Bacterial Levans Research Articles

Coculture of bacterial levans and evaluation of its anti-cancer activity against hepatocellular carcinoma cell lines

This research represents a novel study to assess how coculture affects levan yield, structure, bioactivities, and molecular weight. Among the 16 honey isolates, four bacterial strains recorded the highest levan yield. The Plackett–Burman design showed that the coculture (M) of isolates G2 and K2 had the maximum levan yield (52 g/L) and the effective factors were sucrose, incubation time, and sugarcane bagasse. The CCD showed that the most proper concentrations for maximum levan yield (81 g/L): were 130 g/L of sucrose and 6 g/f of sugarcane bagasse. Levan’s backbone was characterized, and the molecular weight was determined. G2 and K2 isolates were identified based on 16 sRNA as Bacillus megaterium strain YM1C10 and Rhizobium sp. G6-1. M levan had promising antioxidant activity (99.66%), slowed the migration activity to a great extent, and recorded 70.70% inhibition against the hepatoblastoma cell line (HepG2) at 1000 µg/mL. Gene expression analysis in liver cancer cell lines (HePG2) revealed that M levan decreased the expression of CCL20), 2GRB2, and CCR6) genes and was superior to Doxo. While increasing the expression of the IL4R and IL-10 genes. The DNA damage values were significantly increased (P < 0.01) in treated liver cancer cell lines with levan M and Doxo. The results referred to the importance of each of the hydroxyl and carboxyl groups and the molecular weight in levans bioactivities.

Prebiotic-mediated gastroprotective potentials of three bacterial levans through NF-κB-modulation and upregulation of systemic IL-17A

This study aimed to investigate whether the gastroprotective effects of three types of bacterial levans are correlated with their prebiotic-associated anti-inflammatory/antioxidant potentials. Three levans designated as LevAE, LevP, and LevZ were prepared from bacterial honey isolates; purified, and characterized using TLC, NMR, and FTIR. The anti-inflammatory properties of levan preparations were assessed in LPS-stimulated RAW 264.7 cell lines, while their safety and gastroprotective potentials were assessed in Wistar rats. The three levans significantly reduced ulcer number (22.29–70.05 %) and severity (31.76–80.54 %) in the ethanol-induced gastric ulcer model compared to the control (P < 0.0001/each), with the highest effect observed in LevAE and levZ (200 mg/each) (P < 0.0001). LevZ produced the highest levels of glutathione; catalase activity, and the lowest MDA levels (P = 0.0001/each). The highest anti-inflammatory activity was observed in LevAE and levZ in terms of higher inhibitory effect on IL-1β and TNF-α production (P < 0.0001 each); COX2, PGE2, and NF-κB gene expression. The three levan preparations also proved safe with no signs of toxicity, with anti-lipidemic properties as well as promising prebiotic activity that directly correlated with their antiulcer effect. This novel study highlights the implication of prebiotic-mediated systemic immunomodulation exhibited by bacterial levans that directly correlated with their gastroprotective activity.

A Highly Active Endo-Levanase BT1760 of a Dominant Mammalian Gut Commensal Bacteroides thetaiotaomicron Cleaves Not Only Various Bacterial Levans, but Also Levan of Timothy Grass.

Bacteroides thetaiotaomicron, an abundant commensal of the human gut, degrades numerous complex carbohydrates. Recently, it was reported to grow on a β-2,6-linked polyfructan levan produced by Zymomonas mobilis degrading the polymer into fructooligosaccharides (FOS) with a cell surface bound endo-levanase BT1760. The FOS are consumed by B. thetaiotaomicron, but also by other gut bacteria, including health-promoting bifidobacteria and lactobacilli. Here we characterize biochemical properties of BT1760, including the activity of BT1760 on six bacterial levans synthesized by the levansucrase Lsc3 of Pseudomonas syringae pv. tomato, its mutant Asp300Asn, levansucrases of Zymomonas mobilis, Erwinia herbicola, Halomonas smyrnensis as well as on levan isolated from timothy grass. For the first time a plant levan is shown as a perfect substrate for an endo-fructanase of a human gut bacterium. BT1760 degraded levans to FOS with degree of polymerization from 2 to 13. At optimal reaction conditions up to 1 g of FOS were produced per 1 mg of BT1760 protein. Low molecular weight (<60 kDa) levans, including timothy grass levan and levan synthesized from sucrose by the Lsc3Asp300Asn, were degraded most rapidly whilst levan produced by Lsc3 from raffinose least rapidly. BT1760 catalyzed finely at human body temperature (37°C) and in moderately acidic environment (pH 5–6) that is typical for the gut lumen. According to differential scanning fluorimetry, the Tm of the endo-levanase was 51.5°C. All tested levans were sufficiently stable in acidic conditions (pH 2.0) simulating the gastric environment. Therefore, levans of both bacterial and plant origin may serve as a prebiotic fiber for B. thetaiotaomicron and contribute to short-chain fatty acids synthesis by gut microbiota. In the genome of Bacteroides xylanisolvens of human origin a putative levan degradation locus was disclosed.

Levanase from Bacillus subtilis hydrolyses β-2,6 fructosyl bonds in bacterial levans and in grass fructans

A Levanase, LevB, from Bacillus subtilis 168, was expressed as a His6-tagged protein in Escherichia coli. The enzyme was purified and characterised for its activity and substrate specificity. LevB has a pH optimum of 6.0-6.5 and a maximum observed specific activity of 3Umg−1 using levan from Erwinia herbicola as substrate. Hydrolysis products were analysed by HPAEC, TLC, and NMR using chicory root inulin, mixed linkage fructans purified from ryegrass (Lolium perenne) and levan from E. herbicola as substrates. This revealed that LevB is an endolevanase that selectively cleaves the (β-2,6) fructosyl bonds and does not hydrolyse inulin. Ryegrass fructans and bacterial levan was hydrolysed partially releasing oligosaccharides, but together with exoinulinase, LevB hydrolysed both ryegrass fructans and bacterial levan to near completion. We suggest that LevB can be used as a tool to achieve more structural information on complex fructans and to achieve complete degradation and quantification of mixed linkage fructans.

Review on production, characterization and applications of microbial levan

Levan is a homopolymer of fructose naturally obtained from both plants and microorganisms. Microbial levans are more advantageous, economical and industrially feasible with numerous applications. Bacterial levans are much larger than those produced by plants with multiple branches and molecular weights ranging from 2 to 100 million Da. However levans from plants generally have molecular weights ranging from about 2000 to 33,000 Da. Microbial levans have wide range of applications in food, medicine, pharmaceutical, cosmetic and commercial industrial sectors. With excellent polymeric medicinal properties and ease of production, microbial levan appear as a valuable and versatile biopolymer of the future. The present article summarizes and discusses the most essential properties of bioactive microbial levan and recent developments in its production, characterization and the emerging applications in health and industry.

Kinetic behaviour and specificity of β-fructosidases in the hydrolysis of plant and microbial fructans

Fructosidases, in particular exo-β-fructosidases, may act on fructans such as inulins and levans of plant and bacterial origin to produce fructose. In this paper, the kinetic properties of a commercial preparation (Fructozyme L) and a recombinant exoinulinase (BfrA) from Thermotoga maritima, were studied using fructan polymer substrates from various sources. Both enzymatic preparations preferentially hydrolyzed β2-1 linkages and low molecular weight fructans. We show that chicory inulin is degraded most efficiently by both preparations, followed by bacterial inulin, in spite of its high molecular weight and branching in β2-6 positions. All bacterial levans were more slowly hydrolyzed. Michaelis–Menten kinetics describe the hydrolysis of sucrose and low molecular weight fructans (≤8.3 kDa) by both enzyme preparations, while first order kinetics were observed with respect to bacterial fructans due to the high molecular weight and, therefore, low molar concentrations. Comparison of second order rate constants indicates that bacterial inulin ( Leuconostoc citreum CW28) is hydrolyzed more slowly with both enzyme preparations than chicory inulin by approximately one order of magnitude. For Leuconostoc mesenteroides NRRL B-512F levan, the second order rate constant for Fructozyme L is 200-fold lower than for chicory inulin. However, the second order rate constant for BfrA is only 22-fold lower than for chicory inulin. Taken together, our studies characterize the kinetics of fructan hydrolysis and also suggest that the kinetic parameters may be used to differentiate between fructan structures.

Biochemical and molecular characterization of a levansucrase from Lactobacillus reuteri.

Lactobacillus reuteri strain 121 employs a fructosyltransferase (FTF) to synthesize a fructose polymer [a fructan of the levan type, with beta(2-->6) linkages] from sucrose or raffinose. Purification of this FTF (a levansucrase), and identification of peptide amino acid sequences, allowed isolation of the first Lactobacillus levansucrase gene (lev), encoding a protein (Lev) consisting of 804 amino acids. Lev showed highest similarity with an inulosucrase of L. reuteri 121 [Inu; producing an inulin polymer with beta(2-->1)-linked fructosyl units] and with FTFs from streptococci. Expression of lev in Escherichia coli resulted in an active FTF (Lev Delta 773His) that produced the same levan polymer [with only 2-3 % beta(2-->1-->6) branching points] as L. reuteri 121 cells grown on raffinose. The low degree of branching of the L. reuteri levan is very different from bacterial levans known up to now, such as that of Streptococcus salivarius, having up to 30 % branches. Although Lev is unusual in showing a higher hydrolysis than transferase activity, significant amounts of levan polymer are produced both in vivo and in vitro. Lev is strongly dependent on Ca(2+) ions for activity. Unique properties of L. reuteri Lev together with Inu are: (i) the presence of a C-terminal cell-wall-anchoring motif causing similar expression problems in Escherichia coli, (ii) a relatively high optimum temperature for activity for FTF enzymes, and (iii) at 50 degrees C, kinetics that are best described by the Hill equation.

Structure of fructan polymers from tuberous roots of Gomphrena macrocephala (Amaranthaceae) from the cerrado

summaryTwo fructo‐polysaccharides, FP‐I and FP‐II, were separated from a soluble extract from tuberous roots of Gomphrena macrocephala St.‐Hil. by gel permeation chromatography on Toyopearl HW‐40S. FP‐I and FP‐II were mixtures of saccharides with a wide‐ranging degree of polymerization; their acid hydrolysis products were fructose and glucose. The ratios of fructose to glucose in the hydrolysates of FP‐I and FP‐II were 38 and 60, respectively. The average molecular weights of FP‐I and FP‐II were estimated to be 5500 and 10000 respectively, by gel permeation HPLC. The structural confirmation of FP‐I and FP‐II was made by 13C‐NMR analysis. Intense signals corresponding to carbon‐1 (C1), C2, C3, C4, C5 and C6 of fructose residues in FP‐I and FP‐II were observed at δ 60.60, 104.93, 76.99, 75.91, 81.02 and 64.11, respectively. These chemical shifts coincided with those of timothy and bacterial levans. The less intense resonances were tentatively assigned to carbons of terminal fructosyl residues and a fructosyl residue linked to a glucose residue by a β‐2,1 bond in FP‐I and FP‐II. Also, weak signals attributed to carbons of the glucose residue in both polysaccharides were separated from those of other glycosyl residues. These findings were supported by GLC analysis of methanolysate from permethylated FP‐I and FP‐II. Thus FP‐I and FP‐II comprise fructose residues with β‐2,6 linkages, and a terminal glucose bound with fructose residues at position C1, although both polysaccharides possibly contained a non‐terminal glucose residue in the molecule.

Action of Arthrobacter ureafaciens inulinase II on several oligofructans and bacterial levans

1. 1. Arthrobacter ureafaciens inulinase II which converts inulin to di- d-fructofuranose 1,2′ : 2,3′ dianhydride (difructose anhydride III) leaving a small amount of oligosaccharides, was investigated in order to characterize its mode of action. 2. 2. After the enzymatic reaction on the glucose-terminated inulin molecules had been completed, the oligosaccharides left in the enzyme digest were isolated, and identified to be the fructose-glucose oligosaccharides; O-β- d-fructofuranosyl -(2 → 1)-O-β- d-fructofuranosyl α- d-glucopyranoside (1-kestose), O-β- d-fructofuranosyl -[(2 → 1)-O-β- d-fructofuranosyl ] 2 α- d-glucopyranoside and O-β- d-fructofuranosyl -[(2 → 1)-O-β- d-fructofuranosyl ] 3 α- d-glucopyranoside. The difructose anhydride formation from the three fructose-glucose oligosaccharides in the separate reaction system with an increased substrate concentration was observed only with the latter two substrates, but not with the first one. 3. 3. The difructose anhydride formation with several (2 → 1)- β-linked fructose oligosaccharides and bacterial (2 → 6)- β-fructans was examined. The (2 → 1)- β-linked fructose oligosaccharides were effective as substrates for the enzyme with the exception of inulobiose, but the (2 → 6)- β-fructans remained unaffected. 4. 4. It was concluded that the enzyme attacks (2 → 1)- β-linked fructan molecules from the nonreducing fructose ends and requires the presence of at least two adjacent (2 → 1)- β-fructofuranosyl linkages.

Sucrose utilization by Zymomonas mobilis: formation of a levan.

1. Molar growth-yield coefficients of Zymomonas mobilis for glucose, fructose, glucose plus fructose, and sucrose are reported. Yield coefficients for sucrose are appreciably lower than those for the equivalent concentrations of glucose plus fructose. 2. Only 2.6% of [U-(14)C]glucose supplied in the growth medium is incorporated into cell substance by Z. mobilis utilizing glucose as the energy source. 3. During growth on sucrose a levan is formed. It has been characterized and shown to resemble other bacterial levans. 4. Levan formation from sucrose could be demonstrated with both washed cell suspensions and cell extracts of Z. mobilis. 5. Sucrose phosphorylase could not be demonstrated in extracts of the organism.

Protein-carbohydrate interaction: III. Agar gel-diffusion studies on the interaction of concanavalin A, a lectin isolated from jack bean, with polysaccharides

Concanavalin A, a protein isolated from the jack bean ( Canavalia ensiformis), has previously been shown to form a precipitate with glycogen and yeast mannan, and more recently with dextrans and amylopectin. Double-diffusion precipitation studies in agar gel have been found to afford a very sensitive and satisfactory method for investigating the interaction of concanavalin A with certain types of branched, glucose- and mannose- containing polysaccharides. In this manner concanavalin A can be used as a reagent for the detection and preliminary characterization of various types of polysaccharides. Bacterial levans as well as certain plant fructans also form precipitation bands with concanavalin A. This observation represents the first report of such an interaction. The method may also provide an indication as to the heterogeneity of polysaccharide preparations that react with concanavalin A as well as affording preliminary information on the relative molecular size of a series of reactive polysaccharides. Inhibition of the precipitation bands by low molecular weight carbohydrate molecules may be demonstrated. These inhibition studies reveal the highly specific nature of the combining sites of the concanavalin A molecule.

Studies on a beta-fructosidase (inulinase) produced bySaccharomyces fragilis

A study was made of a β-fructosidase, which is produced extracellularly and intracellularly bySaccharomyces fragilis. The enzyme catalyzes the hydrolysis of inulin, bacterial levans, sucrose, and the fructose portion of raffinose, by splitting off terminal fructosyl units. It attacks β-2,1 as well as β-2,6 linkages. The enzyme content of inulin-grown cells is sufficient to allow fermentation of inulin at the same rate as glucose. The ratio of hydrolysis rates with sucrose and inulin was about 25 for the β-fructosidase ofS. fragilis and about 14,000 for invertase.S. fragilis does not contain significant amounts of invertase and it ferments inulin, sucrose and raffinose with the aid of a related, but different enzyme, inulinase.

Bacterial levans of intermediate molecular weight.

Bacterial levans of intermediate molecular weight.

Experiments concerning the renal clearances of some fructosans in the dog.

1. In continuance of previous work, experiments have been carried out on the renal clearance in the female dog of certain fructosans, namely (a) irisin isolated from Iris pseudacorus, and (b) and (c) the high molecular weight levans synthesised respectively by exocellular enzyme systems of Ps. prunicola and B. subtilis.2. From the pattern of the renal clearances over a period of up to three hours, it seems clear that while irisin and Ps. prunicola levan may be, by present standards, chemically homogeneous, they are, in fact, physically heterogeneous, i.e. each consists of an assembly of similarly constituted polymers.3. While intravenous administration of low molecular weight grass levan and irisin is unaccompanied by vasomotor and respiratory disturbances, the higher molecular weight bacterial levans, despite vigorous measures to remove any traces of alkali‐hydrolysable material (e.g. protein), induced such disturbances. B. subtilis levan produced more intense responses than did Ps. prunicola levan.4. Ps. prunicola levan is presumed to be a “very large molecule”. Its extremely low clearance by the kidney indicates that the components of the assembly of molecules in the material pass through the renal glomerular membrane only with great difficulty and are not hydrolysed to any appreciable extent within the animal body.5. B. subtilis levan, which from chemical evidence can be considered to be a polymer of similar chemical units to those comprising Ps. prunicola levan, behaved in a markedly different manner. The high clearance values were not those to be expected from a high molecular weight fructosan. The discovery of considerable quantities of D‐fructose in the urine after injection with this levan indicated that the dog is able to hydrolyse this levan at a rapid rate. The rate of liberation of fructose was so rapid that it exceeded the rate of utilisation in the tissues and/or absorbtion of fructose by the kidney. D‐fructose was identified by (a) paper chromatography, and (b) the formation of the methylphenyl osazone and its acetyl derivative.

Serological Reactions of Levans Formed from Sucrose and Raffinose by Certain Bacilli.

SummaryLevans formed in sucrose and raffinose broth by B. subtilis, B. panis, 5 other strains of Bacillus, and 3 strains of non-sporulating Gram-positive rod-shaped bacteria possessed serological properties similar to those previously described for the levans of several strains of S. salivarius and of one strain of Bacillus. This evidence extends the serological information on the levari class of bacterial polysaccharide and gives a better basis for the application of serological procedures to the detection of bacterial levans both in laboratory culture fluids and In the various materials in which they occur in nature.

Serological Reactions of Two Bacterial Levans

Summary The levan polysaccharides synthesized from sucrose by a spore-forming bacillus of plant origin and by a strain of Streptococcus salivarius were proved to be serologically reactive and serologically similar substances. The levans gave both precipitation and complement fixation with antiserums produced by immunization with either the streptococcus or the bacillus which had been grown in media containing sucrose, and also reacted with some samples but not with most type 20 antipneumococcus serums. The carbohydrate in the culture medium was an important factor. The serologically reactive levans were produced in abundant amount in broth containing sucrose or raffinose whereas none could be found in cultures grown with 8 other sugars. Similarly, antiserums reactive with the levans were readily produced by immunization with suspensions of either the bacillus or streptococcus which had been grown in sucrose media but not by immunization with the same bacteria grown in glucose media.