Glucosidic Bonds Research Articles

Effects of protein in wheat flour on retrogradation of wheat starch.

Albumins, globulins, gliadins, and glutenins were isolated from wheat flour and the effects of those proteins on retrogradation of wheat starch were investigated. The results showed that only glutenins retarded retrogradation of wheat starch and other 3 proteins promoted it. The results of IR spectra proved that no S-S linkage formed during retrogradation of wheat starch blended with wheat proteins. Combination of wheat starch and globulins or gliadins through glucosidic bonds hindered the hydrolysis of wheat starch by α-amylase. The melting peak temperatures of retrograded wheat starch attached to different proteins were 128.46, 126.14, 132.03, 121.65, and 134.84 °C for the control with no protein, albumins, glutenins, globulins, gliadins groups, respectively, and there was no second melting temperature for albumins group. Interaction of wheat proteins and starch in retrograded wheat starch greatly decreased the endothermic enthalpy (△H) of retrograded wheat starch. Retrograded wheat starch bound to gliadins might be a new kind of resistant starch based on glycosidic bond between starch and protein.

Structural features underlying the selective cleavage of a novel exo-type maltose-forming amylase from Pyrococcus sp. ST04.

A novel maltose-forming α-amylase (PSMA) was recently found in the hyperthermophilic archaeon Pyrococcus sp. ST04. This enzyme shows <13% amino-acid sequence identity to other known α-amylases and displays a unique enzymatic property in that it hydrolyzes both α-1,4-glucosidic and α-1,6-glucosidic linkages of substrates, recognizing only maltose units, in an exo-type manner. Here, the crystal structure of PSMA at a resolution of 1.8 Å is reported, showing a tight ring-shaped tetramer with monomers composed of two domains: an N-domain (amino acids 1-341) with a typical GH57 family (β/α)7-barrel fold and a C-domain (amino acids 342-597) composed of α-helical bundles. A small closed cavity observed in proximity to the catalytic residues Glu153 and Asp253 at the domain interface has the appropriate volume and geometry to bind a maltose unit, accounting for the selective exo-type maltose hydrolysis of the enzyme. A narrow gate at the putative subsite +1 formed by residue Phe218 and Phe452 is essential for specific cleavage of glucosidic bonds. The closed cavity at the active site is connected to a short substrate-binding channel that extends to the central hole of the tetramer, exhibiting a geometry that is significantly different from classical maltogenic amylases or β-amylases. The structural features of this novel exo-type maltose-forming α-amylase provide a molecular basis for its unique enzymatic characteristics and for its potential use in industrial applications and protein engineering.

Ectopic expression of bacterial amylopullulanase enhances bioethanol production from maize grain

Heterologous expression of amylopullulanase in maize seeds leads to partial starch degradation into fermentable sugars, which enhances direct bioethanol production from maize grain. Utilization of maize in bioethanol industry in the United States reached ±13.3billion gallons in 2012, most of which was derived from maize grain. Starch hydrolysis for bioethanol industry requires the addition of thermostable alpha amylase and amyloglucosidase (AMG) enzymes to break down the α-1,4 and α-1,6 glucosidic bonds of starch that limits the cost effectiveness of the process on an industrial scale due to its high cost. Transgenic plants expressing a thermostable starch-degrading enzyme can overcome this problem by omitting the addition of exogenous enzymes during the starch hydrolysis process. In this study, we generated transgenic maize plants expressing an amylopullulanase (APU) enzyme from the bacterium Thermoanaerobacter thermohydrosulfuricus. A truncated version of the dual functional APU (TrAPU) that possesses both alpha amylase and pullulanase activities was produced in maize endosperm tissue using a seed-specific promoter of 27-kD gamma zein. A number of analyses were performed at 85°C, a temperature typically used for starch processing. Firstly, enzymatic assay and thin layer chromatography analysis showed direct starch hydrolysis into glucose. In addition, scanning electron microscopy illustrated porous and broken granules, suggesting starch autohydrolysis. Finally, bioethanol assay demonstrated that a 40.2±2.63% (14.7±0.90g ethanol per 100g seed) maize starch to ethanol conversion was achieved from the TrAPU seeds. Conversion efficiency was improved to reach 90.5% (33.1±0.66g ethanol per 100g seed) when commercial amyloglucosidase was added after direct hydrolysis of TrAPU maize seeds. Our results provide evidence that enzymes for starch hydrolysis can be produced in maize seeds to enhance bioethanol production.

Cloning, purification and biochemical characterisation of an organic solvent-, detergent-, and thermo-stable amylopullulanase from Thermococcus kodakarensis KOD1

Thermostable amylopullulanases can catalyse the hydrolysis of both α-1,4 and α-1,6 glucosidic bonds and are of considerable interest in the starch saccharification industry. In this study, the gene Apu-Tk encoding an extracellular amylopullulanase was cloned from an extremely thermophilic anaerobic archaeon Thermococcus kodakarensis KOD1. Apu-Tk encodes an 1100-amino acid protein with a 27-residue signal peptide, which has a predicted mass of 125kDa after signal peptide cleavage. Sequence alignments showed that Apu-Tk contains the five regions conserved in all GH57 family proteins. Full-length Apu-Tk was expressed in Escherichia coli and purified to homogeneity. The purified enzyme displayed both pullulanase and amylase activity. The optimal temperature for Apu-Tk to hydrolyse pullulan and soluble starch was >100°C. Apu-Tk was also active at a broad range of pH (4–7), with an optimum pH of ~5.0–5.5. Apu-Tk also retained >30% of its original activity and partially folded globular structure in the presence of 8% SDS or 10% β-mercaptoethanol. The high yield, broad pH range, and stability of Apu-Tk implicate it as a potential enzyme for industrial applications.

Recombinant Expression and Characterization of an Organic-Solvent-Tolerant α-Amylase from Exiguobacterium sp. DAU5

The enzyme from halophilic microorganisms often has unique properties such as organic-solvent-tolerance. In this study, a novel organic-solvent-tolerant α-amylase gene was cloned from the mild halophile Exiguobacterium sp. DAU5. The open reading frame (ORF) of the enzyme consisted of 1,545bp and encoded 514 amino acids, the primary sequence revealed that it belongs to the glycoside hydrolase (GH) family 13. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed an AmyH monomer of 57kDa. The enzyme exhibited maximal activity at 40°C in pH 8.5 glycine-NaOH buffer, and the activity was strongly inhibited by Zn(2+), Cu(2+), and Fe(2+). The α-amylase AmyH exhibited high hydrolysis activity toward soluble starch, and the major hydrolysis products were maltose, maltotriose, and maltopentaose; the AmyH could not efficiently hydrolyze oligosaccharides smaller than maltoheptaose, nor could it act on the β-1,4 or α-1,6 glucosidic bonds in xylan or pullulan, respectively. In addition, the α-amylase exhibited better tolerance to organic solvents, as it was stable in the presence of dimethylsulfoxide (DMSO), methanol, ethanol, and acetone. Base on all of these results, the enzyme could be useful for practical application in the bakery industry and in biotechnological processes that occur in the presence of organic solvents.

Self‐Assembly and Intermolecular Forces When Cellulose and Water Interact Using Molecular Modeling

Cellulose chains are linear and aggregation occurs via both intra‐ and intermolecular hydrogen bonds. Cellulose has a strong affinity to itself and toward materials containing hydroxyls groups. Based on the preponderance of hydroxyl functional groups, cellulose is very reactive with water. At room temperature, cellulose chains will have at least a monomolecular layer of water associated to it. The formation of hydrogen bonds at the cellulose/water interface is shown to depend essentially on the adsorption site, for example, the equatorial hydroxyls or OH moieties pointing outward from the cellulose chains. The vdW forces also contribute significantly to the adsorption energy. They are a considerable cohesive energy into the cellulose network. At the surface of the cellulose chains, many intermolecular hydrogen bonds of the cellulose chains are lost. However, they are compensated by hydrogen bonds with water molecules. Electronic clouds can be distorted and create electrostatic dipoles. The large antibonding electron cloud that exists around the glucosidic bonds produces an induced polarization at the approach of water molecules. The electron cloud can be distorted and create an electrostatic dipole. It applies to the total displacement of the atoms within the material. Orbitals play a special role in reaction mechanism. Hydrophilic/hydrophobic nature of cellulose is based on its structural anisotropy. Cellulose‐water interactions are exothermic reactions. These interactions may occur spontaneously and result in higher randomness of the system. They are denoted by a negative heat flow (heat is lost to the surroundings). Energy does not need to be inputted in order for cellulose‐water interactions to occur.

Design, synthesis and biological evaluation of glucose-containing scutellarein derivatives as neuroprotective agents based on metabolic mechanism of scutellarin in vivo

Based on metabolic mechanism of scutellarin in vivo that scutellarin could be hydrolyzed into scutellarein by β-glucuronide enzyme, some glucose-containing scutellarein derivatives were designed and synthesized through the introduction of glucose moiety at C-7 position of scutellarein via a glucosidic bond. Biological activity evaluation showed that these glucose-containing scutellarein derivatives exhibited potent DPPH radical scavenging activities. Furthermore, the improvement of physicochemical properties such as anticoagulant and neuroprotective activities alongside with the water solubility was achieved by introducing glucose. These findings suggest that the introduction of the glucose moiety to scutellarein wattants further development of this kind of compounds as neuroprotective agents.

An evolutionary analysis of the GH57 amylopullulanases based on the DOMON_glucodextranase_like domains

Thermostable amylopullulanase (TAPU) is valuable in starch saccharification industry for its capability to catalyze both α-1,4 and α-1,6 glucosidic bonds under the industrial starch liquefication condition. The majority of TAPUs belong to glycoside hydrolase family 57 (GH57). In this study, we performed a phylogenetic analysis of GH57 amylopullulanase (APU) based on the highly conserved DOMON_glucodextranase_like (DDL) domain and classified APUs according to their multidomain architectures, phylogenetic analysis and enzymatic characters. This study revealed that amylopullulanase, pullulanase, andα-amylase had passed through a long joint evolution process, in which DDL played an important role. The phylogenetic analysis of DDL domain showed that the GH57 APU is directly sharing a common ancestor with pullulanase, and the DDL domains in some species undergo evolution scenarios such as domain duplication and recombination.

High-resolution structures ofNeotermes koshunensisβ-glucosidase mutants provide insights into the catalytic mechanism and the synthesis of glucoconjugates

NkBgl, a β-glucosidase from Neotermes koshunensis, is a β-retaining glycosyl hydrolase family 1 enzyme that cleaves β-glucosidic linkages in disaccharide or glucose-substituted molecules. β-Glucosidases have been widely used in several applications. For example, mutagenesis of the attacking nucleophile in β-glucosidase has been conducted to convert it into a glycosynthase for the synthesis of oligosaccharides. Here, several high-resolution structures of wild-type or mutated NkBgl in complex with different ligand molecules are reported. In the wild-type NkBgl structures it was found that glucose-like glucosidase inhibitors bind to the glycone-binding pocket, allowing the buffer molecule HEPES to remain in the aglycone-binding pocket. In the crystal structures of NkBgl E193A, E193S and E193D mutants Glu193 not only acts as the catalytic acid/base but also plays an important role in controlling substrate entry and product release. Furthermore, in crystal structures of the NkBgl E193D mutant it was found that new glucoconjugates were generated by the conjugation of glucose (hydrolyzed product) and HEPES/EPPS/opipramol (buffer components). Based on the wild-type and E193D-mutant structures of NkBgl, the glucosidic bond of cellobiose or salicin was hydrolyzed and a new bond was subsequently formed between glucose and HEPES/EPPS/opipramol to generate new glucopyranosidic products through the transglycosylation reaction in the NkBgl E193D mutant. This finding highlights an innovative way to further improve β-glucosidases for the enzymatic synthesis of oligosaccharides.

Synthetic Studies toward C‐Glucosidic Ellagitannins: A Biomimetic Total Synthesis of 5‐O‐Desgalloylepipunicacortein A

C-glucosidic ellagitannins constitute a subclass of bioactive polyphenolic natural products with strong antioxidant properties, as well as promising antitumoral and antiviral activities that are related to their capacity to interact with both functional and structural proteins. To date, most synthetic efforts toward ellagitannins have concerned glucopyranosic species. The development of a synthetic strategy to access C-glucosidic ellagitannins, whose characteristic structural feature includes an atropoisomeric hexahydroxydiphenoyl (HHDP) or a nonahydroxyterphenoyl (NHTP) unit that is linked to an open-chain glucose core by a C-aryl glucosidic bond, is described herein. The total synthesis of the biarylic HHDP-containing 5-O-desgalloylepipunicacortein A (1 β) was achieved by either using the natural ellagic acid bis-lactone as a precursor of the requested HHDP unit or by implementing an atroposelective intramolecular oxidative biarylic coupling to forge this HHDP unit. Both routes converged in the penultimate step of this synthesis to enable a biomimetic formation of the key C-aryl glucosidic bond in the title compound.

A novel soluble β-1,3-d-glucan Salecan reduces adiposity and improves glucose tolerance in high-fat diet-fed mice

Salecan is a recently identified water-soluble viscous extracellular β-1,3-D-glucan polysaccharide from an Agrobacterium species. It is a high-molecular-mass polymer (about 2 × 10⁶ Da) and composed of a linear chain of glucosyl residues linked through a repeat unit of seven β-(1,3) and two α-(1,3) glucosidic bonds. In the present study, we examined the effects of dietary Salecan fed at 2 and 5 % in a high-fat diet (64 % energy) in C57BL/6J mice. After 6 weeks, mice fed 2 and 5 % Salecan had significantly lower body weight, fat mass and percentage of body fat mass compared with those fed a high-fat cellulose (control) diet. Both the Salecan groups significantly and dose-dependently improved glucose tolerance, with a 9 and 26 % reduction of glucose AUC, respectively. Liver and adipose tissue weights were also significantly decreased by the Salecan treatment. Supplementation with 5 % Salecan led to lower serum TAG, total cholesterol (TC) and HDL-cholesterol (52, 18 and 19 %, respectively) and lower hepatic TAG by 56 % and TC by 22 % compared with the high-fat cellulose control group. Dietary Salecan intake caused an obvious elevation of fat in the faeces. Supplementation with Salecan disturbed bile acid-promoted emulsification and reduced the size of emulsion droplets in vitro. These results indicate that Salecan decreases fat absorption, improves glucose tolerance and has biologically important, dose-related effects on reducing high-fat diet-induced obesity.

Molecular pathogenesis of a new glycogenosis caused by a glycogenin-1 mutation

Glycogenin-1 initiates the glycogen synthesis in skeletal muscle by the autocatalytic formation of a short oligosaccharide at tyrosine 195. Glycogenin-1 catalyzes both the glucose-O-tyrosine linkage and the α1,4 glucosidic bonds linking the glucose molecules in the oligosaccharide. We recently described a patient with glycogen depletion in skeletal muscle as a result of a non-functional glycogenin-1. The patient carried a Thr83Met substitution in glycogenin-1. In this study we have investigated the importance of threonine 83 for the catalytic activity of glycogenin-1. Non-glucosylated glycogenin-1 constructs, with various amino acid substitutions in position 83 and 195, were expressed in a cell-free expression system and autoglucosylated in vitro. The autoglucosylation was analyzed by gel-shift on western blot, incorporation of radiolabeled UDP-14C-glucose and nano-liquid chromatography with tandem mass spectrometry (LC/MS/MS). We demonstrate that glycogenin-1 with the Thr83Met substitution is unable to form the glucose-O-tyrosine linkage at tyrosine 195 unless co-expressed with the catalytically active Tyr195Phe glycogenin-1. Our results explain the glycogen depletion in the patient expressing only Thr83Met glycogenin-1 and why heterozygous carriers without clinical symptoms show a small proportion of unglucosylated glycogenin-1.

Stereochemical Properties of Glucosyl Sulfoxides in Solution

A series of alkyl β-glucosyl sulfoxides were synthesized and characterized in order to study their stereochemical properties. The dependence of the aglycon, solvent and absolute configuration of the sulfinyl group on the conformational properties around the glucosidic and C5-C6 (hydroxymethyl group) bonds were studied. The results for R(S) sulfoxides show linear correlations between the rotamer populations of the hydroxymethyl group and the corresponding Taft's steric parameter (E(S)) of the alkyl group attached to the sulfinyl group in polar and apolar solvents, an increase in the absolute value of E(S) leading to an increase in the gt population. In addition, NOE experiments reveal that as the bulkiness of the alkyl group increases the population of the g- rotamer increases, the latter stabilized by the exo-anomeric effect. These results are in complete agreement with the participation of the exo-anomeric effect in both conformational properties of R(S) sulfoxides. Sulfoxides with the S(S) configuration show different behavior to their R(S) epimers; thus, an increase in the E(S) value of the alkyl group leads to similar or lower gt populations in apolar solvents and to increases in gt in polar solvents. Their NOE studies reveal a conformational equilibrium (in polar and apolar solvents) between g- and g+, dependent on the size of the alkyl group R attached to the sulfinyl group. All these results for both epimers support the general hypothesis that the exo-anomeric effect modifies the conformation of the hydroxymethyl group, fulfills the stereoelectronic requirements, and shows dependence on the solvent.

Modification of carboxymethyl cellulose through oxidation.

The periodate oxidation reaction of carboxymethyl cellulose involve the primary and secondary hydroxyl groups of the pyranose ring. The reaction is accompanied by the opening of the pyranose ring and resulting product is dialdehyde carboxymethyl cellulose along with some hydrated forms. In this process the glucosidic bond becomes weaker; the formation of carboxyl groups induces a depolymerization, thus reducing the polymerization degree and the physical and mechanical strength of the material. The reaction has been has been carried out at pH 3.5, temperature 45°C for 0.5-4h.

A novel hydrogen peroxide biosensor based on the specific binding of horseradish peroxidase with polymeric thiophene-3-boronic acid monolayer in hydrophilic room temperature ionic liquid

In the present work, we have developed a novel hydrogen peroxide (H 2O 2) biosensor based on electropolymerization of thiophene-3-boronic acid film (TBA) in ionic liquid via glucosidic bond with glycoprotein horseradish peroxidase (HRP). A thin film of poly (thiophene-3-boronic acid) (PTBA) has been synthesized by electropolymerization using room temperature ionic liquids (RTIL) as the growth medium and the supporting electrolyte. Horseradish peroxidase (HRP) in this study shows affinity interaction with the formed PTBA thin film. With the aid of hydroquinone (H 2Q) mediator, the biosensor has a fast response of less than 7 s with linear range of 5.0 × 10 −6 to 1.75 × 10 −4 M and a detection limit of 0.5 μM. The developed biosensor exhibits fast response, a low detection limit, high stability and very good reproducibility.

Genome-wide analysis of the beta-glucosidase gene family in maize (Zea mays L. var B73)

The hydrolysis of beta-D: -glucosidic bonds which is required for the liberation of many physiologically important compounds is catalyzed by the enzyme beta-glucosidase (BGLU, EC 3.2.1.21). BGLUs are implicated in several processes in plants, such as the timely response to biotic and abiotic stresses through activation of phytohormones and defense compounds. We identified 26 BGLU isozymes in the genome of the maize inbred B73 and propose a standardized nomenclature for all Zea mays BGLU paralogs (Zmbglu1-Zmbglu26). We characterized their intron-exon structure, protein features, phylogenetic relationships, and measured their expression and activity in various tissues under different environmental conditions. Sequence alignments revealed some characteristic motifs (conserved amino acids) and specific differences among different isozymes. Analysis of putative signal peptides suggested that some BGLUs are plastidic, whereas others are mitochondrial, cytosolic, vacuolar or secreted. Microarray and RT-PCR analysis showed that each member of the Zmbglu family had a characteristic expression pattern with regard to tissue specificity and response to different abiotic conditions. The source of variance for gene expression was highest for the type of organ analyzed (tissue variance) than for the growth conditions (environmental variance) or genotype (genetic variance). Analysis of promoter sequences revealed that each Zmbglu paralog possesses a distinct set of cis elements and transcription factor binding sites. Since there are no two Zmbglu paralogs that have identical molecular properties, we conclude that gene subfunctionalization in maize occurs much more rapidly than gene duplication.

Apo- and Cellopentaose-bound Structures of the Bacterial Cellulose Synthase Subunit BcsZ

Cellulose, a very abundant extracellular polysaccharide, is synthesized in a finely tuned process that involves the activity of glycosyl-transferases and hydrolases. The cellulose microfibril consists of bundles of linear β-1,4-glucan chains that are synthesized inside the cell; however, the mechanism by which these polymers traverse the cell membrane is currently unknown. In Gram-negative bacteria, the cellulose synthase complex forms a trans-envelope complex consisting of at least four subunits. Although three of these subunits account for the synthesis and translocation of the polysaccharide, the fourth subunit, BcsZ, is a periplasmic protein with endo-β-1,4-glucanase activity. BcsZ belongs to family eight of glycosyl-hydrolases, and its activity is required for optimal synthesis and membrane translocation of cellulose. In this study we report two crystal structures of BcsZ from Escherichia coli. One structure shows the wild-type enzyme in its apo form, and the second structure is for a catalytically inactive mutant of BcsZ in complex with the substrate cellopentaose. The structures demonstrate that BcsZ adopts an (α/α)(6)-barrel fold and that it binds four glucan moieties of cellopentaose via highly conserved residues exclusively on the nonreducing side of its catalytic center. Thus, the BcsZ-cellopentaose structure most likely represents a posthydrolysis state in which the newly formed nonreducing end has already left the substrate binding pocket while the enzyme remains attached to the truncated polysaccharide chain. We further show that BcsZ efficiently degrades β-1,4-glucans in in vitro cellulase assays with carboxymethyl-cellulose as substrate.

ChemInform Abstract: First and Biomimetic Total Synthesis of a Member of the C‐Glucosidic Subclass of Ellagitannins, 5‐O‐Desgalloylepipunicacortein A.

The first total synthesis of a member of the C-glucosidic subclass of ellagitannins, 5-O-desgalloylepipunicacortein A, was accomplished by relying on a biomimetic aldol-type formation of its characteristic C-aryl glucosidic bond through the exploitation of the inherent chemical reactivity of a glucopyranosic hemiacetal precursor.

The continuing debate over the structure of cellulose: historical perspective and outlook

The crystalline structure of native cellulose, with its unique microfibrillar morphology resulting from a complex biogenesis mechanism, has not only been the most studied subject in polymer science, but is still generating great interest along with lively debates and controversies. This issue of Cellulose features such a debate in the form of two articles dealing with the conformational analysis of the glucosidic bond in the cellulose chains. This debate is but the latest incident in a long string of events, which represent the foundation of our current understanding of this important material. This makes this an opportune moment to reflect on some of the major milestones in the history of cellulose structure evaluation, which also often involved debates and controversies. It was in 1913, i.e. close to one century ago, that Nishikawa and Ono showed the first diffraction patterns of two cellulose-based plant materials, namely bamboo and hemp, taking advantage of the discovery of the interference phenomena of X-rays reported 1 year earlier by Laue, Friedrich and Knipping. Following this early report, diffraction diagrams of a large number of cellulose materials were duly recorded and subjected to analysis, as it was felt that the knowledge of the crystalline structure of cellulose was of great importance for the development and improvement of cellulose-based products. While recording diffraction patterns of these specimens, it was soon realized that even the X-ray data sets of the most crystalline samples remained limited and it was therefore hard to deduce from them alone the required structural details. It is only when the diffraction data could be complemented by the occurrence of new techniques that substantial advances toward the structural description of cellulose could be made. The sequential emergence of such techniques explains the succession of steps leading to the present understanding. In the progress toward the molecular description of the crystal structure of cellulose from X-ray diffraction data, a first landmark was the incorporation of the stereochemistry of the glucosyl moieties in the molecular models built by, among others, Meyer and Mark (1928), later revised by Meyer and Misch (1937), who presented a model where the crystal of cellulose consisted of two antiparallel chains. This last model, which remained the established standard for at least two decades, was confronted with a series of conflicting data, resulting from new techniques. In 1958, the recording of electron diffraction diagrams of frozen Valonia cell wall fragments allowed Honjo and Watanabe to obtain diffraction data sets far better H. Chanzy—affiliated with the Universite Joseph Fourier and member of the Institut de Chimie Moleculaire de Grenoble.

Isolation of a Gene from Leuconostoc citreum B/110-1-2 Encoding a Novel Dextransucrase Enzyme

The amplicon encoding dextransucrase DSR-F from Leuconostoc citreum B/110-1-2, a novel sucrose glucosyltransferase (GTF)-specific for α-1,6 and α-1,3 glucosidic bond synthesis, with α-1,4 branching was cloned, sequenced, and expressed into Escherichia coli JM109. Recombinant enzyme catalyzed oligosaccharides synthesis from sucrose as donor and maltose acceptor. The dsrF gene encodes for a protein (DSR-F) of 1,528 amino acids, with a theoretical molecular mass of 170447.72 Da (~170 kDa). From amino acid sequence comparison, it appears that DSR-F possesses the same domains as those described for GTFs. However, the variable region is longer than in other GTFs (by 100 amino acids) and two APY repeats (a 79 residue long motif with a high number of conserved glycine and aromatic residues, characterized by the presence of the three consecutive residues Ala, Pro, and Tyr) were identified in the glucan binding domain. The DSR-F catalytic domain possesses the catalytic triad involved in the glucosyl enzyme formation. The amino acid sequence of this domain shares a 56% identity with catalytic domain of the alternansucrase ASR from L. citreum NRRL B-1355 and with the catalytic domain of a putative alternansucrase sequence found in the genome of L. citreum KM20. A truncated active variant DSR-F-∆SP-∆GBD of 1,251 amino acids, with a molecular mass of 145 544 Da (~145 kDa), was obtained.