Hemoglobin-dialdehyde dextran conjugates: Improvement of their oxygen-binding properties with anionic groups

We studied the conjugates formed between hemoglobin and sulfated or unsulfated oxidized dextran. It appears that the presence of sulfated groups favors imino bond formation between the protein and the polymer, as the average molecular size of the conjugates is larger in this case. Under neutral conditions, the oxygen-binding properties of the conjugates depend on the presence or absence of oxygen during the coupling reaction. With unsulfated dextran, oxyhemoglobin leads to conjugates with increased oxygen affinity (P ₅₀/P ₅₀ native hemoglobin 0.5) compared to that of free hemoglobin (P ₅₀=4 mm Hg), whereas deoxyhemoglobin leads to conjugates with decreased oxygen affinity (P ₅₀/P ₅₀ native hemoglobin 3). The use of sulfated dextran reinforces this lowering in oxygen affinity, which indicates that sulfated dextran acts as a permanent macromolecular effector of hemoglobin (P ₅₀/P ₅₀ native hemoglobin 4). Moreover, it can be assumed that some of the linkages involve the 2,3-diphosphoglycerate binding site, as the strong effector inositol hexaphosphate has only a slight effect on the oxygen-binding properties of the conjugate prepared in the deoxy state (P ₅₀/P ₅₀ native hemoglobin close to 4.4 and 6, respectively, for unsulfated and sulfated conjugates). Although dextran substituted with benzenehexacarboxylic acid (BHC) leads to a low-oxygen-affinity conjugate when linked to oxyhemoglobin through amide bonds (P ₅₀/P ₅₀ native hemoglobin 5), oxidized dextran modified with BHC leads, with oxyhemoglobin, to a conjugate whose oxygen affinity is close to that of free hemoglobin (P ₅₀/P ₅₀ native hemoglobin 1.2).     

Impact of aldehyde content on amphotericin B-dextran imine conjugate toxicity

The biocompatibility of oxidized dextran (40 kDa) was investigated in vitro. The contribution of aldehyde groups to the toxicity of polymer-drug conjugates, such as dextran-amphotericin B (AmB) was evaluated. Oxidized dextran was proved to be toxic against the RAW 264.7 cell line with an IC50 of 3 micromol/mL aldehydes. Modification of aldehyde groups and their reaction with ethanolamine reduced the toxicity at least 15-fold. Accordingly, the antifungal and antileishmanial dextran-AmB imine conjugate, which contains unreacted aldehyde groups, was modified with ethanolamine and compared to dextran-AmB amine and imine conjugates. Modification of the imine conjugate with ethanolamine reduced its toxicity toward the RAW cell line by 100%. The effect on Leishmania major parasites was 5 times higher than that of the dextran-AmB amine conjugate. The dextran-AmB-ethanolamine conjugate was at least 15 times less hemolytic than free AmB. Stability and drug release profiles in buffer solution were investigated. The imine conjugates released free AmB while the amine conjugate did not. It is concluded that aldehyde groups may contribute to cell toxicity. This toxicity is reduced by converting the aldehyde groups into imine conjugates with ethanolamine. The results have direct implications toward the safety of AmB-polysaccharide conjugates used against fungal and leishmanial infections.     

Covalent Fixation of Soluble Derivatized Dextrans to Model Proteins in Low-Concentration Medium: Application to Factor IX and Protein C

Factor IX and protein C are zymogens implicated in blood clotting, and an increase in their plasmatic residence time would be of interest for the treatment of the disorders caused by their deficiency. In this context, the conjugation of these proteins to polymers such as modified dextrans could be used to approach the problem. Conjugate formation in concentrated medium ([protein]>50 g/L) is well documented, whereas drastic dilution ([protein] <1 g/L) is quite unfavorable. Before studying the binding of factor IX and protein C to polymers, the coupling of model proteins (human hemoglobin, Hb; human serum albumin, HSA) in low-concentration medium to benzenetetracarboxylate dextran (BTC-dextran) and dialdehyde dextran was investigated. To obtain soluble benzenetetracarboxylate dextran-based conjugates, the conditions of coupling were optimized; the use of sulfo-NHS was necessary to form a conjugate with benzenetetracarboxylate dextran. In fact, the O-acylurea intermediate formed between coupling agent [l-ethyl-3(3-dimethylaminopropyl) carbodiimide, EDC] and BTC-dextran must be stabilized. Concerning dialdehyde dextran, a more oxidized polymer and a higher pH of the buffer of coupling than for highly concentrated solution must be used to obtain a conjugate. Whatever polymer is used, HSA appeared clearly less reactive than Hb, which can be attributed to the better reactivity of N-terminal amino groups in this latter protein and to the marked affinity of benzenetetracarboxylate dextran for it. No soluble conjugate was formed between the same dextran derivatives and factor IX or protein C. Moreover, the activity of both coagulation factors was dramatically decreased by contact with EDC and glutaraldehyde, a small molecule. Thus, bad accessibility of protein amino groups is probably responsible for this lack of reactivity. Nevertheless, it could be shown that carboxylate and amino groups were essential to the activity of factor IX and protein C.     

Covalent fixation of hemoglobin to dextran phosphates decreases its oxygen affinity.

The interactions between various dextran phosphates and Hb (hemoglobin) were studied by measuring the oxygen-binding parameters of the mixtures. The effector properties of polymers were found to depend on the concentration of monoalkylmonophosphate groups on the polymers and also on their molecular weights. The covalent fixation of dextran phosphates bearing aldehydic groups to oxyHb and deoxyHb was carried out. The oxygen-binding properties of the conjugates thus obtained depended upon the initial form of the protein. Thus, only the conjugates synthesized from deoxyHb exhibited a low oxygen affinity, which means that, in this case, the linkages between the dextran phosphate and the protein allow a permanent interaction of the phosphate groups with amines of the 2,3-diphosphoglycerate binding site. The Hill coefficient values of these conjugates were smaller than that of free Hb, corresponding to a loss of the cooperativity of the protein upon fixation of polymers. However, as these new conjugates are capable of unloading more O2 than blood when subjected to oxygen pressures corresponding to physiological conditions, they can be regarded as potential erythrocyte substitutes.     

Effect of combination of high-intensity ultrasound treatment and dextran glycosylation on structural and interfacial properties of buckwheat protein isolates

This study investigated the effects of high-intensity ultrasound and glycosylation on the structural and interfacial properties of the Maillard reaction conjugates of buckwheat protein isolate (BPI). The covalent attachment of dextran to BPI was confirmed by examination of the Fourier-transform infrared spectra. Emulsifying properties of the conjugates obtained by ultrasound treatment were improved as compared to those obtained by classical heating. Structural feature analyses suggested that conjugates obtained by ultrasound treatment had less α-helix and more random coil, higher surface hydrophobicity and less compact tertiary structure as compared to those obtained by classical heating. The surface activity measurement revealed that the BPI–dextran conjugates obtained by ultrasound treatment were closely packed and that each molecule occupied a small area of the interface. Combination of ultrasonic treatment and glycosylation was proved to be an efficient way to develop new stabilizers and thickening agents for food in this study.     

Effect of sulfated glycoconjugates on capacitation and the acrosome reaction of bovine and hamster spermatozoa

The effects of sulfated glycoconjugates on the preparation of mammalian sperm for fertilization were investigated. The three sulfated glycoconjugates tested were heparin, dextran sulfate, and the fucose sulfate glycoconjugate (FSG) from the sea urchin egg jelly coat. In vivo, FSG induces the acrosome reaction in sea urchin sperm. Bovine sperm were found to be capacitated by heparin and FSG as judged both by ability of lysophosphatidylcholine (LC) to induce an acrosome reaction and by ability to fertilize bovine oocytes in vitro. The mechanism by which heparin or FSG capacitated bovine sperm appeared similar, since glucose inhibited capacitation by both glycoconjugates. In contrast to effects on bovine sperm, heparin and FSG induced the acrosome reaction in capacitated hamster sperm. When hamster sperm were incubated under noncapacitating conditions, heparin had no effect on capacitation or the acrosome reaction. Three molecular weights (MW) of dextran sulfate (5,000, 8,000, 500,000) were found to capacitate bovine sperm as judged by the ability of LC to induce an acrosome reaction. Whereas bovine sperm incubated with 5,000 or 8,000 M W dextran sulfate fertilized more bovine oocytes than control sperm (P <0.05), sperm treated with 500,000 M W dextran sulfate failed to penetrate oocytes. The high-MW dextran sulfate appeared to interact with the zona pellucida and/or sperm to prevent sperm binding. Results suggest that sulfated glycoconjugates may prepare sperm for fertilization across a wide range of species.     

Soybean Bowman–Birk Inhibitor Conjugates with Clinical Dextran. Synthesis and Antiproteolytic Activity

Conjugates of the classical soybean Bowman-Birk inhibitor (BBI) with clinical dextran were synthesized. Clinical dextran was preliminarily oxidized with periodate to dialdehydedextran (DAD). The effect of the degree of oxidation of DAD on coupling of the inhibitor was evaluated. The binding of the protein was shown to increase with increasing degree of DAD oxidation (5, 10, 20%). Total coupling of the inhibitor occurred when the degree of oxidation of the dextran was 20%. The BBI-DAD (20%) conjugate contained 13% protein with BBI/DAD molar ratio 1 : 1. The conjugates retained the ability to inhibit trypsin (Ki = 0.2-0.3 nM) and alpha-chymotrypsin (Ki = 15-30 nM). Thus, the coupling of BBI with the polymeric carrier caused practically no decrease in the antiproteolytic activity of the inhibitor.     

Effect of covalent labeling of dextran-benzenehexacarboxylate on hemoglobin

The covalent fixation of benzenehexacarboxylate (BHC) onto dextran was carried out according to several reaction schemes. The polyanionic polymers thus synthesized were capable of decreasing the oxygen affinity of hemoglobin by specifically interacting with the 2,3-diphosphoglycerate (2,3-DPG) binding site of the protein. The intensity of this effect was correlated to both the chemical structure of the polyanionic polymers and the BHC content in polymer. The polyanionic polymer, containing 0.035 mol BHC/g and presenting no cross-linking between its polymer chains, possessed the best effector properties. These properties were used to direct the covalent fixation of the dextran-benzenehexacarboxylate onto the phosphate binding site of the protein. The resulting hemoglobin was mainly substituted at the same time by one or more linked BHC onto both dimers in the vicinity of the 2,3-DPG site. Thus, the modification of hemoglobin led to an increase in the hydrodynamic volume of each dimer sufficient to limit the diffusion of the conjugates through the kidney membrane, even if the conjugates had dissociated into dimers. Compared to that of free hemoglobin, the oxygen affinity of the conjugates was significantly decreased. This type of covalent conjugate exhibited general properties quite suitable for use as blood substitutes.Abbreviations used Hb hemoglobin - 2,3-DPG 2,3-diphosphoglycerate - BHC benzenehexacarboxylate - IHP inositolhexaphosphate - EDCI 3-ethyl-1-(3-dimethylaminopropyl) carbodiimide hydrochloride     

The inhibition of boar acrosin amidase activity by sulfated polysaccharides

Carbohydrate-protein interactions are known to be important in gamete interactions. We therefore investigated the inhibition of boar sperm acrosin amidase activity by carbohydrates. The sulfated polysaccharides fucoidan and dextran sulfate inhibited amide hydrolysis whereas dextran and various monosaccharides did not inhibit acrosin amidase activity. The kinetics of the inhibition corresponded to those characteristic when multiple forms of an enzyme are present. Such a kinetic result was consistent with the presence of the known autolytically produced forms of acrosin. It was previously shown that sulfated polysaccharides inhibit sperm-egg binding and that acrosin binds carbohydrate. We propose that the sulfated polysaccharide inhibition of acrosin amidase activity observed here is causally related to the previously observed sulfated polysaccharide inhibition of sperm binding to the zona pellucida.     

Fixation of aldehydic dextrans onto human deoxyhemoglobin.

A procedure commonly used to transform native adult human hemoglobin (Hb) into a physiological oxygen carrier consists of a pyridoxylation of the protein to lower its oxygen affinity, followed by its polymerization in the presence of glutaraldehyde, with or without further reduction, to increase its circulating half-life. This series of reactions yields derivatives presenting a great molecular heterogeneity that have to be fractionated for use in vivo. Hemoglobin derivatives with low oxygen affinity and a narrow distribution of molecular weights were obtained by linking a dextran polyaldehydic derivative to deoxyhemoglobin at pH 8. From oxygen-binding measurements carried out in the presence of inositolhexaphosphate, a strong effector of hemoglobin, it appeared that the allosteric site of hemoglobin was blocked, probably by crosslinking bonds, which stabilizes its deoxy structure. On the other hand, when the reaction was performed in the presence of inositolhexaphosphate, the resulting conjugates exhibited an oxygen affinity identical to that of unmodified hemoglobin. After treatment with NaBH4, the polymer-hemoglobin derivatives were stable and possessed a reversible oxygen-carrying capacity similar to that of blood. The conjugates prepared from oxyhemoglobin all possessed a lower P50 than native hemoglobin whatever the reaction conditions.     

Influence of rheopolyglucin and dextran dialdehyde on physicochemical properties and thermostability of human hemoglobin

A study was made of the influence of rheopolyglucin and dextran dialdehyde derived therefrom on the structural characteristics and thermostability of human hemoglobin. The effects of solution pH, incubation time and temperature, and the degree of dextran oxidation on the conjugation between human hemoglobin and dextran dialdehyde were assessed. Formation of the hemoglobin-dextran dialdehyde complex resulted in shielding of the protein chromophore groups by the polysaccharide and transition of a part of hemoprotein molecules from a low-spin (HbO₂) to a high-spin (Hb and MetHb) state. It was found that the temperature of denaturation transition for the native protein and hemoglobin in the presence of rheopolyglucin was 60°C versus 80°C for the hemoprotein-dextran dialdehyde conjugate. Presumably, the latter is determined by the enhancement of hydrophobic interactions within the protein globule caused by dextran dialdehyde and the ability of the surface-bound carbohydrate components to prevent the association of hemoglobin molecules.     

Pyridoxal-5'-phosphate as the catalyst for radical isomerization in reactions of PLP-dependent aminomutases

PLP catalyzes the 1,2 shifts of amino groups in free radical-intermediates at the active sites of amino acid aminomutases. Free radical forms of the substrates are created upon H atom abstractions carried out by the 5'-deoxyadenosyl radical. In most of these enzymes, the 5'-deoxyadenosyl radical is generated by an iron-sulfur cluster-mediated reductive cleavage of S-adenosyl-(S)-methionine. However, in lysine 5,6-aminomutase and ornithine 4,5-aminomutase, the radical is generated by homolytic cleavage of the cobalt-carbon bond of adenosylcobalamin. The imine linkages in the initial radical forms of the external aldimines undergo radical addition to form azacyclopropylcarbinyl radicals as central intermediates in the catalytic cycles. In the case of lysine 2,3-aminomutase, the multistep catalytic mechanism is corroborated by direct spectroscopic observation and characterization of a product radical trapped during steady-state turnover. Analogues of the substrate-related radical having substituents adjacent to the radical center to stabilize the unpaired electron are also observed and characterized spectroscopically. A functional allylic analogue of the 5'-deoxyadenosyl radical is observed spectroscopically. A high-resolution crystal structure fully supports the mechanistic proposals. Evidence for a similar free radical mediated amino group transfer in the adenosylcobalamin-dependent lysine 5,6-aminomutase is provided by spectroscopic detection and characterization of radicals generated from the 4-thia analogues of the lysine substrates. This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology.     

Alpha-synuclein and parkin contribute to the assembly of ubiquitin lysine 63-linked multiubiquitin chains

Mutations in alpha-synuclein, Parkin, and UCH-L1 cause heritable forms of Parkinson disease. Unlike alpha-synuclein, for which no precise biochemical function has been elucidated, Parkin functions as a ubiquitin E3 ligase, and UCH-L1 is a deubiquitinating enzyme. The E3 ligase activity of Parkin in Parkinson disease is poorly understood and is further obscured by the fact that multiubiquitin chains can be formed through distinct types of linkages that regulate diverse cellular processes. For instance, ubiquitin lysine 48-linked multiubiquitin chains target substrates to the proteasome, whereas ubiquitin lysine 63-linked chains control ribosome function, protein sorting and trafficking, and endocytosis of membrane proteins. It is notable in this regard that ubiquitin lysine 63-linked chains promote the degradation of membrane proteins by the lysosome. Because both Parkin and alpha-synuclein can regulate the activity of the dopamine transporter, we investigated whether they influenced ubiquitin lysine 63-linked chain assembly. These studies revealed novel biochemical activities for both Parkin and alpha-synuclein. We determined that Parkin functions with UbcH13/Uev1a, a dimeric ubiquitin-conjugating enzyme, to assemble ubiquitin lysine 63-linked chains. Our results and the results of others indicate that Parkin can promote both lysine 48- and lysine 63-linked ubiquitin chains. alpha-Synuclein also stimulated the assembly of lysine 63-linked ubiquitin chains. Because UCH-L1, a ubiquitin hydrolase, was recently reported to form lysine 63-linked conjugates, it is evident that three proteins that are genetically linked to Parkinson disease can contribute to lysine 63 multiubiquitin chain formation.     

Properdin binds to sulfatide [Gal(3-SO4)beta 1-1 Cer] and has a sequence homology with other proteins that bind sulfated glycoconjugates

Properdin, which stabilizes the C3 convertase during the activation of the alternate complement pathway, contains amino acid sequence homologies with several proteins that bind sulfated glycoconjugates, including the adhesive protein thrombospondin and the leech salivary protein antistasin. This homology is based around the sequence Cys-Ser-Val-Thr-Cys-Gly-X-Gly-X-X-X-Arg-X-Arg. To determine if these homologous amino acid sequences are sulfated glycoconjugate-binding domains, purified native properdin, as well as activated properdin (a high molecular weight form of properdin), were examined for binding to various lipids in solid phase radioimmunoassays. Of the lipids tested, both native and activated properdin bind with high affinity only to sulfatide [Gal(3-SO4)beta 1-1 Cer], but not to comparable levels of cholesterol-3-SO4, or several neutral glycolipids, gangliosides, and phospholipids. Sulfatide binding by both forms of properdin is inhibited by dextran sulfate (Mr = 500,000) or fucoidan, whereas only the activated form is inhibited by dextran sulfate (Mr = 5,000) or heparin. Comparable levels of chondroitin sulfates A, B, and C, keratan sulfate, dextran (Mr = 90,000), or hyaluronic acid do not inhibit binding. Taken together, these data suggest that properdin, like antistasin and thrombospondin, binds sulfated glycoconjugates and supports the conclusion that the homologous sequences are sulfated glycoconjugate-binding domains.     

Sulfated glycoconjugates are powerful modulators of bovine sperm adhesion and release from the oviductal epithelium in vitro

The mechanisms of sperm adhesion and release within the mammalian oviduct are still poorly understood. In this in vitro study, a previously developed adhesion assay was used to analyze the effects of heparin, N-desulfated heparin, fucoidan, dextran sulfate, and dextran on bovine sperm-oviductal cell adhesion and release. Results showed that 1) all sulfated glycoconjugates were powerful inhibitors of sperm binding to oviductal monolayers in a dose-dependent manner, whereas N-desulfated heparin and dextran had no effect; 2) sperm pretreatment with heparin and fucoidan markedly inhibited adhesion; 3) treatment of oviductal monolayers with heparinase I, II, or sodium chlorate (an inhibitor of sulfation) had no effect on sperm adhesion; 4) sulfated glycoconjugates were also powerful and quick inducers of sperm release from oviductal monolayers; and 5) addition of sulfated glycoconjugates to the cocultures caused a sudden increase of bound-sperm flagellar beat frequencies, followed by a release of highly motile sperm. In conclusion, these data support the hypothesis that sulfated glycoconjugates may act as signals that induce sperm release and migration from the oviductal reservoir.     

Role of dextran-specific suppressor T cells in the regulation of the immune response by a reactive form of dextran

Reactive forms of antigens or haptens have been shown to induce a state of hyporesponsiveness mediated in part by suppressor T cells. Injection of Balb/c x C57B16 F1 (CB6F1) mice with a reactive form of dextran B1355S (periodate oxidized dextran, dex-P) specifically reduced responses to dextran immunization within 1 day after dex-P treatment. This unresponsiveness lasted at least 23 days and required a reactive form of dextran for its induction since native dextran and oxidized/reduced dextran failed to induce tolerance. Furthermore, hyporesponsiveness could be induced by iv injection of dextran-coupled cells, especially peripheral blood lymphocytes, a result which suggests that in vivo coupling to cellular antigens is involved in dex-P-induced hyporesponsiveness. Suppression of the anti-dextran response could be transferred to normal mice with T-cell-enriched spleen cell populations from dex-P-injected mice. Interestingly, the presence of B cells in the transferred cell preparations interfered with detection of suppression. Both Lyt 1+2- and Lyt 1-2+ cells were involved in the dex-P-induced suppression; indeed, mixtures of these types of T cells led to the most profound degree of suppression. The suppressive activity of spleen cells from dex-P-injected mice could be removed by passage over dextran-coated plates. Moreover, cells eluted from the plates specifically suppressed anti-dextran responses of normal mice, indicating that dex-P injection induces a population of antigen-binding suppressor cells. This system will allow the study of the suppressor-T-cell receptors in a well-defined idiotypic system.     

Polysaccharides with sulfate groups are human T cell mitogens and murine polyclonal B cell activators (PBAs) II. Cellulose sulfate and dextran sulfate with two different lower molecular weights

In our previous paper, we reported that various types of carrageenan, dextran sulfate and fucoidan, which are sulfated homopolysaccharides with high molecular weights, were human T cell mitogens and murine polyclonal B cell activators (PBAs) and that heparin, a sulfated heteropolysaccharide, was a very weak human mitogen and mouse PBA. Here we used cellulose sulfate (Mr 7-9 X 10(3], dextran sulfate with two different low molecular weights (Mr 5 X 10(3) and 8 X 10(3], two different condroitin sulfates (Mr 3.5 X 10(4], polyvinyl sulfate and polygalacturonic acid to investigate mitogenic activities of polysaccharides in detail. The following results were obtained. Low-molecular-weight sulfated homopolysaccharides, dextran sulfate and cellulose sulfate, were very weak or not human T cell mitogens. However, they were better murine PBAs. Sulfated heteropolysaccharides, chondroitin 4-sulfate and chondroitin 6-sulfate, hardly induced mitogenic changes in human T cells and mouse B cells, even though the molecular weight of these substances was more than 1 X 10(4). There were no other polymers examined so far which activated both human T cells and murine B cells. The relationship among molecular size, sulfate groups and lymphocyte activation is discussed in detail.     

Microscale synthesis of dextran-based multivalent N-linked oligosaccharide probes

We developed a convenient method for the synthesis of dextran-based multivalent probes containing N-linked oligosaccharides which is efficient even in a small scale. Oligosaccharides were derivatized with succinic dihydrazide and dimethylamine borane under a mild acidic condition. The derivatized oligosaccharides were then conjugated in a good yield to periodate-oxidized dextran (500 kDa). Thus, the conjugates containing 120 to 140 oligosaccharide chains per dextran molecule were successfully synthesized. Their practical advantage was shown by the example that the asialofetuin oligosaccharide-dextran conjugate has much higher affinity to Ricinus communis agglutinin (RCA-I) than asialofetuin oligosaccharide itself or asialofetuin. The conjugates were further labeled with fluorescent reagent or biotinylation reagent containing a hydrazino group by the use of the unreacted aldehyde groups of the oxidized dextran, yielding probes with similar densities of fluorophores or biotin groups. Direct binding of the biotinylated asialofetuin oligosaccharide-dextran probe to RCA-I coated on the titer plate at a concentration of 50 ng/50 microl was easily detected using 50 fmol (as oligosaccharides) of the probe. The method for the synthesis of dextran-based oligosaccharide probes will facilitate the investigation of carbohydrate-mediated molecular interactions based on the native oligosaccharide structures.     

Molecular characterization and expression analysis of the glucansucrase DSRWC from Weissella cibaria synthesizing a α(1→6) glucan

Weissella cibaria isolated from human saliva produces a soluble glucan that predominantly has α-1,6-glucosidic type linkages. Using degenerated primers that were selected based on the amino acid sequences of conserved regions from known glucansucrases, a single 2.7-kb fragment was isolated. In subsequent steps, a 4969-bp product was obtained using inverse PCR. The coding region for the glucansucrase gene ( dsrWC ) consisted of a 4419-bp ORF that encoded a 1472-amino acid protein with a calculated molecular mass of 161.998 Da. The produced DSRWC glucansucrases exhibited similarity with the enzymes of the glucosylhydrolase family 70, which includes the Lactobacillus fermentum glucansucrase. The expressed recombinant DSRWC (rDSRWC) synthesized oligosaccharides in the presence of maltose or isomaltose as an acceptor and the synthesized products included α-1,6-linked glucosyl residues in addition to the maltosyl or isomaltosyl residue. rDSRWC synthesized water-soluble polymers using sucrose as substrate. According to the ¹³C-nuclear magnetic resonance analysis, the polymer that was synthesized by rDSRWC was a linear dextran, which formed predominately α-1,6-glucosidic linkages. This is the first report on the molecular characterization of glucansucrase from a W. cibaria strain.     

Dextran dextrinase and dextran of Gluconobacter oxydans

Certain strains of Gluconobacter oxydans have been known since the 1940s to produce the enzyme dextran dextrinase (DDase; EC2.4.1.2)—a transglucosidase converting maltodextrins into (oligo)dextran. The enzyme catalyses the transfer of an α1,4 linked glucosyl unit from a donor to an acceptor molecule, forming an α1,6 linkage: consecutive glucosyl transfers result in the formation of high molecular weight dextran from maltodextrins. In the early 1990s, the group of K. Yamamoto in Japan revived research on DDase, focussing on the purification and characterisation of the intracellular DDase produced by G. oxydans ATCC 11894. More recently, this was taken further by Y. Suzuki and coworkers, who investigated the properties and kinetics of the extracellular DDase formed by the same strain. Our group further elaborated on fermentation processes to optimise DDase production and dextran formation, DDase characterisation and its use as a biocatalyst, and the physiological link between intracellular and extracellular DDase. Here, we present a condensed overview of the current scientific status and the application potential of G. oxydans DDase and its products, (oligo)dextrans. The production of DDase as well as of dextran is first described via optimised fermentation processes. Specific assays for measuring DDase activity are also outlined. The general characteristics, substrate specificity, and mode of action of DDase as a transglucosidase are described in detail. Two forms of DDase are produced by G. oxydans depending on nutritional fermentation conditions: an intracellular and an extracellular form. The relationship between the two enzyme forms is also discussed. Furthermore, applications of DDase, e.g. production of (oligo)dextran, transglucosylated products and speciality oligosaccharides, are summarized.