Leuconostoc Mesenteroides NRRL B-1299 Research Articles

Dextran: Sources, Structures, and Properties

Dextran is an exopolysaccharide (EPS) synthesized by lactic acid bacteria (LAB) or their enzymes in the presence of sucrose. Dextran is composed of a linear chain of d-glucoses linked by α-(1→6) bonds, with possible branches of d-glucoses linked by α-(1→4), α-(1→3), or α-(1→2) bonds, which can be low (<40 kDa) or high molecular weight (>40 kDa). The characteristics of dextran in terms of molecular weight and branches depend on the producing strain, so there is a great variety in its properties. Dextran has commercial interest because its solubility, viscosity, and thermal and rheological properties allow it to be used in food, pharmaceutical, and research areas. The aim of this review article is to compile the latest research (in the past decade) using LAB to synthesize high or low molecular weight dextran. In addition, studies using modified enzymes to produce dextran with specific structural characteristics (molecular weights and branches) are addressed. On the other hand, special attention is paid to LAB extracted from unconventional sources to expose their capacities as dextran producers and their possible application to compete with the only commercial strain (Leuconostoc mesenteroides NRRL B512).

Functional and Structural Characterization of α-(1→2) Branching Sucrase Derived from DSR-E Glucansucrase

ΔN(123)-glucan-binding domain-catalytic domain 2 (ΔN(123)-GBD-CD2) is a truncated form of the bifunctional glucansucrase DSR-E from Leuconostoc mesenteroides NRRL B-1299. It was constructed by rational truncation of GBD-CD2, which harbors the second catalytic domain of DSR-E. Like GBD-CD2, this variant displays α-(1→2) branching activity when incubated with sucrose as glucosyl donor and (oligo-)dextran as acceptor, transferring glucosyl residues to the acceptor via a ping-pong bi-bi mechanism. This allows the formation of prebiotic molecules containing controlled amounts of α-(1→2) linkages. The crystal structure of the apo α-(1→2) branching sucrase ΔN(123)-GBD-CD2 was solved at 1.90 Å resolution. The protein adopts the unusual U-shape fold organized in five distinct domains, also found in GTF180-ΔN and GTF-SI glucansucrases of glycoside hydrolase family 70. Residues forming subsite -1, involved in binding the glucosyl residue of sucrose and catalysis, are strictly conserved in both GTF180-ΔN and ΔN(123)-GBD-CD2. Subsite +1 analysis revealed three residues (Ala-2249, Gly-2250, and Phe-2214) that are specific to ΔN(123)-GBD-CD2. Mutation of these residues to the corresponding residues found in GTF180-ΔN showed that Ala-2249 and Gly-2250 are not directly involved in substrate binding and regiospecificity. In contrast, mutant F2214N had lost its ability to branch dextran, although it was still active on sucrose alone. Furthermore, three loops belonging to domains A and B at the upper part of the catalytic gorge are also specific to ΔN(123)-GBD-CD2. These distinguishing features are also proposed to be involved in the correct positioning of dextran acceptor molecules allowing the formation of α-(1→2) branches.

Transglucosylation potential of six sucrose phosphorylases toward different classes of acceptors

In this study, the transglucosylation potential of six sucrose phosphorylase (SP) enzymes has been compared using eighty putative acceptors from different structural classes. To increase the solubility of hydrophobic acceptors, the addition of various co-solvents was first evaluated. All enzymes were found to retain at least 50% of their activity in 25% dimethylsulfoxide, with the enzymes from Bifidobacterium adolescentis and Streptococcus mutans being the most stable. Screening of the enzymes’ specificity then revealed that the vast majority of acceptors are transglucosylated very slowly by SP, at a rate that is comparable to the contaminating hydrolytic reaction. The enzyme from S. mutans displayed the narrowest acceptor specificity and the one from Leuconostoc mesenteroides NRRL B1355 the broadest. However, high activity could only be detected on l-sorbose and l-arabinose, besides the native acceptors d-fructose and phosphate. Improving the affinity for alternative acceptors by means of enzyme engineering will, therefore, be a major challenge for the commercial exploitation of the transglucosylation potential of sucrose phosphorylase.

Characterization of the Dextran Produced by Leuconostoc mesenteroides from Date Fruit Extract

In the present study, the production of dextran from date extract and sucrose as carbon sources by the bacterium Leuconostoc mesenteroides NRRL B512 (f) was investigated. In comparison to blue dextran (Mw≈2000 Da), dextran molecular weight was reduced (Mw2000 KDa). Flow behavior indices of dextran solutions from date extracts were investigated and rheological parameters were evaluated at concentrations of 1.5, 2.5, 5, together with 10% total solids at 25˚C using a Brookfield rotational viscometer as well as a cone and plate geometry. The experimental results followed the power law model for the best fit and the values of flow behavior index (n) were less than unity (0.49 .93) at all concentrations, revealing the shear thinning nature of the produced dextran. Furthermore, dependence of apparent viscosity on concentration was confirmed using the power law model.

Synthesis of dextrans with controlled amounts of α-1,2 linkages using the transglucosidase GBD–CD2

GBD-CD2 is an alpha-1,2 transglucosidase engineered from DSR-E, a glucansucrase naturally produced by Leuconostoc mesenteroides NRRL B-1299. This enzyme catalyses from sucrose, the alpha-1,2 transglucosylation of glucosyl moieties onto alpha-1,6 dextran chains. Steady-state kinetic studies showed that hydrolysis and transglucosylation reactions occurred at the early stage of the reaction in the presence of 70 kDa dextran as acceptor and sucrose. The transglucosylation reaction catalysed by GBD-CD2 follows a Ping Pong Bi Bi mechanism with a high kcat value of 970 s(-1). The amount of the synthesised alpha-1,2 side chains was found to be directly dependent on the initial molar ratio [Sucrose]/[Dextran]. Dextrans with controlled alpha-1,2 linkage contents ranging from 13% to 40% were synthesised. The procedure resulted in the production of dextrans with the highest content of alpha-1,2 linkages ever reported.

Expression of alternansucrase in potato plants

Alternan, which consists of alternating α-(1→3)/α-(1→6)-linked glucosyl residues, was produced in potato tubers by expressing a mature alternansucrase (Asr) gene from Leuconostoc mesenteroides NRRL B-1355 in potato. Detection of alternan was performed by enzyme-linked immunosorbent assay in tuber juices, revealing a concentration between 0.3 and 1.2 mg g-1 fresh wt. The Asr transcript levels correlated well with alternan accumulation in tuber juices. It appeared that the expression of sucrose-regulated starch-synthesizing genes (ADP-glucose pyrophosphorylase subunit S and granule-bound starch synthase I) was down-regulated. Despite this, the physico-chemical properties of the transgenic starches were unaltered. These results are compared to those obtained with other transgenic potato plants producing mutan [α-(1→3)-linked glucosyl residues] and dextran [α-(1→6)-linked glucosyl residues].

Identification and functional characterization of levS, a gene encoding for a levansucrase from Leuconostoc mesenteroides NRRL B-512 F

A Leuconostoc mesenteroides NRRL B-512 F levansucrase gene, ( levS), was isolated, sequenced and cloned in Escherichia coli. The recombinant enzyme was shown to be a fructosyltransferase producing a polymer identified by 13C-NMR as levan. Based on sequence analysis, we found that this levansucrase is a mosaic protein, bearing structural features of glucosyltransferases in the amino and carboxy terminal regions similarly to inulosucrase from Leuconostoc citreum. The phylogenetic analysis of the C-terminal region domain of levansucrases from L. mesenteroides demonstrates that they group together into a novel putative sub-family of genes and evolved long before all other glucosyltransferases, while their catalytic domain structure is species related.

Synthesis of methyl α-d-glucooligosaccharides by entrapped dextransucrase from Leuconostoc mesenteroides B-1299

The synthesis of methyl alpha-D-glucooligosaccharides, using sucrose as glucosyl donor and methyl alpha-D-glucopyranoside as acceptor, was studied with dextransucrase from Leuconostoc mesenteroides NRRL B-1299. The enzyme was immobilized by entrapment in alginate. By NMR and mass spectrometry we identified three homologous series (S1-S3) of methyl alpha-D-glucooligosaccharides. Series S2 and S3 were characterized by the presence of alpha(1-->2) linkages, in combination with alpha(1-->6) bonds. Two parameters, sucrose to acceptor concentration ratio (S/A) and the total sugar concentration (TSC) determined the yield of methyl alpha-D-glucooligosaccharides. The maximum concentration achieved of the first acceptor product, methyl alpha-D-isomaltoside, was 65 mM using a S/A 1:4 and a TSC of 336 g l(-1). When increasing temperature, a shift of selectivity towards compounds containing alpha(1-->2) bonds was observed. The formation of leucrose as a side process was very significant (reaching values of 32 g l(-1)) at high sucrose concentrations.

Production of dextran and fructose from carob pod extract and cheese whey by Leuconostocmesenteroides NRRL B512(f)

The production of dextran and fructose from carob pod extract (CPE) and cheese whey (CW) as carbon source by the bacterium Leuconostoc mesenteroides was investigated. The influence of secondary carbon sources (maltose, lactose and galactose) on dextran molecular weight and fermented broth viscosity were also studied. Significant changes were not observed in broth viscosity during dextran production at initial sucrose concentration of 20 and 120 g/l. Complementary sugars maltose, lactose and galactose together with sucrose promote production of dextran with fewer glucose units. Dextran molecular weight decreases from the range 1,890,000–10,000,000 to 240,000–400,000 Da when complementary sugars are present. Polydispersity was improved when complementary sugars were used. Fermentation using mixtures of carob pod extract and cheese whey confirm these results obtained for production of dextran. Final concentrations of dextran and fructose indicate that reaction yields were not affected. Carob pod and cheese whey can be successfully used as raw material in the fermentation system described. The maximum concentrations of dextran and fructose obtained using carob pod extract resulted in 8.56 and 7.78 g/l, respectively. Combined carob pod extract and cheese whey resulted in dextran and fructose concentrations of 7.23 and 6.98 g/l, respectively. The corresponding dextran mean molecular weight was 1,653,723 and 325,829.

Production of Cyclic Tetrasaccharide with 6-.ALPHA.-Glucosyltransferase and .ALPHA.-Isomaltosyltransferase

Cyclic tetrasaccharide (CTS; cyclo{→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→}) is a cyclic oligosaccharide four D-glucosyl residues linked in an alternating α-1,3- and α-1,6-fashion. CTS has been reported for enzymatic synthesis from alternan, an α-(1→3)-α-(1→6)-D-glucan synthesized from sucrose by Leuconostoc mesenteroides NRRL B-1355 alternansucrase, by endo-glucanase (alternanase) from Bacillus sp. NRRL B-21195. While searching for nonreducing oligosaccharides that can be produced from α-1,4-glucan as the substrate, we screened bacteria isolated from soil in our own way, and obtained Bacillus globisporus C11, which produces CTS from starch. Two kinds of glycosyltransferase, 6-α-glucosyltransferase (6GT) and α-isomaltosyltransferase (IMT), were purified from the culture supernatant of this strain. It was found that CTS is produced by the sequential action of both enzymes. The genes for IMT (CtsY) and 6GT (CtsZ) were linked together on the chromosome, forming ctsYZ. Both of the gene products showed similarities to α-glucosidases belonging to glycoside hydrolase family 31 and conserved two aspartic acids corresponding to the putative catalytic residues of these enzymes. CtsYZ and four open reading frames upstream of ctsYZ form a gene cluster, ctsUVWXYZ. The reaction conditions for CTS synthesis were examined using 6GT and IMT from B. globisporus C11. The optimum reaction conditions to obtain CTS from Pinedex #100 (partial hydrolyzate of starch, 1.3%-hydrolysis) were the following: substrate concentration, 3%; pH, 6-7; temperature, 30°C; enzyme dosages, 1 U/g-dry solid 6GT, 10 U/g-dry solid IMT. In these optimum conditions the CTS yields reached 62% at the reaction time of 48 h. A mass-production method of highly purified CTS crystals at a reasonable cost was established, and the functions and characteristics of CTS were studied.

Encapsulation in LentiKats of Dextransucrase from Leuconostoc mesenteroides NRRL B-1299, and its Effect on Product Selectivity

Insoluble (cell-bound) dextransucrase from Leuconostoc mesenteroides B-1299 was encapsulated in highly elastic and stable hydrogels formed by polyvinyl alcohol. The gelation was carried out by controlled partial drying at room temperature, resulting in lens-shaped particles, called LentiKats. A similar recovery of activity (approximately 55%) was achieved when compared with entrapment in calcium alginate gels. Under reaction conditions, the protein leakage in LentiKats was reduced from 18% to 4% by pre-treatment of the dextransucrase with glutaraldehyde. The immobilized dextransucrases were tested in the acceptor reaction with methyl α-D-glucopyranoside. The conversion to oligosaccharides using Lentikat-dextransucrase was higher than that obtained for alginate-dextransucrase, probably due to the reduction of diffusional limitations derived from its lenticular shape. In addition, a shift of selectivity towards the synthesis of oligosaccharides containing α(1→2) bonds was observed for the Lentikat-biocatalysts. These non-digestible compounds are supposed to be specifically fermented by beneficial species of the human microflora (prebiotic effect). The Lentikat-entrapped dextransucrase can be efficiently reused in this process at least for five cycles of 24 h.

Molecular characterization of DSR-E, an alpha-1,2 linkage-synthesizing dextransucrase with two catalytic domains.

A novel Leuconostoc mesenteroides NRRL B-1299 dextransucrase gene, dsrE, was isolated, sequenced, and cloned in Escherichia coli, and the recombinant enzyme was shown to be an original glucansucrase which catalyses the synthesis of alpha-1,6 and alpha-1,2 linkages. The nucleotide sequence of the dsrE gene consists of an open reading frame of 8,508 bp coding for a 2,835-amino-acid protein with a molecular mass of 313,267 Da. This is twice the average mass of the glucosyltransferases (GTFs) known so far, which is consistent with the presence of an additional catalytic domain located at the carboxy terminus of the protein and of a central glucan-binding domain, which is also significantly longer than in other glucansucrases. From sequence comparison with family 70 and alpha-amylase enzymes, crucial amino acids involved in the catalytic mechanism were identified, and several original sequences located at some highly conserved regions in GTFs were observed in the second catalytic domain.

Dextran and fructose separation on an SMB continuous chromatographic unit

A dextran/fructose mixture was obtained by fermentation of a sucrose rich media using Leuconostoc mesenteroides NRRL B512(f). The dextran and fructose mixture obtained by fermentation as well as a synthetic mixture of pure components were separated using simulated moving bed (SMB) chromatography. In the case of the synthetic feed, the purity in the raffinate (dextran) and extract (fructose) was 96 and 81%, respectively. For the fermented mixture the purity values were 87.2% for the extract and 100% for the raffinate.

Factors affecting alpha,-1,2 glucooligosaccharide synthesis by Leuconostoc mesenteroides NRRL B-1299 dextransucrase.

The optimization of alpha-1,2 glucooligosaccharide (GOS) synthesis from maltose and sucrose by Leuconostoc mesenteroides NRRL B-1299 dextransucrase was achieved using experimental design and consecutive analysis of the key parameters. An increase of the pH of the reaction from 5.4 to 6.7 and of the temperature from 25 to 40 degrees C significantly favored alpha-1,2 GOS synthesis, thanks to a significant decrease of the side reactions, i.e., dextran and leucrose synthesis. These positive effects were not sufficient to compensate for the decrease of enzyme stability caused by the use of high pH and temperature. However, the critical parameters were the sucrose to maltose concentration ratio (S/M) and the total sugar concentration (TSC). Alpha1,2 GOS synthesis was favored at high S/M ratios. But using these conditions also led to an increase of side reactions which could be modulated by choosing the appropriate TSC. Finally, with S/M = 4 and TSC = 45% w/v, dextran and leucrose productions were limited and the final alpha-1,2 GOS yield reached 56.7%, the total GOS yield being 88%.

Sequence analysis of the gene encoding alternansucrase, a sucrose glucosyltransferase from Leuconostoc mesenteroides NRRL B-1355.

The gene encoding alternansucrase (ASR) from Leuconostoc mesenteroides NRRL B-1355, an original sucrose glucosyltransferase (GTF) specific to alternating alpha-1,3 and alpha-1,6 glucosidic bond synthesis, was cloned, sequenced and expressed into Escherichia coli. Recombinant enzyme catalyzed oligoalternan synthesis from sucrose and maltose acceptor. From sequence comparison, it appears that ASR possesses the same domains as those described for GTFs specific to either contiguous alpha-1,3 osidic bond or contiguous alpha-1,6 osidic bond synthesis. However, the variable region and the glucan binding domain are longer than in other GTFs (by 100 and 200 amino acids respectively). The N-catalytic domain which presents 49% identity with the other GTFs from L. mesenteroides possesses the three determinants potentially involved in the glucosyl enzyme formation.

Sequence analysis of the gene encoding alternansucrase, a sucrose glucosyltransferase from Leuconostoc mesenteroides NRRL B-1355

The gene encoding alternansucrase (ASR) from Leuconostoc mesenteroides NRRL B-1355, an original sucrose glucosyltransferase (GTF) specific to alternating α-1,3 and α-1,6 glucosidic bond synthesis, was cloned, sequenced and expressed into Escherichia coli. Recombinant enzyme catalyzed oligoalternan synthesis from sucrose and maltose acceptor. From sequence comparison, it appears that ASR possesses the same domains as those described for GTFs specific to either contiguous α-1,3 osidic bond or contiguous α-1,6 osidic bond synthesis. However, the variable region and the glucan binding domain are longer than in other GTFs (by 100 and 200 amino acids respectively). The N-catalytic domain which presents 49% identity with the other GTFs from L. mesenteroides possesses the three determinants potentially involved in the glucosyl enzyme formation.

Production of dextransucrase, dextran and fructose from sucrose using Leuconostoc mesenteroides NRRL B512(f)

The production of dextransucrase, dextran and fructose by sucrose fermentation using Leuconostoc mesenteroides NRRL-B512(F) was studied in batch operation in a bioreactor with total working volume of 1.5 dm 3. The effect of temperature (20 to 40°C), pH (5.5 and 6.7) and sucrose concentration (10 to 120 g/l) on process performance was studied. The optimum conditions for dextran and fructose production were T = 35°C and pH = 5.5. Cell growth is not inhibited by high sucrose concentrations; however, for sucrose concentration higher than 40 g/dm 3 separation of products from cells is difficult. Biomass ( X), enzyme ( E), dextran ( D), fructose ( F) and sucrose ( S) rate equations were considered in order to derive a simple fermentation kinetic model from batch experimental data. The logistic equation provided a reasonable description for cell concentration, X. The Luedeking and Piret equation was used to describe the enzyme production rate, by considering only the growth associated term. The concentrations of products (dextran and fructose) were reasonably described by a first order kinetic law with respect to both substrate and enzyme concentrations; the substrate, S was consumed for cell growth and for dextran and fructose production. Model parameters μ m and X o were calculated from cell growth as a function of time. The yield Y E/X were calculated from X max and E max and Y X/S was estimated from X max and the sucrose consumed by the bacteria. The remaining parameter k′ was obtained by fitting the experimental data of substrate, dextran and fructose concentrations versus time.

Influence of nitrogen/carbon ratio and complementary sugars on dextransucrase production by Leuconostoc mesenteroides NRRL B512(f).

The influence of the nitrogen/carbon ratio was studied during fermentation with Leuconostoc mesenteroides NRRL B512(f) using sucrose as substrate. The enzyme concentration was measured through activity tests and radiotracer tests with [ 14C]phenylalanine. In batch fermentation a slowing down in the rate of enzyme synthesis was observed with a decrease in the nitrogen/carbon ratio. Addition of pulses of nitrogen when the nitrogen/carbon ratio decreased allowed a constant production rate of enzyme and lower fermentation time. The influence of complementary sugars was also addressed. Lactose inhibited enzyme production. When galactose was used the yield was the same as in the fermentation with sucrose alone, but with a different production rate. Maltose favoured the synthesis of dextransucrase.

Some structural features of an insoluble alpha-D-glucan from a mutant strain of Leuconostoc mesenteroides NRRL B-1355.

Leuconostoc mesenteroides strain NRRL B-1355 produces two soluble extracellular alpha-D-glucans from sucrose: alternan and dextran. An unusual mutant strain derived from NRRL B-1355 has recently been isolated which produces practically no soluble polysaccharide, but significant amounts of an insoluble D-glucan. Methylation analysis shows it contains linear (1-->3) and (1-->6) linkages as well as (1-->2) and (1-->3) branch linkages. The insoluble glucan was partially digestible by endodextranase, giving rise to a series of oligosaccharides, a high-molecular weight soluble fraction and an insoluble residue. Treatment of the soluble dextranase-limit fraction with an alpha(1-->2) debranching enzyme led to further dextranase susceptibility. Methylation, FTIR and NMR analyses of the dextranase-treated fractions indicate a non-uniform structure with domains bearing similarities to L. mesenteroides strain NRRL B-1299 dextran and to insoluble streptococcal D-glucans.

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.