Development and Physical Characterization of α-Glucan Nanoparticles.

α-Glucans that were enzymatically synthesized from sucrose using glucansucrase cloned from Leuconostoc mesenteroides NRRL B-1118 were found to have a glass transition temperature of approximately 80 °C. Using high-pressure homogenization (~70 MPa), the α-glucans were converted into nanoparticles of ~120 nm in diameter with a surface potential of ~-3 mV. Fluorescence measurements using 1,6-diphenyl-1,3,5-hexatriene (DPH) indicate that the α-glucan nanoparticles have a hydrophobic core that remains intact from 10 to 85 °C. α-Glucan nanoparticles were found to be stable for over 220 days and able to form at three pH levels. Accelerated exposure measurements demonstrated that the α-glucan nanoparticles can endure exposure to elevated temperatures up to 60 °C for 6 h intervals.

Structural analysis of the α-d-glucan produced by the sourdough isolate Lactobacillus brevis E25.

Cereal-associated Lactic Acid Bacteria (LAB) are well known for homopolymeric exopolysaccharide (EPS) production. Herein, the structure of an EPS isolated from sourdough isolate Lactobacillus brevis E25 was determined. A modified BHI medium was used for production of EPS-E25 in order to eliminate potential contaminants. Analysis of sugar monomers in EPS revealed that glucose was the only sugar present. Structural characterisation of EPS by NMR and methylation analysis revealed that E25 produced a highly branched α-glucan with (α1→3) and (α1→6) glycosidic linkages, and was similar in structure to a previously reported EPS from Lactobacillus reuteri 180. The 1H and 13C NMR data were contrasted with newly recorded data for known polysaccharides (alternan, commercial dextran) which also contain α-(1,3,6)Glc branch points. It was found in both E25 EPS and alternan that NMR parameters could be used to distinguish glucose residues that had the same substitution pattern but occupied different positions in the structure.

Isomelezitose formation by glucansucrases.

Several glucansucrases were surveyed for their ability to produce isomelezitose, a trisaccharide with the structure α-D-glucopyranosyl (1 â†’ 6) ß-D-fructofuranosyl (2 â†” 1) α-D-glucopyranoside. Nearly all strains tested, with one exception, produced at least trace levels of isomelezitose. Yields were low but significant, ranging from less than 1% to approximately 5% based on sucrose. This trisaccharide may arise in either of two ways: glucopyranosyl transfer to the 6Fru-OH position of sucrose, or to the anomeric OH position of isomaltulose. This study indicates that isomelezitose formation may be a general phenomenon of many glucansucrase reactions.

Effect of a single point mutation on the interaction of glucans with a glucansucrase from Leuconostoc mesenteroides NRRL B-1118.

Our previous work showed that substitution of an amino acid that is coupled with the +2 subsite adjacent to the transition stabilizer of a glucansucrase, which produces a water-insoluble glucan, resulted in significant changes in the structures and yields of the water-insoluble glucans produced. We now describe how these changes affect the ability of the glucansucrase to bind to exogenous glucans, and how these glucans can influence the yield, product structures, and kinetics of the mutant glucansucrases. The activity of the wild-type enzyme, with threonine at position 654, is not significantly activated by added dextran, and the yield of water-insoluble glucan from sucrose is only slightly increased by dextran. Mutant T654Y is not affected at all by the addition of dextran. However, several mutant enzymes exhibit markedly lower yields of glucan relative to the wild type; these lower yields can be partially or completely overcome by the addition of water-soluble dextran. Although evidence indicates that the soluble dextran is incorporated into water-insoluble glucan, the increased yields cannot be accounted for solely by incorporation of the dextran into insoluble product. Furthermore, these DsrI mutants are significantly activated by exogenous glucans. The addition of dextran does not markedly change the KM for sucrose in the mutant enzymes, but does increase the Vmax of the reaction. These effects apparently depend on the presence of unbranched sequences of α1→6-linked D-glucose units in the glucan.

Secreted expression of Leuconostoc mesenteroides glucansucrase in Lactococcus lactis for the production of insoluble glucans.

We expressed a glucansucrase, DsrI, from Leuconostoc mesenteroides that catalyzes formation of water-insoluble glucans from sucrose using a nisin-controlled gene expression system in Lactococcus lactis. These polymers have potential for production of biodegradable gels, fibers, and films. We optimized production of DsrI using several different background vectors, signal peptides, strains, induction conditions, and bioreactor parameters to increase extracellular accumulation. Optimal production of the enzyme utilized a high-copy plasmid, pMSP3535H3, which contains a nisin immunity gene, L. lactis LM0230, and bioreactors maintained at pH 6.0 to stabilize the enzyme. We were able to significantly improve growth using the lactic acid inhibitor heme and by continuous removal of lactic acid with anion exchange resins, but enzyme production was less than the controls. The recombinant enzyme under optimized conditions accumulated in the culture medium to approximately 380 mg/L, which was over 150-fold higher compared to the native L. mesenteroides strain. Methods are also included for purification of DsrI utilizing the glucan-binding domain of the enzyme.

Effects of mutations at threonine-654 on the insoluble glucan synthesized by Leuconostoc mesenteroides NRRL B-1118 glucansucrase.

Twelve different amino acids were each substituted for threonine-654 in a cloned glucansucrase from Leuconostoc mesenteroides NRRL B-1118. Both the native and the cloned enzyme with threonine at position 654 produced a water-insoluble glucan containing approximately 44 mol% 1,3-disubstituted α-D-glucopyranosyl units and 29 mol% 1,6-disubstituted α-D-glucopyranosyl units. Several substitutions yielded an enzyme that produced an increased percentage of 1,3-disubstituted α-D-glucopyranosyl units, with corresponding decreases in 1,6-disubstituted α-D-glucopyranosyl units. Only one substitution, tyrosine, resulted in a significant increase in the percentage of 1,6-disubstituted α-D-glucopyranosyl units, with a concomitant increase in glucan yield. The mutated enzymes that produced the highest levels of 1,3-disubstituted α-D-glucopyranosyl units were also significantly activated by the addition of dextran, but glucan yields were also lower in these mutants.

Glucansucrase acceptor reactions with d-mannose.

The main acceptor product of glucansucrases with d-mannose has not previously been identified. We used glucansucrases that form water-insoluble α-d-glucans to produce increased yields of acceptor products from d-mannose, and identified the major product as 6-O-α-d-glucopyranosyl-d-mannose. Glucansucrases that synthesize insoluble α-d-glucans produced higher yields of the disaccharide compared to typical dextransucrases.

Automated UV-C mutagenesis of Kluyveromyces marxianus NRRL Y-1109 and selection for microaerophilic growth and ethanol production at elevated temperature on biomass sugars.

The yeast Kluyveromyces marxianus is a potential microbial catalyst for fuel ethanol production from a wide range of biomass substrates. To improve its growth and ethanol yield at elevated temperature under microaerophilic conditions, K. marxianus NRRL Y-1109 was irradiated with UV-C using automated protocols on a robotic platform for picking and spreading irradiated cultures and for processing the resulting plates. The plates were incubated under anaerobic conditions on xylose or glucose for 5 mo at 46 °C. Two K. marxianus mutant strains (designated 7-1 and 8-1) survived and were isolated from the glucose plates. Both mutant strains, but not wild type, grew aerobically on glucose at 47 °C. All strains grew anaerobically at 46 °C on glucose, galactose, galacturonic acid, and pectin; however, only 7-1 grew anaerobically on xylose at 46 °C. Saccharomyces cerevisiae NRRL Y-2403 did not grow at 46 °C on any of these substrates. With glucose as a carbon source, ethanol yield after 3 d at 46 °C was higher for 8-1 than for wild type (0.51 and 0.43 g ethanol/g glucose, respectively). With galacturonic acid as a carbon source, the ethanol yield after 7 d at 46 °C was higher for 7-1 than for wild type (0.48 and 0.34 g ethanol/g galacturonic acid, respectively). These mutant strains have potential application in fuel ethanol production at elevated temperature from sugar constituents of starch, sucrose, pectin, and cellulosic biomass.

The production of glucans via glucansucrases from Lactobacillus satsumensis isolated from a fermented beverage starter culture.

Several starter cultures used in the production of fermented beverages were screened for lactic acid bacteria that produced water-insoluble polysaccharides from sucrose. The strain producing the greatest amount was identified as Lactobacillus satsumensis by its 16S RNA sequence and was deposited in the ARS culture collection as NRRL B-59839. This strain produced at least two α-D-glucans from sucrose. One was a water-soluble dextran, consisting of predominantly α-(1 → 6)-linked D-glucose units, and the other was a water-insoluble glucan containing both α-(1 → 6)-linked and α-(1 → 3)-linked D-glucose units. The culture fluid was found to contain glucansucrases responsible for the two glucans, and no significant level of fructansucrase was detected. Glucansucrase activity was not present in the culture fluid when the bacteria were grown on glucose, fructose, or raffinose as the carbon source. Although the water-soluble glucans produced by cell-free enzyme and by cell suspensions were essentially identical, the same was not true for the water-insoluble glucans. The water-insoluble glucan produced by cell-free culture fluid contained a higher proportion of α-(1 → 3)-linked D-glucose units than the water-insoluble glucan produced by cell suspensions.

Cloning, expression, and characterization of an insoluble glucan-producing glucansucrase from Leuconostoc mesenteroides NRRL B-1118.

We have cloned a glucansucrase from the type strain of Leuconostoc mesenteroides (NRRL B-1118; ATCC 8293) and successfully expressed the enzyme in Escherichia coli. The recombinant processed enzyme has a putative sequence identical to the predicted secreted native enzyme (1,473 amino acids; 161,468 Da). This enzyme catalyzed the synthesis of a water-insoluble α-D-glucan from sucrose (K(M) 12 mM) with a broad pH optimum between 5.0 and 5.7 in the presence of calcium. Removal of calcium with dialysis resulted in lower activity in the acidic pH range, effectively shifting the pH optimum to 6.0-6.2. The enzyme was quickly inactivated at temperatures above approximately 45°C. The presence of dextran offered some protection from thermal inactivation between room temperature and 40°C but had little effect above 45°C. NMR and methylation analysis of the water-insoluble α-D-glucan revealed that it had approximately equal amounts of α(1 → 3)-linked and α(1 → 6)-linked D-glucopyranosyl units and a low degree of branching.

In vitro fermentation of alternansucrase raffinose-derived oligosaccharides by human gut bacteria.

In this work, in vitro fermentation of alternansucrase raffinose-derived oligosaccharides, previously fractionated according to their degree of polymerization (DP; from DP4 to DP10), was carried out using small-scale pH-controlled batch cultures at 37 °C under anaerobic conditions with human feces. Bifidogenic activity of oligosaccharides with DP4-6 similar to that of lactulose was observed; however, in general, a significant growth of lactic acid bacteria Bacteroides , Atopobium cluster, and Clostridium histolyticum group was not shown during incubation. Acetic acid was the main short chain fatty acid (SCFA) produced during the fermentation process; the highest levels of this acid were shown by alternansucrase raffinose acceptor pentasaccharides at 10 h (63.11 mM) and heptasaccharides at 24 h (54.71 mM). No significant differences between the gas volume produced by the mixture of raffinose-based oligosaccharides (DP5-DP10) and inulin after 24 h of incubation were detected, whereas lower gas volume was generated by DP4 oligosaccharides. These findings indicate that novel raffinose-derived oligosaccharides (DP4-DP10) could be a new source of prebiotic carbohydrates.

Corrosion protection of low-carbon steel using exopolysaccharide coatings from Leuconostoc mesenteroides.

Corrosion of metals is a serious and challenging problem faced worldwide by industry. Purified Leuconostoc mesenteroides exopolysaccharide (EPS) coatings, cast from aqueous solution, inhibited the corrosion of low-carbon steel as determined by electrochemical impedance spectroscopy (EIS). There were two different corrosion behaviors exhibited when EPS films from different strains were cast onto the steel. One EPS coating reacted immediately with the steel substrate to form an iron (III) oxide layer ("rust") during the drying process while another did not. The samples that did not flash corrode had higher corrosion inhibition and formed an iron (II) passivation layer during EIS testing that persisted after the cells were disassembled. Corrosion inhibition was strain-specific as polysaccharides with similar structure did not have the same corrosion potential.

Hydrodynamic versus size exclusion chromatography characterization of alternan and comparison to off-line MALS.

Alternan is an ultrahigh molar mass polysaccharide composed of alternating α-(1→3) and α-(1→6) repeat units and that also possesses long-chain branching. Its molar mass distribution (MMD) can extend into the hundreds of millions of grams per mole. Characterizing alternan by means of size exclusion chromatography (SEC) is a lengthy process and an incomplete one because even under the best possible experimental conditions, the polysaccharide appears to degrade during its passage through the SEC columns. As an alternative to SEC, we have investigated the use of hydrodynamic chromatography (HDC) as a possible characterization technique for alternan. Results from packed-column HDC with multiangle static light scattering (MALS) detection compare favorably to results from off-line MALS analysis, and HDC results are obtained in a fraction of the time needed for SEC. The largest molar mass alternan did appear to undergo some degradation during HDC as a result of interstitial stresses in the column bed. This could be remedied by the use of columns with larger packing particles. Evidence of long-chain branching in alternan is provided via a comparison of the intrinsic viscosities and viscometric radii of the polysaccharide to those of a pullulan standard and of a theoretical pullulan of molar mass equal to those of the alternans examined.

Glucosylation of raffinose via alternansucrase acceptor reactions.

The glucansucrase known as alternansucrase [EC 2.4.1.140] can transfer glucosyl units from sucrose to raffinose to give good yields of oligosaccharides, which may serve as prebiotics. The main products were the tetrasaccharides alpha-d-Glcp-(1-->3)-alpha-d-Galp-(1-->6)-alpha-d-Glcp-(1<-->2)-beta-d-Fruf and alpha-d-Glcp-(1-->4)-alpha-d-Galp-(1-->6)-alpha-d-Glcp-(1<-->2)-beta-d-Fruf in ratios ranging from 4:1 to 9:1, along with lesser amounts of alpha-d-Glcp-(1-->6)-alpha-d-Galp-(1-->6)-alpha-d-Glcp-(1<-->2)-beta-d-Fruf. Ten unusual pentasaccharide structures were isolated. Three of these arose from glucosylation of the major tetrasaccharide product, two each from the minor tetrasaccharides, and three were the result of glucosylations of the fructose acceptor product leucrose or isomaltulose. The major pentasaccharide product arose from glucosylation of the major tetrasaccharide at position 4 of the fructofuranosyl unit, to give a subunit structure analogous to that of maltulose. A number of hexasaccharides and higher oligosaccharides were also produced. Unlike alternansucrase, dextransucrase [EC 2.4.1.5] gave only a single tetrasaccharide product in low yield, and no significant amounts of higher oligosaccharides. The tetrasaccharide structure from dextransucrase was found to be alpha-d-Glcp-(1-->4)-alpha-d-Galp-(1-->6)-alpha-d-Glcp-(1<-->2)-beta-d-Fruf, which is at odds with the previously published structure.

An SEC/MALS study of alternan degradation during size-exclusion chromatographic analysis.

Ultrahigh-molar-mass (M) polymers such as DNA, cellulose, and polyolefins are routinely analyzed using size-exclusion chromatography (SEC) to obtain molar mass averages, distributions, and architectural information. It has long been contended that high-M polymers can degrade during SEC analysis; if true, the inaccurate molar mass information obtained can adversely affect decisions regarding processing and end-use properties of the macromolecules. However, most evidence to the effect of degradation has been circumstantial and open to alternative interpretation. For example, the shift in SEC elution volume as a function of increased chromatographic flow rate, observed using only a concentration-sensitive detector, may be the result of degradation or of elution via a nondegradatory slalom chromatography mechanism. Here, using both concentration-sensitive and multiangle static light-scattering detection, we provide unambiguous evidence that the polysaccharide alternan actually degrades during SEC analysis. The decrease in molar mass and size of alternan with increasing flow rate, measured using light scattering, allows ruling out an SC mode of elution and can only be interpreted as due to degradation. These findings demonstrate the extreme fragility of ultrahigh-M polymers and the care that must be taken for accurate characterization.

Insoluble glucans from planktonic and biofilm cultures of mutants of Leuconostoc mesenteroides NRRL B-1355.

Leuconostoc mesenteroides strain NRRL B-1355 produces the soluble exopolysaccharides alternan and dextran in planktonic cultures. Mutants of this strain are available that are deficient in the production of alternan, dextran, or both. Our recent work demonstrated that biofilms from mutant strains contained insoluble polysaccharides. We now find that the insoluble polysaccharides are composed of D-glucose polymers with contiguous sequences of alpha(1-->3) and alpha(1-->6) linkages. In addition, planktonic cultures of the wild type also produce this insoluble mixture in association with the cell mass. This material is similar to the insoluble glucan matrix known as mutan formed by cariogenic strains of streptococci. The production of insoluble mutan-like glucans may be more widespread among Leuconostoc spp. than previously recognized.

Modification of alternan by dextranase.

Alternan is a unique glucan with a backbone structure of alternating alpha-(1 --> 6) and alpha-(1 --> 3) linkages. Previously, we isolated strains of Penicillium sp. that modify native, high molecular weight alternan in a novel bioconversion process to a lower molecular weight form with solution viscosity properties similar to those of commercial gum arabic. The mechanism of this modification was unknown. Here, we report that these Penicillium sp. strains secrete dextranase during germination on alternan. Furthermore, alternan is modified in vitro by commercial dextranases, and dextranase-modified alternan appears to be identical to bioconversion-modified alternan. This is surprising, since alternan has long been considered to be resistant to dextranase. Results suggest that native alternan may have localized regions of consecutive alpha-(1 --> 6) linkages that serve as substrates for dextranase. Dextranase treatment of native alternan, particularly with GRAS enzymes, may have practical advantages for the production of modified alternan as a gum arabic substitute.

Acceptor products of alternansucrase with gentiobiose. Production of novel oligosaccharides for food and feed and elimination of bitterness.

In the presence of suitable acceptor molecules, dextransucrase makes a homologous series of oligosaccharides in which the isomers differ by a single glucosyl unit, whereas alternansucrase synthesizes one trisaccharide, two tetrasaccharides, etc. Previously, we showed that alternansucrase only forms certain isomers of DP>4 from maltose in measurable amounts, and that these oligosaccharides belong to the oligoalternan series rather than the oligodextran series. We now demonstrate that the acceptor products from gentiobiose, also formed in good yields (nearly 90% in unoptimized reactions), follow a pattern similar to those formed from maltose. The initial product is a single trisaccharide, alpha-d-Glcp-(1-->6)-beta-d-Glcp-(1-->6)-d-Glc. Two tetrasaccharides were formed in approximately equal quantities: alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->6)-beta-d-Glcp-(1-->6)-d-Glc and alpha-d-Glcp-(1-->6)-alpha-d-Glcp-(1-->6)-beta-d-Glcp-(1-->6)-d-Glc. Just one pentasaccharide was isolated from the reaction mixture, alpha-d-Glcp-(1-->6)-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->6)-beta-d-Glcp-(1-->6)-d-Glc. Our hypothesis that the enzyme is incapable of forming two consecutive alpha-(1-->3) linkages, and does not form products with more than two consecutive alpha-(1-->6) linkages, apparently applies to other acceptors as well as to maltose. The glucosylation of gentiobiose reduces or eliminates its bitter taste.

Biofilm formation by exopolysaccharide mutants of Leuconostoc mesenteroides strain NRRL B-1355.

Leuconostoc mesenteroides strain NRRL B-1355 produces the soluble exopolysaccharides alternan and dextran in planktonic cultures. Mutants of this strain are available that are deficient in the production of alternan, dextran, or both. Another mutant of NRRL B-1355, strain R1510, produces an insoluble glucan in place of alternan and dextran. To test the effect of exopolysaccharide production on biofilm formation, these strains were cultured in a biofilm reactor. All strains grew well as biofilms, with comparable cell densities, including strain NRRL B-21414, which produces neither alternan nor dextran in planktonic cultures. However, the exopolysaccharide phenotype clearly affected the appearance of the biofilms and the sloughed-off biofilm material produced by these biofilms. For all strains, soluble glucansucrases and soluble polysaccharides produced by biofilm cultures appeared to be similar to those produced by planktonic cultures. Biofilms from all strains also contained insoluble polysaccharides. Strain R1510 biofilms contained an insoluble polysaccharide similar to that produced by planktonic cultures. For most other strains, the insoluble biofilm polysaccharides resembled a mixture of alternan and dextran.

Influence of glycosidic linkages and molecular weight on the fermentation of maltose-based oligosaccharides by human gut bacteria.

A structure-function study was carried out to increase knowledge of how glycosidic linkages and molecular weights of carbohydrates contribute toward the selectivity of fermentation by gut bacteria. Oligosaccharides with maltose as the common carbohydrate source were used. Potentially prebiotic alternansucrase and dextransucrase maltose acceptor products were synthesized and separated into different molecular weights using a Bio-gel P2 column. These fractions were characterized by matrix-assisted laser desorption/ionization time-of-flight. Nonprebiotic maltooligosaccharides with degrees of polymerization (DP) from three to seven were commercially obtained for comparison. Growth selectivity of fecal bacteria on these oligosaccharides was studied using an anaerobic in vitro fermentation method. In general, carbohydrates of DP3 showed the highest selectivity towards bifidobacteria; however, oligosaccharides with a higher molecular weight (DP6-DP7) also resulted in a selective fermentation. Oligosaccharides with DPs above seven did not promote the growth of "beneficial" bacteria. The knowledge of how specific structures modify the gut microflora could help to find new prebiotic oligosaccharides.