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.     

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.

     

Levan-type FOS production using a Bacillus licheniformis endolevanase

In the present work we describe an enzymatic production method to obtain β2-6 fructose oligosaccharides (levan-type FOS) through a sequential reaction in which a bacterial endolevanase is applied to levan produced from sucrose by bacterial levansucrases. A putative gene encoding an endolevanase, designated as LevBl, was identified through a bioinformatics search, isolated from a strain of Bacillus licheniformis IBt1 from our own collection and expressed in Escherichia coli. LevB1 showed a specific activity of 1.8 U/mg protein at 35 °C in 50 mM phosphate buffer pH 6.0. A first order kinetic behavior was found when up to 150 g/L of low molecular weight levan (8.3 kDa) was used as the substrate. The product profile was determined by HPAEC-PAD and consisted of levan-type FOS with a polymerization degree between 2 and 8, with levanbiose as the major product after long reaction times. Yields of 97% of levan-type FOS were obtained when 1.0 U/mL of LevB1 reacted with 100 g/L of levan produced by the levansucrase from Bacillus subtilis. Finally, it was observed that levan-type FOS are efficiently fermented by probiotic lactic acid bacteria.     

Levan and fructosyl derivatives formation by a recombinant levansucrase from Rahnella aquatilis

Levan, fructo-oligosaccharides and fructosyl derivatives were formed from sucrose using recombinant levansucrase from Rahnella aquatilis. Levan formation was optimal at 30 °C resulting 57 % of the theoretical yield. The more suitable substrate concentration for levan formation was 200 g sucrose/L. Oligosaccharides was accumulated selectively at high substrate concentration. The increase of levan and oligosaccharides formation was not achieved by adding water-miscible organic solvents. Alkyl fructosides were synthesized from various alcohols as fructosyl acceptors by R. aquatilis levansucrase. © Rapid Science Ltd. 1998     

Pseudomonas asturiensis sp. nov., isolated from soybean and weeds

Five strains of gram negative bacteria, isolated from soybean (LPPA 221^T, 222 and 223) and weeds (LPPA 816 and 1442), were analyzed by a polyphasic approach. The isolates showed variation in their phenotypic traits and were placed in the Pseudomonas fluorescens lineage, based on 16S rRNA gene sequence phylogeny, as a single but well separated cluster. MLSA analysis based on gyrB and rpoD sequences clustered the strains in a single branch in the Pseudomonas syringae group, and revealed P. viridiflava as closest relative. DNA–DNA hybridizations showed medium levels of DNA–DNA relatedness with the type strain of P. viridiflava (50%) and lower levels (<32%) with other type strains of the P. syringae group, supporting classification within a novel species of the genus Pseudomonas. The strains can be distinguished from species of the P. syringae group by the fatty acid C_17:0 cyclo that is present in a low amount (2.5%) and from P. viridiflava by their inability to assimilate d-tartrate and d-sorbitol, and by the formation of red colonies on TTC medium. For this new species, the name Pseudomonas asturiensis sp. nov. is proposed. The type strain is LPPA 221^T (=LMG 26898^T = CECT 8095^T).     

Characterization and mutational analysis of three allelic lsc genes encoding levansucrase in Pseudomonas syringae

In the plant pathogen Pseudomonas syringae pv. glycinea PG4180 and other bacterial species, synthesis of the exopolysaccharide levan is catalyzed by the extracellular enzyme levansucrase. The results of Southern blotting and PCR analysis indicated the presence of three levansucrase-encoding genes in strain PG4180: lscA, lscB, and lscC. In this study, lscB and lscC were cloned from a genomic library of strain PG4180. Sequence analysis of the two lsc genes showed that they were virtually identical to each other and highly similar to the previously characterized lscA gene. lscA and lscC had a chromosomal location, whereas lscB resided on an indigenous plasmid of PG4180. Mutants with impaired expression of individual lsc genes and double mutants were generated by marker exchange mutagenesis. Determination of levansucrase activities in these mutants revealed that the lscB gene product was secreted but not that of lscA or lscC. Our results indicated that lscB and lscC but not lscA contributed to periplasmic levan synthesis of PG4180. The lscB lscC double mutant was completely defective in levan formation and could be complemented by either lscB or lscC. Our data suggested a compartment-specific localization of two lsc gene products, with LscB being the secreted, extracellular enzyme and LscC being the predominantly periplasmic levansucrase. Results of Western blot analyses indicated that lscA was not expressed and that lscA was not associated with levansucrase activities in any particular protein fraction. LscA could be detected in PG4180 only when transcribed from the vector-borne P(lac) promoter. PCR screening in various P. syringae strains with primers derived from the three characterized lsc genes demonstrated the presence of multiple Lsc isoenzymes in other P. syringae pathovars.     

Structure and functions of exopolysaccharide produced by gut commensal Lactobacillus reuteri 100-23

Lactobacillus reuteri strain 100-23 together with a Lactobacillus-free mouse model, provides a system with which the molecular traits underpinning bacterial commensalism in vertebrates can be studied. A polysaccharide was extracted from sucrose-containing liquid cultures of strain 100-23. Chemical analysis showed that this exopolysaccharide was a levan (β-2, 6-linked fructan). Mutation of the fructosyl transferase (ftf) gene resulted in loss of exopolysaccharide production. The ftf mutant was able to colonise the murine gastrointestinal tract in the absence of competition, but colonisation was impaired in competition with the wild type. Biofilm formation by the mutant on the forestomach epithelial surface was not impaired and the matrix between cells was indistinguishable from that of the wild type in electron micrographs. Colonisation of the mouse gut by the wild-type strain led to increased proportions of regulatory T cells (Foxp3+) in the spleen, whereas colonisation by the ftf mutant did not. Survival of the mutant in sucrose-containing medium was markedly reduced relative to the wild type. Comparison of the genomic ftf loci of strain 100-23 with other L. reuteri strains suggested that the ftf gene was acquired by lateral gene transfer early in the evolution of the species and subsequently diversified at accelerated rates. Levan production by L. reuteri 100-23 may represent a function acquired by the bacterial species for life in moderate to high-sucrose extra-gastrointestinal environments that has subsequently been diverted to novel uses, including immunomodulation, that aid in colonisation of the murine gut.     

Purification and characterization of an extracellular levansucrase from Pseudomonas syringae pv. phaseolicola.

Levansucrase (EC 2.4.1.10), an exoenzyme of Pseudomonas syringae pv. phaseolicola, was purified to homogeneity from the cell supernatant by chromatography on TMAE-Fraktogel and butyl-Fraktogel. The enzyme has molecular masses of 45 kDa under denaturing conditions and 68 kDa during gel filtration of the native form. In isoelectric focusing, active bands appeared at pH 3.55 and 3.6. Maximum sucrose cleaving activities were measured at pH 5.8 to 6.6 and 60 degrees C. The enzyme was highly tolerant to denaturing agents, proteases, and repeated freezing and thawing. The molecular weight of the produced levan depended on temperature, salinity, and sucrose concentration. The enzyme had levan-degrading activity and did not accept raffinose as a substrate. Comparison of the N-terminal amino acid sequence with the predicted amino acid sequence of levansucrases from Erwinia amylovora and Zymomonas mobilis showed 88 and 69% similarity, respectively, in amino acids 5 to 20. No similarity could be detected to levansucrases of gram-positive bacteria in the first 20 amino acids. By comparison of all levansucrases which have been sequenced to date, the enzyme seems to be conserved in the gram-negative bacteria. The rheological behavior of the product levan prompted a new assessment of the enzyme's role in pathogenesis. Depending on formation conditions, levan solutions exclude other polymer solutions. This behavior supports the presumption that the levansucrase is important in the early phase of infection by creating a separating layer between bacteria and plant cell wall to prevent the pathogen from recognition.     

Intrinsic Levanase Activity of Bacillus subtilis 168 Levansucrase (SacB)

Levansucrase catalyzes the synthesis of fructose polymers through the transfer of fructosyl units from sucrose to a growing fructan chain. Levanase activity of Bacillus subtilis levansucrase has been described since the very first publications dealing with the mechanism of levan synthesis. However, there is a lack of qualitative and quantitative evidence regarding the importance of the intrinsic levan hydrolysis of B. subtilis levansucrase and its role in the levan synthesis process. Particularly, little attention has been paid to the long-term hydrolysis products, including its participation in the final levan molecules distribution. Here, we explored the hydrolytic and transferase activity of the B. subtilis levansucrase (SacB) when levans produced by the same enzyme are used as substrate. We found that levan is hydrolyzed through a first order exo-type mechanism, which is limited to a conversion extent of around 30% when all polymer molecules reach a structure no longer suitable to SacB hydrolysis. To characterize the reaction, Isothermal Titration Calorimetry (ITC) was employed and the evolution of the hydrolysis products profile followed by HPLC, GPC and HPAEC-PAD. The ITC measurements revealed a second step, taking place at the end of the reaction, most probably resulting from disproportionation of accumulated fructo-oligosaccharides. As levanase, levansucrase may use levan as substrate and, through a fructosyl-enzyme complex, behave as a hydrolytic enzyme or as a transferase, as demonstrated when glucose and fructose are added as acceptors. These reactions result in a wide variety of oligosaccharides that are also suitable acceptors for fructo-oligosaccharide synthesis. Moreover, we demonstrate that SacB in the presence of levan and glucose, through blastose and sucrose synthesis, results in the same fructooligosaccharides profile as that observed in sucrose reactions. We conclude that SacB has an intrinsic levanase activity that contributes to the final levan profile in reactions with sucrose as substrate.     

Enzymatic synthesis of fructosyl oligosaccharides by levansucrase from Microbacterium laevaniformans ATCC 15953

Levansucrase from Microbacterium laevaniformans ATCC 15953 produced in a 3% sucrose medium was purified to homogeneity from cell-free extracts by ammonium sulfate precipitation, DEAE-Sepharose Fast Flow and Sephacryl S-100 HR chromatographies. The molecular mass of the purified enzyme was 64 kDa as measured by SDS–PAGE. The optimum pH and temperature for the levan formation were 6.0 and 30 °C, respectively. The levan-forming activity was strongly inhibited by CuSO₄ and HgCl₂, and moderately inhibited by ZnSO₄. The enzyme synthesized a variety of fructosyl oligosaccharides from various saccharides as fructosyl acceptors. Disaccharides were more favorable fructosyl acceptors than monosaccharides. The structure of the transfer product when melibiose was used as an acceptor was determined by enzyme hydrolysis and ¹³C NMR spectroscopy. The chemical structure of the resulting fructosyl melibiose was identified as O-- -galactopyranosyl-(1→6)-- -glucopyranosyl-(1→2)-β- -fructofranoside. This result suggests that levansucrase from M. laevaniformans specifically transferred the fructose moiety of sucrose to the C1---OH position of the glucose residue of melibiose.     

Synthesis of Fructooligosaccharides by IslA4, a truncated inulosucrase from Leuconostoc citreum

Background

IslA4 is a truncated single domain protein derived from the inulosucrase IslA, which is a multidomain fructosyltransferase produced by Leuconostoc citreum. IslA4 can synthesize high molecular weight inulin from sucrose, with a residual sucrose hydrolytic activity. IslA4 has been reported to retain the product specificity of the multidomain enzyme.

Results

Screening experiments to evaluate the influence of the reactions conditions, especially the sucrose and enzyme concentrations, on IslA4 product specificity revealed that high sucrose concentrations shifted the specificity of the reaction towards fructooligosaccharides (FOS) synthesis, which almost eliminated inulin synthesis and led to a considerable reduction in sucrose hydrolysis. Reactions with low IslA4 activity and a high sucrose activity allowed for high levels of FOS synthesis, where 70% sucrose was used for transfer reactions, with 65% corresponding to transfructosylation for the synthesis of FOS.

Conclusions

Domain truncation together with the selection of the appropriate reaction conditions resulted in the synthesis of various FOS, which were produced as the main transferase products of inulosucrase (IslA4). These results therefore demonstrate that bacterial fructosyltransferase could be used for the synthesis of inulin-type FOS.     

Cloning,expression and functional validation of a β-fructofuranosidase from Lactobacillus plantarum

Fructooligosaccharides (FOS) are prebiotics that selectively stimulate the growth and activity of lactobacilli and bifidobacteria. These strains metabolize FOS with endogenous β-fructofuranosidase. In this study, a β-fructofuranosidase gene from Lactobacillus plantarum ST-III designated sacA was cloned into Escherichia coli, and the properties of the recombinant protein (SacA) were examined. The sacA gene encodes a peptide of 501 amino acids with a predicted molecular weight of 56.7 kDa. Sequence alignment revealed the presence of three highly conserved motifs, NDPNG, RDP and EC, indicating that the enzyme belongs to glycoside hydrolase family 32. The predicted three-dimensional structure of the SacA enzyme was similar to β-fructofuranosidases of bifidobacteria, such that it contained a five-blade β-propeller module and a β-sandwich domain with one additional N-terminal α-helix. The optimal reaction temperature and pH of the enzyme were 37 °C and 6.0, respectively. Substrate hydrolysis and kinetic parameters demonstrated that β-fructofuranosidase from L. plantarum ST-III liberated fructosyl residues from the non-reducing terminus of fructans, such as sucrose, FOS, levan or inulin, and FOS was the preferred substrate. The expression of the sacA gene in a non-FOS-fermenting strain, Lactobacillus rhamnosus GG, enabled the recombinant strain to metabolize FOS and sucrose.     

Phenolic Glycosides with antiproteasomal activity from Centaurea urvillei DC. subsp. urvillei

A new flavanone glycoside, naringenin-7-O-β-d-glucuronopyranoside, and a new flavonol glycoside, 6-hydroxykaempferol-7-O-β-d-glucuronopyranoside were isolated together with 12 known compounds, 5 flavone glycoside; hispidulin-7-O-β-d-glucuronopyranoside, apigenin-7-O-β-d-methylglucuronopyranoside, hispidulin-7-O-β-d-methylglucuronopyranoside, hispidulin-7-O-β-d-glucopyranoside, apigenin-7-O-β-d-glucopyranoside, a flavonol; kaempferol, two flavone; apigenin, and luteolin, a flavanone glycoside; eriodictyol-7-O-β-d-glucuronopyranoside, and three phenol glycoside; arbutin, salidroside, and 3,5-dihydroxyphenethyl alcohol-3-O-β-d-glucopyranoside from Centaurea urvillei subsp. urvillei. The structure elucidation of the new compounds was achieved by a combination of one- (¹H and ¹³C) and two-dimensional NMR techniques (G-COSY, G-HMQC, and G-HMBC) and LC-ESI-MS. The isolated compounds were tested for their antiproteasomal activity. The results indicated that kaempferol, a well known and widely distributed flavonoid in the plant kingdom, was the most active antiproteasomal agent, followed by apigenin, eriodictyol-7-O-β-d-glucuronopyranoside, 3,5-dihydroxyphenethyl alcohol-3-O-β-d-glucopyranoside, and salidroside, respectively.     

Physicochemical characterization of fructooligosaccharides and evaluation of their suitability as a potential sweetener for diabetics

Fructooligosaccharides (FOSs) were prepared from sucrose using fungal fructosyl transferase (FTase) obtained from Aspergillus oryzae MTCC 5154. The resulting mixture consisted of glucose (28-30%), sucrose (18-20%) and fructooligosaccharides (50-54%) as indicated by HPLC analysis. Identification of oligomers present in the mixture of fructooligosaccharides was carried out using NMR spectroscopy and LC-MS. No compounds other than mono-, di-, tri-, tetra- and pentasaccharides were identified in the FOS mixture prepared using FTase. NMR and LC-MS spectra proved the absence of any toxic microbial metabolites of Aspergillus species in FOS thereby emphasizing its safe use as a food ingredient. Animal studies conducted on streptozotocin-induced diabetic rats suggested that the use of FOS as an alternative non-nutrient sweetener is without any adverse effects on various diabetes-related metabolic parameters. Despite the high free-sugar content associated with it, FOS did not further aggravate the hyperglycemia and glucosuria in diabetic animals, even at 10% levels. On the other hand, by virtue of its soluble fibre effect, it has even alleviated diabetic-related metabolic complications to a certain degree.     

Isolation and characterization of a furostanol glycoside from fenugreek

From the seed of fenugreek, a new glycoside has been isolated and shown to have the structure, (25S)-22-O- methyl-5α-furostan-3β,22,26-triol 3-O-α-rhamnopyranosyl(1→2)[-β-d-glucopyranosyl (1→3)]-β-d- glucopyranoside-26-O-β-d-glucopyranoside.     

热带假丝酵母(Candida tropiclis)去除蔗渣木聚糖酶解副产物的研究

该文研究了木糖、木糖醇对木聚糖酶Shearzyme 500L酶解蔗渣木聚糖的影响。通过热带假丝酵母(Candida tropiclis)转化酶解副产物木糖,解除木糖对木聚糖酶的抑制作用,从而获得高木二糖含量的低聚木糖。结果表明:木糖是Shearzyme 500L的酶活性抑制物,其抑制作用与溶液中的木糖量成正比;木糖醇对木聚糖酶无抑制作用;热带假丝酵母可将蔗渣木聚糖酶解液中的木糖转化为木糖醇而不利用低聚木糖,木二糖占总糖比例由53.09%升高到62.92%,经二次酶解后,木二糖比例可达78.90%。     

Association study of the ABCC8 gene variants with type 2 diabetes in south Indians

BACKGROUND:

The ABCC8 gene which encodes the sulfonylurea receptor plays a major role in insulin secretion and is a potential candidate for type 2 diabetes. The -3c → t (rs1799854) and Thr759Thr (C → T, rs1801261) single nucleotide polymorphisms (SNPs) of the ABCC8 gene have been associated with type 2 diabetes in many populations. The present study was designed to investigate the association of these two SNPs in an Asian Indian population from south India.

MATERIALS AND METHODS:

A total of 1,300 subjects, 663 normal glucose tolerant (NGT) and 637 type 2 diabetic subjects were randomly selected from the Chennai Urban Rural Epidemiology Study (CURES). The -3c → t and Thr759Thr were genotyped in these subjects using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and a few variants were confirmed by direct sequencing.

RESULTS:

The frequency of the ‘t’ allele of the -3c → t SNP was found to be 0.27 in NGT and 0.29 in type 2 diabetic subjects (P = 0.44). There was no significant difference in the genotypic frequency between the NGT and type 2 diabetic group (P = 0.18). Neither the genotypic frequency nor the allele frequency of the Thr759Thr polymorphism was found to differ significantly between the NGT and type 2 diabetic groups.

CONCLUSION:

The -3c → t and the Thr759Thr polymorphisms of the ABCC8 gene were not associated with type 2 diabetes in this study. However, an effect of these genetic variants on specific unidentified sub groups of type 2 diabetes cannot be excluded.     

In vitro study of Prebiotic Properties of Levan-type Exopolysaccharides from Lactobacilli and Non-digestible Carbohydrates Using Denaturing Gradient Gel Electrophoresis

Batch cultures inoculated with human faeces were used to study the prebiotic properties of levan-type exopolysaccharides (EPS) from Lactobacillus sanfranciscensis as well as levan, inulin, and fructooligosaccharide (FOS). Denaturing gradient gel electrophoresis of 16S rDNA fragments generated by PCR with universal primers was used to analyse the cultures. Characteristic changes were revealed in the composition of the gut bacteria during fermentation of the carbohydrates. An enrichment of Bifidobacterium spp. was found for the EPS and inulin but not for levan and FOS. The bifidogenic effect of the EPS was confirmed by culturing on selective medium. In addition, the use of EPS and FOS resulted in enhanced growth of Eubacterium biforme and Clostridium perfringens, respectively.     

Phenolic glycosides from Agrimonia pilosa

Phytochemical investigation of the methanolic extract from the aerial parts of Agrimonia pilosa led to the isolation of three compounds, (−)-aromadendrin 3-O-β-d-glucopyranoside (1), desmethylagrimonolide 6-O-β-d-glucopyranoside (2), and 5,7-dihydroxy-2-propylchromone 7-O-β-d-glucopyranoside (3), together with nine known compounds, agrimonolide 6-O-glucoside, takanechromone C, astragalin, afzelin, tiliroside, luteolin, quercetin, isoquercetrin, and quercitrin. Their structures were determined by various spectroscopic analysis and chemical transformations.     

Unusual glycosides of pyrrole alkaloid and 4'-hydroxyphenylethanamide from leaves of Moringa oleifera

Glycosides of pyrrole alkaloid (pyrrolemarumine 4″-O-α-l-rhamnopyranoside) and 4′-hydroxyphenylethanamide (marumosides A and B) were isolated from leaves of Moringa oleifera along with eight known compounds; niazirin, methyl 4-(α-l-rhamnopyranosyloxy)benzylcarbamate, benzyl β-d-glucopyranoside, benzyl β-d-xylopyranosyl-(1 → 6)-β-d-glucopyranoside, kaempferol 3-O-β-d-glucopyranoside, quercetin 3-O-β-d-glucopyranoside, adenosine and l-tryptophan. Structure elucidations were based on analyses of chemical and spectroscopic data including 1D- and 2D-NMR.