B-512F Dextransucrase Research Articles

A novel method for covalent immobilization of dextransucrase

B-512F dextransucrase is an industrial enzyme used in commercial production of dextran and prebiotics. Several researchers have studied the covalent immobilization of this enzyme with little success due to dextran masking the reactive groups on the enzyme and inactivation of the enzyme during immobilization. A novel dextransucrase was designed and produced successfully to eliminate problems faced in covalent immobilization. In production of the novel enzyme, B-512F dextransucrase was truncated at N and C terminals and fused to glutathione S-transferase. The novel dextransucrase was fully active and carried out dextran biosynthesis and acceptor reactions effectively. The novel enzyme was immobilized covalently onto Eupergit C 250L, giving rise to 100% immobilization and 83.3% activity yields. Immobilized enzyme was used successfully for the production of acceptor products and low molecular weight dextran. The immobilized enzyme showed no decrease in activity for 15 batch reactions and retained its initial activity at storage (4 °C) for 35 days. Optimal conditions were not affected by the immobilization. The kinetic parameters for the free and immobilized enzyme were determined. The acceptor reactions using fructose, glucose, maltose, and lactose were also studied. The novel method developed could also be used in immobilization of some other biomolecules.

Synthesis and Characterization of a Potential Prebiotic Trisaccharide from Cheese Whey Permeate and Sucrose by Leuconostoc mesenteroides Dextransucrase

The production of new bioactive oligosaccharides is currently garnering much attention for their potential use as functional ingredients. This work addresses the enzymatic synthesis and NMR structural characterization of 2-α-D-glucopyranosyl-lactose derived from sucrose:lactose and sucrose:cheese whey permeate mixtures by using a Leuconostoc mesenteroides B-512F dextransucrase. The effect of synthesis conditions, including concentration of substrates, molar ratio of donor/acceptor, enzyme concentration, reaction time, and temperature, on the formation of transfer products is evaluated. Results indicated that cheese whey permeate is a suitable material for the synthesis of 2-α-D-glucopyranosyl-lactose, giving rise to yields around 50% (in weight respect to the initial amount of lactose) under the optimum reaction conditions. According to its structure, this trisaccharide is an excellent candidate for a new prebiotic ingredient, due to the reported high resistance of α-(1→2) linkages to the digestive enzymes in humans and animals, as well as to its potential selective stimulation of beneficial bacteria in the large intestine mainly attributed to the two linked glucose units located at the reducing end that reflects the disaccharide kojibiose (2-α-D-glucopyranosyl-D-glucose). These findings could contribute to broadening the use of important agricultural raw materials, such as sucrose or cheese whey permeates, as renewable substrates for enzymatic synthesis of oligosaccharides of nutritional interest.

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.

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.

Alternansucrase acceptor products

The regioselectivity of alternansucrase (EC 2.4.1.140) differs from dextransucrase (EC 2.4.1.5) in ways that can be useful for the synthesis of novel oligosaccharide structures. For example, it has been recently shown that the major oligosaccharides produced when maltose is the acceptor include one trisaccharide structure, two tetrasaccharides, one pentasaccharide, two hexasaccharides, one heptasaccharide, and at least two octasaccharides, containing no adjacent α-(1→3) linkages and no more than two consecutive α-(1→6) linkages. This may shed some light on how the enzyme works to produce the alternating structure. Another characteristic of alternansucrase that distinguishes it from dextransucrase is its greater ability to use leucrose as an acceptor. Leucrose, produced by glucosyl transfer to fructose released from the initial sucrose substrate, represents a very poor substrate for Leuconostoc mesenteroides NRRL B-512F dextransucrase. Alternansucrase, however, continues to transfer glucosyl units to leucrose, resulting in some unusual glucosyl-fructose oligosaccharides.

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 (14 kDa 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 2.8 × 10 −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.

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.

Proteolytic modification of Leuconostoc mesenteroides 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.

Proteolytic processing of dextransucrase ofLeuconostoc 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.

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°C, various dextransucrase forms with molecular masses as low as 120 kDa conserving dextransucrase activity were obtained.

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.

Large-scale preparation of highly purified dextransucrase from a high-producing constitutive mutant of Leuconostoc mesenteroides B-512FMC

Conditions for the cultivation of Leuconostoc mesenteroides B-512FMC-16 (formerly L. mesenteroides B-512FMC/6HG8), a high-producing constitutive mutant of dextransucrase, has been studied in a jar fermentor by varying the amount of nitrogen (Bactopeptone and yeast extract) and the amount of carbon (glucose) with pH control. The optimum conditions were found to be 3.76 g l −1 Bactopeptone, 3.76 g l −1 yeast extract, 33.8 g l −1 glucose with the pH controlled at 6.0 for 23 h of fermentation. The mutant produced 20–25 IU ml −1 of the enzyme under the optimum conditions. The enzyme was concentrated using polysulfone ultrafiltration hollow fiber cartridges in the presence of 0.1% Tween 80 and 1 m m CaCl 2. It can be concentrated not only by a hollow fiber cartridge with molecular weight cut-off of 100,000, but also by one with a particle cut-off size of 0.1 μm. The enzyme that was concentrated using the later hollow fiber column gave a pure protein band with a molecular mass of 190 kDa on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and the highest specific activity (183 IU mg −1) ever reported for B-512F dextransucrase. The role of Tween 80 and CaCl 2 in the concentration procedure was studied and it was found that Tween 80 dissociates inactive enzyme aggregates and produces active enzyme molecules. CaCl 2 produces aggregation; in the presence of both Tween 80 and CaCl 2, active enzyme aggregates are formed that do not pass through the hollow fiber membrane, permitting concentration and purification. Using the methods described, B-512FMC dextransucrase can be purified on a 10 g or larger scale using only three operation steps.

Chemical modification of dextransucrase from Leuconostoc mesenteroides NRRL B-512F by pyridoxal 5'-phosphate: evidence for the presence of an essential lysine residue at the active site.

The treatment of Leuconostoc mesenteroides NRRL B-512F dextransucrase with lysine specific reagent, pyridoxal 5'-phosphate (PLP) at pH 5.2 and 30 degrees C resulted in the loss of enzyme activity. The inactivation by PLP could be reversed completely by dilution or dialysis. Sucrose as well as acceptor substrates, glucose and dextran protected the enzyme against inactivation by PLP. A statistical, kinetic analysis of the inactivation by PLP showed that one lysine residue is essential for the enzyme activity. All these results showed that one lysine residue present at the active is essential for the activity of dextransucrase.

Studies on the inactivation of Leuconostoc mesenteroides NRRL B-512F dextransucrase by o-phthalaldehyde: evidence for the presence of an essential lysine residue at the active site.

The kinetics of inactivation of Leuconostoc mesenteroides NRRL B-512F dextransucrase by o-phthalaldehyde showed that the reaction followed pseudo-first order reaction. The loss of enzyme activity was concomitant with an increase in fluorescence at 417 nm indicating that the inhibition involved the reaction of an epsilon-amino and a thiol group of the enzyme leading to the formation of an isoindole derivative. The stoichiometry of inactivation showed that one isoindole derivative was formed per enzyme molecule. The substrates sucrose and glucose provided protection against o-phthalaldehyde inactivation which was also corroborated by fluorescence studies. Dextransucrase was not inactivated by 5,5'-dithiobis(2-nitrobenzoic acid), showing that the cysteine present in close proximity to the lysine is not essential for enzyme activity. Denaturation of dextransucrase by urea or heat treatment prior to o-phthalaldehyde addition resulted in a decrease of fluorescence intensity indicating that the native conformation of the enzyme is essential for isoindole derivative formation. These results established that a lysine residue is present at the active site and is essential for the activity of dextransucrase.

Cloning and sequencing of a gene coding for a novel dextransucrase from Leuconostoc mesenteroides NRRL B-1299 synthesizing only α(1–6) and α(1–3) linkages

The coding region for a Leuconostoc mesenteroides NRRL B-1299 dextransucrase gene ( dsrA) was isolated and sequenced. Using a pair of primers designed on the basis of two highly conserved amino-acid (aa) sequences in L. mesenteroides NRRL B-512F dextransucrase and streptococcal glucosyltransferases (GTFs), a fragment of dsrA was amplified by the polymerase chain reaction (PCR). This PCR product was used as an hybridization probe to isolate a 1.8-kb fragment identified as the central region of dsrA. Cleavage by Sac I of this fragment allowed two probes to be obtained to isolate the 5′ and the 3′ ends of dsrA. The nucleotide sequence of the dsrA gene was determined and found to consist of an open reading frame (ORF) of 4870 base pairs (bp) coding for a 1290-aa protein with an M r of 145 590. The aa sequence exhibited a high similarity with other GTFs. The two domains previously described in GTFs are conserved in DSRA: an N-terminal conserved domain and a C-terminal domain composed of a series of repeats. Surprisingly, the expected signal peptide was not detected. The entire gene was reconstructed and the activity of DSRA was investigated. The dextran produced appeared to be composed of 85% α(1–6) and 15% α(1–3) linkages and the oligosaccharides synthesized in the presence of maltose were mainly composed of α(1–6) linkages. This enzyme is a novel dextransucrase from L. mesenteroides NRRL B-1299 producing no α(1–2) linkages and is the first glucosyltransferase having no signal peptide described.

Fractionation of Leuconostoc mesenteroides NRRL B-512F dextran sucrase by polyethylene glycol: a simple and effective method purification

Leuconostoc mesenteroides NRRL B-512F dextran sucrase (sucrose: 1,6-α- d-glucan 6-α- d-glucosyltransferase EC 2.4.1.5) (1.4 U/ml culture supernatant, specific activity 0.58 U/mg protein) was subjected to fractionation by polyethylene glycol (PEG) of different molecular weights. PEG 400 selectively gave greater specificity of precipitation than PEG of higher molecular weights. In a single step precipitation, at a final concentration of 33% (v/v) PEG 400, the enzyme gave the maximum specific activity (8.7 U/mg protein) and in the three successive steps of precipitation it gave 29 U/mg protein specific activity with 50 fold purification. Multiple forms of the extracellular dextran sucrase were characterized by SDS-polyacryline gel electrophoresis. The enzyme exhibited oligomeric forms containing 64, 126 and 188 kDa proteins. Purified dextran sucrase after three successive steps of fractionation by 33% (v/v) PEG 400 gave a single band of protein corresponding to 188 kDa.

An active-site peptide containing the second essential carboxyl group of dextransucrase from Leuconostoc mesenteroides by chemical modifications

The treatment of Leuconostoc mesenteroides B-512F dextransucrase with 10 mM 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) and glycine ethyl ester (GEE) inactivated the enzyme almost completely within 24 min where the modification of one carboxyl group/mol of the enzyme by EDC was attained. Though 30 mM diethyl pyrocarbonate (DEP) also inactivated the enzyme, about 35% of the activity remained during a 36-min incubation. When 10 mol of imidazole residues/mol of the enzyme was modified by DEP, 50% of the activity was still retained. The addition of the substrate sucrose greatly retarded the enzyme inactivation by EDC. However, the addition of dextran slightly protected the inactivation of the glucosyl-transferring activity and accelerated the inactivation of the sucrose-cleaving activity. In the case of DEP, the addition of sucrose or dextran gave no influence on the inactivation of the enzyme. Therefore, the carboxyl group seemed to play a more important role in the substrate binding and in the catalytic activity of the dextransucrase than the imidazolium group. Differential labeling of Leuconostoc dextransucrase by EDC was conducted in the presence of a sucrose analog, sucrose monocaprate. The fluorescent probe N-(1-naphthyl)ethylenediamine (EDAN) was used as the nucleophile instead of GEE. A fluorescent labeled peptide was isolated from a trypsin digest of the EDC-EDAN modified enzyme. The amino acid sequence of the isolated peptide was Leu-Gln-Glu-Asp-Asn-Ser-Asn-Val-Val-Val-Glu-Ala.(ABSTRACT TRUNCATED AT 250 WORDS)

Large-scale purification of Leuconostoc mesenteriodes NRRL B512F dextransucrase for use in the biosynthesis of dextran by batch and continuous chromatography

Large-scale purification of Leuconostoc mesenteriodes NRRL B512F dextransucrase for use in the biosynthesis of dextran by batch and continuous chromatography

Large-scale purification of Leuconostoc mesenteriodes NRRL B512F dextransucrase for use in the biosynthesis of dextran by batch and continuous chromatography

The extracellular enzyme dextransucrase was produced from Leuconostoc mesenteriodes NRRL B512F and purified by ultracentrifugation and cross-flow ultrafiltration for use in the biosynthesis of the macromolecule dextran by ion exchange chromatographic reaction—separation techniques. The two-stage purification process yielded over 90% pure dextransucrase with overall enzyme recovery of over 60%. A second stage of centrifugation was required to achieve complete cell removal. The purified enzyme contained 1–2 g l −1 of solute ions, which affected the operation of the chromatographic system. Gel filtration removed over 93% of the remaining ions but resulted in high activity losses. Two-phase separation with polyethylene glycol (PEG) and purification by ion exchange chromatography were less successful in desalting the enzyme. PEG precipitation was successful in concentrating the enzyme, but the ions remained predominantly with the enzyme portion of the two phases. The purified enzyme was found to be unstable during storage. Use of the enzyme in chromatographic reactor—separators for the production of dextran resulted in over 33% more high molecular weight dextran (the desired product) and a useful pure fructose byproduct being obtained than for a conventional reactor. Sodium and potassium ions in the enzyme hampered continuous operation by displacing calcium ions from the resin and thus reducing the separation efficiency of the system. Partial regeneration of the resin with calcium nitrate rather than complete enzyme desalting, which was very expensive and resulted in high activity losses, helped overcome this effect.

Specificity of acceptor binding to Leuconostoc mesenteroides B-512F dextransucrase: Binding and acceptor-product structure of α-methyl- d-glucopyranoside analogs modified at C-2, C-3, and C-4 by inversion of the hydroxyl and by replacement of the hydroxyl with hydrogen

The specificity of acceptor binding to the active site of dextransucrase was studied by using α-methyl- d-glucopyranoside analogs modified at C-2, C-3, and C-4 positions by (a) inversion of the hydroxyl group and (b) replacement of the hydroxyl group with hydrogen. 2-Deoxy-α-methyl- d-glucopyranoside was synthesized from 2-deoxyglucose; 3- and 4-deoxy-α-methyl- d-glucopyranosides were synthesized from α-methyl- d-glucopyranoside; and α-methyl- d-allopyranoside was synthesized from d-glucose. The analogs were incubated with [ 14C]sucrose and dextransucrase, and the products were separated by thin-layer chromatography and quantitated by liquid scintillation spectrometry. Structures of the acceptor products were determined by methylation analyses and optical rotation. The relative effectiveness of the acceptor analogs in decreasing order were 2-deoxy, 2-inverted, 3-deoxy, 3-inverted, 4-inverted, and 4-deoxy. The enzyme transfers d-glucopyranose to the C-6 hydroxyl of analogs modified at C-2 and C-3, to the C-4 hydroxyl of 4-inverted, and to the C-3 hydroxyl of 4-deoxy analogs of α-methyl- d-glucopyranoside. The data indicate that the hydroxyl group at C-2 is not as important for acceptor binding as the hydroxyl groups at C-3 and C-4. The hydroxyl group at C-4 is particularly important as it determines the binding orientation of the α-methyl- d-glucopyranoside ring.