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.     

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.     

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.     

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.     

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.     

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.     

Acceleration of Dextransucrase Activity of Streptococcus mutans by Secretory Immunoglobulin A

The effect of immunoglobulins on the activity of dextransucrase purified from Streptococcus mutans strain HS-6 is described. When human salivary immunoglobulin A (IgA) or colostral IgA, either natured or denatured, was incubated with dextransucrase, the rate of the dextran synthesis was markedly accelerated, whereas human serum IgA or IgG neither accelerated nor inhibited the enzyme activity. The results suggest that a portion unique for secretory IgA, the secretory component, might be related to the enzyme acceleration. On the other hand, specific rabbit antiserum against the dextransucrase inhibited completely dextran synthesis by the enzyme.     

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.     

Dextransucrase constitutive mutants of Leuconostoc mesenteroides B-1299

Leuconostoc mesenteroides B-1299 dextrans are separated into two kinds: fraction L, which is precipitated by an ethanol concentration of 38%, and fraction S, which is precipitated at an ethanol concentration of 40%. Fraction S dextran contained 35% of -1,2 branch linkages, and fraction L contained 27% -1,2 branch linkage with 1% -1,3 branch linkages. We have isolated mutants constitutive for dextransucrase from L. mesenteroides NRRL B-1299 using ethyl methane sulfonate. The mutants produced extracellular as well as cell-associated dextransucrases on glucose media with higher activities (2.5–4.5 times) than what the parental strain produced on sucrose. Based on Penicillium endo-dextranase hydrolysis, mutant B-1299C dextransucrases produced slightly different dextrans when they were elaborated on a glucose medium and on a sucrose medium. Mutant B-1299CA dextransucrase elaborated on a glucose medium and on a sucrose medium synthesized the same dextran, although the dextran was different from those of other mutants and the parental strain. Mutant B-1299CB dextransucrase, elaborated on a glucose medium and on a sucrose medium, formed different dextrans. Differences in water solubility, susceptibility to endo-dextranase hydrolysis, and the physical appearance of the ethanol precipitated dextrans elaborated by different mutants grown on glucose media and sucrose media were found. All mutant dextransucrases elaborated on a glucose medium bound to Sephadex G-200. After activity staining of nondenaturing sodium dodecyl sulfate—polyacrylamide gel electrophoresis activity bands, 184 and 240 Kd for each enzyme preparation, although each dextransucrase formed different dextran(s).     

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.     

Proteolytic processing of dextransucrase of Leuconostoc mesenteroides

Various dextransucrase molecular mass forms found in enzyme preparations may sometimes be products of proteolytic activity. Extracellular protease in Leuconostoc mesenteroides strains NRRL B-512F and B-512FMC dextransucrase preparations was identified. Protease had a molecular mass of 30 kDa and was the predominant form derived from a high molecular mass precursor. The production and activity of protease in culture medium was strongly dependent on pH. When L. mesenteroides dextransucrase (173 kDa) was hydrolyzed by protease, at pH 7 and 37 degrees C, various dextransucrase forms with molecular masses as low as 120 kDa conserving dextransucrase activity were obtained.     

Effect of dextran and ammonium sulphate on the reaction catalysed by a glucosyltransferase complex from Streptococcus mutans

The highly aggregated proteins precipitated by (NH4)2SO4 from the culture fluid of three strains of Streptococcus mutans gradually released less aggregated glucosyltransferase activities - dextransucrase and mutansucrase - which catalysed the synthesis of water-soluble and insoluble glucans from sucrose. Mutansucrase was eluted from a column of Sepharose 6B before dextransucrase. This activity was lost during subsequent dialysis and gel filtration, but there was a corresponding increase in dextransucrase activity which catalysed the formation of soluble glucan when incubated with sucrose alone, and insoluble glucan when incubated with sucrose and 1.55 M-(NH4)2SO4. Relative rates of synthesis of soluble and insoluble glucan in the presence of 1.55 M-(MH4)2SO4 were dependent upon the enzyme concentration: high concentrations favoured insoluble glucan synthesis. Insoluble glucans synthesized by mutansucrase or by dextransucrase in the presence of 1.55 M-(NH4)2SO4 were more sensitive to hydrolysis by mutanase than by dextranse, but soluble glucans were more extensively hydrolysed by dextranase than by mutanase. Partially purified dextransucrase sedimented through glycerol density gradients as a single symmetrical peak with an apparent molecular weight in the range 100000 to 110000. In the presence of 1.55 M-(NH4)2SO4, part of the activity sedimented rapidly as a high molecular weight aggregate. The results strongly suggest that soluble and insoluble glucans are synthesized by interconvertible forms of the same glucosyltransferase. The aggregated form, mutansucrase, preferentially catalyses (1 leads to 3)-alpha bond formation but dissociates during gel filtration to the dextransucrase form which catalyses (1 leads to 6)-alpha bond formation.     

Stabilization of dextransucrase from Leuconostoc mesenteroides NRRL B-512F by nonionic detergents, poly(ethylene glycol) and high-molecular-weight dextran

Dextransucrase (sucrose: 1,6-alpha-D-glucan 6-alpha-D-glucosyltransferase, EC 2.4.1.5) (3 IU/ml culture supernatant) was obtained by a modification of the method of Robyt and Walseth (Robyt, J.F. and Walseth, T.F. (1979) Carbohydr. Res. 68, 95-111) from a nitrosoguanidine mutant of Leuconostoc mesenteroides NRRL B-512F selected for high dextransucrase production. Dialyzed, concentrated culture supernatant (crude enzyme) was treated with immobilized dextranase (EC 3.2.1.11) and chromatographed on a column of Bio-Gel A-5m. The resulting, purified enzyme lost activity rapidly at 25 degrees C or on manipulation, as did the crude enzyme when diluted below 1 U/ml. Both enzyme preparations could be stabilized by low levels of high-molecular-weight dextran (2 micrograms/ml), poly(ethylene glycol) (e.g., 10 micrograms/ml PEG 20 000), or nonionic detergents (e.g., 10 micrograms/ml Tween 80). The stabilizing capacity of poly(ethylene glycol) and of dextran increased with molecular weight. Calcium had no stabilizing action in the absence of other additions, but reduced the inactivation that occurred in the presence of 0.5% bovine serum albumin or high concentrations (greater than 0.1%) of Triton X-100. In summary, dextransucrase could be stabilized against activity losses caused by heating or by dilution through the addition of low concentrations of nonionic polymers (dextran, PEG 20000, methyl cellulose) or of nonionic detergents at or slightly below their critical micelle concentrations.     

Gene encoding a dextransucrase-like protein in Leuconostoc mesenteroides NRRL B-512F

A gene, dsrT, encoding a dextransucrase-like protein was isolated from the genomic DNA libraries of Leuconostoc mesenteroides NRRL B-512F dextransucrase-like gene. The gene was similar to the intact open reading frames of the dextransucrase gene dsrS of L. mesenteroides NRRL B-512F, dextransucrase genes of strain NRRL B-1299 and streptococcal glucosyltransferase genes, but was truncated after the catalytic domain, apparently by the deletion of five nucleotides. dsrT mRNA was produced in this strain L. mesenteroides when cells were grown in a sucrose medum, but at a level of 20% of that of dsrS mRNA. The molecular weight of the dsrT gene product was 150,000 by SDS-PAGE. The product did not synthesize dextran, but had weak sucrose cleaving activity. The insertion of five nucleotides at the putative deletion point in dsrT resulted in an enzyme with a molecular weight of 210,000 and with dextransucrase activity.     

The mechanism of acceptor reactions of leuconostoc mesenteroides B-512F dextransucrase

Reactions of dextransucrase and sucrose in the presence of sugars (acceptors) of low molecular weight have been observed to give a dextran of low molecular weight and a series of oligosaccharides. The acceptor reaction of dextransucrase was examined in the absence and presence of sucrose by using d-[¹⁴C]glucose, d-[¹⁴C]fructose, and ¹⁴C-reducing-end labeled maltose as acceptors. A purified dextransucrase was pre-incubated with sucrose, and the resulting d-fructose and unreacted sucrose were removed from the enzyme by chromatography on columns of Bio-Gel P-6. The enzyme, which migrated at the void volume, was collected and referred to as “charged enzyme”. The charged enzyme was incubated with ¹⁴C-acceptor in the absence of sucrose. Each of the three acceptors gave two fractions of labeled products, a high molecular weight product, identified as dextran, and a product of low molecular weight that was an oligosaccharide. It was found that all three of the acceptors were incorporated into the products at the reducing end. Similar results were obtained when the reactions were performed in the presence of sucrose, but higher yields of labeled products were obtained and a series of homologous oligosaccharides was produced when d-glucose or maltose was the acceptor. We propose that the acceptor reaction proceeds by nucleophilic displacement of glucosyl and dextranosyl groups from a covalent enzyme-complex by a specific, acceptor hydroxyl group, and that this reaction effects a glycosidic linkage between the d-glucosyl and dextranosyl groups and the acceptor. We conclude that the acceptor reactions serve to terminate polymerization of dextran by displacing the growing dextran chain from the active site of the enzyme; the acceptors, thus, do not initiate dextran polymerization by acting as primers.     

Origin and function of the multiple extracellular glucosyltransferase species from cultures of a serotype c strain of Streptococcus mutans

Two methods were used to purify the bifunctional extracellular enzyme sucrose: (1-6)- and (1-3)-alpha-D-glucan-6-alpha-D-glucosyltransferase (EC 2.4.1.5; dextransucrase) from continuous cultures of a serotype c strain of Streptococcus mutans. The first method, based on a previously published report, involved Sepharose 6B gel filtration and DEAE cellulose anion exchange chromatography. This resulted in a dextransucrase preparation with an apparent molecular mass of 162 kDa and a specific activity of 125 mg of glucan formed from sucrose h-1 (mg of protein)-1, at 37 degrees C. It was almost homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The ratio of carbohydrate to protein was 0.14 and the recovery was 14% relative to the total glucosyltransferase activity in the original culture fluid. In the subsequently preferred method, hydroxyapatite-Ultrogel was used to purify dextransucrase with a 24% yield. The specific activity, 197 mg of glucan formed h-1 (mg of protein)-1, was the highest yet reported and this preparation contained less than 0.5 glucose-equivalent per subunit of molecular mass 162 kDa. Dextransucrase is therefore not a glycoprotein. Exogenous dextran stimulated activity, but was not essential for activity. The purified protein slowly degraded to multiple lower molecular mass forms during storage at 4 degrees C and 87% of the activity was lost after 20 days. The molecular mass of the most prominent, active degradation product was 140 kDa, similar to that of one of the multiple forms of dextransucrase detected in other laboratories. Preparations in which either the 140-kDa or the 162-kDa species predominated catalyzed the synthesis of a water-soluble glucan with sucrose alone, but catalyzed that of an insoluble glucan with sucrose and a high concentration of either (NH4)2SO4 or polyethylene glycol. The water-insoluble glucan was shown to lack sequences of 1,3-alpha-linked glycosyl residues typical of the insoluble glucan, mutan, which has been implicated in dental caries. We conclude that mutan is synthesized by the concerted action of two independent glucosyltransferases rather than by interconvertible forms of a single enzyme, as was proposed previously.     

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.     

Purification and some properties of free and cell-associated dextransucrase from Streptococcus sanguis.

Dextransucrase of Streptococcus sanguis occurred in cell-free and cell-associated forms. Cell-free dextransucrase was purified by four successive chromatographies on Bio-Gel P 60, DEAE-cellulose, and Bio-Gel P 200 from the culture supernatant. The purification of cell-associated dextransucrase was made from the pellet of Streptococcus sanguis culture. Bacterial pellet was extracted with 1 M phosphate buffer (pH 6.0) and chromatographied by using an immunosorbent column. The two enzymes gave single bands in polyacrylamide gel electrophoresis. The molecular weight determined by sodium dodecyl sulfate polyacrylamide gel was about 100 000 daltons for the two forms of dextransucrases. The optimum pH of the cell-free and cell-associated enzymes was around 6 and the temperature optimum was broad for the two enzymes. The KM values for sucrose were respectively 2 mM and 3 mM for cell-free and cell-associated enzymes. When primer dextran was added, the reaction velocity increased but the KM for sucrose remained the same, and the KA for dextran was 200 muM for the two dextransucrases. Trehalose and maltose acted also as glucosyl residue acceptors. Purified enzymes had dextran synthesising activity and invertase-like activity. The same properties of the two forms of enzymes and the positive cross reaction against anti free and anti cell-associated globulins stongly suggest the identity of the two enzymes.     

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.