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.     

Molecular cloning and expression in Escherichia coli of an exo-levanase gene from the endophytic bacterium Gluconacetobacter diazotrophicus SRT4

Gluconacetobacter diazotrophicus produces levan from sucrose by a secreted levansucrase (LsdA). A levanase-encoding gene (lsdB), starting 51 bp downstream of the lsdA gene, was cloned from strain SRT4. The lsdB gene (1605 bp) encodes a protein (calculated molecular mass 58.4 kDa) containing a putative 36-amino-acid signal peptide at the N-terminus. The deduced amino acid sequence shares 34%, 33%, 32%, and 29% identities with levanases from Actinomyces naeslundii, Bacillus subtilis, Paenibacillus polymyxa, and Bacteroides fragilis, respectively. The lsdB expression in Escherichia coli under the control of the T7 RNA polymerase promoter resulted in an active enzyme which hydrolyzed levan, inulin, 1-kestose, raffinose, and sucrose, but not melezitose. Levanase activity was maximal at pH 6.0 and 30°C, and it was not inhibited by the metal ion chelator EDTA or the denaturing agents dithiothreitol and β-mercaptoethanol. The recombinant LsdB showed a fourfold higher rate of hydrolysis on levan compared to inulin, and the reaction on both substrates resulted in the successive liberation of the terminal fructosyl residues without formation of intermediate oligofructans, indicating a non-specific exo-levanase activity. Received: 27 August 2001 / Accepted: 15 October 2001     

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.     

Gene cloning, characterization, and heterologous expression of levansucrase from Bacillus amyloliquefaciens

Although levan produced by Bacillus amyloliquefaciens is known to have efficient immunostimulant property which gives 100% survival of common carp when infected with Aeromonas hydrophila, no detailed reports are available describing kinetic studies of d-glucose production and levan formation. In this study, we cloned and characterized the enzymatic kinetics using levansucrase expressed in Escherichia coli. Optimum pH for d-glucose production and levan formation was 6.0 and 8.0, respectively, whereas optimum temperature was 30°C and 4°C, respectively. The K _m and V _max values for levansucrase were calculated to be 47.81 mM sucrose and 57.47 μmole/min mg protein, respectively. Prominent expression of levansucrase was obtained through xylose induction in Bacillus megaterium, where most of the His₆-tagged protein was secreted into the culture broth, giving levansucrase activity of 12,906 U/l. Response-surface methodology (RSM) was further employed to optimize the fermentation conditions and improve the level of levansucrase production. Maximum levansucrase activity of 20,251 U/l was obtained in 12 h of fermentation carried out at 28°C, starting induction with 0.735% xylose when A ₆₀₀ was 1.2, which was 1.6- and 62-fold higher than those obtained in the nonoptimized conditions for the recombinant strain and the native strain, respectively.     

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.     

Molecular and functional characterization of a levansucrase from the sourdough isolate Lactobacillus sanfranciscensis TMW 1.392

Exopolysaccharides (EPS) produced in situ by sourdough lactobacilli affect rheological properties of dough as well as bread quality and may serve as prebiotics. The aim of this study was to characterize EPS-formation by Lactobacillus sanfranciscensis TMW 1.392 at the molecular level. A levansucrase gene from L. sanfranciscensis TMW 1.392 encompassing 2,300 bp was sequenced. This levansucrase is predicted to be a cell-wall associated protein of 879 amino acids with a relative molecular weight (M_R) of 90,000. The levansucrase gene was heterologously expressed in Escherichia coli and purified to homogeneity. The recombinant enzyme exhibited transferase and hydrolase activities and produced glucose, fructose, 1-kestose and levan from sucrose; truncation of the N-terminal domain did not affect catalytic activity. Kestose formation was enhanced relative to fructose and levan formation by low temperature or high sucrose levels. During growth in wheat doughs, strain TMW 1.392 utilized sucrose to form fructose, 1-kestose, and fructan, whereas a levansucrase deletion mutant, L. sanfranciscensis TMW 1392lev, lost the ability to hydrolyze sucrose, and did not produce fructan or 1-kestose. These results indicate that, in L. sanfranciscensis TMW 1.392, sucrose metabolism and formation of fructan and 1-kestose is dependent on the activity of a single enzyme, levansucrase.     

Donor substrate recognition in the raffinose-bound E342A mutant of fructosyltransferase <Emphasis Type="Italic">Bacillus subtilis</Emphasis> levansucrase

Background  

Fructans – β-D-fructofuranosyl polymers with a sucrose starter unit – constitute a carbohydrate reservoir synthesised by a considerable number of bacteria and plant species. Biosynthesis of levan (αGlc(1–2)βFru [(2–6)βFru]_n), an abundant form of bacterial fructan, is catalysed by levansucrase (sucrose:2,6-β-D-fructan-6-β-D-fructosyl transferase), utilizing sucrose as the sole substrate. Previously, we described the tertiary structure of Bacillus subtilis levansucrase in the ligand-free and sucrose-bound forms, establishing the mechanistic roles of three invariant carboxylate side chains, Asp86, Asp247 and Glu342, which are central to the double displacement reaction mechanism of fructosyl transfer. Still, the structural determinants of the fructosyl transfer reaction thus far have been only partially defined.     

Comparison of characteristics of levan produced by different preparations of levansucrase from Zymomonas mobilis

The characteristics of levan formation by different preparations of levansucrase (free and immobilized enzyme and toluene-permeabilized whole cells), derived from recombinant levansucrase from Zymomonas mobilis expressed in Escherichia coli, were investigated. The maximal yield of levan by the three preparations were similar and were about 70–80% on a fructose-released basis with sucrose as nutrient at 100 g l^–1. Immobilized enzyme and toluene-permeabilized whole cells produced low molecular weight levan (2–3 × 10⁶), as determined by HPLC while high molecular weight levan (>6 × 10⁶) was the major product with the free levansucrase. The size of levan can thus be controlled by immobilized levansucrase and toluene-permeabilized whole cells in high yield.     

Production of Levansucrase from Bacillus subtilis NRC 33a and Enzymic Synthesis of Levan and Fructo-Oligosaccharides

Bacillus subtilis NRC33a was able to produce both inducible and constitutive extracellular levansucrase, respectively, using sucrose and glucose as carbon source. The optimal production of the levansucrase was at 30°C. The effect of different nitrogen sources showed that baker’s yeast with 2% concentration gave the highest levansucrase activity. Addition of 0.15 g/L MgSO₄ was the most favorable for levansucrase production. The enzymic synthesis of levan was studied using 60% acetone fraction. The results indicated that high enzyme concentrations produced increasing amounts of levan, and hence conversion of fructose to levan reached 84% using 1000 μg/ml enzyme protein. Sucrose concentration was the most effective factor controlling the molecular weight of the synthesized levan. The conversion of fructose to levan was maximal at 30°C. The time of reaction clearly affected the conversion of fructose to levan, which reached its maximum productivity at 18 hours (92%). Identification of levan indicated that fructose was the building unit of levan.     

Identification and characterisation of the extracellular sucrases of Zymomonas mobilis UQM 2716 (ATCC 39676)

An investigation was conducted to isolate, and characterise the extracellular sucrases of Zymomonas mobilis UQM 2716. Levansucrase (EC 2.4.1.10) was the only extracellular sucrase produced by this organism. This enzyme was responsible for sucrose hydrolysis, levan formation, and oligosaccharide production. It had a molecular mass of 98 kDa, a Michaelis constant (K _m) of 64 mm, and a pH optimum of 5.5. It was inhibited by glucose, but not by fructose, ethanol, sorbitol, NaCl, TRIS or ethylenediaminetetraacetic acid (EDTA). The formation of levan was the principal reaction catalysed by this enzyme at low temperatures. However, levan formation was thermolabile, being irreversibly lost when levansucrase was heated to 35°C. S This did not effect sucrose hydrolysis or oligosaccharide formation, which were optimal at 45°C. Sucrose concentration greatly influenced the type of acceptor molecule used in the transfructosylation reactions catalysed by levansucrase. At low sucrose concentration, the predominant reaction catalysed was the hydrolysis of sucrose to free glucose and fructose. At high sucrose concentrations, oligosaccharide production was the major reaction catalysed.     

Serine substitution for cysteine residues in levansucrase selectively abolishes levan forming activity

Levansucrase is responsible for levan formation during sucrose fermentation of Zymomonas mobilis, and this decreases the efficiency of ethanol production. As thiol modifying agents decrease levan formation, a role for cysteine residues in levansucrase activity has been examined using derivatives of Z. mobilis levansucrase that carry serine substitutions of cysteine at positions 121, 151 or 244. These substitutions abolished the levan forming activity of levansucrase whilst only halving its activity in sucrose hydrolysis. Thus, polymerase and hydrolase activities of Z. mobilis levansucrase are separate and have different requirements for the enzyme's cysteine residues.     

Evaluation of cross-linked aggregates from purified Bacillus subtilis levansucrase mutants for transfructosylation reactions

Background  

Increasing attention has been focused on inulin and levan-type oligosaccharides, including fructosyl-xylosides and other fructosides due to their nutraceutical properties. Bacillus subtilis levansucrase (LS) catalyzes the synthesis of levan from sucrose, but it may also transfer the fructosyl moiety from sucrose to acceptor molecules included in the reaction medium. To study transfructosylation reactions with highly active and robust derivatives, cross-linked enzyme aggregates (CLEAs) were prepared from wild LS and two mutants. CLEAs combine the catalytic features of pure protein preparations in terms of specific activity with the mechanical behavior of industrial biocatalysts.     

Possible correlation between levansucrase production and probiotic activity of <Emphasis Type="Italic">Bacillus</Emphasis> sp. isolated from honey and honey bee

Five bacterial isolates from honey and bee gut were selected based on their high levansucrase activity and levan yield which were strongly positively correlated. All isolates showed good tolerance to temperature up to 70?°C, to NaCl up to 3 M and to 0.1% H₂O₂. They maintained over 59 and 64% survival at pH 9.0 and 2.0 respectively, but showed varying tolerance to 0.1% bile salts and pancreatic enzymes. Most isolates were susceptible to widely used antibiotics, but demonstrated diverse antimicrobial activity. Non hemolytic isolates were identified on the basis of 16S rRNA sequencing as Bacillus subtilis HMNig-2 and B. subtilis MENO2 with 97% homology. They exhibited promising probiotic characteristics and achieved highest levansucrase activity of 94.1 and 81.5 U/mL respectively. Both exhibited highest biofilm formation ability in static microtiter plate assay. Also, they achieved 34 and 26% adhesion respectively to Caco-2cells and had highest free radical scavenging activity of 30.8 and 26.2% respectively. The levans of the two isolates showed good antimicrobial activity against some pathogens and exhibited positive prebiotic effect (prebiotic index >1) with Lactobacillus casei and Lactobacillus reuteri. Results suggest a correlation between levansucrase production, levan yield and pre-probiotic activities of the studied strains.     

Levansucrase from Halomonas smyrnensis AAD6T: first halophilic GH-J clan enzyme recombinantly expressed,purified, and characterized

Fructans, homopolymers of fructose produced by fructosyltransferases (FTs), are emerging as intriguing components in halophiles since they are thought to be associated with osmotic stress tolerance and overall fitness of microorganisms and plants under high-salinity conditions. Here, we report on the full characterization of the first halophilic FT, a levansucrase from Halomonas smyrnensis AAD6^T (HsLsc; EC 2.4.1.10). The encoding gene (lsc) was cloned into a vector with a 6xHis Tag at its C-terminus, then expressed in Escherichia coli. The purified recombinant enzyme (47.3 kDa) produces levan and a wide variety of fructooligosaccharides from sucrose, but only in the presence of high salt concentrations (> 1.5 M NaCl). HsLsc showed Hill kinetics and pH and temperature optima of 5.9 and 37 °C, respectively. Interestingly, HsLsc was still very active at salt concentrations close to saturation (4.5 M NaCl) and was selectively inhibited by divalent cations. The enzyme showed high potential in producing novel saccharides derived from raffinose as both fructosyl donor and acceptor and cellobiose, lactose, galactose, and ʟ-arabinose as fructosyl acceptors. With its unique biochemical characteristics, HsLsc is an important enzyme for future research and potential industrial applications in a world faced with drought and diminishing freshwater supplies.

     

Fructan Biosynthesis by Intra‐ and Extracellular Zymomonas mobilis Levansucrase after Simultaneous Production of Ethanol and Levan

The chemical composition of the Zymomonas mobilis biomass and the culture liquid after ethanol and levan synthesis were studied. The activities of intra‐ and extracellular levansucrase produced by the Z. mobilis strain 113 “S” under optimum conditions both for levan and fructooligosaccharide (FOS) synthesis were also determined. It was shown that levan production relates to the reduction of the carbohydrate and lipid content in the biomass by increasing the nucleic acid and protein content. The levan producing activity of cellular levansucrase after ethanol and levan synthesis was approximately 30–40% of the total activity in the second fermentation stage. It was established that the cell free culture liquid, containing ethanol, levan, gluconic acid and sucrose (15%) at 25 °C, did not show any additional levan synthesising activity. At optimum FOS synthesis conditions (45 °C and 70% sucrose), the cell‐free culture liquid exhibited a high FOS synthesising activity (31% from total carbohydrates), with slightly reduced biomass activity. It was concluded that as a result of the simultaneous ethanol and levan production, the remaining biomass as well as the cell‐free culture liquid could be used for FOS production.     

In vitro selection and identification of sweetpotato (<Emphasis Type="Italic">Ipomoea batatas</Emphasis> (L.) Lam.) plants tolerant to NaCl

In vitro selection of sweetpotato (Ipomoea batatas (L.) Lam.) plants tolerant to NaCl was achieved using embryogenic suspension cultures of sweetpotato cv. Lizixiang and gamma-ray induced mutation. Cell aggregates from embryogenic suspension cultures of Lizixiang were irradiated with 80 Gy gamma-ray, and 1 week after irradiation they were cultured in a selective medium containing 342 mM NaCl for in vitro selection. A total of 276 plants were regenerated from the irradiated 2,783 cell aggregates by a two-step in vitro selection procedure. After the regenerated plants were propagated into plant lines on the basal medium, they were cultured on the medium supplemented with 86, 171, 257 and 342 mM NaCl, respectively, in order to evaluate their in vitro salt tolerance. Of them 18 plant lines showed significantly higher in vitro salt tolerance than control plants. Proline and superoxide dismutase (SOD) were more accumulated in these 18 plant lines than in control plants when both were exposed to NaCl. Salt tolerance of the 18 plant lines was further evaluated with Hoalgland solution containing different concentrations of NaCl in a greenhouse. The results indicated that 3 of them had significantly better growth and rooting ability than the remaining 15 plant lines and control plants at 171 mM NaCl.     

Cloning and expression of levansucrase from Leuconostoc mesenteroides B-512 FMC in Escherichia coli

Leuconostoc mesenteroides B-512 FMC produces dextran and levan using sucrose. Because of the industrial importance of dextrans and oligosaccharides synthesized by dextransucrase (one of glycansucrases from L. mesenteroides), much is known about the dextransucrase, including expression and regulation of gene. However, no detailed report about levansucrase, another industrially important glycansucrase from L. mesenteroides, and its gene was available. In this paper, we report the first-time isolation and molecular characterization of a L. mesenteroides levansucrase gene (m1ft). The gene m1ft is composed of 1272-bp nucleotides and codes for a protein of 424 amino acid residues with calculated molecular mass of 47.1 kDa. The purified protein was estimated to be about 51.7 kDa including a His-tag based on SDS-PAGE. It showed an activity band at 103 kDa on a non-denaturing SDS-PAGE, indicating a dimeric form of the active M1FT. M1FT levan structure was confirmed by NMR and dot blot analysis with an anti-levan-antibody. M1FT converted 150 mM sucrose to levan (18%), 1-kestose (17%), nystose (11%) and 1,1,1-kestopentaose (7%) with the liberation of glucose. The M1FT enzyme produced erlose [O-alpha-D-glucopyranosyl-(1-->4)-O-alpha-D-glucopyranosyl-(1-->2)-beta-D-fructofuranoside] as an acceptor product with maltose. The optimum temperature and pH of this enzyme for levan formation were 30 degrees C and pH 6.2, respectively. M1FT levansucrase activity was completely abolished by 1 mM Hg2+ or Ag2+. The Km and Vmax values for levansucrase were calculated to be 26.6 mM and 126.6 micromol min-1 mg-1.     

Biosynthesis of Levan,a Bacterial Extracellular Polysaccharide,in the Yeast Saccharomyces cerevisiae

Levans are fructose polymers synthesized by a broad range of micro-organisms and a limited number of plant species as non-structural storage carbohydrates. In microbes, these polymers contribute to the formation of the extracellular polysaccharide (EPS) matrix and play a role in microbial biofilm formation. Levans belong to a larger group of commercially important polymers, referred to as fructans, which are used as a source of prebiotic fibre. For levan, specifically, this market remains untapped, since no viable production strategy has been established. Synthesis of levan is catalysed by a group of enzymes, referred to as levansucrases, using sucrose as substrate. Heterologous expression of levansucrases has been notoriously difficult to achieve in Saccharomyces cerevisiae. As a strategy, this study used an invertase (Δsuc2) null mutant and two separate, engineered, sucrose accumulating yeast strains as hosts for the expression of the levansucrase M1FT, previously cloned from Leuconostoc mesenteroides. Intracellular sucrose accumulation was achieved either by expression of a sucrose synthase (Susy) from potato or the spinach sucrose transporter (SUT). The data indicate that in both Δsuc2 and the sucrose accumulating strains, the M1FT was able to catalyse fructose polymerisation. In the absence of the predicted M1FT secretion signal, intracellular levan accumulation was significantly enhanced for both sucrose accumulation strains, when grown on minimal media. Interestingly, co-expression of M1FT and SUT resulted in hyper-production and extracellular build-up of levan when grown in rich medium containing sucrose. This study presents the first report of levan production in S. cerevisiae and opens potential avenues for the production of levan using this well established industrial microbe. Furthermore, the work provides interesting perspectives when considering the heterologous expression of sugar polymerizing enzymes in yeast.     

Synthesis and characterization of hydroquinone fructoside using <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> levansucrase

Hydroquinone (HQ) functions as a skin-whitening agent, but it has the potential to cause dermatitis. We synthesized a HQ fructoside (HQ-Fru) as a potential skin-whitening agent by reacting levansucrase from Leuconostoc mesenteroides with HQ as an acceptor and sucrose as a fructofuranose donor. The product was purified using 1-butanol partition and silica-gel column chromatography. The structure of the purified HQ-Fru was determined by ¹H and ¹³C nuclear magnetic resonance, and the molecular ion of the product was observed at m/z 295 (C12 H16 O7 Na)⁺. The HQ-Fru was identified as 4-hydroxyphenyl-β-d-fructofuranoside. The optimum condition for HQ-Fru synthesis was determined using a response surface method (RSM), and the final optimum condition was 350 mM HQ, 115 mM sucrose, and 0.70 U/ml levansucrase, and the final HQ-Fru produced was 1.09 g/l. HQ-Fru showed anti-oxidation activities and inhibition against tyrosinase. The median inhibition concentration (IC₅₀) of 1,1-diphenyl-2-picrylhydrazyl scavenging activity was 5.83 mM, showing higher antioxidant activity compared to β-arbutin (IC₅₀ = 6.04 mM). The K _i value of HQ-Fru (1.53 mM) against tyrosinase was smaller than that of β-arbutin (K _i  = 2.8 mM), indicating that it was 1.8-times better as an inhibitor. The inhibition of lipid peroxidation by HQ-Fru was 105.3% that of HQ (100%) and 118.9 times higher than that of β-arbutin (0.89% of HQ).