The molecular structure of low and high molecular weight levans synthesized by levansucrase

The levan synthesized by Bacillus subtilis levansucrase in the presence of alcohols was of only high molecular weight, while in solutions of high ionic strength only low molecular weight (MW) levan was produced. The addition of low MW levan to the enzyme reaction mixture at low ionic strength stimulated synthesis of a high MW levan, but the levan added was not incorporated into this high MW levan. Methylation analysis revealed that low MW levans contained glucose, which was isolated as 2,3,46-tetra-O-methyl alditol acetate showing that the glucose units existed as terminal residues. The molecular weight of levan estimated on the basis of glucose content coincided with that determined by the gel filtration method. Methylation analysis also revealed that the number of fructose residues of the linear fraction linked by leads to 6(F)2 leads to type bonds was 22 for levan with a molecular weight of (8.4(-22)) x 10(3), while it was 11 for that of 2,000 x 10(3). The number of (formula: see text) type branched residues increased with increase in the molecular weight of the levan synthesized.     

Two active forms of Zymomonas mobilis levansucrase. An ordered microfibril structure of the enzyme promotes levan polymerization

Fructansucrases, members of glycoside hydrolase family 68, catalyze both sucrose hydrolysis and the polymerization of fructose to beta-d-fructofuranose polymers. The resulting fructan polymers are distinguished by the nature of the glycosidic bond: inulin (beta-(2-1)-fructofuranose) and levan (beta-(2-6)-fructofuranose). In this study we demonstrate that Zymomonas mobilis levansucrase exists in two active forms, depending on the pH and ionic strength. At pH values above 7.0, the enzyme is mainly a dimer, whereas at pH values below 6.0, the protein forms well ordered microfibrils that precipitate out of the solution. These two forms are readily interchangeable simply by changing the pH. Surprisingly the manner in which the enzyme is arranged strongly affects its product specificity and kinetic properties. At pH values above 7.0, the activity of the enzyme as a dimer is mainly sucrose hydrolysis and the synthesis of short fructosaccharides (degree of polymerization, 3). At pH values below 6.0, in its microfibril form, the enzyme catalyzes almost exclusively the synthesis of levan (a degree of polymerization greater than 20,000). This difference in product specificity appears to depend on the form of the enzyme, dimer versus microfibril, and not directly on the pH. Images made by negative stain transmission electron microscopy reveal that the enzyme forms a very ordered structure of long fibrils that appear to be composed of repeating rings of six to eight protein units. A single amino acid replacement of H296R abolished the ability of the enzyme to form microfibrils with organized fibril networks and to synthesize levan at pH 6.0.     

Production of levan using recombinant levansucrase immobilized on hydroxyapatite

Levansucrase of Zymomonas mobilis was immobilized onto the surface of hydroxyapatite by ionic binding. Optimum conditions for the immobilization were: pH 6.0, 4 h of immobilization reaction time, and 20 U of enzyme/g of matrix. The enzymatic and biochemical properties of the immobilized enzyme were similar to those of the native enzyme, especially towards the effect of salts and detergents. The immobilized enzyme showed sucrose hydrolysis activity higher as that of the native enzyme, but levan formation activity was 70% of the native enzyme. HPLC analysis of levan produced by immobilized enzyme showed the presence of two different types of levan: high-molecular-weight levan and low-molecular-weight levan. The proportion of low-molecular-weight levan to total levan produced by the immobilized enzyme was much higher than that with the native enzyme, indicating that immobilized levansucrase could be applied to produce low-molecular-weight levan. Immobilized levansucrase retained 65% of the original activity after 6 times of repeated uses and 67% of the initial activity after 40 d when stored at 4 °C.     

Intermolecular fructosyl and levanbiosyl transfers by levan fructotransferase of Arthrobacter ureafaciens

A purified levan fructotransferase preparation from the culture of the bacterium Arthrobacter ureafaciens, which produces di-D-fructose 2,6':6,2' dianhydride (difructose anhydride IV) from levan by an intramolecular levan fructosyl transfer (ILFT) reaction, was found to produce a trioligofructan and a tetraoligofructan from levan in the presence of levanbiose, indicating the intermolecular fructosyl and levanbiosyl transfer (LFT and LBT) reactions. The tri- and tetraoligofructans were identified to be levantriose and -tetraose respectively. Increase in the levanbiose concentration brought about increased production of both oligofructans with decreased formation of difructose anhydride IV, supporting the previous theory proposed by Tanaka et al. (1983) that the ILFT, LFT, and LBT reactions are catalyzed by the same enzyme. In addition, there existed a roughly stoichiometric relationship between the increase and decrease in the productions of these oligofructans, and the LBT reaction was found to occur more intensively than the LFT reaction. Acceptor specificity of the LFT and LBT reactions was studied using fifteen sugars including mono-, di-, and trisaccharides. The enzyme showed both of the reactions only with levanbiose, -triose, and kestose, indicating that the exposed non-reducing levanbiosyl residue was essential for the acceptor and suggesting the existence of a levanbiosyl acceptor site on the enzyme molecule.     

Synthesis of levan in water-miscible organic solvents

The synthesis of levan using a levansucrase from a strain of Bacillus subtilis was studied in the presence of the water-miscible solvents: acetone, acetonitrile and 2-methyl-2-propanol (2M2P). It was found that while the enzyme activity is only slightly affected by acetone and acetonitrile, 2M2P has an activating effect increasing the total activity 35% in 40-50% (v/v) 2M2P solutions at 30 degrees C. The enzyme is highly stable in water at 30 degrees C; however, incubation in the presence of 15 and 50% (v/v) 2M2P reduced the half-life time to 23.6 and 1.8 days, respectively. This effect is reversed in 83% 2M2P, where a half-life time of 11.8 days is observed. The presence of 2M2P in the system increases the transfer/hydrolysis ratio of levansucrase. As the reaction proceeds with 10% (w/v) sucrose in 50/50 water/2M2P sucrose is converted to levan and an aqueous two-phase system (2M2P/Levan) is formed and more sucrose can be added in a fed batch mode. It is shown that high molecular weight levan is obtained as an hydrogel and may be easily recovered from the reaction medium. However, when high initial sucrose concentrations (40% (w/v) in 50/50 water/2M2P) are used, an aqueous two-phase system (2M2P/sucrose) is induce, where the synthesized levan has a similar molecular weight distribution as in water and remains in solution.     

Water soluble levan polysaccharide biopolymer electrospun fibers

Electrospinning has been used to prepare nanofibers from diverse biopolymers. Here we report on preparation of fibers by electrospinning of levan (a polysaccharide) from distilled water. A high concentration of levan was required for fiber formation. This suggests that higher concentrations enable the formation of chain entanglements required to maintain the jet strength. In general, fiber diameter decreased with increased voltage, distance between collector plate and needle and decreased pump flow rate. X-ray diffraction of the fibers showed a highly amorphous character in levan formed from solution compared to the levan powder.     

In vitro digestibility and fermentability of levan and its hypocholesterolemic effects in rats

This study describes the in vitro digestibility and fermentability of high molecular weight (ca. 2,000,000) levan and its effect on the metabolism of lipids in growing rats fed cholesterol-free diets. Levan was synthesized from sucrose using bacterial levansucrase immobilized on a honeycomb-shaped ceramic support. Although body weight gain, weight of visceral organs, morphologic changes in the digestive tract, and the serum triacylglycerol and glucose concentrations were not affected by feeding levan diets for 4 weeks, a significant hypocholesterolemic effect was observed. Serum cholesterol level was decreased to 83% or 59% by feeding a 1% or 5% levan diet, respectively. The hypocholesterolemic effect was accompanied by a significant increase in fecal excretion of sterols and lipids. High molecular weight levan, though not hydrolyzed by the salivary amylases, was hydrolyzed by artificial gastric juice and was changed to a low molecular weight (ca. 4,000) levan with a small amount of fructose, but did not produce any fructooligosaccharides. Low molecular weight (ca. 6,000) levan was not hydrolyzed by either pancreatic juice or small intestinal enzymes. This suggests that, in vivo, low molecular weight levan derived from the high molecular weight material is not further digested and reaches the colon intact. The fermentation of low molecular weight levan (ca. 6,000) by several strains of bifidobacteria was not observed. These results showed that the hypocholesterolemic effect of levan may result from the prevention of intestinal sterol absorption, and not from the action of the fermentation products of levan.     

Characterization of levan produced by Serratia sp.

Levan was produced by a newly isolated bacterium from soil, taxonomically identified as a Serratia sp. This is the first report of levan production by Serratia sp. The levan was digested by levanase, which cannot hydrolyze β-2,1 linkages and the remaining substrate was analyzed by NMR. It was found that this levan had less β-2,1 linkage than other microbial levans, and that the structure was quite different from the levan produced by other bacteria such as genus Bacillus.     

Biosynthesis of levan and a new method for the assay of levansucrase activity

The polysaccharide levan was synthesized in a solidified agar medium containing sucrose as a source of fructose. The biosynthesis was achieved by the enzyme levansucrase (2,6-fructan–d-glucose 6-fructosyltransferase, EC 2.4.1.10), a small quantity of which was placed in circular wells cut in the agar gel. The enzyme slowly diffused through the agar–sucrose medium and the synthesis of levan was observed as circular white areas, the size of which was dependent on the time of incubation and the concentration of enzyme used.     

Influence of some physicochemical factors on the viscosity of aqueous levan solutions of Zymomonas mobilis

Zymomonas mobilis strain 113 “S” produces levan – an extracellular, viscous, biologically active, non-toxic fructose polymer with a unique structure and extraordinary properties. This polysaccharide was isolated at two different degrees of purity by alcohol precipitation from aqueous solutions and was characterized with respect to some rheological properties and stability of viscous solutions. The effects of temperature, pH and salt concentration on the viscosity of 1–3% levan solutions were examined. The viscosity of levan solutions was found to be quite stable and reversible at room temperature over a wide range of pH from 4 to 11. The viscosity was slightly affected by increased salt concentration. Levan solutions were rather stable at high temperatures (up to 70°C, 1 h, pH 6), where the viscosity could be almost completerly restored (up to 80–100%). Therefore, the degradation of the polymer structure under these conditions is probably insignificant. Temperatures of 70–100°C with a pH of less than 3.5 caused irreversible degradation of the levan structure. The above-mentioned properties of levan, obtained from Zymomonas mobilis 113 “S”, demonstrated the potential for the development of various therapeutic forms of pharmacologically-active levan and their application in medicine as well as in the food and other industries.     

Reuteran and levan as carbohydrate sinks in transgenic sugarcane

The present study reports the effect of high molecular weight bacterial fructan (levan) and glucan (reuteran) on growth and carbohydrate partitioning in transgenic sugarcane plants. These biopolymers are products of bacterial glycosyltransferases, enzymes that catalyze the polymerization of glucose or fructose residues from sucrose. Constructs, targeted to different subcellular compartments (cell wall and cytosol) and driven by the Cauliflower mosaic virus-35S: maize-ubiquitin promoter, were introduced into sugarcane by biolistic transformation. Polysaccharide accumulation severely affected growth of callus suspension cultures. Regeneration of embryonic callus tissue into plants proved problematic for cell wall-targeted lines. When targeted to the cytosol, only plants with relative low levels of biopolymer accumulation survived. In internodal stalk tissue that accumulate reuteran (max 0.03?mg/g FW), sucrose content (ca 60?mg/g FW) was not affected, while starch content (<0.4?mg/g FW) was increased up to four times. Total carbohydrate content was not significantly altered. On the other hand, starch and sucrose levels were significantly reduced in plants accumulating levan (max 0.01?mg/g FW). Heterologous expression resulted in a reduction in total carbohydrate assimilation rather than a simple diversion by competition for substrate.     

Properties of Geobacillus stearothermophilus levansucrase as potential biocatalyst for the synthesis of levan and fructooligosaccharides

The production of levansucrase (LS) by thermophilic Geobacillus stearothermophilus was investigated. LS production was more effective in the presence of sucrose (1%, w/v) than fructose, glucose, glycerol or raffinose. The results (T_op 57°C; stable for 6 h at 47°C) indicate the high stability of the transfructosylation activity of G. stearothermophilus LS as compared with LSs from other microbial sources. Contrary to temperature, the pH had a significant effect on the selectivity of G. stearothermophilus LS‐catalyzed reaction, favoring the transfructosylation reaction in the pH range of 6.0–6.5. The kinetic parameter study revealed that the catalytic efficiency of transfructosylation activity was higher as compared with the hydrolytic one. In addition to levan, G. stearothermophilus LS synthesized fructooligosaccharides in the presence of sucrose as the sole substrate. The results also demonstrated the wide acceptor specificity of G. stearothermophilus LS with maltose being the best fructosyl acceptor. This study is the first on the catalytic properties and the acceptor specificity of LS from G. stearothermophilus. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1405–1415, 2013     

Antidiabetic activity of levan polysaccharide in alloxan-induced diabetic rats

This study aims to examine the effects of polysaccharide levan on oxidative stress and hyperglycemia in alloxan-induced diabetic rats. Levan, used in this study, was a microbial levan synthetisized by a non pathogenic bacteria recently isolated and identified as Bacillus licheniformis. Animals were allocated into four groups of six rats each: a control group (Control), diabetic group (Diab.), normal rats received levan (L) and diabetic rats fed with levan (DL). Treated diabetic rats were administrated with levan in drinking water through oral gavage for 60 days. The administration of polysaccharide levan in diabetic rats caused a significant increase in glycogen level by 52% and a decrease in glucose level in plasma by 52%. Similarly, the administration of polysaccharide levan in diabetic rats caused a decrease in the thiobarbituric acid-reactive substances (TBARS) by 31%, 41%, 39% and 25%, an increase in superoxide dismutase (SOD) by 40%, 50%, 44% and 34%, and in catalase (CAT) by 18%, 20%, 12% and 18% in liver, kidney, pancreas and heart, respectively. Furthermore, a significant decrease in hepatic and renal indices toxicity was observed, i.e. alkalines phosphatases (ALP), aspartate and lactate transaminases (AST and ALT) activities, total bilirubin, creatinine and urea levels by 19%, 31%, 32%, 36%, 37% and 23%, respectively. The results show that administration of polysaccharide levan can restore abnormal oxidative indice near normal levels. This study demonstrates, for the first time, that polysaccharide levan is efficient in inhibiting hyperglycemia and oxidative stress induced by diabetes and suggests that levan supplemented to diet may be helpful in preventing diabetic complications in adult rats.     

High levan accumulation in transgenic tobacco plants expressing the Gluconacetobacter diazotrophicus levansucrase gene

Bacterial levansucrase (EC 2.4.1.10) converts sucrose into non-linear levan consisting of long β(2,6)-linked fructosyl chains with β(2,1) branches. Bacterial levan has wide food and non-food applications, but its production in industrial reactors is costly and low yielding. Here, we report the constitutive expression of Gluconacetobacter diazotrophicus levansucrase (LsdA) fused to the vacuolar targeting pre-pro-peptide of onion sucrose:sucrose 1-fructosyltransferase (1-SST) in tobacco, a crop that does not naturally produce fructans. In the transgenic plants, levan with degree of polymerization above 10⁴ fructosyl units was detected in leaves, stem, root, and flowers, but not in seeds. High levan accumulation in leaves led to gradual phenotypic alterations that increased with plant age through the flowering stage. In the transgenic lines, the fructan content in mature leaves varied from 10 to 70% of total dry weight. No oligofructans were stored in the plant organs, although the in vitro reaction of transgenic LsdA with sucrose yielded β(2,1)-linked FOS and levan. Transgenic lines with levan representing up to 30 mg g⁻¹ of fresh leaf weight produced viable seeds and the polymer accumulation remained stable in the tested T1 and T2 progenies. The lsdA-expressing tobacco represents an alternative source of highly polymerized levan.     

Levan果聚糖的应用与生产研究进展

Levan是一类果聚糖,由大量的果糖单元以β-（2,6）果糖苷键连接构成聚糖主链并含有少量β-（2,1）果糖苷键连接的支链组成。部分微生物来源的Levan具有抗肿瘤、抗病毒、降血糖、降血脂、免疫增强等重要的生物活性,在医药和功能性食品方面具有巨大的应用潜能。微生物发酵液提取和酶法合成是目前大量获得Levan果聚糖的两种方法,其中微生物发酵液提取的Levan果聚糖产量和蔗糖转化率一般较低,且发酵液中同时存在的其他高聚物不利于Levan的规模化纯化;而利用Levan蔗糖酶以蔗糖为底物转果糖基合成的Levan果聚糖产量已经高达200g/L、蔗糖转化率高达50%,并且Levan蔗糖酶合成Levan过程中酶的活性受到pH值、温度、螯合剂、金属离子等多种因素的影响,可以通过控制反应条件促进多糖合成反应的进行。因此,酶法合成将是工业化获得Levan果聚糖的主要方式。     

Enhanced levan production using chitin-binding domain fused levansucrase immobilized on chitin beads

Levan is a homopolymer of fructose which can be produced by the transfructosylation reaction of levansucrase (EC 2.4.1.10) from sucrose. In particular, levan synthesized by Zymomonas mobilis has found a wide and potential application in the food and pharmaceutical industry. In this study, the immobilization of Z. mobilis levansucrae (encoded by levU) was attempted for repeated production of levan. By fusion levU with the chitin-binding domain (ChBD), the hybrid protein was overproduced in a soluble form in Escherichia coli. After direct absorption of the protein mixture from E. coli onto chitin beads, levansucrase tagged with ChBD was found to specifically attach to the affinity matrix. Subsequent analysis indicated that the linkage between the enzyme and chitin beads was substantially stable. Furthermore, with 20% sucrose, the production of levan was enhanced by 60% to reach 83 g/l using the immobilized levansucrase as compared to that by the free counterpart. This production yield accounts for 41.5% conversion yield (g/g) on the basis of sucrose. After all, a total production of levan with 480 g/l was obtained by recycling of the immobilized enzyme for seven times. It is apparent that this approach offers a promising way for levan production by Z. mobilis levansucrase immobilized on chitin beads.     

Enzymic synthesis of levan and fructo-oligosaccharides by <Emphasis Type="Italic">Bacillus circulans</Emphasis> and improvement of levansucrase stability by carbohydrate coupling

Bacillus circulans was able to produce extracellular levansucrase using sucrose as carbon source optimally at 35°C. The enzymic synthesis of levan and fructo-oligosaccharides was studied using a 50% ethanol fraction of crude extract. The molecular weight of the synthesized levan was markedly affected by sucrose concentration, the molecular weight of levan decreased with increased sucrose concentration up to 32% whereby fructo-oligosaccharides were isolated. Temperature and the reaction time clearly affected the conversion of fructose to levan with molecular weight values ranging from 10 to 38 kDa. Identification of levan indicated that fructose was the building unit of the levan obtained. Thermal and pH stabilities of B. circulans levansucrase could be improved by enzyme glycosylation using sodium metaperiodate treatment. Chemical modification provides additional points of attachment of the enzyme to the support which offered the modified enzyme greater stabilization than did the free enzyme. The modified enzyme exhibited thermal tolerance up to 50°C, where it retained 88.25% of its activity, while the free enzyme only retained 64.55% of its original activity. The half-life significantly increased from 130 min for the free enzyme to 347 min for the modified enzyme at 50°C, however, it increased from 103 min for the free enzyme to 210 min for the modified enzyme at 60°C. Other properties i.e., the response to some metal ions as well as the ability to convert higher substrate levels and tolerance to an extension of the reaction periods were also improved upon modification. Obviously, the results obtained outlined the conditions leading to the formation of important high or low molecular weight or levan and fructo-oligosaccharides suitable for different industrial applications.     

Rheological characterization of levan polysaccharides from Microbacterium laevaniformans

Levan polysaccharides were produced from Microbacterium laevaniformans and its rheological behaviors were characterized as a function of concentration and temperature. The intrinsic viscosity of the purified levan was determined to be 0.38dL/g at 25 degrees C which was relatively higher than that of levans from other microbial sources. The flow behaviors of the levan solutions were characterized by the increase in the shear stress, giving more increments in the shear rate. Thus, the levan solutions exhibited the pseudoplastic behavior, which was characterized by the power law model. In addition, the flow behaviors of the levans were satisfactorily fitted to the Arrhenius equation where the activation energy of flow (Ea) decreased from 24.07 to 13.53kJ/mol (R2=0.98-0.99) with increasing concentrations. Moreover, the exponential equation was favorably applied to describe the effect of concentration on the apparent viscosity of the levan polysaccharides.     

Partial purification of a Bacillus licheniformis levansucrase producing levan with antitumor activity

The extracellular fructosyltransferase (FTase) of a novel strain of Bacillus licheniformis capable of producing fructooligosaccharides (FOS) and a polysaccharide type levan was obtained and partially purified. The purification was achieved by ammonium sulfate precipitation, DEAE cellulose and gel filtration chromatographies. The enzyme was partially purified as determined by SDS-PAGE, and the specific activity reached was 67.5, representing a purification factor of 177 and yield of 40%. Levan was isolated from the cultures of B. licheniformis. The levan was composed mainly of fructose residues when analyzed by TLC after acid hydrolysis and NMR analysis. In a previous study, the levan produced exhibited a hypoglycemiant activity. The present paper deals with the study of the antitumor and anti-cytotoxic effect of levan produced by B. licheniformis strain. In the in vitro antitumor activity test of levan against some tumor cell lines, relatively the significantly high activity was observed against the HepG(2).