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 to 3)-linked and α(1 to 6)-linked d-glucopyranosyl units and a low degree of branching.
•A sourdough isolate Lactobacillus brevis E25 produced a highly branched α-glucan.•A modified medium was used to isolate the E25 exopolysaccharide (EPS).•E25 EPS structure was determined and compared ...with alternan and commercial dextran.•E25 EPS contains a higher proportion of (1,6)Glc→6 units.•E25 EPS is more highly branched than alternan.
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.
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.
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.
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.
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.
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. Keywords: Maltose-based oligosaccharides; molecular weight; structure−function relationship; gut microflora
α-Glucans that were enzymatically synthesized from sucrose using glucansucrase cloned from
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.
Several glucansucrases were surveyed for their ability to produce isomelezitose, a trisaccharide with the structure α-D-glucopyranosyl (1 → 6) β-D-fructofuranosyl (2 ↔ 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.
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•Isomelezitose is formed at detectable levels in 11 of 12 randomly selected glucansucrases.•Isomelezitose yield varies, depending on the enzyme source.•Leuconostoc mesenteroides NRRL B-1297 is the best isomelezitose producer found.•Higher sucrose concentrations enhance isomelezitose yields.
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