The dextran acceptor reaction of dextransucrase from Streptococcus mutans K1-R

Soluble dextransucrase activity(ies) was eluted with a solution of clinical dextran from the insoluble dextran-cell complex produced by Streptococcus mutans K1-R grown in the presence of sucrose. Studies of the dextran acceptor-reaction of the soluble enzyme-preparation indicate that it is highly specific for dextran of high molecular weight. Increased dextran synthesis in the presence of dextran acceptor and the apparent inhibition of this stimulation by higher concentrations of dextran result from product modification rather than a direct effect on the level of enzyme activity. The results demonstrate that the potentially water-insoluble structure synthesized by dextransucrase on exogenous, soluble dextran acts as a more-efficient acceptor than the soluble dextran. The role of the acceptor reaction in the biosynthesis of complex dextrans is discussed.     

On the production of dextran by free and immobilized dextransucrase

Dextransucrase from Leuconostoc mesenteroides was produced in a semicontinuous culture with slow addition of a concentrated sucrose solution. The resulting high activity of the fermentation broth allowed a one-step purification method, by gel permeation chromatography (GPC) in 96.4% yield. This procedure resulted in 140-fold purification, with specific activity of 122 U/mg. The enzyme was immobilized onto an amino-Spherosil support activated with glutaraldehyde. Preparations with dextransucrase activities as high as 40.5 U/g of support were obtained, when low specific area supports were used and maltose was added during the enzyme coupling. Diffusional limitations were found during enzyme reaction, as shown by a kinetic study. As a consequence of immobilization, the average molecular weight of dextrans seems to increase. Immobilized dextransucrase looks promising for low-molecular-weight dextran production. Clinical dextran was synthesized when the polysaccharides produced in the presence of maltose were used as acceptor of a second synthesis reaction. The molecular weight distribution of the resulting production was less disperse than when clinical dextran was produced by acid hydrolysis of high-molecular-weight dextran.     

The mechanism of dextransucrase action: Biosynthesis of branch linkages by acceptor reactions with dextran

Dextransucrase from Leuconostoc mesenteroides B-512 catalyzes the polymerization of dextran from sucrose. The resulting dextran has 95% α-1 → 6 linkages and 5% α-1 → 3 branch linkages. A purified dextransucrase was insolubilized on Bio-Gel P-2 beads (BGD, Bio-Gel-dextransucrase). The BGD was labeled by incubating it with a very low concentration of [¹⁴C]sucrose or it was first charged with nonlabeled sucrose and then labeled with a very low concentration of [¹⁴C]sucrose. After extensive washings with buffer, the ¹⁴C label remained attached to BGD. This labeled material was previously shown to be [¹⁴C]dextran and was postulated to be attached covalently at the reducing end to the active site of the enzyme. When the labeled BGD was incubated with a low molecular weight nonlabeled dextran (acceptor dextran) all of the BGD-bound label was released as [¹⁴C]dextran whereas essentially no [¹⁴C]dextran was released when the labeled BGD was incubated in buffer alone under comparable conditions. The released [¹⁴C]dextran was shown to be a slightly branched dextran by hydrolysis with an exodextranase. Acetolysis of the released dextran gave 7.3% of the radioactivity in nigerose. Reduction with sodium borohydride, followed by acid hydrolysis, gave all of the radioactivity in glucose, indicating that the nigerose was exclusively labeled in the nonreducing glucose unit. These results indicated that [¹⁴C]dextran was being released from BGD by virtue of the action of the low molecular weight dextran and that this action gave the formation of a new α-1 → 3 branch linkage. A mehanism for branching is proposed in which a C₃-OH on an acceptor dextran acts as a nucleophile on C₁ of the reducing end of a dextranosyl-dextransucrase complex, thereby displacing dextran from dextransucrase and forming an α-1 → 3 branch linkage. It is argued that the biosynthesis of branched linkages does not require a separate branching enzyme but can take place by reactions of an acceptor dextran with a dextranosyl-dextransucrase complex.     

Dextransucrase and the mechanism for dextran biosynthesis

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

Comparative study of the efficacies of nine assay methods for the dextransucrase synthesis of dextran

A comparative study of nine assay methods for dextransucrase and related enzymes has been made. A relatively widespread method for the reaction of dextransucrase with sucrose is the measurement of the reducing value of d-fructose by alkaline 3,5-dinitrosalicylate (DNS) and thereby the amount of d-glucose incorporated into dextran. Another method is the reaction with ¹⁴C-sucrose with the addition of an aliquot to Whatman 3MM paper squares that are washed three times with methanol to remove ¹⁴C-d-fructose and unreacted ¹⁴C-sucrose, followed by counting of ¹⁴C-dextran on the paper by liquid scintillation counting (LSC). It is shown that both methods give erroneous results. The DNS reducing value method gives extremely high values due to over-oxidation of both d-fructose and dextran, and the ¹⁴C-paper square method gives significantly low values due to the removal of some of the ¹⁴C-dextran from the paper by methanol washes. In the present study, we have examined nine methods and find two that give values that are identical and are an accurate measurement of the dextransucrase reaction. They are (1) a ¹⁴C-sucrose/dextransucrase digest in which dextran is precipitated three times with three volumes of ethanol, dissolved in water, and added to paper and counted in a toluene cocktail by LSC; and (2) precipitation of dextran three times with three volumes of ethanol from a sucrose/dextransucrase digest, dried, and weighed. Four reducing value methods were examined to measure the amount of d-fructose. Three of the four (two DNS methods, one with both dextran and d-fructose and the other with only d-fructose, and the ferricyanide/arsenomolybdate method with d-fructose) gave extremely high values due to over-oxidation of d-fructose, d-glucose, leucrose, and dextran.     

Inhibition of dextran synthesis by glucoamylase and endodextranase

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

Search for a dextransucrase minimal motif involved in dextran binding

Fourteen truncated forms of Leuconostoc mesenteroides NRRL B512-F dextransucrase, involving N-, C- or N- plus C-terminal domain truncations were tested for their ability to bind dextrans. The shortest fragment (14kDa molecular weight) that still exhibited a strong interaction with dextran was localized between amino acids N1397 and A1527 of the C-terminal domain (GBD-7) and consists of six YG repeats. With a dissociation constant K(d) of 2.8x10(-9)M, this motif shows a very high affinity for isomaltohexaose and longer dextrans, supporting the proposed role of GBD in polymer formation. The potential application of GBD-7 as an affinity tag onto cheap resins like Sephacryl S300HR for rapid purification was evaluated and is discussed.     

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

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

Characterization of a novel dextransucrase from Weissella confusa isolated from sourdough

Weissella confusa and Weissella cibaria isolated from wheat sourdoughs produce, from sucrose, linear dextrans due to a single soluble dextransucrase. In this study, the first complete gene sequence encoding dextransucrase from a W. confusa strain (LBAE C39-2) along with the one from a W. cibaria strain (LBAE K39) were reported. Corresponding gene cloning was achieved using specific primers designed on the basis of the draft genome sequence of these species. Deduced amino acid sequence of W. confusa and W. cibaria dextransucrase revealed common structural features of the glycoside hydrolase family 70. Notably, the regions located in the vicinity of the catalytic triad (D, E, D) are highly conserved. However, comparison analysis also revealed that Weissella dextransucrases form a distinct phylogenetic group within glucansucrases of other lactic acid bacteria. We then cloned the W. confusa C39-2 dextransucrase gene and successfully expressed the mature corresponding enzyme in Escherichia coli. The purified recombinant enzyme rDSRC39-2 catalyzed dextran synthesis from sucrose with a K _m of 8.6 mM and a V _max of 20 μmol/mg/min. According to ¹H and ¹³C NMR analysis, the polymer is a linear class 1 dextran with 97.2 % α-(1→6) linkages and 2.8 % α-(1→3) branch linkages, similar to the one produced by W. confusa C39-2 strain. The enzyme exhibited optimum catalytic activity for temperatures ranging from 35 to 40 °C and a pH of 5.4 in 20 mM sodium acetate buffer. This novel dextransucrase is responsible for production of dextran with predominant α-(1→6) linkages that could find applications as food hydrocolloids.     

Characterization of the multiple forms and main component of dextransucrase from Leuconostoc mesenteroides NRRL B-512F

Multiple forms of dextransucrase (sucrose:1.6-alpha-D-glucan 6-alpha-D-glucosyltransferae EC 2.4.1.5) from Leuconostoc mesenteroides NRRL B-512F strain were shown by gel filtraton and electrophoretic analyses. Two components of enzyme, having different affinities for dextran gel, were separated by a column of Sephadex G-100. The major component voided from the Sephadex column was treated with dextranase and purified to an electrophoretically homogeneous state. The ]urified enzyme had a molecular weight of 64 000-65 000, pI value of 4.1, and 17% of carbohydrate in a molecule. EDTA showed a characteristic inhibition on the enzyme while stimulative effects were observed by the addition of exogenous dextran to the incubation mixture. The enzyme activity was stimulated by various dextrans and its Km value was decreased with increasing concentration of dextran. The purified enzyme showed no affinity for a Sephadex G-100 gel, and readily aggregated after the preservation at 4 degrees C in a concentrated solution.     

One-step synthesis of isomalto-oligosaccharide syrups and dextrans of controlled size using engineered dextransucrase

Isomalto-oligosaccharides and dextrans of controlled molecular weight of about 10 and 40 kDa were produced using a simple one-step process using engineered L. mesenteroides NRRL B-512F dextransucrase variants. Isomalto-oligosaccharides were produced in a 58% yield by the acceptor reaction with glucose, and reached a degree of polymerization of at least 27 glucosyl units. Reaction conditions for optimal synthesis of dextrans of controlled molecular weight were defined, in respect of initial sucrose concentration and reaction temperature. Thus, we achieved synthesis with impressive yields of 69 and 75% for the 40 and 10 kDa dextran species, respectively. These two dextran sizes are particularly suitable for clinical applications, and are of great industrial demand. Compared with the traditional processes based on chemical hydrolysis and fractionation, which achieve only low yields, the new enzymatic methods offer improvement in quantity, quality and efficiency.     

Proteolytic modification of <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> B-512F dextransucrase

Multiple active lower molecular weight forms from Leuconostoc mesenteroides B512F dextransucrase have been reported. It has been suggested that they arise from proteolytic processing of a 170 kDa precursor. In this work, the simultaneous production of proteases and dextransucrase was studied in order to elucidate the dextransucrase proteolytic processing. The effect of the nitrogen source on protease and dextransucrase production was studied. Protease activity reaches a maximum early in the logarithmic phase of dextransucrase synthesis using the basal culture medium but the nitrogen source plays an important effect on growth: the highest protease concentration was obtained when ammonium sulfate, casaminoacids or tryptone were used. Two active forms of 155 and 129 kDa were systematically obtained from dextransucrase precursor by proteolysis. The amino termini of these forms were sequenced and the cleavage site deduced. Both forms of the enzyme obtained had the same cleavage site in the amino terminal region (F209–Y210). From dextransucrase analysis, various putative cleavage sites with the same sequence were found in the variable region and in the glucan binding domain. Although no structural differences were found in dextrans synthesized with both the precursor and the proteolyzed 155 kDa form under the same reaction conditions, their rheological behaviour was modified, with dextran of a lower viscosity yielded by the smaller form.Martha Argüello-Morales and Mónica Sánchez-González equally contributed to this work.     

Kinetic studies on dextransucrase from the cariogenic oral bacterium Streptococcus mutans

The kinetic mechanism of dextransucrase was studied using the Streptococcus mutans enzyme purified by affinity chromatography to a specific activity of 36.9 mumol/min/mg of enzyme. In addition to dextran synthesis, the enzyme catalyzed sucrose hydrolysis and isotope exchange between fructose and sucrose. The rates of sucrose hydrolysis and dextran synthesis were partitioned as a function of dextran concentration such that exclusive sucrose hydrolysis was observed in the absence of dextran and exclusive dextran synthesis at high dextran concentrations. An analogous situation was observed with fructose-dependent partitioning of sucrose hydrolysis and fructose exchange. Steady state dextran synthesis and fructose isotope exchange kinetics were simplified by assay at dextran or fructose concentrations high enough to eliminate significant contributions from sucrose hydrolysis. This limited dextran synthesis assays to dextran concentrations above apparent saturation. The limitation was diminished by establishing conditions in which the enzyme does not distinguish between dextran as a substrate and product which allowed initial discrimination among mechanisms on the basis of the presence or absence of dextran substrate inhibition. No inhibition was observed, which excluded ping-pong and all but three common sequential mechanisms. Patterns of initial velocity fructose production inhibition and fructose isotope exchange at equilibrium were consistent with dextran synthesis proceeding by a rapid equilibrium random mechanism. A nonsequential segment was apparent in the exchange reaction between fructose and sucrose assayed in the absence of dextran. However, the absence of detectable glucosyl exchange between dextrans and the lack of steady state dextran substrate inhibition indicate that glucosyl transfer to dextran must occur almost exclusively through the sequential route. A review of the kinetic constants from steady state dextran synthesis, fructose product inhibition, and fructose isotope exchange showed a consistency in constants derived from each reaction and revealed that dextran binding increases the affinity of sucrose and fructose for dextransucrase.     

Structural characterization of enzymatically synthesized dextran and oligosaccharides from Leuconostoc mesenteroides NRRL B-1426 dextransucrase

Leuconostoc mesenteroides NRRL B-1426 dextransucrase synthesized a high molecular mass dextran (>2 × 10⁶ Da) with ～85.5% α-(1→6) linear and ～14.5% α-(1→3) branched linkages. This high molecular mass dextran containing branched α-(1→3) linkages can be readily hydrolyzed for the production of enzyme-resistant isomalto-oligosaccharides. The acceptor specificity of dextransucrase for the transglycosylation reaction was studied using sixteen different acceptors. Among the sixteen acceptors used, isomaltose was found to be the best, having 89% efficiency followed by gentiobiose (64%), glucose (30%), cellobiose (25%), lactose (22.5%), melibiose (17%), and trehalose (2.3%) with reference to maltose, a known best acceptor. The β-linked disaccharide, gentiobiose, showed significant efficiency for oligosaccharide production that can be used as a potential prebiotic.     

One-step synthesis of isomalto-oligosaccharide syrups and dextrans of controlled size using engineered dextransucrase

Isomalto-oligosaccharides and dextrans of controlled molecular weight of about 10 and 40 kDa were produced using a simple one-step process using engineered L. mesenteroides NRRL B-512F dextransucrase variants. Isomalto-oligosaccharides were produced in a 58% yield by the acceptor reaction with glucose, and reached a degree of polymerization of at least 27 glucosyl units. Reaction conditions for optimal synthesis of dextrans of controlled molecular weight were defined, in respect of initial sucrose concentration and reaction temperature. Thus, we achieved synthesis with impressive yields of 69 and 75% for the 40 and 10 kDa dextran species, respectively. These two dextran sizes are particularly suitable for clinical applications, and are of great industrial demand. Compared with the traditional processes based on chemical hydrolysis and fractionation, which achieve only low yields, the new enzymatic methods offer improvement in quantity, quality and efficiency.     

Reactor optimization for alpha-1,2 glucooligosaccharide synthesis by immobilized dextransucrase

The immobilization of dextransucrase in Ca-alginate beads relies on the close association between dextran polymer and dextransucrase. However, high amounts of dextran in the enzyme preparation drastically limit the specific activity of the immobilized enzyme (4 U/mL of alginate beads). Moreover, even in the absence of diffusion limitation at the batch conditions used, the enzyme behavior is modified by entrapment so that the dextran yield increases and the alpha-1,2 glucooligosaccharides (GOS) are produced with a lower yield (46.6% instead of 56.7%) and have a lower mean degree of polymerization than with the free dextransucrase. When the immobilized catalyst is used in a continuous reaction, the reactor flow rate necessary to obtain high conversion of the substrates is very low, leading to external diffusion resistance. As a result, dextran synthesis is even higher than in the batch reaction, and its accumulation within the alginate beads limits the operational stability of the catalyst and decreases glucooligosaccharide yield and productivity. This effect can be limited by using reactor columns with length to diameter ratio > or =20, and by optimizing the substrate concentrations in the feed solution: the best productivity obtained was 3.74 g. U(-1). h(-1), with an alpha-1,2 GOS yield of 36%.     

Volume changes during enzyme reactions. The influence of pressure on the action of invertase, dextranase and dextransucrase.

The pressure dependence of the maximum velocities and the Michaelis constants for the enzymes invertase and dextranase was measured up to 1400 bar. The corresponding activation volumes deltaV not equal to c and deltaV not equal to Km proved to be independent of pressure. Together with data from other sources the meaning of deltaV not equal to c and deltaV not equal to Km is established and the volume profiles of the reactions are constructed. These profiles are similar in contour to the volume profile of the dextran formation catalyzed by the enzyme dextransucrase, but the amount of the volume changes is very much larger for dextransucrase. The evaluation of salt effects shows, that for all three enzymes solvent interactions are not important in explaining the results. The reaction mechanisms seem to be governed by conformation changes of the enzymes. The larger effects in dextransucrase are explained by the produced dextran chain remaining tightly bound to the enzyme and being transported relative to the enzymes position in each reaction cycle.     

Mechanism of chain initiation by dextransucrase: Absence of terminal ‘sucrose’ linkage in newly synthesized dextran chains

Dextran was synthesized using dextransucrase from Streptococus sanguis 10558 and (F)-[¹⁴C]sucrose as substrate to test the possibility that sucrose may be the initial acceptor for glucose. If sucrose is the initial acceptor, then dextran chains should have [¹⁴C] fructose in a terminal ‘sucrose’ linkage which can be cleaved under mild conditions. Although incorporation of [¹⁴C]fructose into dextran was observed, the label was not released by mild hydrolysis, indicating that sucrose is not the initiator for dextran synthesis. Incorporation of [¹⁴C]fructose into dextran might represent its ability to act as an acceptor, as suggested by the isolation of leucrose as a by-product in the reaction.     

Properties of Leuconostoc mesenteroides B-512FMC constitutive dextransucrase

Leuconostoc mesenteroides B-512FMC, a constitutive mutant for dextransucrase, was grown on glucose, fructose, or sucrose. The amount of cell-associated dextransucrase was about the same for the three sugars at different concentrations (0.6% and 3%). Enzyme produced in glucose medium was adsorbed on Sephadex G-100 and G-200, but much less enzyme was adsorbed when it was produced in sucrose medium. Sephadex adsorption decreased when the glucose-produced enzyme was preincubated with dextrans of molecular size greater than 10 kDa. The release of dextransucrase activity from Sephadex by buffer (20 mM acetate, pH 5.2) was the highest at 28°–30°C. The addition of dextran to the enzyme stimulated dextran synthesis but had very little effect on the temperature or pH stability. Dextransucrase purified by ammonium sulfate precipitation, hydroxyapatite chromatography, and Sephadex G-200 adsorption did not contain any carbohydrate, and it synthesized dextran, showing that primers are not necessary to initiate dextran synthesis. The purified enzyme had a molecular size of 184 kDa on SDS-PAGE. On standing at 4°C for 30 days, the native enzyme was dissociated into three inactive proteins of 65, 62, and 57 kDa. However, two protein bands of 63 and 59 kDa were obtained on SDS-PAGE after heat denaturation of the 184-kDa active enzyme at 100°C. The amount of 63-kDa protein was about twice that of 59-kDa protein. The native enzyme is believed to be a trimer of two 63-kDa and one 59-kDa monomers.     

Entrapment of purified novel dextransucrase obtained from newly isolated Acetobacter tropicalis and its comparative study of kinetic parameters with free enzyme

Abstract

Purified Acetobacter tropicalis dextransucrase was immobilized in different matrices viz. calcium-alginate, κ-carrageenan, agar, agarose and polyacrylamide. Calcium-alginate was proved to be superior to the other matrices for immobilization of dextransucrase enzyme. Standardization of immobilization conditions in calcium-alginate resulted in 99.5% relative activity of dextransucrase. This is the first report with such a large amount of relative activity as compared to the previous reports. The immobilized enzyme retained activity for 11 batch reactions without a decrease in activity which suggested that enzyme can be used repetitively for 11 cycles. The dextransucrase was also characterized, which revealed that enzyme worked best at pH 5.5 and 37?°C for 30?min in both the free as well as immobilized state. Calcium-alginate immobilized dextransucrase of A. tropicalis showed the K_m and V_max values of 29?mM and 5000?U/mg, respectively. Free and immobilized enzyme produced 5.7?mg/mL and 2.6?mg/mL of dextran in 2?L bench scale fermenter under optimum reaction conditions. This immobilization method is very unconventional for purified large molecular weight dextran-free dextransucrase of A. tropicalis as this method is used usually for cells. Such reports on entrapment of purified enzyme are rarely documented.