucosidase BGL1, was isolated to apparent homogeneity from the thermophilic ascomycete Talaromyces thermophilus CBS 236.58. The monomeric enzyme has a molecular mass of 50 kDa (SDS-PAGE) and an isoelectric point of 4.5-4.6. The enzyme is active with both glucosides such as cellobiose and galactosides including lactose; based on the catalytic efficiencies determined glucosides are the preferred substrates. beta-Galactosidase activity of BGL1 is activated by various mono and divalent cations including Na+, K+ and Mg2+, and it is moderately inhibited by its reaction products glucose and galactose. Its pH optimum for the hydrolysis of galactosides is in the range of 5.5-6.0, and its optimum temperature was found to be 50 degrees C (15 min assay). In addition to its hydrolytic activity, BGL1 shows a significant transferase activity which results in the formation of galacto-oligosaccharides. These have recently attracted interest because of possible applications in food industry. The highest yields of oligosaccharides was approximately 20% when using 38 gl(-1) lactose as the starting material.

Enzymatic hydrolysis of chitin in the production of oligosaccharides using Lecanicillium fungicola chitinases

Lecanicillium fungicola was selected among 15 strains as chitinase producer and it was used to obtain the crude enzyme (UFL) in submerged fermentation (SF) with added chitin. The UFL displayed at pH 6 and 40 °C the highest endochitinase (Endo) and N-acetylhexosaminidase (NHase) activities, 747 and 410 U/mg, respectively. Four bands of proteins with molecular weights of 123.1, 85.5, 33.1 and 23 kDa were detected in UFL by SDS-PAGE. In order to increase solubilities of the substrates, α and β-chitins were treated with alkali; degrees of deacetylation (DD) were determined 55 and 50%, respectively. Thereafter, chitin hydrolysis with UFL was carried out at 40 °C and pH 5, Endo and NHase at these conditions were 619 and 355 U/mg of protein, respectively. The partial deacetylation as well as the use of acidified reaction media improved significantly the enzyme efficiency in terms of yields of chitin oligosaccharides produced and process time. The maximum chitin oligosaccharides concentration ( P max) obtained from α- and β-deacetylated chitins were 2.77 and 4.44 mmol/l, respectively; whereas for α-chitin it was determined a very small amount of product (0.17 mmol/l). Despite of these results, the maximum production rate ( V max 0.0836 mmol/l h) for α-deacetylated chitin was significantly higher than β-deacetylated chitin (0.0363 mmol/l h).

Galacto-oligosaccharide synthesis by immobilized Aspergillus oryzae β-galactosidase

Aspergillus oryzae β-galactosidase was immobilized by three different techniques, namely adsorption on celite, covalent coupling to chitosan and aggregation by cross-linking. These techniques were compared in terms of the yield of immobilized preparation, enzymatic characteristics, stability and efficiency in oligosaccharide synthesis. Immobilization led to increase in K m in each case. Immobilization on chitosan gave maximum enzyme yield and oligosaccharide synthesis. At 60 °C, the chitosan-immobilized enzyme was stabilized (by 1.6-fold) due to protection effect of the matrix. However, at 65 °C, the t 1/2 of cross-linked enzyme aggregates (CLEA) of β-galactosidase was 1.07 h as compared to 0.79 h in the case of free enzyme. Both chitosan-immobilized enzyme and CLEA were used for oligosaccharide synthesis. Using 20% (w/v) lactose, the chitosan-immobilized enzyme gave maximum oligosaccharide yield (17.3% of the total sugar) as compared to free enzyme (10.0%) in 2 h at 40 °C. CLEA were instead found effective in lactose hydrolysis yielding 78% monosaccharide in 12 h.

Structural characterization of 2‐aminobenzamide‐derivatized oligosaccharides using a matrix‐assisted laser desorption/ionization two‐stage time‐of‐flight tandem mass spectrometer

Oligosaccharides were derivatized by reductive amination using 2-aminobenzamide (2-AB) and analyzed by matrix-assisted laser desorption/ionization two-stage time-of-flight (MALDI-TOF/TOF) tandem mass spectrometry (MS/MS) in the positive ion mode. The major signals were obtained under these conditions from the [M+Na]+ ions for all 2-AB-derivatized oligosaccharides. A systematic study was conducted on a series of 2-AB-derivatized oligosaccharides to allow rationalization of the fragmentation processes. The MALDI-TOF/TOF-MS/MS spectra of the [M+Na]+ ions of 2-AB-derivatized oligosaccharides were dominated by glycosidic cleavages. These fragments originating both from the reducing and the non-reducing ends of the oligosaccharide yield information on sequence and branching. Moreover, the MALDI-TOF/TOF-MS/MS spectra were also characterized by abundant cross-ring fragments which are very informative on the linkages of the monosaccharide residues constituting these oligosaccharides. MALDI-TOF/TOF-MS/MS analysis of 2-AB-derivatized oligosaccharides, by providing structural information at the low-picomole level, appears to be a powerful tool for carbohydrate structural analysis.

Enzyme glycation influences product yields during oligosaccharide synthesis by reverse hydrolysis

Possible evidence is presented for Maillard glycation of enzymes during oligosaccharide synthesis by reverse hydrolysis. In 70% (w/v) mannose solutions, 1,2-α-mannosidase from Penicillium citrinum lost 40% and α-mannosidase from almonds lost 60% activity at 55 °C over 2 weeks. Oligosaccharide yields were 15 and 45% respectively. Higher molecular weight glycation adducts were formed in a time-dependent manner as seen by MALDI-TOF. Inhibitors of the Maillard reaction were able to partially alleviate these effects resulting in reduced loss of enzyme activity and oligosaccharide yield increases of 27–53% relative to the control.

Product monitoring and quantitation of oligosaccharides composition in agar hydrolysates by precolumn labeling HPLC

Since the discovery of multiple bioactivities for agarobiose oligomers, a quantitative method has been in great need to monitor the agarobiose oligomers. This report demonstrates that agarobiose oligomers can be separated with high resolution in HPLC after introducing α-naphthylamine into compounds. Agarobiose oligomers ranged from biose to decaose were isolated by Sephadex column. HPLC analysis indicated that each oligomer could be quantified with good linearity and a low detection limit of 0.1–4μg/ml. The chromatographic profiles of agaro-oligosaccharides with different hydrolysis modes (hydrochloride, citric acid, solid acid, and hydroxyl radical degradation) showed that agarobiose could be obtained more than 57.8% using solid acid mediated hydrolysis, while hydrochloride acid could degrade agar into a series of agaro-oligosaccharides from biose to decaose. The yield of oligosaccharides was low if hydrolyzed by citric acid. The Fenton degradation can increase the speed of hydrolysis, but the product was complex.

Synthesis of FimH receptor-active manno-oligosaccharides by reverse hydrolysis using alpha-mannosidases from Penicillium citrinum, Aspergillus phoenicis and almond.

Recombinant Penicillium citrinum alpha-1,2-mannosidase, expressed in Aspergillus oryzae, was employed to carry out regioselective synthesis of alpha- d-mannopyranosyl-(1-->2)- d-mannose. Yields (w/w) of 16.68% disaccharide, 3.07% trisaccharide and 0.48% tetrasaccharide were obtained, with alpha1-->2 linkages present at 98.5% of the total linkages formed. Non-specific alpha-mannosidase from almond was highly efficient in reverse hydrolysis and oligosaccharide yields of 45-50% were achieved. The products of the almond mannosidase were a mixture of disaccharides (30.75%, w/w), trisaccharides (12.26%, w/w) and tetrasaccharides (1.89%, w/w) with 1-->2, 1-->3 and 1-->6 isomers. alpha-1,2-linkage specific mannosidase from P. citrinum and alpha-1,6-linkage-specific mannosidase from Aspergillus phoenicis were used in combination to hydrolyse the respective linkages from the mixture of isomers, resulting in alpha- d-mannopyranosyl-(1-->3)- d-mannose in 86.4% purity. The synthesised oligosaccharides can potentially inhibit the adhesion of pathogens by acting as "decoys" of receptors of type-1 fimbriae carried by enterobacteria.

Extraction of hemicellulosic oligosaccharides from spruce using microwave oven or steam treatment.

This paper describes the extraction of hemicellulosic oligosaccharides from spruce, using microwave or steam treatment that can be used for the production of polymers, replacing fossil-based polymers, e.g., hydrogels. The highest yield of oligosaccharides, measured as mannan, was 70% obtained with treatment in the microwave oven at 200 degrees C for 5 min. The amount of oligosaccharides extracted was 12.5 g per 100 g of dry wood. The molecular weights of some selected samples were analyzed using fast protein liquid chromatography and size exclusion chromatography and time-of-flight matrix-assisted laser desorption ionization. Recovered oligosaccharides following steam treatment at 200 degrees C for 2 min had a mean molecular weight of 3400 g/mol with a maximum weight of 12000 g/mol. Higher severity, i.e., increased temperature (>200 degrees C) and residence time, resulted in lower mean molecular weights and yield. Oligosaccharides with higher mean molecular weights were obtained at lower severity, but the yield was considerably lower. The feasibility of using the extracted hemicellulosic oligosaccharides from spruce for the synthesis of hydrogels was demonstrated.

Maillard reactions and increased enzyme inactivation during oligosaccharide synthesis by a hyperthermophilic glycosidase.

The thermostable Pyrococcus furiosus beta-glycosidase was used for oligosaccharide production from lactose in a kinetically controlled reaction. Our experiments showed that higher temperatures are beneficial for the absolute as well as relative oligosaccharide yield. However, at reaction temperatures of 80 degrees C and higher, the inactivation rate of the enzyme in the presence of sugars was increased by a factor of 2 compared to the inactivation rate in the absence of sugars. This increased enzyme inactivation was caused by the occurrence of Maillard reactions between the sugar and the enzyme. The browning of our reaction mixture due to Maillard reactions was modeled by a cascade of a zeroth- and first-order reaction and related to enzyme inactivation. From these results we conclude that modification of only a small number of amino groups already gives complete inactivation of the enzyme.

Enzyme inactivation due to Maillard reactions during oligosaccharide synthesis by a hyperthermophilic glycosidase: influence of enzyme immobilisation

The extremely thermostable β-glycosidase from Pyrococcus furiosus was used for the production of oligosaccharides with lactose as a substrate. Using a thermozyme made it possible to operate at higher reaction temperatures, and thus to increase the substrate concentration. This increased the substrate concentration and the subsequent lower water concentration suppressed hydrolysis and therefore improved the oligosaccharide yield. During the reaction, brown pigments were formed, caused by Maillard reactions. This changes the structure of the enzyme and causes faster inactivation of the enzyme, compared to normal inactivation by temperature. This faster inactivation is the main design criterion for the reaction system. Reduction of Maillard reactions can be done by altering the process conditions or through modification of the enzyme, either chemically or by altering the enzyme structure through genetic modifications. In this work, chemical modification of the enzyme was chosen by covalent immobilisation on Eupergit. Unfortunately, the immobilisation did not reduce Maillard reactivity.

Synthesis and Fermentation Properties of Novel Galacto-Oligosaccharides by β-Galactosidases from Bifidobacterium Species

beta-Galactosidase enzymes were extracted from pure cultures of Bifidobacterium angulatum, B. bifidum BB-12, B. adolescentis ANB-7, B. infantis DSM-20088, and B. pseudolongum DSM-20099 and used in glycosyl transfer reactions to synthesize oligosaccharides from lactose. At a lactose concentration of 30% (wt/wt) oligosaccharide yields of 24.7 to 47.6% occurred within 7 h. Examination of the products by thin-layer chromatography and methylation analysis revealed distinct product derived spectra from each enzyme. These were found to be different to that of Oligomate 55, a commercial prebiotic galacto-oligosaccharide. Fermentation testing of the oligosaccharides showed an increase in growth rate, compared to Oligomate 55, with products derived from B. angulatum, B. bifidum, B. infantis, and B. pseudolongum. However B. adolescentis had a lower growth rates on its oligosaccharide compared with Oligomate 55. Mixed culture testing of the B. bifidum BS-4 oligosaccharide showed that the overall prebiotic effect was equivalent to that of Oligomate 55.

Improved oligosaccharide synthesis by protein engineering of beta-glucosidase CelB from hyperthermophilic Pyrococcus furiosus.

Enzymatic transglycosylation of lactose into oligosaccharides was studied using wild-type beta-glucosidase (CelB) and active site mutants thereof (M424K, F426Y, M424K/F426Y) and wild-type beta-mannosidase (BmnA) of the hyperthermophilic Pyrococcus furiosus. The effects of the mutations on kinetics, enzyme activity, and substrate specificity were determined. The oligosaccharide synthesis was carried out in aqueous solution at 95 degrees C at different lactose concentrations and pH values. The results showed enhanced synthetic properties of the CelB mutant enzymes. An exchange of one phenylalanine to tyrosine (F426Y) increased the oligosaccharide yield (45%) compared with the wild-type CelB (40%). Incorporation of a positively charged group in the active site (M424K) increased the pH optimum of transglycosylation reaction of CelB. The double mutant, M424K/F426Y, showed much better transglycosylation properties at low (10-20%) lactose concentrations compared to the wild-type. At a lactose concentration of 10%, the oligosaccharide yield for the mutant was 40% compared to 18% for the wild-type. At optimal reaction conditions, a higher ratio of tetrasaccharides to trisaccharides was obtained with the double mutant (0.42, 10% lactose) compared to the wild-type (0.19, 70% lactose). At a lactose concentration as low as 10%, only trisaccharides were synthesized by CelB wild-type. The beta-mannosidase BmnA from P. furiosus showed both beta-glucosidase and beta-galactosidase activity and in the transglycosylation of lactose the maximal oligosaccharide yield of BmnA was 44%. The oligosaccharide yields obtained in this study are high compared to those reported with other transglycosylating beta-glycosidases in oligosaccharide synthesis from lactose.

Production of galactooligosaccharides from lactose using a beta-glucosidase from Thermus sp. Z-1.

A thermostable beta-glucosidase from Thermus sp. Z-1 that not only hydrolyzes beta-glucosides but also beta-galactosides was shown to efficiently produce oligosaccharides during hydrolysis of lactose. The yield of oligosaccharides was more than 40% for 0.88 M lactose solution at 70 degrees C at pH 7.0. The major product was a trisaccharide, 3'-galactosyllactose, formed by a galactosyltransfer reaction.