Streptococcus mutans Dextransucrase: Requirement for Primer Dextran

Dextran stimulation (priming) of the dextransucrase (EC 2.4.1.5) from Streptococcus mutans strain 6715 was studied. The dextransucrase activity in supernatant fluids from glucose-grown cultures was shown to be partially primer dependent. During extended storage at 4 C the enzyme retained its activity. However, the ability to make dextran became increasingly primer dependent. Hydroxylapatite-chromatographed enzyme preparations were completely dependent upon added dextran for rapid synthesis of methanol-insoluble glucan from sucrose. Half-maximal stimulation of new dextran synthesis occurred with dextran at a concentration of 2 to 3 muM and with a molecular weight of about 2,600. Neither glycogen, amylose, inulin, nor isomaltose functioned as primer. Studies with the dextransucrase activities detectable by in situ assay in polyacrylamide gels subjected to electrophoresis under nondenaturing conditions revealed that the major activity was detectable in the presence of sucrose alone and was stimulated by addition of primer dextran. The minor activity was only detected when primer dextran was present. Homogeneous preparations of both enzymes contained 30 to 40% carbohydrate.     

Effectors Differently Modulating the Dextransucrase Activity of Leuconostoc mesenteroides

Effects of various compounds on the dextransucrase (EC 2.4.1.5) from Leuconostoc mesenteroides was evaluated based on the two catalytic activities of enzyme, that is the hydrolase activity for the substrate, sucrose, and the transferase activity of a d-glucosyl group to an acceptor molecule. The effectors were grouped into six categories by their activation or inhibition of the sucrase and transferase activities of dextransucrase. Type I-A inhibited both activities, type I-B inhibited the sucrase activity, and type I-C inhibited the transferase activity. Type A-A activated both the hydrolase and transferase, and type A-B activated only the transferase. Antagonistic modulation (type IA-A), was shown by methyl α-d-glucoside and glycerol, which activated the sucrase and inhibited the transferase. A double reciprocal plot for dextran gave a biphasic pattern which led to Ki values for each limb. Based on the biphasic kinetics and the action of antagonistic effectors, the regulation of dextran synthesis was discussed.     

Milligram to gram scale purification and characterization of dextransucrase from Leuconostoc mesenteroides NRRL B-512F

A sequence of dextranase treatment, DEAE-cellulose chromatography, affinity chromatography on Sephadex G-200, and chromatography on DEAE-Trisacryl M has been optimized to give a dextransucrase preparation with low carbohydrate content (1-100 micrograms/mg protein) and high specific activity (90-170 U/mg protein) relative to previous procedures, in 30-50% yield. Levansucrase was absent after DEAE-cellulose chromatography, and dextranase was undetectable after Sephadex G-200 chromatography. The method could be scaled up to produce gram quantities of purified enzyme. The purified dextransucrase had a pH optimum of 5.0-5.5, a Km of 12-16 mM, and produced the same lightly branched dextran as before purification. The purified enzyme was not activated by added dextran, but the rate of dextran synthesis increased abruptly during dextran synthesis at a dextran concentration of approximately 0.1 mg/mL. The enzyme had two major forms, of molecular weight 177,000 and 158,000. The 177,000 form predominated in fresh preparations of culture supernatant or purified enzyme, whereas the amount of the 158,000 form increased at the expense of the 177,000 form during storage of either preparation.     

Electrophoretic analysis of the multiple forms of dextransucrase from Leuconostoc mesenteroides

Multiple forms of the extracellular dextransucrase [EC 2.4.1.5] from Leuconostoc mesenteroides NRRL B-512F strain were characterized by polyacrylamide gel electrophoresis. Based on the Rm (Relative mobility) values, a newly devised simple plot of log (Rm X 10/(1-Rm)) vs. degree of association of the enzyme showed a good correlation with the results obtained by the Hedrick-Smith method. Both results indicated that the B-512F dextransucrase aggregates were a mixture of two types of forms, i.e., oligomers of a 65 kDa protomer and their charge isomers. Boiling and treatment of the enzyme at pH 10.5 suggested that enzyme aggregates contained dextran or its fragments bound to the enzyme and the enzyme-dextran complex showed the charge isomerism. Since the highly aggregated forms showed higher activity for dextran synthesis than the dissociated forms, the endogenous dextran may serve as a source of primer and may stabilize the enzyme molecule. Besides allosteric regulation of the activity, the occurrence of oligomeric forms of the enzyme may play an important role in the control of dextran synthesis in vivo.     

Influence of secretory immunoglobulin A and purified secretory component on dextran-sucrase activity of Streptococcus mutans.

The net stimulation of dextransucrase EC 2.4.1.5) activity from Streptococcus mutans HS6 by dextran, secretory immunoglobulin A, or secretory component was investigated. Approximately equal stimulation resulted from treatment with these three components.     

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.     

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.     

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.     

Activation and inhibition of dextransucrase by calcium

Initial rate kinetics of dextran synthesis by dextransucrase (sucrose:1,6-alpha-D-glucan-6-alpha-D-glucosyltransferase, EC 2.4.1.5) from Leuconostoc mesenteroides NRRL B-512F showed that below 1 mM, Ca2+ activated the enzyme by increasing Vmax and decreasing the Km for sucrose. Above 1 mM, Ca2+ was a weak competitive inhibitor (Ki = 59 mM). Although it was an activator at low concentration, Ca2+ was not required for dextran synthesis, either of main chain or branch linkages. Neither was it required for sucrose hydrolysis, acceptor reactions, or enzyme renaturation after SDS-polyacrylamide gel electrophoresis. A model for dextran synthesis is proposed in which dextransucrase has two Ca2+ sites, one activating and one inhibitory. Ca2+ at the inhibitory site prevents the binding of sucrose.     

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.     

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.     

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%.     

Characterization of dextransucrase immobilized on calcium alginate beads from Leuconostoc mesenteroides PCSIR-4

Immobilization of dextransucrase from Leuconostoc mesenteroides PCSIR-4 on alginate is optimized for application in the production of dextran from sucrose. Dextransucrase was partially purified by ethanol upto 2.5 fold. Properties of dextransucrase were less affected by immobilization on alginate beads from soluble enzyme. Highest activities of both soluble and immobilized dextransucrase found to be at 35 degrees C and optimum pH for activity remain 5.00. Substrate maxima for immobilized enzyme changed from 125 mg/ml to 200 mg/ml. Incubation time for enzyme-substrate reaction for maximum enzyme activity was increased from 15 minutes to 60 minutes in case of immobilized enzyme. Maximum stability of immobilized dextransucrase was achieved at 25 degrees C with respect to time.     

Production,purification, and properties of dextransucrase from Leuconostoc mesenteroides NRRL B-512F

The production of dextransucrase fromLeuconostoc mesenteroides NRRL B-512F was stimulated 2-fold by the addition of 0.005% of calcium chloride to the medium; levansucrase levels were unaffected. Dextransucrase was purified by concentration and dialysis of the culture supernatant with a Bio-Fiber 80 miniplant, and by treatment with dextranase followed by chromatography on Bio-Gel A-5m. A 240-fold purification, with a specific activity of 53 U/mg, was obtained. Contaminating enzyme activities of levansucrase, invertase, dextranase, glucosidase, and sucrose phosphorylase were decreased to non-detectable levels. Poly(acrylamide)-gel electrophoresis of the purified enzyme showed only two protein bands, both of which had dextransucrase activity. These bands also gave a carbohydrate stain, indicating that the dextransucrase could be a glycoprotein. Acid hydrolysis, followed by paper chromatography, of the purified enzyme showed that the major carbohydrate was mannose. ConcanavaIin A completely removed dextransucrase activity from solution, confirming the mannoglycoprotein character of the enzyme. Dextransucrase activity was not altered by the addition of 0.008?4 mg/ml of dextran, but its storage stability was increased by the addition of 4 mg/ml of dextran. As previously shown by others, the activity of dextransucrase was decreased by EDTA, and was restored by the addition of calcium ions. Zinc, cadmium, lead, mercury, and copper ions were inhibitory to various degrees.     

An overview of purification methods of glycoside hydrolase family 70 dextransucrase

The enzyme dextransucrase (sucrose:1, 6-α-D-glucan 6-α-glucosyltransferase, EC 2.4.1.5) catalyses the synthesis of exopolysaccharide, dextran from sucrose. This class of polysaccharide has been extensively exploited in pharmaceutical industry as blood volume expander, as stabiliser in food industry and as a chromatographic medium in fine chemical industry because of their nonionic nature and stability. Majority of the dextrans are synthesized from sucrose by dextransucrase secreted mainly by bacteria belonging to genera Leuconostoc, Streptococcus and Lactobacillus. Bulk of the information on purification of extracellular dextransucrase has been generated from Leuconostoc species. Various methods such as precipitation by ammonium sulphate, ethanol or polyethylene glycol, phase partitioning, ultrafiltration and chromatography have been used to purify the enzyme. Purification of dextransucrase is rendered difficult by the presence of viscous dextran in the medium. However, processes like ultra-filtration, salt and PEG precipitation, chromatography and phase partitioning have been standardized and successfully used for higher scale purification of the enzyme. A recombinant dextransucrase from Leuconostoc mesenteroides B-512F with a histidine tag has been expressed in E. coli cells and purifi ed by immobilized metal ion chromatography. This review reports the available information on purifi cation methods of dextransucrase from Leuconostoc mesenteroides strains.     

Structural Characteristics of Dextrans Synthesized by Dextransucrases from Leuconostoc mesenteroides NRRL B-1299

Four major dextransucrase (EC 2.4.1.5) preparations from Leuconostoc mesenteroides were studied in relation to their reaction products. The extracellular enzyme II, a highly aggregated form of enzyme I, synthesized the largest amount of dextran per 1 unit of enzyme. Moreover, this dextran emerged at the void volume by Sepharose 6B chromatography. Dextran produced by the enzyme I was composed almost exclusively of water-soluble form having a molecular weight (MW) smaller than that of the product with enzyme II. Although soluble dextran produced by the intracellular enzyme (enzyme III or IV) had a low MW, ratio of insoluble dextran to total dextran was higher than that of the products with extracellular enzyme. Dextran produced by the enzyme II contained a large amount of non-α-l,6-linkages whereas dextran produced by the enzyme I was rich in linear α-l,6-linked structure. The structural analyses of various dextrans showed that each enzyme seemed to be responsible for the synthesis of both α-1,6 and non-α-l,6-linkages. Difference in the amounts and structures of dextrans suggests that the extracellular enzymes may play a major role for the dextran synthesis in vivo.     

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.     

The mechanism of dextransucrase action: Activation of dextransucrase from Streptococcus mutans OMZ 176 by dextran and modified dextran and the nonexistence of the primer requirement for the synthesis of dextran

Streptococcus mutans OMZ 176 was grown in a sucrose-free medium containing fructose as a carbohydrate source. Dextransucrase was precipitated from the culture supernatant with 40% saturated ammonium sulfate. The activity of dextransucrase was shown to be stimulated by exogenous dextran. Maximum activity was reached when the concentration of exogenous dextran was 2 mg/ml. Dextrans modified at the nonreducing ends by reaction with tripsyl chloride and/or by hydrolysis with an exodextranase also activated dextransucrase four to six times over that of a control. The exodextranasemodified dextrans have nonreducing chains that are very short in comparison with unmodified dextran and the tripsyl-modified dextrans have chains that are blocked at the nonreducing ends with a triisopropylbenzenesulfonyl group on the C₆ hydroxyl group. Because the nonreducing ends of the modified dextrans are not available for reaction, the activation of dextransucrase by these modified dextrans cannot be due to primer reactions with the nonreducing ends. The activation of dextransucrase, thus, must be by an alternate mechanism. Two alternative mechanisms discussed are an allosteric effect and nucleophilic displacement reactions by the added dextran. It was also found that the addition of increasing amounts of dextran shifted the synthesis from an insoluble dextran to a soluble dextran.     

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.     

Regulation of glucosyl- and fructosyltransferase synthesis by continuous cultures of Streptococcus mutans

Streptococcus mutans strains Ingbritt, and its derivative B7 which had been passaged through monkeys, have been used to investigate how the synthesis of extracellular glucosyl- and fructosyltransferases is regulated. The most active enzyme from carbon-limited continuous cultures was a fructosyltransferase; enzymes catalysing the formation of water-insoluble glucans from sucrose were relatively inactive. Dextransucrase (EC 2.4.1.5), which catalyses soluble glucan synthesis, was most active in the supernatant fluid from cultures grown with excess glucose, fructose or sucrose, but full activity was detected only when the enzyme was incubated with both sucrose and dextran. Little dextransucrase activity was detected in carbon-limited cultures. It is concluded that glucosyl- and fructosyltransferases are constitutive enzymes in that they are synthesized at similar rates during growth with an excess of the substrate or of the products of the reactions which they catalyse. Although the Ingbritt strain was originally isolated from a carious lesion, it is now a poor source of glucosyltransferase activity. Glucosyltransferases were extremely active in cultures of a recent clinical isolate, strain 3209, and were apparently induced during growth with excess glucose.