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Co-immobilization is a groundbreaking technique for enzymatic catalysis, sometimes strategic, as for dextransucrase and dextranase. In this approach, dextranase hydrolytic action removes the dextran layer that covers dextransucrase reactive groups, improving the immobilization. Another advantage is the synergic effect of the two enzymes towards prebiotic oligosaccharides production. Thus, both enzymes were co-immobilized onto the heterobifunctional support Amino-Epoxy-Glyoxyl-Agarose (AMEG) and the ion exchanger support monoaminoethyl-N-ethyl-agarose (Manae) at pH 5.2 and 10, followed or not by glutaraldehyde treatment. This work is the first attempt to immobilize dextransucrase under alkaline conditions. The immobilized dextransucrase on AMEG support at pH 10 (12.78 ± 0.70 U/g) presents a similar activity of the biocatalyst produced at pH 5.2 (14.95 ± 0.82 U/g). The activity of dextranase immobilized onto Manae was 5-fold higher than the obtained onto AMEG support. However, the operational stability test showed that the biocatalyst produced on AMEG at pH 5.2 kept >60% of both enzyme activities for five batches. The glutaraldehyde treatment was not worthwhile to improve the operational stability of this biocatalyst.

Although the extracellular polysaccharides have been analyzed in the previous period, the biochemical, enzymological characters and stimulation and inhibition effect on glucansucrase are not fully understood. After three steps purification, salting out, DEAE-Sepharose and Sephadex G-75, the final specific activity was 264.84 U/mg protein with 4.31-fold. The SDS-PAGE analysis of fraction gave a single band 170.35 kDa in the stained gel. The active band was analyzed with LC–MS/MS to identify glucansucrase. The highest coverage rate of dextransucrase from Leu. citreum (ACY92456.2) was 55.60%, the results were speculated that the glucansucrase secreted from Leu. citreum B-2 may be a novel glucansucrase. The purified enzyme was optimally active at 20–30 °C and pH 6.0–8.0. Metal ions K+, Na+, Ca2+, Mn2+, Mg2+, and Cr+ had an apparent stimulating effect on enzyme activity, especially in divalent ions Ca2+ and Mn2+, the residual activities were higher than 200%. In a reverse, Hg+, acetonitrile, SDS, salt, and guanidine expressed inhibition effect on enzyme residual activity. The KM and Vmax were detected to be 4.82 mM and 0.97 U/mg, respectively. All these data collectively indicate that B-2 glucansucrase is a novel one, which have good properties and may applied to new food areas.

Non-digestible oligosaccharides have wide food industrial applications as dietary fibers and prebiotics. The aim of this study is to realize the effective biosynthesis of isomalto-oligosaccharides (IMOs) and reduce the production of by-product dextran. In the presence of acceptors improved the dextransucrase reaction shifting to oligosaccharides formation but a number of by-products dextran appeared. Maltose acceptor performed stronger inhibition behaviors in dextran synthesis than lactose and glucose acceptor due to its higher efficiencies. Acceptors had no influence on the structure of by-product dextran which mainly composed of α-(1,6)-glycosidic linkages and low α-(1,3)-glycosidic branch. In addition, the Mw and contents of IMOs and oligodextrans synthesized by dual-enzyme were hard to control. Addition of maltose acceptor in the dual-enzyme reaction, the adequate dextranase preferentially degraded dextran than the acceptor products to yield the IMOs. Results indicated that the combined use of the dual-enzyme and the maltose acceptor is a simple and effective method to promote the high-quality of functional IMOs.

The lactic acid bacteria (LAB) have great potential to produce homoexopolysaccharides (HoPS), have been the subject of extensive research efforts, given their health benefits and physicochemical properties. The HoPS functional properties are determined by structural characteristics of varied molecular weights, types of glycosidic linkages, degrees of branching and chemical composition. The dextransucrases (DSases) are responsible of the synthesis of a kind of HoPS (dextran polymers), which are among the first biopolymers produced at industrial scale with applications in medicine and biotechnology. The concept of glycodiversification opens additional applications for DSases. In that sense the design and characterization of new DSases is of prime importance. Previously, we described the isolation and characterization of a novel extracellular dextransucrase (DSR-F) encoding gene. In this study, from DSR-F, we design a novel chimeric dextransucrase DSR-F-∆SP-∆GBD-CBM2a, where DSR-F-∆SP-∆GBD is fused to the carbohydrate-binding module (CBM2a) of the β-1-4 exoglucanase/xylanase Cex (Xyn10A) of Cellulomonas fimi ATCC 484. This dextransucrase variant is active and without alteration in its specificity. The DSR-F-∆SP-∆GBD-CBM2a is purified by cellulose affinity chromatography for the very first time. Our results indicate that new hybrids and chimeric DSases with novel binding capacity to cellulose can be designed to obtain glyco-biocatalysts from renewable lignocellulosic materials.

Lactic acid bacteria are known to produce extracellular polysaccharides such as α-glucan, levan, and inulin, which are promising for applications in food systems because of their prebiotic properties. In this work, a novel glucansucrase (GS) Gtf-DSM from Lactobacillus ingluviei DSM 14792 was recombinantly expressed and biochemically characterized as a dextransucrase capable of producing a dextran polysaccharide with four types of linkages, including 69% (α1 → 6), 24% (α1 → 3), 6% (α1 → 4), and 1% (α1 → 2). Intriguingly, the dextransucrase Gtf-DSM had a sequence identity of 99.3% with the reuteransucrase GtfO producing a reuteran with 21% (α1 → 6) and 79% (α1 → 4) linkages. Thus, the dextransucrase Gtf-DSM is a unique target for understanding the linkage specificity of GSs and the investigation of site-directed mutagenesis using Gtf-DSM and GtfO as templates is underway.

Dextransucrases are extracellular enzymes, which are exclusively expressed by lactic acid bacteria (LAB) and produce α−1→6 linked glucose polymers from sucrose. In this study, two dextransucrases derived from water kefir borne Lactobacillus hordei TMW 1.1822 and Lactobacillus nagelii TMW 1.1827 were identified and comparatively investigated. Differences between both proteins mainly arise from an additional C-terminal glucan-binding domain and the presence of a signal motif in the L. nagelii TMW 1.1827 dextransucrase. L. hordei TMW 1.1822 released the enzyme only in the presence of its substrate sucrose in contrast to L. nagelii TMW 1.1827, while both strains functionally expressed the dextransucrases independently of sucrose. Both enzymes could be recovered as crude protein extracts in culture supernatants, as they are not covalently bound to the cell surface. This enabled the formation of dextrans at equal reaction conditions as well as their subsequent structural analysis in terms of molecular structure and molecular weight. The volumetric transglycosylation and hydrolysis activities were distinctly different for both enzymes, which produced O3-branched dextrans with a comparable degree of branching. Moreover, identical oligosaccharides were obtained for both dextrans upon endo-dextranase digestion, while some differences in the polysaccharide fine structures could be identified from the varying portions of certain oligosaccharides. Dextrans synthesized by the dextransucrase released by L. nagelii exhibited an averaged molecular weight (M w) of 7.9×107 Da, while those produced by the dextransucrase released by L. hordei exhibited an M w of 6.1×107 Da. Moreover, glycosylation of glucansucrases by LAB was identified for the first time for the released dextransucrase of L. nagelii TMW 1.1827. Our study therefore reveals new molecular insights into how dextransucrases released by water kefir borne L. hordei TMW 1.1822 and L. nagelii TMW 1.1827 contribute to the complex formation of the traditional beverage water kefir.

The genes for dextransucrase and dextranase were cloned from the genomic regions of Leuconostoc mesenteroides MTCC 10508 and Streptococcus mutans MTCC 497, respectively. Heterologous expression of genes was performed in Escherichia coli. The purified enzyme fractions were entrapped in the alginate–pectin beads. A high immobilization yield of dextransucrase (~ 96%), and dextranase (~ 85%) was achieved. Alginate–pectin immobilization did not affect the optimum temperature and pH of the enzymes; rather, the thermal tolerance and storage stability of the enzymes was improved. The repetitive batch experiments suggested substantially good operational stability of the co-immobilized enzyme system. The synergistic catalytic reactions of alginate–pectin co-entrapped enzyme system were able to produce 7–10 g L−1 oligosaccharides of a high degree of polymerization (DP 3–9) from sucrose (~ 20 g L−1) containing feedstocks, e.g., table sugar and cane molasses. The alginate–pectin-based co-immobilized enzyme system is a useful catalytic tool to bioprocess the agro-industrial bio-resource for the production of prebiotic biomolecules.

The best support reported for dextransucrase (DS) and dextranase (DN) immobilization was Eupergit C, but this carrier was discontinued and is no longer available in the market. Thus, epoxy-agarose support is suggested as an alternative to co-immobilize DS from Leuconostoc mesenteroides B-512F and DN from Chaetomium erraticum. The co-immobilization approach improved DS performance, compared to immobilized DS, due to the removal of the dextran layer accumulated on DS by DN, and enhanced both enzymes catalytic activities under a wide range of pH and temperature. Since DS and DN have the same optimum pH and temperature, the co-immobilization was done in a single step. The EPA-DS-DN0.5 (co-immobilized DS (26.16 UI) and DN (2.76 UI) in epoxy-agarose) showed the highest recovered activity (59.54%). The biocatalyst was stable at 4 °C, retaining above 70% of activity for 60 days and was efficient producing oligosaccharides, yielding 12.68 ± 0.09 g/L of oligosaccharides with a degree of polymerization up to 5.

Purified Acetobacter tropicalis dextransucrase was immobilized in different matrices viz. calcium-alginate, κ-carrageenan, agar, agarose and polyacrylamide. Calcium-alginate was proved to be superi...

Plant polyphenols have been extensively studied for their chemopreventive properties for human health. Dextransucrase plays an essential role in synthesizing exopolysaccharides from its exclusive substrate sucrose in Streptococcus mutans. In the present study, the effect of polyphenols gallic acid and tannic acid was investigated on the dextransucrase activity. The enzyme was purified by ethanol precipitation followed by column chromatography by Sephadex G-200 gel chromatography, followed by PEG-400 treatment. The purified enzyme exhibited 52 fold enrichment with 17.5% yield and specific activity of 3.54 Units/mg protein. On SDS-PAGE enzyme protein gave a single band with a molecular weight of 160 kDa. Dextransucrase activity was inhibited 80–90% by 0.04 mM tannic acid (TA) or 0.4 mM gallic acid (GA) suggesting that tannic acid has 10- fold more inhibitory potential than gallic acid on the activity of dextransucrase. CD/ORD studies revealed modifications in the tertiary structure of enzyme protein in presence of tannic acid and gallic acid, which were further confirmed by fluorescence spectra of the protein in presence of tannic acid. These results suggest that inhibition of dextransucrase activity in S. mutans by polyphenols may have potential applications in the prevention and control of dental caries., Graphical abstract Image 1, Highlights i• Dextransuccrase an important enzyme of S. mutans is involved in the metabolism of sucrose. • Purified enzyme is inhibited (80-90%) by plant polyphenols. Observed inhibition is due to change in teritary structure. • S. mutans is an important cariogenic agent. • Plant polyphenols are good anticariogenic agents.

Lactic acid bacteria (LAB) are capable of producing a variety of exopolysaccharide α-glucans, such as dextran, mutan, reuteran, and alternan. Their structural diversity allows LAB-derived α-glucans to hold vast commercial value and application potential in the food, cosmetic, medical, and biotechnology fields, garnering much attention in recent years. Glycoside Hydrolase 70 family (GH70) enzymes are efficient tools for the biosynthesis of α-glucans with various sizes, linkage compositions, and degrees of branching, using renewable and low-cost sucrose and starch as substrates. To date, plenty of various LAB-derived GH70 glucansucrases (especially dextransucrase) have been biochemically characterized to synthesize α-glucans from sucrose with a variety of structural organizations. This review mainly aimed at the biotechnological synthesis of α-glucans using GH70 family enzymes and their diverse (potential) applications. The purification, structural analysis and physicochemical properties of α-glucan polysaccharides were reviewed in detail. Synchronously, some new insights and future perspectives of LAB-derived α-glucans enzymatic synthesis and applications were also discussed. To expand the range of applications, the physicochemical properties and bioactivities of LAB-derived α-glucans, other than dextran, should be further explored. Additionally, screening novel GH70 subfamily starch-acting enzymes is conducive to expanding the repertoire of α-glucans.

Liquorilactobacillus (L.) hordei (formerly Lactobacillus hordei) is one of the dominating lactic acid bacteria within the water kefir consortium, being highly adapted to survive in this environment, while producing high molecular weight dextrans from sucrose. In this work, we extensively studied the physiological response of L. hordei TMW 1.1822 to sucrose compared to glucose, applying label-free, quantitative proteomics of cell lysates and exoproteomes. This revealed the differential expression of 53 proteins within cellular proteomes, mostly associated with carbohydrate uptake and metabolism. Supported by growth experiments, this suggests that L. hordei TMW 1.1822 favors fructose over other sugars. The dextransucrase was expressed irrespectively of the present carbon source, while it was significantly more released in the presence of sucrose (log2FC = 3.09), being among the most abundant proteins within exoproteomes of sucrose-treated cells. Still, L. hordei TMW 1.1822 expressed other sucrose active enzymes, predictively competing with the dextransucrase reaction. While osmolysis appeared to be unlikely, sucrose led to increased release of a multitude of cytoplasmic proteins, suggesting that biofilm formation in L. hordei is not only composed of a polysaccharide matrix but is also of proteinaceous nature. Therefore, our study highlights the intrinsic adaptation of water kefir-borne L. hordei to sucrose-rich habitats and provides fundamental knowledge for its use as a starter culture in plant-based food fermentations with in situ dextran formation.

High molecular weight dextrans improve the rheological properties of fermented products and have immunomodulatory and antiviral activity. We report on 5.84 × 10–2.61 × 10 Da dextrans produced by Leuconostoc lactis AV1n, Weissella cibaria AV2ou and Weissella confusa V30 and FS54 strains. Dextransucrases catalyze dextran synthesis by sucrose hydrolysis concomitant with fructose generation. The four bacteria have dextransucrases with molecular weight of about 160 kDa detected by zymograms. Each bacterium showed different interplay of dextran production and metabolic fluxes. All bacteria produced lactate, and AV2ou apart, synthesized mannitol from fructose. FS54 hydrolyzed dextran blue and the concentration of dextran produced by this bacterium decreased during the stationary phase. The AV1n binding to Caco-2 cells and polystyrene plates was higher under conditions for dextran synthesis. Thus, this is the first instance of a Weissella dextranase, associated with a dextransucrase ability, and of a positive influence of dextran on adhesion and aggregation properties of a bacterium., This work was supported by the Spanish Ministry of Science, Innovation and Universities (grant RTI2018-097114-B-I00) and the TunisianMinistry of Higher Education and Scientific research.

Unfortunately, M. M. Vrvic name has been published incorrectly in the original publication as M. M. Vrivic, corrected name appears in this erratum.

Dextrans are α-(1,6)-linked glucose polymers, which are exclusively produced by lactic acid bacteria from sucrose via extracellular dextransucrases. Previous studies suggested that the environmental pH and the presence of sucrose can impact the release and activity of these enzymes. To get deeper insight into this phenomenon, the dextransucrase expressed by water kefir borne Liquorilactobacillus (L.) nagelii TMW 1.1827 (formerly Lactobacillus nagelii) was recovered in supernatants of buffered cell suspensions that had been incubated with or without sucrose and at different pH. The obtained secretomes were used to time-dependently produce and recover dextrans, whose molecular and macromolecular structures were determined by methylation analysis and AF4-MALS-UV measurements, respectively. The initial pH of the buffered cell suspensions had solely a minor influence on the released dextransucrase activity. When sucrose was present during incubation, the secretomes contained significantly higher dextransucrase activities, although the amounts of totally released proteins obtained with or without sucrose were comparable. However, the dextransucrase appeared to be released in lower amounts into the environment if sucrose was not present. The amount of isolable dextran increased up to 24 h of production, although the total sucrose was consumed within the first 10 min of incubation. Furthermore, the sucrose isomer leucrose had been formed after 10 min, while its concentrations decreased over time and the portions of longer isomaltooligosaccharides (IMOs) increased. This indicated that leucrose can be used by L. nagelii TMW 1.1827 to produce more elongated and branched dextran molecules from presynthesized IMOs, while disproportionation reactions on short IMOs may appear additionally. This leads to increasing amounts of high molecular weight dextran in a state of sucrose depletion. These findings reveal new insights into the pH- and sucrose-dependent kinetics of extracellular dextran formation and may be useful for optimization of fermentative and enzymatic dextran production processes.

Dextransucrase is one of the important extracellular enzyme produced by majority of lactic acid bacterial species. The enzyme produces variety of polysaccharides which are having diverse biological functions with potential application in food, pharma and bio medical sectors. The enzyme in presence of sucrose and acceptor sugar produces prebiotic oligosaccharides which has wide application in food and baking industry. The present chapter embodies about the different bacterial species and their sources involved in the production of dextransucrase. A brief outlook of the reaction mechanism involved in the production of exopolysaccharides and oligosaccharides upon reaction with different sugars has been elaborated. Production of dextransucrase using various statistical optimization techniques is mentioned. An overview of various purification and downstream processing aspects with special emphasis on strain development and enhancing the reusability of enzyme by immobilization techniques has been discussed. Further, the application of enzyme in food, dairy and pharmaceutical industries has been described.

The properties of the glucopolymer dextran are versatile and linked to its molecular size, structure, branching, and secondary structure. However, suited strategies to control and exploit the variable structures of dextrans are scarce. The aim of this study was to delineate structural and functional differences of dextrans, which were produced in buffers at different conditions using the native dextransucrase released by Liquorilactobacillus (L.) hordei TMW 1.1822. Rheological measurements revealed that dextran produced at pH 4.0 (MW = 1.1 * 108 Da) exhibited the properties of a viscoelastic fluid up to concentrations of 10% (w/v). By contrast, dextran produced at pH 5.5 (MW = 1.86 * 108 Da) was gel-forming already at 7.5% (w/v). As both dextrans exhibited comparable molecular structures, the molecular weight primarily influenced their rheological properties. The addition of maltose to the production assays caused the formation of the trisaccharide panose instead of dextran. Moreover, pre-cultures of L. hordei TMW 1.1822 grown without sucrose were substantial for recovery of higher dextran yields, since the cells stored the constitutively expressed dextransucrase intracellularly, until sucrose became available. These findings can be exploited for the controlled recovery of functionally diverse dextrans and oligosaccharides by the use of one dextransucrase type.

Crocin, one of the major carotenoid pigments of Crocus sativus (saffron), is responsible for antioxidant activity, neuroprotection, and the inhibition of tumor cell proliferation. In order to improve the functionality of crocin, α-glucosyl-(1→6)-trans-crocins (C-Gs) were synthesized using sucrose and dextransucrase from Leuconostoc mesenteroides. High hydrostatic pressure (HHP) technique was applied to the synthesis process of C-Gs in order to improve its transglucosylation yield. A 100 MPa HHP condition enhanced the production yield of C-Gs by 1.95 times compared to that of 0.1 MPa atmospheric pressure. Novel C-Gs were purified by HPLC, and their chemical structures were determined using NMR analysis. Novel C-Gs increased water solubility 4.6-5.7 times and antioxidant activity 1.5-2.6 times, respectively, compared to crocin, and their neuroprotections (cell viability 92.5-100.4 %) on HT22 mouse hippocampal neuronal cells were significantly higher than that of crocin (cell viability 84.6 %). This advanced neuroprotection of novel C-Gs could be highly associated with their enhanced antioxidant activity. Thus, the enhanced water solubility and functionality of novel C-Gs can induce better clinical efficacy of neuroprotection than trans-crocin.

We describe the development of cross-linked enzyme aggregates (CLEA) of dextransucrase of Leuconostoc mesenteroides B-512 F. Treatments of enzyme preparation using dextranase were evaluated varying incubation time to remove the dextran layer involving the enzyme molecule, turning it suitable for immobilization. Results showed 21 days of treatment as the best outcome. Subsequently, we tested different water-miscible organic solvents as precipitants, the reaction and centrifugation times, and the concentration of cross-linker agent (glutaraldehyde) in the preparation of CLEA. Optimal conditions were: the use of isopropanol as solvent, 30 min of centrifugation time, 3 h of cross-linking time, and 100 mM of glutaraldehyde. A central composite design was carried out to optimize conditions to obtain the highest enzymatic activity, testing the pH (3.0–7.0) and temperature (20 °C-60 °C). Results showed that dextransucrase CLEA operate at optimal pH of 3.0 and temperature of 60 °C. The operational stability of the immobilized biocatalyst showed up to 30% of residual activity after 10 cycles of reuse, in a solution of 100 mM of sucrose and 600 mM of maltose. The preparation of dextransucrase CLEA is described for the first time and results suggest that this novel immobilized biocatalyst has potential in many industrial applications.

Arbutin, a glucoside of hydroquinone, is used as a powerful skin lightening agent in the cosmeceutical industry because of its strong inhibitory effect on the human tyrosinase activity. It is a natural compound occurring in a number of plants, with a β-anomeric form of the glycoside bond between glucose and hydroquinone. α-Arbutin, which glycoside bond is generated with α-anomeric form, is the isomer of natural arbutin. α-Arbutin is generally produced by transglucosylation of hydroquinone by microbial glycosyltransferases. It is interesting that α-arbutin is found to be over 10 times more effective than arbutin, and thus biological production of α-arbutin attracts increasing attention. Seven different microbial enzymes have been identified to be able to produce α-arbutin, including α-amylase, sucrose phosphorlase, cyclodextrin glycosyltransferase, α-glucosidase, dextransucrase, amylosucrase, and sucrose isomerase. In this work, enzymatic and microbial production of α-arbutin is reviewed in detail.

Dextran produced by dextransucrase hold strong potential for industrial applications. The exact determinants of the linkage specificity of glucansucrase enzymes have remained largely unknown. Previous studies have investigated the relationships between structure and linkage specificity of the dextransucrase DSR from Leuconostoc mesenteroides by the site-directed mutagenesis of the catalytic pocket. The glycosidic linkage of dextran produced by mutant enzymes changed slightly by 3% to 20%. The mutagenesis dextransucrases were constructed by inserting an amino acid into a catalytic pocket to investigate the product specificities of dextransucrase thoroughly. The sequence and structural analysis of glycoside-hydrolase family 70 enzymes led to two sequences (Motif II and Motif IV) being targeted, which were inserted by saturation mutagenesis and simultaneously recombined between A552 and V553, D662, and S663. Variants with catalytic activity were screened of the library, which synthesizes high molecular weight α-glucans with different proportions of α(1–4) linkages, which ranges from 0% to 52%. Mutant sequence analysis, biochemical characterization, and molecular modeling studies revealed the mechanism of product specificities. The mutant dextransucrase, which synthesizes hyperbranched dextran, were obtained by the novel mutagenesis method. The different properties of dextran provide the foundation for subsequent studies and application.

Non-digestible isomaltooligosaccharides (NDIMOS) are functional food and beverage ingredients that contributed to human health benefits through metabolism of gastrointestinal microorganism. In this study, NDIMOS were synthesized by combine dextransucrase from Leuconostoc mesenteroides B512F/KM and alternansucrase from L. mesenteroides NRRL 1355CF10/KM using sucrose as substrate and maltose as acceptor. Their digestibility was confirmed by using digestive enzymes including α-amylase and amyloglucosidase. NDIMOS inhibited insoluble glucan formation through mutansucrase from Streptococcus mutans. The bifidogenic effect of NDIMOS was investigated by growth of four strains of Bifidobacterium in MRS broth containing NDIMOS, compared with MRS broth contain glucose and negative control. Additionally, Bifidobacterium bifidum or Bifidobacterium adolescentis inhibited the growth of Salmonella enterica serovar typhimurium when they were co-cultivation in MRS broth containing NDIMOS. These results suggested that NDIMOS is potential functional ingredient for food, beverage, and pharmaceutical application.

Improving enzyme stability is very important for enzyme applications. Structural modification is a reliable and effective method to improve the characteristics of protein. By artificially extending the C-terminus, 6 domain modification variants of different sizes were constructed, and a new enzyme species with high stability was obtained. Experimental results affirmed that high stability can be achieved by decreasing the degree of domain freedom. The optimum temperatures of domain modification variants were improved by 10 °C compared with the original enzyme. Specifically, compared with the original enzyme, the half-life of the variant dexYG-fdx (D-F) was increased to 280% under 35 °C and 200% under 45 °C, and the pH tolerance range was wider. Further structural simulations and molecular docking studies provided a reasonable explanation (The increased domain reduced the degree of freedom of the enzyme terminal to some extent) for this variant to increase stability and produce dextran. This study can provide valuable information for increasing the characteristics of recombinant dextransucrase.

In this study Lactobacillus sp. has been isolated form Sauerkraut for the production of dextransucrase. The fermentation process is continued by the mixed activity of lactic acid bacteria (LAB) present in the medium. Morphological, physiological and biochemical analysis deciphered the presence of acid producing bacteria belongs to the genera Lactobacilli. Characteristically the isolates were catalase producing but oxidase negative significant to the Lactobacilli genera. The isolated bacterium was tested to be positive in the production of dextransucrase in Tsuchiya medium. Maximum protein production containing dextransucrase was achieved to be 400g/l which is significantly high in terms of concentration. Partially purified enzyme exhibited Vmax=2.9±0.2µmole/min, Km=1.1±0.2mg/ml and Kcat=2.75s-1 and the enzyme followed first order rate kinetics. Better exploration strategy for higher dextransucrase activity from Lactobacillus sp. by inducing operon to higher dextran production can be used later as potent nutraceuticals.

The gut microbiota plays an essential role in host health. The ingestion of prebiotic foods can improve the balance and composition of this microbiota. In the present study, the prebiotic cashew apple juice was produced by enzyme synthesis using its sugars as substrate. The oligosaccharides synthesis was carried out with and without agitation by dextransucrase enzyme (30 °C/17 h). Tagatose restored the juice sweetness. An in vitro digestion was carried out at 37 °C/6 h to evaluate the functional juice samples' prebiotic effect on the probiotic strain Lactobacillus casei NRRL B-442, Bifidobacterium breve NRRL B-41408, and Bifidobacterium infantis NRRL B-41661 growth. Oligosaccharides, dextran, and tagatose were resistant to gastrointestinal conditions and stimulated the probiotic strains' growth. However, each microorganism had a different behavior and preferably consumed some carbohydrates. Tagatose proved to be a suitable substrate for the growth of Bifidobacterium infantis NRRL B-41661 with a maximum consumption rate of 0.045 h−1. On the other hand, Lactobacillus casei NRRL B-442 and Bifidobacterium breve NRRL B-41408 showed maximum growth rates associated with high consumption rates of dextran and oligosaccharides. This study pointed out an excellent alternative for consumers and the functional food market. When metabolized by beneficial microorganisms, the prebiotic juice in the gut promoted these probiotics' growth.

Leucrose is a sucrose isomer which has an α-1,5-linkage, and slowly hydrolyzed to glucose and fructose by small intestinal α-glucosidases. Leucrose can be produced by an isomerization reaction of dextransucrase on a sucrose substrate. In this study, the recombinant dextransucrase from Streptococcus mutans (SmDS) was applied to optimize the reaction conditions for leucrose production. With a substrate mixture of 0.5 M sucrose + 1.0 M fructose, the greatest yield (ca. 24.5%) of leucrose was obtained by SmDS treatment at 30 °C for 120 h. When preadipocyte 3T3-L1 cells were treated with leucrose, this disaccharide inhibited intracellular lipid accumulation in a dose-dependent manner and significantly suppressed mRNA levels of major adipogenic genes, including CCAT/enhancer-binding protein α (C/EBPα), peroxisome proliferator-activated receptor-γ (PPARγ), fatty acid synthase (FAS), and sterol regulatory element-binding protein-1C (SREBP-1C). Phosphorylation of PI3 kinase/Akt/mTOR was also reduced with leucrose treatment. These results suggest that leucrose has a potential in regulating adipogenesis.

Dextransucrase is the enzyme that catalyzes the synthesis of dextran from sucrose. Though dextran is an unwanted deterioration product in the sugar industry, it is being widely used in medicine, cosmetics, and several food areas. Currently, the natural enzyme activity of dextransucrase is low. Thus, it is of great interest to understand how to improve the dextransucrase activity. In the study, Leuconostoc mesenteroides (L.M.CICC-20724) were cultured in a glucose-based solid medium (glucose was the sole carbon source) and then transferred to a sucrose-based solid medium (sucrose was the sole carbon source) for rejuvenation. It was observed that the dextransucrase production was inhibited when L.M.CICC-20724 using the glucose-based solid medium. After 3 months of acclimation, the activity of dextransucrase produced by the acclimated L. mesenteroides (Glu-L.M.) achieved a stable level at approximately 4.20 U/mL. The dextransucrase activity (approximately 1–2 U/mL) increased more than two times with L.M.CICC-20724 in comparision with original one. Dextran products generated from Glu-L.M. and L.M.CICC-20724 were studied using Fourier transform infrared spectroscopy and results showed that both dextrans had a consistent structural composition. The kinetics of sucrose consumption was studied. Under the condition of 0.5 U/mL dextransucrase activity, the consumption of relative low initial sucrose concentrations (0.1, 0.2 and 0.4 M) more fitted to the first-order reaction kinetics, while the consumption of relative high initial sucrose concentrations (0.6 and 0.8 M) was fitted better to the second-order reaction kinetics.

Dextransucrase from Leuconostoc mesenteroides NRRL B-512F was subjected to immobilization and co-immobilization with dextranase from Chaetomium erraticum. Immobilization has enhanced the operational and storage stability of dextransucrase. Two hundred milligrammes (2.4 IU/mg) of alginate beads (immobilized and co-immobilized) were found to be optimum for the production of gluco-oligosaccharides (GOS) in orange juice with a high degree of polymerization. The pulp of the orange juice did not interfere in the reaction. In the batch process, co-immobilized dextransucrase (41 g/L) produced a significantly higher amount of GOS than immobilized dextransucrase (37 g/L). Alginate entrapment enhanced the thermal stability of dextransucrase for up to 3 days in orange juice at 30 °C. The production of GOS in semi-continuous process was 39 g/L in co-immobilized dextransucrase and 33 g/L in immobilized dextransucrase. Thus, immobilization technology offers a great scope in terms of reusability and efficient production of a value added functional health drink.

Caffeic acid was modified via transglucosylation using sucrose and dextransucrase from Leuconostoc mesenteroides B-512FMCM. Following enzymatic modification, a caffeic acid glucoside was isolated by butanol separation, silica gel chromatography, and preparative HPLC. The synthesized caffeic acid glucoside had a molecular mass-to-charge ratio of 365 m/z, and its structure was identified as caffeic acid-3-O-α-d-glucopyranoside. The production of this caffeic acid-3-O-α-d-glucopyranoside at a concentration of 153 mM was optimized using 325 mM caffeic acid, 355 mM sucrose, and 650 mU mL–1 dextransucrase in the synthesis reaction. In comparison with the caffeic acid, the caffeic acid-3-O-α-d-glucopyranoside displayed 3-fold higher water solubility, 1.66-fold higher antilipid peroxidation effect, 15% stronger inhibition of colon cancer cell growth, and 11.5-fold higher browning resistance. These results indicate that this caffeic acid-3-O-α-d-glucopyranoside may be a suitable functional component of food and ph...

Dextransucrase (EC2.4.5.1) from strain Leuconostoc mesenteroides 0326, which synthesizes dextran and oligosaccharides, which act as prebiotics, are popularly used in such industries as food and medicine. A novel dextransucrase efficient in synthesizing oligosaccharides was designed. We constructed the truncation mutant DSR-S1-ΔA (residues 1-3087bp) by deleting the 1494bp fragment of the C-terminal. The novel enzyme (MW: 110kDa) loss activity, when sucrose was used as only substrate. After adding an acceptor, DSR-S1-ΔA was fully activated but with heavily impaired polysaccharide synthesis ability. Instead, the enzyme produced a large amount of oligosaccharides. DSR-S1-ΔA showed transglycosylation for synthesizing more oligosaccharides of lower degree of polymerization (DP) with different acceptors, and it also improved the selection range of dextransucrase acceptor response to acceptors. The enzyme developed in this study can be applied in glycodiversifcation studies.

Dextransuccrase (E.C 2.4.1.5) is a key enzyme in S. mutans for the metabolism of sucrose which helps in the adherence and accumulation of bacteria on tooth surface leading to the formation of dental caries. Dextransuccrase resembles in its catalytic properties with the brush boarder sucrase and exhibits pH dependent inhibitory and stimulatory effects in response to Na+. In this communication we studied the effect of monovalent cations on the activity of dextransuccrase from S. mutans. The percentage inhibition of dextransuccrase was 65% at 0.5 mM NaCl which enhanced to 90% at 20 mM sodium concentration. However there was no effect on dextransucrase activity in presence of other monovalent cations (Rb+, Cs+, and K+) tested. Enzyme activity was enhanced 20–24% in acidic pH but was strongly inhibited (59–89%) around neutral and alkaline pH by 0.5–2.0 mM sodium chloride. Upon dialysis, 86% of enzyme activity was restored to control values. There was no effect of 2 mM NaCl on glucosyltransferase activity of the enzyme. Kinetic studies revealed that enzyme showed biphasic effects in response to Na+ ions. At acidic pH the enzyme exhibited mixed type of activation affecting both Vmax and Km, while in alkaline pH, the enzyme showed V- type effect reducing Vmax by 74% without affecting Km. The effects of sodium ions on dextransuccrase activity were specific, thus it can be useful to block its catalytic activity, and reducing the cariogenic potential of S. mutans., Highlights • This is the first report describing the effects of sodium ions on dextransucrase activity in S. mutans, a gram positive bacteria. • Dextransucrase activity in S. mutans is essential for metabolism of sucrase a primary substrate for the growth of organism. • Data presented herein shows pH dependent biphasic effects of sodium ions on dextransucrase activity similar to that reported in mammalian brush border sucrose. • Enzyme activity was stimulated at acidic pH but is strongly inhibited by sodium ions. this may help in reducing the cariogenic potential of S. mutans.

International audience; The factors that define the resulting polymer length of distributive polymerases are poorly understood. Here, starting from the crystal structure of the dextransucrase DSR-M in complex with an isomaltotetraose, we define different anchoring points for the incoming acceptor. Mutation of one of these, Trp624, decreases the catalytic rate of the enzyme but equally skews the size distribution of the resulting dextran chains toward shorter chains. Nuclear magnetic resonance analysis shows that this mutation influences both the dynamics of the active site and the water accessibility. Monte Carlo simulation of the elongation process allows interpretation of these results in terms of enhanced futile encounters, whereby the less effective binding increases the pool of effective seeds for the dextran chains and thereby directly determines the length distribution of the final polymers.

Leuconostoc lactis AV1 strain isolated from a Tunisian avocado was characterized as a dextran producer. The promoter PdsrLL and the dsrLL gene encoding the DsrLL dextransucrase responsible for the dextran synthesis were transcriptionally fused to the mCherry coding gene generating the pRCR20 plasmid. Upon plasmid transfer, both AV1n and the dextran non-producing Leuconostoc mesenteroides CM70 became red due to expression of the mCherry from the PdsrLL-dsr-mrfp transcriptional fusion. Characterization of the polymers present in cultures supernatants revealed that the DsrLL encoded from pRCR20 in the recombinant bacteria was able to synthesize dextran. The production of dextran by the DsrLL in AV1n increased in response to low temperature, reaching 10-fold higher levels at 20∘C than at 37∘C (4.15 g/L versus 0.41 g/L). To analyze if this stress response includes activation at the transcriptional level and if it was only restricted to Leuconostoc, AV1n was transformed with plasmids carrying either the PdsrLL-mrfp fusion or the PdsrLS of Lactobacillus sakei MN1 fused to the mrfp gene, and the influence of temperature and carbon source on expression from the Dsr promoters was monitored by measurement of the mCherry levels. The overall expression analysis confirmed an induction of expression from PdsrLL upon growth at low temperature (20∘C versus 30∘C and 37∘C) in the presence of sugars tested (sucrose, glucose, maltose, and fructose). In addition, the presence of sucrose, the substrate of Dsr, also resulted in activation of expression from PdsrLL. A different behavior was detected, when expression from PdsrLS was evaluated. Similar levels of fluorescence were observed irrespectively of the carbon source or temperature, besides a sequential decrease at 30∘C and 20∘C, when sucrose was present in the growth medium. In conclusion, the two types of regulation of expression of Dsr presented here revealed two different mechanisms for environmental adaptation of Leuconostoc and Lactobacillus that could be exploited for industrial applications., This work was supported by the Spanish Ministry of Economy and Competitiveness (Grants AGL2015-65010-C3-1-R and RTI2018-097114-B-100) and the Tunisian Ministry of Higher Education and Scientific Research.

Epigallocatechin gallate (EGCG) is the most abundant catechin found in the leaves of green tea, Camellia sinensis. In this study, novel epigallocatechin gallate-glucocides (EGCG-Gs) were synthesized by using dextransucrase from Leuconostoc mesenteroides B-1299CB4. Response surface methodology was adopted to optimize the conversion of EGCG to EGCG-Gs, resulting in a 91.43% conversion rate of EGCG. Each EGCG-G was purified using a C18 column. Of nine EGCG-Gs identified by nuclear magnetic resonance analysis, five EGCG-Gs (2 and 4–7) were novel compounds with yields of 2.2–22.6%. The water solubility of the five novel compounds ranged from 229.7 to 1878.5 mM. The 5′-OH group of EGCG-Gs expressed higher antioxidant activities than the 4′-OH group of EGCG-Gs. Furthermore, glucosylation at 7-OH group of EGCG-Gs was found to be responsible for maintaining tyrosinase inhibitory activity and increasing browning-resistant activities.

Dextransucrase from Leuconostoc mesenteroides B-512F was covalently immobilized on glutaraldehyde-actived chitosan particles. The best initial protein loading (400 mg/g of dried support) showed 197 U/g of catalytic activity. The optimal reaction pH and temperature of this new biocatalyst were determined to be 4.5 and 20 °C, respectively. Regarding the thermal stability, the immobilization enhanced enzyme protection against high temperatures, whereas glucose and maltose acted as stabilizers. The biocatalyst was stable under storage at 5 °C for a month. The biocatalyst presented good operational stability, retaining up to 40% of its initial activity after ten batch cycles of reaction to obtain oligosaccharides. These results suggest the use of the immobilized dextransucrase on chitosan particles as a promising novel biocatalyst to produce dextran and oligosaccharides.

Oligosaccharide (OS) is used as a sugar replacement as well as an ingredient in functional foods because of its beneficial effects, mainly on reducing calorie content and promoting intestinal health. By contrast, the effects of OS on inflammation are less well investigated. The purpose of this study was to investigate the effects of sucrose-derived OS on glucose control and inflammation in high fat (HF) diet-fed mice. Male C57BL6 mice were randomly assigned to six treatment groups (n = 10-14 mice per group): 1) lean control (CON), 2) HF control, 3) HF-low sucrose (LS, 100 mg/kg/day), 4) HF-high sucrose (HS, 1000 mg/kg/day), 5) HF-low OS (LOS, 100 mg/kg/day), and 6) HF-high OS (HOS, 1000 mg/kg/day). PBS (vehicle), sucrose, and OS were administered by stomach gavage. Body weight, food intake, and markers of liver function (activities of aspartate aminotransferase and alanine aminotransferase) were not affected by the treatments. HOS treatment decreased levels of serum glucose, insulin, and homeostasis model assessment-insulin resistance compared with sucrose treatment. However, serum adiponectin levels of the HOS group were higher than those of the sucrose groups. Serum levels of the pro-inflammatory cytokines interleukin-6 (IL-6) and fetuin-A were lower in the HOS group than in the sucrose groups. Hepatic gene expression levels of pro-inflammatory cytokines and related factors (fetuin-A, NF-κB, TLR4, TNF-alpha, and IL-6) were decreased and the levels of insulin signaling-related molecules (sirtuin 1, insulin receptor, and Akt) were increased in HOS-treated mice as compared with sucrose-treated mice. These results demonstrate that OS treatment is effective in improving glucose control and inflammation in high fat diet-fed mice.

Sugar beet pulp (SBP) and molasses, as an agro industrial waste material, are produced in large amounts annually. Thus, a major challenge nowadays is to develop procedures that could increase the value of the generated waste. In this study, SBP as a support for cell immobilization and molasses as a source of nutrients were used for a dextransucrase (DS) production by Leuconostoc mesenteroides T3. The influence of SBP in native form (SBP-N) and after treatment with NaOH (SBP-NaOH) on DS production was investigated. The optimal medium composition for the maximum DS production was determined by varying the concentration of molasses, SBP, and sucrose. The maximum DS yield of 2.02 U/ml was obtained in the medium with 2.5 % of molasses, 2.5 % SBP-NaOH, and 4 % of sucrose concentration. Scanning electron microscopy (SEM) showed immobilization of Lc. mesenteroides T3 cells onto SBP-NaOH. According to the obtained results, the production of DS on molasses could be improved by using NaOH-treated SBP as a carrier for whole-cell immobilization. Our study reveals the basis for the development of process for DS production with additional reduction of expenses by using waste materials for obtaining the valuable biotechnological product.

Our previous work showed that substitution of an amino acid that is coupled with the +2 subsite adjacent to the transition stabilizer of a glucansucrase, which produces a water-insoluble glucan, resulted in significant changes in the structures and yields of the water-insoluble glucans produced. We now describe how these changes affect the ability of the glucansucrase to bind to exogenous glucans, and how these glucans can influence the yield, product structures, and kinetics of the mutant glucansucrases. The activity of the wild-type enzyme, with threonine at position 654, is not significantly activated by added dextran, and the yield of water-insoluble glucan from sucrose is only slightly increased by dextran. Mutant T654Y is not affected at all by the addition of dextran. However, several mutant enzymes exhibit markedly lower yields of glucan relative to the wild type; these lower yields can be partially or completely overcome by the addition of water-soluble dextran. Although evidence indicates that the soluble dextran is incorporated into water-insoluble glucan, the increased yields cannot be accounted for solely by incorporation of the dextran into insoluble product. Furthermore, these DsrI mutants are significantly activated by exogenous glucans. The addition of dextran does not markedly change the KM for sucrose in the mutant enzymes, but does increase the Vmax of the reaction. These effects apparently depend on the presence of unbranched sequences of α1→6-linked D-glucose units in the glucan.

Leuconostoc mesenteroides DRP105 isolated from Chinese sauerkraut juice is an intensive producer of dextran. We report the complete genome sequence of Leu. mesenteroides DRP105. This strain contains a dextransucrase gene (dsr) involved in the production of dextran, possibly composed of glucose monomers. To explore the dextran synthesis mechanism of Leu. mesenteroides DRP105, we constructed a dsr-deficient strain derived from Leu. mesenteroides DRP105 using the Cre-loxP recombination system. The secondary structure prediction results showed that Leu. mesenteroides DRP105 dextransucrase (Dsr) was coded by dsr and contained 17.07% &alpha, helices, 29.55% &beta, sheets, 10.18% &beta, turns, and 43.20% random coils. We also analyzed the dextran yield, monosaccharide change, organic acid, and amino-acid content of Leu. mesenteroides DRP105 and Leu. mesenteroides DRP105&minus, &Delta, dsr. The result showed that the lack of dsr changed the Leu. mesenteroides DRP105 sugar metabolism pathway, which in turn affected the production of metabolites.

International audience; The dextransucrase enzymes synthesize dextran, a glucose polymer with broad industrial applications, making the search for new dextransucrases of great interest. The work described aimed at the partial characterizing of a recombinant dextransucrase enzyme from Leuconostoc citreum M-3. From the genomic DNA of strain M-3, an amplicon containing a coding region of a dextransucrase called DSRV was isolated, and deposited in the GenBankTM (Accession number: KF724950). The amino acid sequence alignment of DSR-V with other dextransucrases demonstrated that it shares a 94% identity with the DSR-D of L. mesenteroides Lcc4 and the DSR-S of L. mesenteroides NRRL B-512F. The DSR-V was cloned and expressed in Escherichia coli JM109 facilitating the formation and detection of DSR-V specific dextran. The SDS-PAGE soluble fraction zymography of E. coli DSR-V and the C-13-NMR spectra of dextran polymers synthesized by this clone confirm that L. citreum M-3 dsrV gene codes for a different dextransucrase synthesizing linear dextrans with mainly alpha(1-6) linkages.

14 INTRODUCTION 16 MATERIALS AND METHODS 19 Carbohydrates and reagents 19 Enzyme 19 Enzyme digest conditions 20 Digest conditions to obtain Hanes-Woolf plots 20 Digest conditions to obtain Michaelis plots 20 Determination of the amount of dextran synthesized 21 RESULTS AND DISCUSSION 22 REFERENCES 3 0 iii PAPER II INTERPRETATION OF DEXTRANSUCRASE INHIBITION AT HIGH SUCROSE CONCENTRATIONS 3 9 ABSTRACT 4 0 INTRODUCTION 41 EXPERIMENTAL 44 Chromatography 44 Enzyme 4 4 Enzyme digest conditions 4 5 RESULTS 4 7 DISCUSSION 49 REFERENCES 54

The fructophilic bacterium Lactobacillus kunkeei has promising applications as probiotics promoting the health of both honey bees and humans. Here, we report the synthesis of a highly branched dextran by L. kunkeei DSM 12361 and biochemical characterization of a GH70 enzyme (GtfZ). Sequence analysis revealed that GtfZ harbors two separate catalytic cores (CD1 and CD2), predicted to have glucansucrase and branching sucrase specificity, respectively. GtfZ-CD1 was not characterized biochemically due to its unsuccessful expression. With only sucrose as substrate, GtfZ-CD2 was found to mainly catalyze sucrose hydrolysis and leucrose synthesis. When dextran was available as acceptor substrate, GtfZ-CD2 displayed an efficient transglycosidase activity with sucrose as donor substrate. Kinetic analysis showed that the GtfZ-CD2-catalyzed transglycosylation reaction follows a Ping Pong Bi Bi mechanism, indicating the in-turn binding of donor and acceptor substrates in the active site. Structural characterization of the products revealed that GtfZ-CD2 catalyzes the synthesis of single glucosyl (α1 → 3) linked branches onto dextran, resulting in the production of highly branched comb-like α-glucan products. These (α1 → 3) branches can be formed on adjacent positions, as shown when isomaltotriose was used as acceptor substrate. Homology modeling of the GtfZ-CD1 and GtfZ-CD2 protein structure strongly suggests that amino acid differences in conserved motifs II, III, and IV in the catalytic domain contribute to product specificity. Our present study highlights the ability of beneficial lactic acid bacteria to produce structurally complex α-glucans and provides novel insights into the molecular mechanism of an (α1 → 3) branching sucrase.

The extracellular dextransucrase from Leuconostoc mesenteroides NRRL B-1426 was produced and purified using polyethylene glycol fractionation. In our earlier study, it was reported that L. mesenteroides dextransucrase synthesizes a high-molecular mass dextran (>2 × 10(6) Da) with ∼85.5% α-(1→6) linear and ∼14.5% α-(1→3) branched linkages. Isomalto-oligosaccharides (IMOs) were synthesized through depolymerization of dextran by the action of dextranase. The degree of polymerization of IMOs was 2-10 as confirmed by mass spectrometry. The nuclear magnetic resonance spectroscopic analysis revealed the presence of α-(1→3) linkages in the synthesized IMOs. The IMOs were resistant to dextranase, α-glucosidase, and α-amylase, and therefore can have potential application as food additives in the functional foods.