Production of levan from Bacillus subtilis var. natto and apoptotic effect on SH-SY5Y neuroblastoma cells.

Levan is a high-valued polysaccharide of fructose produced by several microbial species. These polysaccharides have been described as effective therapeutic agents in some human disease conditions, such as cancer, heart diseases and diabetes. The objective of this study was to examine the effect of levan (ß-(2 â†’ 6)-fructan) produced through sucrose fermentation by B. subtilis var. natto on the proliferation rate, cytotoxicity, and apoptosis of human neuroblastoma SH-SY5Y cells. It was obtained 41.44 g/L of levan in 18 h by biotechnological fermentation and SH-SY5Y cells were exposed to 1000 µg/mL of levan. The treatment with 1000 µg/mL of levan induced apoptosis in SH-SY5Y cancer cells by the significant increase in Annexin V/7-AAD and caspase 3/7 activation, but did not decrease proliferation or triggered a cytotoxic effect. 1000 µg/mL levan treatment is a promising therapeutic strategy for SH-SY5Y neuroblastoma cells.

Apoplastic maize fructan exohydrolase Zm-6-FEH displays substrate specificity for levan and is induced by exposure to levan-producing bacteria.

Fructan exohydrolases (FEHs) are structurally related to cell wall invertases. While the latter are ubiquitous in higher plants, the role of FEHs in non-fructan species has remained enigmatic. To explore possible roles of FEHs in maize, a full length putative Zm-6-FEH-encoding cDNA was cloned displaying high sequence similarity with cell wall invertases. For functional characterization, Zm-6-FEH protein was expressed in Picha pastoris and in Nicotiana benthamiana leaves. Enzyme activity of recombinant Zm-6-FEH protein showed a strong preference for levan as substrate. Expression profiling in maize seedlings revealed higher transcript amounts in the more mature leaf parts as compared to the growth zone at the base of the leaf, in good correlation with FEH enzyme activities. Subcellular localization analysis indicated Zm-6-FEH location in the apoplast. Noteworthy, incubation of leaf discs with levan and co-incubation with high levan-producing bacteria selectively up-regulated transcript levels of Zm-6-FEH, accompanied by an increase of 6-FEH enzyme activity. In summary, the results indicate that Zm-6-FEH, a novel fructan exohydrolase of a non-fructan species, may have a role in plant defense against levan-producing bacteria.

Scaling up of levan yield in Bacillus subtilis M and cytotoxicity study on levan and its derivatives.

This study focused on kinetics of levan yield by Bacillus subtilis M, in a 150 L stirred tank bioreactor under controlled pH conditions. The optimized production medium was composed of (g/L): commercial sucrose 100.0, yeast extract 2.0, K2HPO4 3.0 and MgSO4⋅7H2O 0.2; an increase in both carbohydrates consumption and cell growth depended on increasing the size of the stirred tank bioreactor from 16 L to 150 L. The highest levansucrase production (63.4 U/mL) and levan yield of 47 g/L was obtained after 24 h. Also, the specific levan yield (Yp/x) which reflects the cell productivity increased with the size increase of the stirred tank bioreactor and reached its maximum value of about 29.4 g/g cells. These results suggested that B. subtilis M could play an important role in levan yield on a large scale in the future. Chemical modifications of B. subtilis M crude levan (CL) into sulfated (SL), phosphorylated (PL), and carboxymethylated levans (CML) were done. The difference in CL structure and its derivatives was detected by FT-IR transmission spectrum. The cytotoxicity of CL and its derivatives were evaluated by HepGII, Mcf-7 and CaCo-2. In general most tested levans forms had no significant cytotoxicity effect. In fact, the carboxymethylated and phosphrylated forms had a lower anti-cancer effect than CL. On the other hand, SL had the highest cytotoxicity showing SL had a significant anti-cancer effect. The results of cytotoxicity and cell viability were statistically analyzed using three-way ANOVA.

Brachybacterium sp. CH-KOV3 isolated from an oil-polluted environment-a new producer of levan.

Various microorganisms isolated from polluted environments, such as Pseudomonas sp. and Micrococcus sp. can synthesize exopolysaccharides (EPSs) which are natural, non-toxic and biodegradable polymers. EPSs play a key role in protection of microbial cells under various external influences. For humans, these substances have potential use in many industries. EPSs can be applied as a flavor or a fragrance carrier, an emulsifier, a stabilizer, a prebiotic, an antioxidant or an antitumor agent. In this study, we characterized an environmental microorganism that produces EPS, optimized EPS production by this strain and characterized the EPS produced. Isolate CH-KOV3 was identified as Brachybacterium paraconglomeratum. The sucrose level in the growth medium greatly influenced EPS production, and the highest yield was when the microorganism was incubated in media with 500g/L of sucrose. The optimal temperature and pH were 28°C and 7.0, respectively. The nuclear magnetic resonance (NMR) results and GC-MS analysis confirmed that the residues were d-fructofuranosyl residues with ß-configuration, where fructose units are linked by ß-2,6-glycosidic bonds, with ß-2,1-linked branches. All these data indicate that the investigated EPS is a levan-type polysaccharide. Thus, it was concluded that Brachybacterium sp. CH-KOV3 could constitute a new source for production of the bioactive polysaccharide, levan.

Effect of bacteria type and sucrose concentration on levan yield and its molecular weight.

BACKGROUND: Levan has been traditionally produced from microorganism. However, there is a continuous effort in looking for new strains that improve levan production yield and uses alternative sugar sources for growth. Despite having a wide range of data about levan yield, there are not papers which allow controlling molecular weight, and that plays an essential role for further applications. RESULTS: The effect of the sucrose concentration on levan yield (and its molecular weight) from Bacillus atrophaeus and Acinetobacter nectaris (Gram positive and Gram negative respectively) was studied in this work. It was found that A. nectaris growth (from 3 to 1.5 g L-1 in 40 h) and its levan production (from 3 to 1.5 g L-1) decreases by increasing sucrose concentration (best results at a concentration of 120 g L-1) whereas B. atrophaeus growth (3.5 g L-1 in 30 h) and its levan production (also 3.5 g L-1) were not affected by modifying that parameter. Levan molecular weight from A. nectaris decreases by increasing sucrose concentration (from 8000 to 2000 kDa) whereas levan molecular weight from B. Atrophaeus remains always around 50 kDa. By performing a kinetic study, it was shown that A. nectaris growth follows a substrate-inhibition model, whereas Monod equation provided a good fit for B. atrophaeus growth. Finally, wastes from orange juice industry were used as a medium culture to cultivate those microorganism, obtaining good results with B. atrophaeus (growth 3 g L-1 in 30 h). CONCLUSIONS: Levan production kinetics was determined and compared between different bacteria types.

Influence of levan-producing acetic acid bacteria on buckwheat-sourdough breads.

Buckwheat sourdoughs supplemented with molasses as natural sucrose source were fermented with levan-producing Gluconobacter (G.) albidus TMW 2.1191 and Kozakia (K.) baliensis NBRC 16680. Cell growth, concomitant levan and low-molecular-weight metabolite production were monitored. Sourdough breads were prepared with different sourdoughs from both strains (24, 30 and 48 h fermentation, respectively) and analyzed with respect to bread volume, crumb hardness and sensory characteristics. During fermentation, levan, acetic and gluconic acids were increasingly produced, while spontaneously co-growing lactic acid bacteria additionally formed acetic and lactic acids. Sourdoughs from both strains obtained upon 24 h of fermentation significantly improved the bread sensory and quality, including higher specific volume as well as lower crumb hardness. Buckwheat doughs containing isolated levan, with similar molecular size and mass compared to in situ produced levan in the sourdough at 48 h, verified the positive effect of levan on bread quality. However, the positive effects of levan were masked to a certain extent by the impact from the natural acidification during fermentations. While levan-producing acetic acid bacteria are a promising alternative for the development of clean-label gluten-free breads without the need of additives, an appropriate balance between acidification and levan production (amount and structure) must be reached.

Dissection of exopolysaccharide biosynthesis in Kozakia baliensis.

BACKGROUND: Acetic acid bacteria (AAB) are well known producers of commercially used exopolysaccharides, such as cellulose and levan. Kozakia (K.) baliensis is a relatively new member of AAB, which produces ultra-high molecular weight levan from sucrose. Throughout cultivation of two K. baliensis strains (DSM 14400, NBRC 16680) on sucrose-deficient media, we found that both strains still produce high amounts of mucous, water-soluble substances from mannitol and glycerol as (main) carbon sources. This indicated that both Kozakia strains additionally produce new classes of so far not characterized EPS. RESULTS: By whole genome sequencing of both strains, circularized genomes could be established and typical EPS forming clusters were identified. As expected, complete ORFs coding for levansucrases could be detected in both Kozakia strains. In K. baliensis DSM 14400 plasmid encoded cellulose synthase genes and fragments of truncated levansucrase operons could be assigned in contrast to K. baliensis NBRC 16680. Additionally, both K. baliensis strains harbor identical gum-like clusters, which are related to the well characterized gum cluster coding for xanthan synthesis in Xanthomanas campestris and show highest similarity with gum-like heteropolysaccharide (HePS) clusters from other acetic acid bacteria such as Gluconacetobacter diazotrophicus and Komagataeibacter xylinus. A mutant strain of K. baliensis NBRC 16680 lacking EPS production on sucrose-deficient media exhibited a transposon insertion in front of the gumD gene of its gum-like cluster in contrast to the wildtype strain, which indicated the essential role of gumD and of the associated gum genes for production of these new EPS. The EPS secreted by K. baliensis are composed of glucose, galactose and mannose, respectively, which is in agreement with the predicted sugar monomer composition derived from in silico genome analysis of the respective gum-like clusters. CONCLUSIONS: By comparative sugar monomer and genome analysis, the polymeric substances secreted by K. baliensis can be considered as unique HePS. Via genome sequencing of K. baliensis DSM 14400 + NBRC 16680 we got first insights into the biosynthesis of these novel HePS, which is related to xanthan and acetan biosynthesis. Consequently, the present study provides the basis for establishment of K. baliensis strains as novel microbial cell factories for biotechnologically relevant, unique polysaccharides.

Effect of plant growth-promoting bacteria on the growth and fructan production of Agave americana L

ABSTRACT The effect of plant growth-promoting bacteria inoculation on plant growth and the sugar content in Agave americana was assessed. The bacterial strains ACO-34A, ACO-40, and ACO-140, isolated from the A. americana rhizosphere, were selected for this study to evaluate their phenotypic and genotypic characteristics. The three bacterial strains were evaluated via plant inoculation assays, and Azospirillum brasilense Cd served as a control strain. Phylogenetic analysis based on the 16S rRNA gene showed that strains ACO-34A, ACO-40 and ACO-140 were Rhizobium daejeonense, Acinetobacter calcoaceticus and Pseudomonas mosselii, respectively. All of the strains were able to synthesize indole-3-acetic acid (IAA), solubilize phosphate, and had nitrogenase activity. Inoculation using the plant growth-promoting bacteria strains had a significant effect (p < 0.05) on plant growth and the sugar content of A. americana, showing that these native plant growth-promoting bacteria are a practical, simple, and efficient alternative to promote the growth of agave plants with proper biological characteristics for agroindustrial and biotechnological use and to increase the sugar content in this agave species.

Antioxidant and anti-inflammatory levan produced from Acetobacter xylinum NCIM2526 and its statistical optimization.

Levan is a homopolymer of fructose naturally obtained from both the plants and microorganisms. Along with the general properties of a biopolymer like bio-compatibility, bio-degradability, renewability, flexibility, and eco-friendliness, levan also offers some important biomedical properties such as anti-oxidant, anti-inflammatory, anti-carcinogenic, anti-AIDS and hyperglycaemic inhibitor. In this study, we have demonstrated the microbial production of therapeutically potential levan by batch fermentation process in sucrose rich medium using Acetobacter xylinum NCIM 2526. The produced Levan was characterized using various physicochemical techniques such as FTIR, (1)H NMR, (13)C NMR spectroscopy, TGA and HPLC. The biomedical potential of the isolated A. xylinum levan for its anti-oxidant and anti-inflammatory activities was exploited in vitro. Further the present study also focused on the optimization of levan production using one factor at a time approach followed by a statistical method, central composite design (CCD) with selected variables. The yield of levan was increased significantly from 0.54 to 13.25g/L with the optimized variables.

Line differences in Cor/Lea and fructan biosynthesis-related gene transcript accumulation are related to distinct freezing tolerance levels in synthetic wheat hexaploids.

In common wheat, cultivar differences in freezing tolerance are considered to be mainly due to allelic differences at two major loci controlling freezing tolerance. One of the two loci, Fr-2, is coincident with a cluster of genes encoding C-repeat binding factors (CBFs), which induce downstream Cor/Lea genes during cold acclimation. Here, we conducted microarray analysis to study comprehensive changes in gene expression profile under long-term low-temperature (LT) treatment and to identify other LT-responsive genes related to cold acclimation in leaves of seedlings and crown tissues of a synthetic hexaploid wheat line. The microarray analysis revealed marked up-regulation of a number of Cor/Lea genes and fructan biosynthesis-related genes under the long-term LT treatment. For validation of the microarray data, we selected four synthetic wheat lines that contain the A and B genomes from the tetraploid wheat cultivar Langdon and the diverse D genomes originating from different Aegilops tauschii accessions with distinct levels of freezing tolerance after cold acclimation. Quantitative RT-PCR showed increased transcript levels of the Cor/Lea, CBF, and fructan biosynthesis-related genes in more freezing-tolerant lines than in sensitive lines. After a 14-day LT treatment, a significant difference in fructan accumulation was observed among the four lines. Therefore, the fructan biosynthetic pathway is associated with cold acclimation in development of wheat freezing tolerance and is another pathway related to diversity in freezing tolerance, in addition to the CBF-mediated Cor/Lea expression pathway.

Studies on production of fructo-oligosaccharides (FOS) by gamma radiation processing of microbial levan.

Microbial levan, a natural polymer of fructose, was produced and purified by alcohol precipitation from culture supernatants of Bacillus megaterium type 1 grown in an optimized liquid sucrose medium. GPC analysis showed that the yield of the major fraction of levan having molecular weight ~5000 D increased with increase in sucrose concentration in the broth. Levan subjected to (60)Co-gamma radiation as well as acid hydrolysis was investigated by rheometry, UV-visible spectrophotometry and gel permeation chromatography (GPC) techniques. Unlike most of the polysaccharides, levan powder exhibited good radiation degradation stability up to 150 kGy. Gamma irradiation of 10% levan aqueous solution at 250 kGy yielded 63.0% fructo-oligosaccharide (FOS) with an average molecular weight of 1250 D. Acid hydrolysis of levan using 0.5 N HCl for 60 min treatment time gave rise to the desired FOS with lower yield (23.1%) as compared to that obtained in gamma radiolysis process.

Combinational transformation of three wheat genes encoding fructan biosynthesis enzymes confers increased fructan content and tolerance to abiotic stresses in tobacco.

KEY MESSAGE: Seven kinds of transgenic tobacco plants transformed with combinations of three FBE genes were obtained. The transgenic plants transformed with Ta1-SST + Ta6-SFT genes appeared to have the highest fructan or soluble sugar content and the strongest salt tolerance. Fructan is thought to be one of the important regulators involved in plant tolerance to various abiotic stresses. In this study, wheat-derived genes, Ta1-SST, Ta6-SFT, and Ta1-FFT, encoding fructan biosynthesis enzymes (FBE) were isolated and cloned into vectors modified pBI121 or pZP211. Seven different combinations of the three target genes were transformed into tobacco plants through an Agrobacterium-mediated approach, and transgenic tobacco plants were identified by PCR, ELISA, and Southern blotting. Compared with tobacco plants transformed with other six combinations of the three target genes and with wild-type plants, the transgenic plants transformed with Ta1-SST + Ta6-SFT genes contained the highest fructan and soluble sugar content. All seven types of transgenic tobacco plants displayed a much higher level of tolerance to drought, low temperature, and high salinity compared with the wild type. Differences of drought and low temperature tolerance between the transgenic plants containing a single FBE gene and those harboring two or three FBE genes were not significant, but the salt tolerance level of the transgenic plants with different FBE gene combinations from high to low was: Ta1-SST + Ta6-SFT > Ta1-SST + Ta6-SFT + Ta1-FFT > Ta1-SST + Ta1-FFT > Ta1-SFT + Ta1-FFT > single FBE gene. These results indicated that the tolerances of the transgenic tobacco plants to various abiotic stresses were associated with the transformed target gene combinations and the contents of fructan and soluble sugar contained in the transgenic plants.

Molecular and biochemical identification of alien chromosome additions in shallot (Allium cepa L. Aggregatum group) carrying extra chromosome(s) of bunching onion (A. fistulosum L.).

To develop the bunching onion (Allium fistulosum L.; genomes, FF) chromosome-specific genetic markers for identifying extra chromosomes, eight shallot (A. cepa L. Aggregatum group; genomes, AA)--A. fistulosum monosomic addition plants (AA+nF) and 62 shallot--A. fistulosum single-alien deletion plants (AAF-nF) were analyzed by 23 different chromosome-specific genetic markers of shallot. The eight monosomic addition plants consisted of one AA+2F, two AA+6F, and five AA+8F. Of the 62 single-alien deletion plants, 60 could be identified as six different single-alien deletion lines (AAF-1F, -3F, -4F, -6F, -7F, and -8F) out of the eight possible types. Several single-alien deletion lines were classified on the basis of leaf and bulb characteristics. AAF-8F had the largest number of expanded leaves of five deletion plants. AAF-7F grew most vigorously, as expressed by its long leaf blade and biggest bulb size. AAF-4F had very small bulbs. AAF-7F and AAF-8F had different bulbs from those of shallot as well as other types of single-alien deletion lines in skin and outer scale color. Regarding the sugar content of the bulb tissues, the single-alien deletion lines showed higher fructan content than shallot. Moreover, shallot could not produce fructan with degree of polymerization (DP) 12 or higher, although the single-alien deletion lines showed DP 20 or higher. The content of S-alk(en)yl-L-cysteine sulfoxide (ACSO) in the single-alien deletion lines was significantly lower than that in shallot. These results indicated that chromosomes from A. fistulosum might carry anonymous factors to increase the highly polymerized fructan production and inhibit the synthesis of ACSO in shallot bulbs. Accordingly, alien chromosomes from A. fistulosum in shallot would contribute to modify the quality of shallot bulbs.

Developing fructan-synthesizing capability in a plant invertase via mutations in the sucrose-binding box.

Fructans are fructose polymers that are synthesized from sucrose by fructosyltransferases. Fructosyltransferases are present in unrelated plant families suggesting a polyphyletic origin for their transglycosylation activity. Based on sequence comparisons and enzymatic properties, fructosyltransferases are proposed to have evolved from vacuolar invertases. Between 1% and 5% of the total activity of vacuolar invertase is transglycosylating activity. We investigated the nature of the changes that can convert a hydrolysing invertase into a transglycosylating enzyme. Remarkably, replacing 33 amino acids (amino acids 143-175) corresponding to the N-terminus of the mature onion vacuolar invertase with the corresponding region of onion fructan:fructan 6G-fructosyltransferase (6G-FFT) led to a shift in activity from hydrolysis of sucrose towards transglycosylation between two sucrose molecules. The substituted N-terminal region contains the sucrose-binding box that harbours the nucleophile involved in sucrose hydrolysis (Asp164). Subsequent research into the individual amino acids responsible for the enhanced transglycosylation activity revealed that mutations in amino acids Trp161 and Asn166, can give rise to a shift towards polymerase activity. Changing the amino acid at either of these positions in the sucrose-binding box increases the transglycosylation capacity of invertases two- to threefold compared to wild type. Combining the two mutations had an additive effect on transglycosylation ability, resulting in an approximately fourfold enhancement. The mutations generated correspond with natural variation present in the sucrose-binding boxes of vacuolar invertases and fructosyltransferases. These relatively small changes that increase the transglycosylation capacity of invertases might explain the polyphyletic origin of the fructan accumulation trait.

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.

Cloning of sucrose:sucrose 1-fructosyltransferase from onion and synthesis of structurally defined fructan molecules from sucrose.

Sucrose (Suc):Suc 1-fructosyltransferase (1-SST) is the key enzyme in plant fructan biosynthesis, since it catalyzes de novo fructan synthesis from Suc. We have cloned 1-SST from onion (Allium cepa) by screening a cDNA library using acid invertase from tulip (Tulipa gesneriana) as a probe. Expression assays in tobacco (Nicotiana plumbaginifolia) protoplasts showed the formation of 1-kestose from Suc. In addition, an onion acid invertase clone was isolated from the same cDNA library. Protein extracts of tobacco protoplasts transformed with this clone showed extensive Suc-hydrolyzing activity. Conditions that induced fructan accumulation in onion leaves also induced 1-SST mRNA accumulation, whereas the acid invertase mRNA level decreased. Structurally different fructan molecules could be produced from Suc by a combined incubation of protein extract of protoplasts transformed with 1-SST and protein extract of protoplasts transformed with either the onion fructan:fructan 6G-fructosyltransferase or the barley Suc:fructan 6-fructosyltransferase.

Differences in chain length distribution of inulin from Cynara scolymus and Helianthus tuberosus are reflected in a transient plant expression system using the respective 1-FFT cDNAs.

A newly isolated cDNA clone, Cy3, encoding the fructan fructan 1-fructosyltransferase (1-FFT) from artichoke was expressed using tobacco protoplasts as expression system. Analysis of the inulin molecules synthesized upon incubation of protoplast extracts with a mixture of oligofructans (DP3-5) shows the production of inulins with a degree of polymerization (DP) of up to 23, whereas parallel experiments performed using a 1-FFT cDNA from Jerusalem artichoke led to the production of fructans with a DP of up to only 12. The results of in vitro fructan synthesis catalyzed by transiently expressed enzymes therefore reflect the difference of in vivo fructan composition of Jerusalem artichoke (M(DP) = 8-10) and artichoke (M(DP) = 65). These data suggest that the fructan pattern in a given species is mainly defined by the enzymatic characteristics of 1-FFT.

Fructan of the inulin neoseries is synthesized in transgenic chicory plants (Cichorium intybus L.) harbouring onion (Allium cepa L.) fructan:fructan 6G-fructosyltransferase.

Fructan (polyfructosylsucrose) is an important storage carbohydrate in many plant families. fructan:fructan 6G-fructosyltransferase (6G-FFT) is a key enzyme in the formation of the inulin neoseries, a type of fructan accumulated by members of the Liliales. We have cloned the 6G-FFT from onion by screening a cDNA library using barley sucrose:fructan 6-fructosyltransferase (6-SFT) as a probe. The deduced amino acid sequence showed a high homology with plant invertases and 6-SFT. Incubation of protein extracts from transgenic tobacco plants with the trisaccharide 1-kestose and sucrose resulted in the formation of neokestose and fructans of the inulin neoseries with a degree of polymerization up to six. Introduction of the onion 6G-FFT into chicory resulted in the synthesis of fructan of the inulin neoseries, in addition to the synthesis of linear inulin.

Fructan synthesis in transgenic tobacco and chicory plants expressing barley sucrose: fructan 6-fructosyltransferase.

We have recently cloned a cDNA encoding sucrose:fructan 6-fructosyltransferase (6-SFT), a key enzyme of fructan synthesis forming the beta-2,6 linkages typical of the grass fructans, graminans and phleins [Sprenger et al. (1995) Proc. Natl. Acad. Sci. USA 92, 11652-11656]. Here we report functional expression of 6-SFT from barley in transgenic tobacco and chicory. Transformants of tobacco, a plant naturally unable to form fructans, synthesized the trisaccharide kestose and a series of unbranched fructans of the phlein type (beta-2,6 linkages). Transformants of chicory, a plant naturally producing only unbranched fructans of the inulin type (beta-2,1 linkages), synthesized in addition branched fructans of the graminan type, particularly the tetrasaccharide bifurcose which is also a main fructan in barley leaves.

Purification, cloning, and functional expression of sucrose:fructan 6-fructosyltransferase, a key enzyme of fructan synthesis in barley.

Fructans play an important role in assimilate partitioning and possibly in stress tolerance in many plant families. Sucrose:fructan 6-fructosyltransferase (6-SFT), an enzyme catalyzing the formation and extension of beta-2,6-linked fructans typical of grasses, was purified from barley (Hordeum vulgare L.). It occurred in two closely similar isoforms with indistinguishable catalytic properties, both consisting of two subunits with apparent masses of 49 and 23 kDa. Oligonucleotides, designed according to the sequences of tryptic peptides from the large subunit, were used to amplify corresponding sequences from barley cDNA. The main fragment generated was cloned and used to screen a barley cDNA expression library. The longest cDNA obtained was transiently expressed in Nicotiana plumbaginifolia protoplasts and shown to encode a functional 6-SFT. The deduced amino acid sequence of the cDNA comprises both subunits of 6-SFT. It has high similarity to plant invertases and other beta-fructosyl hydrolases but only little to bacterial fructosyltransferases catalyzing the same type of reaction as 6-SFT.