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The glycoside hydrolase family 70 (GH70) contains bacterial extracellular multidomain enzymes, synthesizing α-glucans from sucrose or starch-like substrates. A few dozen have been biochemically characterized, while crystal structures cover only the core domains and lack significant parts of auxiliary domains. Here we present a systematic overview of GH70 enzymes and their 3D structural organization and bacterial origin. A representative set of 234 permuted and 25 nonpermuted GH70 enzymes was generated, covering 12 bacterial families and 3 phyla and containing 185 predicted glucansucrases (GS), 15 branching sucrases (BrS), 8 "twin" GS-BrSs, and 51 α-glucanotransferases (α-GT). Analysis of AlphaFold models of all 259 entries showed that, apart from the core domains, the structural variation regarding auxiliary domains is far greater than anticipated, with nine different domain types. We analyzed the phylogenetic distribution and discuss the possible roles of auxiliary domains as well as possible correlations between enzyme specificity, auxiliary domain type, and bacterial origin.

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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.

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Naringin found in citrus fruits is a flavanone glycoside with numerous biological activities. However, the bitterness, low water-solubility, and low bioavailability of naringin are the main issues limiting its use in the pharmaceutical and nutraceutical industries. Herein, a glucansucrase from isolated Leuconostoc citreum NY87 was used for trans-α-glucosylattion of naringin by using sucrose as substrate. Two naringin glucosides (O-α-D-glucosyl-(1'''' → 6″) naringin (compound 1) and 4'-O-α-D-glucosyl naringin (compound 2)) were purified and determined their structures by nuclear magnetic resonance. The optimization condition for the synthesis of compound 1 was obtained at 10 mM naringin, 200 mM sucrose, and 337.5 mU/mL at 28 °C for 24 h by response surface methodology method. Compound 1 and compound 2 showed 1896- and 3272 times higher water solubility than naringin. Furthermore, the bitterness via the human bitter taste receptor TAS2R39 displayed that compound 1 was reduced 2.9 times bitterness compared with naringin, while compound 2 did not express bitterness at 1 mM. Both compounds expressed higher neuroprotective effects than naringin on human neuroblastoma SH-SY5Y cells treated with 5 mM scopolamine based on cell viability and cortisol content. Compound 1 reduced acetylcholinesterase activity more than naringin and compound 2. These results indicate that naringin glucosides could be utilized as functional material in the nutraceutical and pharmaceutical industries. KEY POINTS: • A novel O-α-D-glucosyl-(1 → 6) naringin was synthesized using glucansucrase from L. citreum NY87. • Naringin glucosides improved water-solubility and neuroprotective effects on SH-SY5Y cells. • Naringin glucosides showed a decrease in bitterness on bitter taste receptor 39.

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Glucansucrase was purified from Leuconostoc pseudomesenteroides. The glucansucrase exhibited maximum activity at pH 5.5 and 30 °C. Ca2+ significantly promoted enzyme activity. An exopolysaccharide (EPS) was synthesized by this glucansucrase in vitro and purified. The molecular weight of the EPS was 3.083 × 106 Da. Fourier transform infrared (FT-IR) and nuclear magnetic resonance (NMR) spectroscopy showed that the main structure of glucan was 97.3% α-(1→6)-linked D-glucopyranose units, and α-(1→3) branched chain accounted for 2.7%. Scanning electron microscopy (SEM) observation of dextran showed that its surface was smooth and flaky. Atomic force microscopy (AFM) of dextran revealed a chain-like microstructure with many irregular protuberances in aqueous solution. The results showed that dextran had good thermal stability, water holding capacity, water solubility and emulsifying ability (EA), as well as good antioxidant activity; thus it has broad prospects for development in the fields of food, biomedicine, and medicine.

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Polyphenols exhibit various beneficial biological activities and represent very promising candidates as active compounds for food industry. However, the low solubility, poor stability and low bioavailability of polyphenols have severely limited their industrial applications. Enzymatic glycosylation is an effective way to improve the physicochemical properties of polyphenols. As efficient transglucosidases, glycoside hydrolase family 70 (GH70) glucansucrases naturally catalyze the synthesis of polysaccharides and oligosaccharides from sucrose. Notably, GH70 glucansucrases show broad acceptor substrate promiscuity and catalyze the glucosylation of a wide range of non-carbohydrate hydroxyl group-containing molecules, including benzenediol, phenolic acids, flavonoids and steviol glycosides. Branching sucrase enzymes, a newly established subfamily of GH70, are shown to possess a broader acceptor substrate binding pocket that acts efficiently for glucosylation of larger size polyphenols such as flavonoids. Here we present a comprehensive review of glucosylation of polyphenols using GH70 glucansucrase and branching sucrases. Their catalytic efficiency, the regioselectivity of glucosylation and the structure of generated products are described for these reactions. Moreover, enzyme engineering is effective for improving their catalytic efficiency and product specificity. The combined information provides novel insights on the glucosylation of polyphenols by GH70 glucansucrases and branching sucrases, and may promote their applications.

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Schisandra chinensis (Omija) is a well-known medicinal plant in East Asia. In this study, Omija oligosaccharide syrup was prepared from sucrose with Omija fruit extract using two glucansucrases of Leuconostoc mesenteroides B-512F/KM and L. mesenteroides B-1355CF10/KM. The degree of polymerization of Omija oligosaccharide syrup was ranged from 2 - 13 by MALDI-TOF-MS analysis. Compared to the Omija syrup, the Omija oligosaccharide syrup reduced 61% calories based on the enzymatic gravimetric method. It also reduced up to 96% insoluble glucan formation from sucrose by mutansucrase of Streptococcus mutans at 500 mg/mL. Additionally, it has 1.78-fold higher oxygen radical absorbance capacity value compared to Omija syrup. Using electronic tongue sensor system, Omija oligosaccharide syrup showed decreased sourness, astringency, and saltiness compared to Omija syrup. Thus, Omija oligosaccharides can be used as functional sweetener in nutraceutical industries. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-022-01061-8.

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Glucansucrases and branching sucrases are classified in the family 70 of glycoside hydrolases. They are produced by lactic acid bacteria occupying very diverse ecological niches (soil, buccal cavity, sourdough, intestine, dairy products, etc.). Usually secreted by their producer organisms, they are involved in the synthesis of α-glucans from sucrose substrate. They contribute to cell protection while promoting adhesion and colonization of different biotopes. Dextran, an α-1,6 linked linear α-glucan, was the first microbial polysaccharide commercialized for medical applications. Advances in the discovery and characterization of these enzymes have remarkably enriched the available diversity with new catalysts. Research into their molecular mechanisms has highlighted important features governing their peculiarities thus opening up many opportunities for engineering these catalysts to provide new routes for the transformation of sucrose into value-added molecules. This article reviews these different aspects with the ambition to show how they constitute the basis for promising future developments.

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Ellagic acid glucoside was synthesized via transglucosylation using sucrose and glucansucrase derived from Leuconostoc mesenteroides B-512 FMCM. After such enzymatic synthesis, the product was purified by 50% ethyl acetate fraction and C18 column chromatography. Modification of ellagic acid glucoside was verified by LC-MS/MS at m/z 485.1 (M + Na)- and m/z 531.1 (M + 3Na)-. The yield of ellagic acid glucoside was 69% (3.47 mM) by response surface methodology using 150 mM sucrose, 300 mU/mL glucansucrase, and 5 mM ellagic acid. The synthesized ellagic acid glucoside showed improved water solubility, up to 58% higher brain nerve cell (SH-SY5Y) protective effect, threefold higher cortisol reducing effect, and fourfold stronger inhibitory effect on acetylcholinesterase (AChE) than ellagic acid. These results indicate that ellagic acid glucoside could be used as a neuroprotective agent.

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Leuconostoc lactis SBC001, isolated from chive, produces glucansucrase and synthesizes oligosaccharides through its enzymatic activity. This study was conducted to optimize oligosaccharide production using response surface methodology, analyze the structure of purified oligosaccharides, and investigate the prebiotic effect on 24 bacterial and yeast strains and the anti-inflammatory activity using RAW 264.7 macrophage cells. The optimal conditions for oligosaccharide production were a culture temperature of 30 °C and sucrose and maltose concentrations of 9.6% and 7.4%, respectively. Based on 1H-NMR spectroscopic study, the oligosaccharides were identified as gluco-oligosaccharides that consisted of 23.63% α-1,4 glycosidic linkages and 76.37% α-1,6 glycosidic linkages with an average molecular weight of 1137 Da. The oligosaccharides promoted the growth of bacterial and yeast strains, including Lactobacillus plantarum, L. paracasei, L. johnsonii, Leuconostoc mesenteroides, L. rhamnosus, and Saccharomyces cerevisiae. When lipopolysaccharide-stimulated RAW 264.7 cells were treated with the oligosaccharides, the production of nitric oxide was decreased; the expression of inducible nitric oxide synthase, tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, and IL-10 was suppressed; and the nuclear factor-kappa B signaling pathway was inhibited. In conclusion, the gluco-oligosaccharides obtained from Leu. lactis SBC001 exhibited a prebiotic effect on six bacterial and yeast strains and anti-inflammatory activity in RAW 264.7 macrophage cells.

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Glycoside hydrolase family 70 (GH70) glucansucrases produce α-d-glucan polysaccharides (e.g. dextran), which have different linkage composition, branching degree and size distribution, and hold potential applications in food, cosmetic and medicine industry. In addition, GH70 branching sucrases add single α-(1→2) or α-(1→3) branches onto dextran, resulting in highly branched polysaccharides with "comb-like" structure. The physico-chemical properties of these α-d-glucans are highly influenced by their linkage compositions, branching degrees and sizes. Among these α-d-glucans, dextran is commercially applied as plasma expander and separation matrix based on extensive studies of its structure and physico-chemical properties. However, such detailed information is lacking for the other type of α-d-glucans. Aiming to stimulate the application of α-d-glucans produced by glucansucrases, we present an overview of the structures, production, physico-chemical properties and (potential) applications of these sucrose-derived α-d-glucan polysaccharides. We also discuss bottlenecks and future perspectives for the application of these α-d-glucan polysaccharides.

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Microbial α-glucans produced by GH70 (glycoside hydrolase family 70) glucansucrases are gaining importance because of the mild conditions for their synthesis from sucrose, their biodegradability, and their current and anticipated applications that largely depend on their molar mass. Focusing on the alternansucrase (ASR) from Leuconostoc citreum NRRL B-1355, a well-known glucansucrase catalyzing the synthesis of both high- and low-molar-mass alternans, we searched for structural traits in ASR that could be involved in the control of alternan elongation. The resolution of five crystal structures of a truncated ASR version (ASRΔ2) in complex with different gluco-oligosaccharides pinpointed key residues in binding sites located in the A and V domains of ASR. Biochemical characterization of three single mutants and three double mutants targeting the sugar-binding pockets identified in domain V revealed an involvement of this domain in alternan binding and elongation. More strikingly, we found an oligosaccharide-binding site at the surface of domain A, distant from the catalytic site and not previously identified in other glucansucrases. We named this site surface-binding site (SBS) A1. Among the residues lining the SBS-A1 site, two (Gln700 and Tyr717) promoted alternan elongation. Their substitution to alanine decreased high-molar-mass alternan yield by a third, without significantly impacting enzyme stability or specificity. We propose that the SBS-A1 site is unique to alternansucrase and appears to be designed to bind alternating structures, acting as a mediator between the catalytic site and the sugar-binding pockets of domain V and contributing to a processive elongation of alternan chains.

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OBJECTIVE: 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. RESULTS: 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. CONCLUSION: All these data collectively indicate that B-2 glucansucrase is a novel one, which have good properties and may applied to new food areas.

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Dental caries is a common cause for tooth loss and Streptococcus mutans is identified as the etiologic pathogen. This study evaluates the inhibitory potential of Epigallocatechin gallate (EGCG) on S.mutans glucansucrase enzyme and its biofilm. Glucansucrase binding and the inhibitory potential of EGCG was validated using AutoDock tool and enzyme inhibitory assay. Biofilm inhibitory potential was also confirmed using Scanning Electron Microscopic (SEM) analysis in human tooth samples. Molecular docking revealed that EGCG interacted with GLU 515 and TRP 517 amino acids and binds to glucansucrase. SEM analysis revealed inhibition of S.mutans biofilm by various concentrations of EGCG on surfaces of tooth samples. Bioinformatics and biological assays confirmed that EGCG potentially binds to the S. mutans glucansucrase and inhibits its enzymatic activity. Enzymatic inhibition of glucansucrase attenuated biofilm formation potential of S. mutans on tooth surface. Thus, we conclude that EGCG inhibitory potential of S. mutans biofilm on the tooth surface is a novel approach in prevention of dental caries.

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The dextransucrase DSR-OK from the Gram-positive bacterium Oenococcus kitaharae DSM17330 produces a dextran of the highest molar mass reported to date (∼109 g/mol). In this study, we selected a recombinant form, DSR-OKΔ1, to identify molecular determinants involved in the sugar polymerization mechanism and that confer its ability to produce a very-high-molar-mass polymer. In domain V of DSR-OK, we identified seven putative sugar-binding pockets characteristic of glycoside hydrolase 70 (GH70) glucansucrases that are known to be involved in glucan binding. We investigated their role in polymer synthesis through several approaches, including monitoring of dextran synthesis, affinity assays, sugar binding pocket deletions, site-directed mutagenesis, and construction of chimeric enzymes. Substitution of only two stacking aromatic residues in two consecutive sugar-binding pockets (variant DSR-OKΔ1-Y1162A-F1228A) induced quasi-complete loss of very-high-molar-mass dextran synthesis, resulting in production of only 10-13 kg/mol polymers. Moreover, the double mutation completely switched the semiprocessive mode of DSR-OKΔ1 toward a distributive one, highlighting the strong influence of these pockets on enzyme processivity. Finally, the position of each pocket relative to the active site also appeared to be important for polymer elongation. We propose that sugar-binding pockets spatially closer to the catalytic domain play a major role in the control of processivity. A deep structural characterization, if possible with large-molar-mass sugar ligands, would allow confirming this hypothesis.

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Glucansucrases catalyse the formation of glucans from sucrose. The glucansucrase-encoding gene from Leuconostoc citreum ABK-1, dex-N, was successfully cloned and expressed in E. coli BL21 Star (DE3). DEX-N produces 2 types of glucans: soluble (S-dextran) and insoluble (I-glucan) glucans. The S-dextran was determined to be ca. 10 kDa in size and contained >90% α-1,6 linkages; along with its water solubility, this is similar to commercial dextran. On the other hand, I-glucan was water-insoluble, harbouring a block-wise pattern of α-1,3 and α-1,6 linkages in its structure. Notably, the FTIR and powder X-ray diffraction pattern of I-glucan exhibited a combination of features found in α-1,6-linked dextran and α-1,3-linked mutan. Although both I-glucan and mutan are insoluble glucans, their physical characteristics are notably dissimilar.

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Mangiferin, a major constituent of Mangifera indica L., has attracted substantial attention due to its anti-oxidant, anti-diabetic, anti-inflammatory, and anti-microbial activities. However, its poor solubility in water limits its use in food and pharmaceutical industries. In this study, novel mangiferin-(1→6)-α-d-glucopyranoside (Mg-G1) was enzymatically synthesized from mangiferin and sucrose using glucansucrase from Leuconostoc mesenteroides B-512F/KM, and optimized using response surface methodology. The water solubility of Mg-G1 was found to be 824.7 mM, which is more than 2300-fold higher than that of mangiferin. Mg-G1 also showed DPPH radical scavenging activity and superoxide dismutase (SOD)-like scavenging activity, which were 4.77- and 3.71-fold higher than that of mangiferin, respectively. Mg-G1 displayed inhibitory activity against human intestinal maltase and COX-2. Thus, the novel glucosylated mangiferin may be used as an ingredient in functional food and pharmaceutical application.

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Glucansucrases can be used to glucosylate various plant-derived phenolic compounds by using sucrose as donor substrate. We applied Lactobacillus reuteri TMW 1.106 dextransucrase to glucosylate the acceptor substrates caffeic acid and gallic acid. Subsequently, monoglucosylated and in particular oligo- and polyglucosylated conjugates were characterized by using different chromatographic techniques and two-dimensional NMR spectroscopy. Both acceptors were substituted at positions O3 and O4. Under the conditions used, two monoglucosylated products were formed for caffeic acid, whereas only one O3-monosubstituted conjugate was detected for gallic acid. However, both acceptors resulted in O4-substituted oligo- and polyglucosylated conjugates, the amount of which was higher from gallic acid than from caffeic acid. Profile analysis tensiometry suggested that, in contrast to unmodified dextrans, oligo- and polymeric glucoconjugates of gallic acid are highly interfacially active. Overall, we provide the first detailed characterization of enzymatically conjugated oligo- and polymeric dextrans, which may have further potential as functional ingredients.

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Gentiobiose-derived oligosaccharides were synthesized by the acceptor reaction of glucansucrase E81 obtained from Lactobacillus reuteri E81 with sucrose and gentiobiose as donor-acceptor sugars, respectively. The reaction products were monitored by TLC analysis and gentiobiose-derived oligosaccharides up to DP 8 were formed during the acceptor reaction as determined by ESI-MS/MS analysis. The glycosylation of the gentiobiose with α-(1 → 6) linkages and α-(1 → 3) linkages was shown by 1H and 13C NMR analysis confirming the structure of these gentiobiose-derived oligosaccharides. The in vitro prebiotic function of the oligosaccharides was determined in which probiotic strains were stimulated whereas no growth was observed in pathogen strains. Gentiobiose-derived oligosaccharides showed immune-modulatory functions in vitro and triggered the production of IL-4, IL12 and TNF-α cytokines in HT29 cells in a dose dependent manner. This study showed the production and functional characterisation of gentiobiose-derived oligosaccharides establishing a promising avenue for future applications.

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Rebaudioside A was modified via glucosylation by recombinant dextransucrase of Leuconostoc lactis EG001 in Escherichia coli BL21 (DE3), forming single O-α-D-glucosyl-(1″→6') rebaudioside A with yield of 86%. O-α-D-glucosyl-(1″→6') rebaudioside A was purified using HPLC and Diaion HP-20 and its properties were characterized for possible use as a food ingredient. Almost 98% of O-α-D-glucosyl-(1″→6') rebaudioside A was dissolved after 15 days of storage at room temperature, compared to only 11% for rebaudioside A. Compared to rebaudioside A, O-α-D-glucosyl-(1″→6') rebaudioside A showed similar or improved acidic or thermal stability in commercial drinks. Thus, O-α-D-glucosyl-(1″→6') rebaudioside A could be used as a highly pure and improved sweetener with high stability in commercial drinks. PRACTICAL APPLICATION: The proposed method can be used to generate glucosyl rebaudioside A by enzymatic glucosylation. Simple glucosyl rebaudioside A exhibited high acid/thermal stability and improved sweetener in commercialized drinks. This method can be applied to obtain high value-added bioactive compounds by enzymatic modification.

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Flavonoids are commonly abundant, plant-derived polyphenolic compounds which are responsible for color, taste, and antioxidant properties of certain plant based foods. Glucosylation by glucansucrases or other glycosyltransferases/glycoside hydrolases has been described to be a promising approach to modify stability, solubility, bioavailability, and taste profile of flavonoids and other compounds. In this study, we modified and applied a recombinant dextransucrase from Lactobacillus reuteri TMW 1.106 to glucosylate various flavonoids and flavonoid glycosides. The glucoconjugates were subsequently isolated and characterized by using two-dimensional NMR spectroscopy. Efficient glucosylation was achieved for quercetin and its glycosides quercetin-3-O-ß-glucoside and rutin. Significant portions of α-glucose conjugates were also obtained for epigallocatechin gallate, dihydromyricetin, and cyanidin-3-O-ß-glucoside, whereas glucosylation efficiency was low for naringin and neohesperidin dihydrochalcone. Most of the flavonoids with a catechol or pyrogallol group at the B-ring were predominantly glucosylated at position O4'. However, glycosyl substituents such as ß-glucose, rutinose, or neohesperidose were glucosylated at varying positions. Therefore, mutant dextransucrase from L. reuteri TMW 1.106 can be applied for versatile structural modification of flavonoids.

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