Dextran Biosynthesis Research Articles

Dextran Used in Blood Transfusion, Hematology, and Pharmaceuticals: Biosynthesis of Diverse Molecular-Specification-Dextrans in Enzyme-Catalyzed Reactions

Dextran is an exopolysaccharide synthesized in reactions catalyzed by enzymes obtained from microbial agents of specific species and strains. Products of dextran polysaccharides with different molecular weights are suitable for diverse pharmaceutical and clinical uses. Dextran solutions have multiple characteristics, including viscosity, solubility, rheological, and thermal properties; hence, dextran has been studied for its commercial applications in several sectors. Certain bacteria can produce extracellular polysaccharide dextran of different molecular weights and configurations. Dextran products of diverse molecular weights have been used in several industries, including medicine, cosmetics, and food. This article aims to provide an overview of the reports on dextran applications in blood transfusion and clinical studies and its biosynthesis. Information has been summarized on enzyme-catalyzed reactions for dextran biosynthesis from sucrose and on the bio-transformation process of high molecular weight dextran molecules to obtain preparations of diverse molecular weights and configurations.

Production of Dextran from Domestic Weissella cibaria Isolated

Dextran is an exopolysaccharide (EPS) of bacterial origin that is readily available in the commercial market. that is used in various industrial applications as an adjuvant, emulsifier, carrier, and stabilizer in the food and biomedical industries. Industrial production of dextran is carried out by fermentation in sucrose-rich media. Studies to optimize dextran production have shown that dextran yield varies depending on specific production conditions. This study aimed to generate dextrans Lactobacillus species were obtained from the vaginal and stool samples of healthy infants. The mucosal technique was employed to determine the biosynthesis of dextran from various isolates of Lactobacillus spp. colony assay and confirmed by isopropanol precipitation. The effects of various parameters such as sucrose concentration, nitrogen concentration, temperature, incubation time, pH, and inoculum size were studied to optimize the maximum dextran yield. In the present study, Weissella cibaria may produce dextran. Optimal conditions for dextran production are a 48-hour incubation at The experimental conditions involved maintaining a temperature of 37°C, with a sugar concentration of 10%, and a yeast extract concentration of 0.5%. The inoculum volume was set at 4%, and the pH level was maintained at 6.5. The experiment was conducted under aerobic conditions. The obtained value for dextran dry weight was 520 mg/100 ml.

Dextran of Diverse Molecular-Configurations Used as a Blood-Plasma Substitute, Drug-Delivery Vehicle and Food Additive Biosynthesized by Leuconostoc, Lactobacillus and Weissella

Dextran, a microbial metabolite of diverse molecular configurations, can be biosynthesized employing selected strains of characterized species of bacteria. Dextran molecules are secreted as an extracellular polysaccharide in the culture medium of the bacterial fermentation system. This microbially produced polymer of glucose possesses multi-faceted characteristics such as its solubility in different solvents and formation of dextran solutions of needed viscosity. Several preparations can be formulated for the desired thermal and rheological properties. Due to such multifunctional characteristics, dextran with different structural specifications is a desired polysaccharide for clinical, pharmaceutical, and food industry commercial applications. Dextran and its derivative products with various molecular weights, in a range of high and low, have established their uses in drug delivery and in analytical devices using columns packed with polysaccharide gel. Therefore, being a neutral raw material, the resourcefulness of dextran preparations of different molecular weights and linkages in their polymer configuration is important. For this purpose, several studies have been performed to produce this commercially important polysaccharide under optimized bacterial cultivation processes. This article aims to overview recently published research reports on some significant applications of dextran in the pharmaceutical and food industries. Studies conducted under optimized conditions in fermentation processes for the biosynthesis of dextran of diverse molecular configurations, which are responsible for its multifunctional properties, have been summarized. Concise information has been presented in three separate tables for each group of specific bacterial species employed to obtain this extracellular microbial polysaccharide.

Bioprocessing of Barley and Lentil Grains to Obtain In Situ Synthesis of Exopolysaccharides and Composite Wheat Bread with Improved Texture and Health Properties.

A comprehensive study into the potential of bioprocessing techniques (sprouting and sourdough fermentation) for improving the technological and nutritional properties of wheat breads produced using barley and lentil grains was undertaken. Dextran biosynthesis in situ during fermentation of native or sprouted barley flour (B or SB) alone or by mixing SB flour with native or sprouted lentil flour (SB-L or SB-SL) by Weissella paramesenteroides SLA5, Weissella confusa SLA4, Leuconostoc pseudomesenteroides DSM 20193 or Weissella confusa DSM 20194 was assessed. The acidification and the viscosity increase during 24 h of fermentation with and without 16% sucrose (on flour weight), to promote the dextran synthesis, were followed. After the selection of the fermentation parameters, the bioprocessing was carried out by using Leuconostoc pseudomesenteroides DSM 20193 (the best LAB dextran producer, up to 2.7% of flour weight) and a mixture of SB-SL (30:70% w/w) grains, enabling also the decrease in the raffinose family oligosaccharides. Then, the SB-SL sourdoughs containing dextran or control were mixed with the wheat flour (30% of the final dough) and leavened with baker’s yeast before baking. The use of dextran-containing sourdough allowed the production of bread with structural improvements, compared to the control sourdough bread. Compared to a baker’s yeast bread, it also markedly reduced the predicted glycemic index, increased the soluble (1.26% of dry matter) and total fibers (3.76% of dry matter) content, giving peculiar and appreciable sensory attributes.

Brewers\u2019 spent grain as substrate for dextran biosynthesis by Leuconostoc pseudomesenteroides DSM20193 and Weissella confusa A16

BackgroundLactic acid bacteria can synthesize dextran and oligosaccharides with different functionality, depending on the strain and fermentation conditions. As natural structure-forming agent, dextran has proven useful as food additive, improving the properties of several raw materials with poor technological quality, such as cereal by-products, fiber-and protein-rich matrices, enabling their use in food applications. In this study, we assessed dextran biosynthesis in situ during fermentation of brewers´ spent grain (BSG), the main by-product of beer brewing industry, with Leuconostoc pseudomesenteroides DSM20193 and Weissella confusa A16. The starters performance and the primary metabolites formed during 24 h of fermentation with and without 4% sucrose (w/w) were followed.ResultsThe starters showed similar growth and acidification kinetics, but different sugar utilization, especially in presence of sucrose. Viscosity increase in fermented BSG containing sucrose occurred first after 10 h, and it kept increasing until 24 h concomitantly with dextran formation. Dextran content after 24 h was approximately 1% on the total weight of the BSG. Oligosaccharides with different degree of polymerization were formed together with dextran from 10 to 24 h. Three dextransucrase genes were identified in L. pseudomesenteroides DSM20193, one of which was significantly upregulated and remained active throughout the fermentation time. One dextransucrase gene was identified in W. confusa A16 also showing a typical induction profile, with highest upregulation at 10 h.ConclusionsSelected lactic acid bacteria starters produced significant amount of dextran in brewers’ spent grain while forming oligosaccharides with different degree of polymerization. Putative dextransucrase genes identified in the starters showed a typical induction profile. Formation of dextran and oligosaccharides in BSG during lactic acid bacteria fermentation can be tailored to achieve specific technological properties of this raw material, contributing to its reintegration into the food chain.

Growth studies of Leuconostoc mesenteroides on corn biomass based substrates

Leuconostoc mesenteroides is a well-known industrial producer of extracellular bacterial polysaccharide dextran. The main raw material for industrial cultivation of these microorganisms is molasses. Sucrose of molasses is the main source of carbon and energy for bacteria, as well as a precursor of dextran biosynthesis. It is known that the bacteria of the genus Leuconostoc are heterofermentative microorganisms and are capable to utilize various carbohydrates. The purpose of this study was to evaluate the of the corn biomass based mediumon the yield of bacterial biomass and the concentration of the extracellular polysaccharide.The hydrolyzate is a product of chemo-enzymatic treatment of plant lignocellulosic corn biomass and contains such monosaccharides as glucose, arabinose and xylose. The parameters of the composition of the medium were the concentration of hydrolyzate carbohydrates in the form of reducing substances (RS), the concentration of nitrogen (protein and non-protein), sucrose and the presence of minerals. The parameters of the cultivation process are pH and temperature. According to experimental data, the maximum yield of bacterial biomass (9.3 and 8.5%) were achieved on combined media with a sucrose content of 0.2% and a sodium salt (sodium phosphate), respectively. The highest specific growth rates of microorganisms (0.0840 and 0.0746) are demonstrated on a combined medium with a content of 0.15 and 0.3% inorganic nitrogen, respectively. The highest concentrations of extracellular polysaccharides (0.840 and 0.583%) are formed when magnesium and manganese salts are present in the nutrient medium. The optimal pH level for effective growth s is about 7, and the temperature in the range of 30-35 ° C.

Biosynthesis of dextran by Weissella confusa and its In vitro functional characteristics

The aim of this study was to monitor the influence of the fermentation conditions on the exopolysaccharides (EPS) biosynthesis. For this, different culture media compositions were tested on an isolated lactic acid bacteria (LAB) strain, identified by 16S rDNA sequence as being Weissella confusa. It was proved that this bacterial strain culture in MRS medium supplemented with 80g/L sucrose and dissolved in UHT milk produced up to 25.2g/L of freeze-dried EPS, in static conditions, after 48h of fermentative process. Using FTIR and NMR analysis, it was demonstrated that the obtained EPS is a dextran. The thermal analysis revealed a dextran structure with high purity while GPC analysis depicted more fractions, which is normal for a biological obtained polymer. A concentration up to 3mg/mL of dextran proved to have no cytotoxic effect on normal human dermal fibroblasts (NHDF). Moreover, at this concentration, dextran breaks up to 70% of the biofilms formed by the Candida albicans SC5314 strain, and has no antimicrobial activity against standard bacterial strains. Due to their characteristics, these EPS are suitable as hydrophilic matrix for controlled release of drugs in pharmaceutical industry.

Designing of a novel dextransucrase efficient in acceptor reactions

Dextransucrase is produced by Leuconostoc, Streptococcus and Lactobacillus Species. The enzyme synthesizes dextran and acceptor products some of which act as prebiotics that are increasingly used in such industries as food, medicine, and cosmetics. B-512F Leuconostoc mesenteroides dextransucrase (DSR-S) is the preferred enzyme in commercial production of dextran and prebiotics. In the present work, a novel dextransucrase which is efficient in prebiotics production was designed. The enzyme was produced at optimal conditions in Escherichia coli by truncation and fusion to glutathione S-transferase (GST) in the gene from Leuconostoc mesenteroides B-512 FMC. The novel enzyme (MW: 119kDa) was active and carried out dextran biosynthesis and acceptor reactions effectively. The novel dextransucrase (fTDSR-S) was produced by truncating signal, variable, and the glucan-binding regions in the gene and fusion of gst gene at the 5′ end. fTDSR-S was characterized in detail and compared to the DSR-S. Truncation and fusion resulted in an increase in fTDSR-S biosynthesis in E. coli BL21 (DE3) by 35 fold. fTDSR-S leads to production of dextran as well as increased acceptor reactions. Due to GST fusion, it was possible to immobilize fTDSR-S covalently onto Eupergit C successfully. It was also found that the size of the active site of dextransucrase is 49 amino acids shorter than that reported previously in the literature.

A novel method for covalent immobilization of dextransucrase

B-512F dextransucrase is an industrial enzyme used in commercial production of dextran and prebiotics. Several researchers have studied the covalent immobilization of this enzyme with little success due to dextran masking the reactive groups on the enzyme and inactivation of the enzyme during immobilization. A novel dextransucrase was designed and produced successfully to eliminate problems faced in covalent immobilization. In production of the novel enzyme, B-512F dextransucrase was truncated at N and C terminals and fused to glutathione S-transferase. The novel dextransucrase was fully active and carried out dextran biosynthesis and acceptor reactions effectively. The novel enzyme was immobilized covalently onto Eupergit C 250L, giving rise to 100% immobilization and 83.3% activity yields. Immobilized enzyme was used successfully for the production of acceptor products and low molecular weight dextran. The immobilized enzyme showed no decrease in activity for 15 batch reactions and retained its initial activity at storage (4 °C) for 35 days. Optimal conditions were not affected by the immobilization. The kinetic parameters for the free and immobilized enzyme were determined. The acceptor reactions using fructose, glucose, maltose, and lactose were also studied. The novel method developed could also be used in immobilization of some other biomolecules.

Dextransucrase and the mechanism for dextran biosynthesis

Remaud-Simeon and co-workers [Moulis, C.; Joucla, G.; Harrison, D.; Fabre, E.; Potocki-Veronese, G.; Monsan, P.; Remaud-Simeon, M. J. Biol. Chem., 2006, 281, 31254–31267] have recently proposed that a truncated Escherichia coli recombinant B-512F dextransucrase uses sucrose and the hydrolysis product of sucrose, d-glucose, as initiator primers for the nonreducing-end synthesis of dextran. Using 14C-labeled d-glucose in a dextransucrase–sucrose digest, it was found that <0.02% of the d-glucose appears in a dextran of M n 84,420, showing that d-glucose is not an initiator primer, and when the dextran was treated with 0.01 M HCl at 80 °C for 90 min and a separate sample with invertase at 50 °C for 24 h, no d-fructose was formed, indicating that sucrose is not present at the reducing-end of dextran, showing that sucrose also was not an initiator primer. It is further shown that both d-glucose and dextran are covalently attached to B-512FMC dextransucrase at the active site during polymerization. A pulse reaction with [ 14C]-sucrose and a chase reaction with nonlabeled sucrose, followed by dextran isolation, reduction, and acid hydrolysis, gave 14C-glucitol in the pulsed dextran, which was significantly decreased in the chased dextran, showing that the d-glucose moieties of sucrose are added to the reducing-ends of the covalently linked growing dextran chains. The molecular size of dextran is shown to be inversely proportional to the concentration of the enzyme, indicating a highly processive mechanism in which d-glucose is rapidly added to the reducing-ends of the growing chains, which are extruded from the active site of dextransucrase. It is also shown how the three conserved amino acids (Asp551, Glu589, and Asp 622) at the active sites of glucansucrases participate in the polymerization of dextran and related glucans from a single active site by the addition of the d-glucose moiety of sucrose to the reducing-ends of the covalently linked glucan chains in a two catalytic-site, insertion mechanism.

Production of insoluble dextran using cell-bound dextransucrase of Leuconostoc mesenteroides NRRL B-523

Water-insoluble, cell-free dextran biosynthesis from Leuconostoc mesenteroides NRRL B-523 has been examined. Cell-bound dextransucrase is used to produce cell-free dextran in a sucrose-rich acetate buffer medium. A comparison between the soluble and insoluble dextrans is made for various sucrose concentrations, and 15% sucrose gave the highest amount of cell-free dextran for a given time. L. mesenteroides B-523 produces more insoluble dextran than soluble dextran. The near cell-free synthesis was validated in a batch reactor, by monitoring the cell growth which is a small (10 6–10 7 CFU/mL) and constant value throughout the synthesis.

Large-scale purification of Leuconostoc mesenteriodes NRRL B512F dextransucrase for use in the biosynthesis of dextran by batch and continuous chromatography

The extracellular enzyme dextransucrase was produced from Leuconostoc mesenteriodes NRRL B512F and purified by ultracentrifugation and cross-flow ultrafiltration for use in the biosynthesis of the macromolecule dextran by ion exchange chromatographic reaction—separation techniques. The two-stage purification process yielded over 90% pure dextransucrase with overall enzyme recovery of over 60%. A second stage of centrifugation was required to achieve complete cell removal. The purified enzyme contained 1–2 g l −1 of solute ions, which affected the operation of the chromatographic system. Gel filtration removed over 93% of the remaining ions but resulted in high activity losses. Two-phase separation with polyethylene glycol (PEG) and purification by ion exchange chromatography were less successful in desalting the enzyme. PEG precipitation was successful in concentrating the enzyme, but the ions remained predominantly with the enzyme portion of the two phases. The purified enzyme was found to be unstable during storage. Use of the enzyme in chromatographic reactor—separators for the production of dextran resulted in over 33% more high molecular weight dextran (the desired product) and a useful pure fructose byproduct being obtained than for a conventional reactor. Sodium and potassium ions in the enzyme hampered continuous operation by displacing calcium ions from the resin and thus reducing the separation efficiency of the system. Partial regeneration of the resin with calcium nitrate rather than complete enzyme desalting, which was very expensive and resulted in high activity losses, helped overcome this effect.

Large-scale purification of Leuconostoc mesenteriodes NRRL B512F dextransucrase for use in the biosynthesis of dextran by batch and continuous chromatography

Large-scale purification of Leuconostoc mesenteriodes NRRL B512F dextransucrase for use in the biosynthesis of dextran by batch and continuous chromatography

A novel approach to the production of clinical‐grade dextran

Efficient unit operations for the production and purification of the enzyme dextransucrase and the biosynthesis and fractionation of dextrans have been developed and tested. An alternative process for the production of clinical-grade dextran has been proposed by integrating these processes to achieve improved dextran product yield and the recovery of fructose as a valuable by-product. The enzyme dextransucrase, which synthesizes dextran from sucrose, has been produced either by non-aerated fed-batch or continuous fermentations, purified by continuous ultracentrifugation and ultrafiltration and used in a bioreactor-separator stage for the biosynthesis of dextran. High molecular weight dextrans have been biosynthesized on continuous counter-current chromatographic bioreactor-separator systems. Membrane filtration and size exclusion continuous chromatography were used for the fractionation of the native dextran and production of clinical-grade dextran. The advantages of the proposed process over the conventional process are related to cost reduction due to the elimination of ethanol usage and recovery, improved product quality control and the production and recovery of fructose as a by-product.

Bioreaction-separation on continuous chromatographic systems

Combined bioreaction and separation has been carried out successfully on a simulated moving column continuous countercurrent chromatographic reactor-separator (SCCR-S) system. The continuous inversion of sucrose to glucose and fructose using invertase and the biosynthesis of dextran from sucrose in the presence of the enzyme dextransucrase have been studied. 100% sucrose conversions were achieved and product purities of up to 95% w/v were obtained in the inversion studies. The operation was carried out on a preparative scale with throughputs in excess of 16 kg of dry sugar solids per cubic metre of resin per hour used. The simultaneous inversion and product separation was found to overcome problems associated with substrate inhibition. Complete reaction and separation was obtained at feed concentrations as high as 55% w/v. Previous studies on the biosynthesis of dextran on batch chromatographic systems showed significant improvements in product yields. The simultaneous removal of the acceptor by product fructose from the reaction mixture led to the formation of greater amounts of high molecular weight dextran (over 80% more than the conventional process at 20% w/v sucrose concentration). To carry out dextran biosynthesis on the SCCR-S system, it was necessary to repack it with larger-size resin to allow for the high pressures caused by the more viscous dextran. Complete conversions have been achieved at sucrose concentrations of 5% w/v with dextran and fructose product purities of up to 100% and 97% respectively obtained under certain conditions. Some levan formation occurred after about 50 h of operation, which is readily removed from the dextran in the normal dextran processing procedures.

Studies on dextran fermentation broth rheology

The rheological properties of dextran fermentation broths of a Leuconostoc mesenteroides culture were investigated. The biosynthesis of dextran is responsible for the non-newtonian flow behavior of the broths in the later stage of dextran fermentation. The rheological behavior can be characterized by a power-law type of equation.

Dextran biosynthesis and dextransucrase production by continuous culture of Leuconostoc mesenteroides

Leuconostoc mesenteroides NRRL B-512(F) was grown in continuous culture under conditions of energy-limited growth. The extracellular enzyme dextransucrase (sucrose: 1,6-alpha-D-glucan 6-alpha-glucosyltransferase EC 2.4.1.5), was not detected in glucose- or maltose-limited cultures. Under conditions of sucrose-limited growth, the enzyme activity of the cell-free culture supernatant increased with increasing dilution rate only after the critical concentration of enzyme inducer (sucrose) in the chemostat had been achieved. The appearance of fructose in the effluent of the sucrose-limited chemostat at higher dilution rates indicated that sucrose was being diverted to dextran biosynthesis. The competition between bacteria and extracellular enzyme for the common substrate sucrose represents an inefficiency in the system of enzyme production. Dextransucrase was isolated from the cell-free culture supernatant by ammonium sulfate precipitation and DEAE-cellulose chromatography. The enzyme preparation exhibited both dextran biosynthetic activity and an invertase-like activity. The biosynthetic efficiency was increased by decreasing the temperature from 30 to 10 degrees C. The enzyme was irreversibly denatured by prolonged incubation in the absence of Ca2+.

Inhibition of dextran synthesis by glucoamylase and endodextranase

In a model experiment, glucoamylase was shown to inhibit α- D-glucan synthesis as catalyzed by potato phosphorylase. Both glucoamylase and endodextranase inhibited dextran synthesis with dextransucrases of Leuconostoc mesenteroides. The inhibition could be ascribed to competition between glucoamylase and dextransucrase for the glucosyl groups at the non-reducing end of dextran. The inhibition caused by endodextranase may result from rapid and random hydrolysis of acceptor dextrans. Moreover, significantly low units of glucoamylase, as compared with endodextranase, effectively inhibited dextran synthesis. These results thus present evidence that bio-synthesis of dextran occurs by the addition of glucosyl groups at the non-reducing end of the growing dextran. The measurement of initial velocity suggested that the ping-pong Bi-Bi mechanism proposed for the levansucrase of Bacillus subtilis is also applicable to dextransucrase.

The mechanism of dextransucrase action: Direction of dextran biosynthesis

The mechanism for the biosynthesis of dextran by dextransucrase from Leuconostoc mesenteroides NRRL-B512F has been studied. Dextransucrase attached to cells and dextransucrase insolubilized on Bio-Gel beads have been obtained. It was found that these forms become labeled when incubated with a pulse of [ 14C]sucrose. The label can be released from cells and from Bio-Gel by adjusting the pH to 2 and heating to 95 °C for 10 min. Two types of labeled carbohydrates were found to be released, glucose and dextran. The direction of the biosynthesis was determined by pulse and chase experiments with [ 14C]sucrose. The labeled dextran was isolated, reduced, and acid hydrolyzed. The hydrolytic products, glucose and sorbitol, were separated by paper chromatography, and their radioactivities were determined. The ratio of the labeled sorbitol to labeled glucose for the pulse of Bio-Gel-dextransucrase was 1:1 and for the chase 1:100. It was concluded that dextran is biosynthesized by the transfer of glucose from sucrose to the reducing end of the growing dextran chain. This was confirmed by the hydrolysis of pulsed dextran by glycoamylase. The total radioactivity in the residual dextran did not change during the hydrolysis and labeled glucose could not be detected. It is proposed that dextransucrase forms an enzymatically active covalent complex with glucose and dextran and that the glucose is inserted between the enzyme and the dextran by a nucleophilic attack of the C 6—OH of glucose onto C 1 of the dextran forming an α-1 → 6 glucosidic bond. This releases one of the nucleophilic groups at the active site which attacks sucrose to give an enzyme-glucosyl complex. The C 6—OH of this glucose then repeats the process of attacking C 1 of dextran. Dextran is built up by extrusion from the enzyme when glucose units are transferred from sucrose to the active site and inserted between the enzyme and the reducing end of the dextran polymer.