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.     

Understanding the polymerization mechanism of glycoside-hydrolase family 70 glucansucrases

Glucan formation catalyzed by two GH-family 70 enzymes, Leuconostoc mesenteroides NRRL B-512F dextransucrase and L. mesenteroides NRRL B-1355 alternansucrase, was investigated by combining biochemical and kinetic characterization of the recombinant enzymes and their respective products. Using HPAEC analysis, we showed that two molecules act as initiator of polymerization: sucrose itself and glucose produced by hydrolysis, the latter being preferred when produced in sufficient amounts. Then, elongation occurs by transfer of the glucosyl residue coming from sucrose to the non-reducing end of initially formed products. Dextransucrase preferentially produces an isomaltooligosaccharide series, whose concentration is always low because of the high ability of these products to be elongated and form high molecular weight dextran. Compared with dextransucrase, alternansucrase has a broader specificity. It produces a myriad of oligosaccharides with various alpha-1,3 and/or alpha-1,6 links in early reaction stages. Only some of them are further elongated. Overall alternan polymer is smaller in size than dextran. In dextransucrase, the A repeats often found in C-terminal domain of GH family 70 were found to play a major role in efficient dextran elongation. Their truncation result in an enzyme much less efficient to catalyze high molecular weight polymer formation. It is thus proposed that, in dextransucrase, the A repeats define anchoring zones for the growing chains, favoring their elongation. Based on these results, a semi-processive mechanism involving only one active site and an elongation by the non-reducing end is proposed for the GH-family 70 glucansucrases.     

Kinetic modeling of oligosaccharide synthesis catalyzed by leuconostoc mesenteroides NRRL B-1299 dextransucrase

The kinetic behavior of soluble and insoluble forms of dextransucrase from Leuconostoc mesenteroides NRRL B-1299 was investigated with sucrose as substrate and maltose as acceptor. To study the parameters involved, a kinetic model was applied that was previously developed for L. mesenteroides NRRL B-512F dextransucrase. There are significant correlations between the parameters of the soluble form of B-1299 dextransucrase and those calculated for the B-512F enzyme; that is, their properties are comparable and differ from those of the insoluble form of B-1299 dextransucrase. Whereas the calculated parameters for high maltose concentrations describe the kinetic behavior very well, the time curves for low maltose concentrations were not described correctly. Therefore, the parameters were calculated separately for the two ranges. Copyright 1999 John Wiley & Sons, Inc.     

Dextran biosynthesis and dextransucrase production by continuous culture of Leuconostoc mesenteroides.

Leuconostoc mesenteroides NRRL B-512(F) was grown in continuous culture under conditions of energy-limited growth. The extracellular enzyme dextransucrase (sucrose: 1,6-alpha-D-glucan 6-alpha-glucosyltransferase EC 2.4.1.5), was not detected in glucose- or maltose-limited cultures. Under conditions of sucrose-limited growth, the enzyme activity of the cell-free culture supernatant increased with increasing dilution rate only after the critical concentration of enzyme inducer (sucrose) in the chemostat had been achieved. The appearance of fructose in the effluent of the sucrose-limited chemostat at higher dilution rates indicated that sucrose was being diverted to dextran biosynthesis. The competition between bacteria and extracellular enzyme for the common substrate sucrose represents an inefficiency in the system of enzyme production. Dextransucrase was isolated from the cell-free culture supernatant by ammonium sulfate precipitation and DEAE-cellulose chromatography. The enzyme preparation exhibited both dextran biosynthetic activity and an invertase-like activity. The biosynthetic efficiency was increased by decreasing the temperature from 30 to 10 degrees C. The enzyme was irreversibly denatured by prolonged incubation in the absence of Ca2+.     

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).     

Characterization of the Different Dextransucrase Activities Excreted in Glucose, Fructose, or Sucrose Medium by Leuconostoc mesenteroides NRRL B-1299

When grown in glucose or fructose medium in the absence of sucrose, Leuconostoc mesenteroides NRRL B-1299 produces two distinct extracellular dextransucrases named glucose glucosyltransferase (GGT) and fructose glucosyltransferase (FGT). The production level of GGT and FGT is 10 to 20 times lower than that of the extracellular dextransucrase sucrose glucosyltransferase (SGT) produced on sucrose medium (traditional culture conditions). GGT and FGT were concentrated by ultrafiltration before sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Their molecular masses were 183 and 186 kDa, respectively, differing from the 195 kDa of SGT. The structural analysis of the dextran produced from sucrose and of the oligosaccharides synthesized by acceptor reaction in the presence of maltose showed that GGT and FGT are two different enzymes not previously described for this strain. The polymer synthesized by GGT contains 30% alpha(1-->2) linkages, while FGT catalyzes the synthesis of a linear dextran only composed of alpha(1-->6) linkages.     

Leuconostoc mesenteroides glucansucrase synthesis of flavonoid glucosides by acceptor reactions in aqueous-organic solvents

The enzymatic glucosylation of luteolin was attempted using two glucansucrases: the dextransucrase from Leuconostoc mesenteroides NRRL B-512F and the alternansucrase from L. mesenteroides NRRL B-23192. Reactions were carried out in aqueous-organic solvents to improve luteolin solubility. A molar conversion of 44% was achieved after 24h of reaction catalysed by dextransucrase from L. mesenteroides NRRL B-512F in a mixture of acetate buffer (70%)/bis(2-methoxyethyl) ether (30%). Two products were characterised by nuclear magnetic resonance (NMR) spectroscopy: luteolin-3'-O-alpha-d-glucopyranoside and luteolin-4'-O-alpha-d-glucopyranoside. In the presence of alternansucrase from L. mesenteroides NRRL B-23192, three additional products were obtained with a luteolin conversion of 8%. Both enzymes were also able to glucosylate quercetin and myricetin with conversion of 4% and 49%, respectively.     

Cloning of a dextransucrase gene (fmcmds) from a constitutive dextransucrase hyper-producing Leuconostoc mesenteroides B-512FMCM developed using VUV

Dextransucrase (FMCMDS) from Leuconostoc mesenteroides B-512FMCM, a dextransucrase constitutive and hyper-producing strain, catalyzes the synthesis of dextran from sucrose. The coding region for fmcmds was isolated and sequenced. It consisted of an open reading frame (ORF) of 4699 bp, coding for a 1527 amino acid protein with a molecular mass of 170 kDa. However, it showed a dextransucrase activity band at 180 kDa in SDS-PAGE. Only one nucleotide changed in the promoter site and two amino acid residues were changed in the structural gene from that of the parent L. mesenteroides NRRL B-512F dsrS; an inducible dextransucrase gene of low productivity.     

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.     

In vitro synthesis of oligosaccharides by acceptor reaction of dextransucrase from Leuconostoc mesenteroides

Glucose was used as acceptor to obtain small chain oligossaccharides from sucrose using dextransucrase from Leuconostoc mesenteroides NRRL B-512F. Better conditions for the synthesis of the oligosaccharides were obtained using experimental design and response surface methodology. Yield of oligosaccharides was increased from 5% to 45% following an increase in both sucrose and glucose/sucrose concentrations, from 58 g/l to 142 g/l and from 0.02 to 0.18, respectively. Molecular weight increased from 2800 to 4500 daltons with a temperature shifting from 10°C to 30°C. © Rapid Science Ltd. 1998     

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 Leuconostoc mesenteroides NRRL B-512F dextransucrase (DSRS) and identification of amino-acid residues playing a key role in enzyme activity

Dextransucrase (DSRS) from Leuconostoc mesenteroides NRRL B-512F is a glucosyltransferase that catalyzes the synthesis of soluble dextran from sucrose or oligosaccharides when acceptor molecules, like maltose, are present. The L. mesenteroides NRRL B-512F dextransucrase-encoding gene (dsrS) was amplified by the polymerase chain reaction and cloned in an overexpression plasmid. The characteristics of DSRS were found to be similar to the characteristics of the extracellular dextransucrase produced by L. mesenteroides NRRL B-512F. The enzyme also exhibited a high homology with other glucosyltransferases. In order to identify critical amino acid residues, the DSRS sequence was aligned with glucosyltransferase sequences and four amino acid residues were selected for site- directed mutagenesis experiments: aspartic acid 511, aspartic acid 513, aspartic acid 551 and histidine 661. Asp-511, Asp-513 and Asp-551 were independently replaced with asparagine and His-661 with arginine. Mutation at Asp-511 and Asp-551 completely suppressed dextran and oligosaccharide synthesis activities, showing that at least two carboxyl groups (Asp-511 and Asp-551) are essential for the catalysis process. However, glucan-binding properties were retained, showing that DSRS has a two-domain structure like other glucosyltransferases. Mutations at Asp-513 and His-661 resulted in greatly reduced dextransucrase activity. According to amino acid sequence alignments of glucosyltransferases, α-amylases or cyclodextrin glucanotransferases, His-661 may have a hydrogen-bonding function. Received: 16 April 1997 / Received revision: 17 June 1997 / Accepted: 23 June 1997     

Enzymatic synthesis and anti-coagulant effect of salicin analogs by using the Leuconostoc mesenteroides glucansucrase acceptor reaction

Glucansucrases from Leuconostoc mesenteroides catalyze the transfer of glucosyl units from sucrose to other carbohydrates by acceptor reaction. We modified salicyl alcohol, phenol and salicin by using various glucansucrases and with sucrose as a donor of glucosyl residues. Salicin, phenyl glucose, isosalicin, isomaltosyl salicyl alcohol, and a homologous series of oligosaccharides, connected to the acceptors and differing from one another by one or more glucose residues, were produced as major reaction products. By using salicin and salicyl alcohol as acceptors, B-1355C2 and B-1299CB-BF563 dextransucrases synthesized most widely diverse products, producing more than 12 and 9 different kinds of saccharides, respectively. With phenol, two acceptor products and oligosaccharides were synthesized by using the B-1299CB-BF563 dextransucrase. Salicyl derivatives, as acceptor products, showed higher anti-coagulation activity compared with that of salicin or salicyl alcohol that were used as acceptors.     

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.     

A facile purification of Leuconostoc mesenteroides B-512FM dextransucrase

Leuconostoc mesenteroides NRRL B-512F has been mutated by treatment with N-nitrosoguanidine. The resulting mutant (designated as B-512FM) produces 300 times as much enzyme as the parent strain. B-512FM dextransucrase was treated extensively with Sigma crude dextranase, followed by column chromatography on Bio-Gel A-5m. The purified dextransucrase had a specific activity of 84 IU/mg, a 100-fold purification with 42% yield, and was shown by SDS-PAGE to have a single protein of molecular weight of 158,000 with dextransucrase activity. The procedure has been used to produce purified enzyme for sequencing. The molecular weight of 158,000 agrees with that calculated from its amino acid sequence.     

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.     

A novel family of glucosyl 1,5-anhydro-d-fructose derivatives synthesised by transglucosylation with dextransucrase from Leuconostoc mesenteroides NRRL B-512F

1,5-Anhydro-d-fructose (AF), a metabolite of starch/glycogen degradation, is a good antioxidant. With the prospect of increasing its applications and use as a food ingredient, AF glucosylation catalysed by the dextransucrase from Leuconostoc mesenteroides NRRL B-512F was performed in the presence of sucrose. This led to AF glucosylated derivatives containing alpha-(1-->6) linkages named 1,5-anhydro-d-fructo-glucooligosaccharides (AFGOS). LC-MS analyses showed that AFGOS with a degree of polymerisation (DP) of up to 7 were synthesised. The amount of AFGOS produced and the average DP increased by using a high sucrose/AF molar ratio and high total sugar concentration. AFGOS were proved to present antioxidant properties quite similar to AF.     

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.     

Immobilization of native and dextran-free dextransucrases from Leuconostoc mesenteroides NRRL B-512F for the synthesis of glucooligosaccharides

Dextransucrase from Leuconostoc mesenteroides NRRL B-512F was immobilized using two different methods: covalent attachment to activated silica and entrapment in calcium alginate. For immobilization on silica, native enzyme and dextran-free enzyme were compared. However, the entrapment in calcium alginate beads gave the best results in terms of immobilization yield and stability. This biocatalyst was employed in the acceptor reaction with maltose showing similar glucooligosaccharide production than the native enzyme but increased operational stability.     

Inactivation of Leuconostoc Mesenteroids NRRL B-512F Dextransucrase by Specific Modification of Lysine Residues with Pyridoxal-5′-Phosphate

Abstract

Dextransucrase from Leuconostoc mesentwoides NRRL B-512F was inactivated by pyridoxal-5′-phosphate (PLP). The inactivation was reversible in as much as the loss of enzyme activity was completely reversed by prolonged dialysis. PLP-modified dextransucrase after reduction with sodium borohydride showed a characteristic fluorescence emission maximum at 397 nm when excited at 325 nm. The stoichiometric results indicated that four lysine residues are modified by PLP under the experimental conditions. These results established for the first time that lysine residues are essential for the activity of dextransucrase.