1,1,1-Kestopentaose

Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.

The non-saccharolytic yeast Pichia pastoris was engineered to express constitutively the mature region of sucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99) from Tall fescue (Schedonorus arundinaceus). The increase of the transgene dosage from one to nine copies enhanced 7.9-fold the recombinant enzyme (Sa1-SSTrec) yield without causing cell toxicity. Secretion driven by the Saccharomyces cerevisiae α-factor signal peptide resulted in periplasmic retention (38%) and extracellular release (62%) of Sa1-SSTrec to an overall activity of 102.1 U/ml when biomass reached (106 g/l, dry weight) in fed-batch fermentation using cane sugar for cell growth. The volumetric productivity of the nine-copy clone PGFT6x-308 at the end of fermentation (72 h) was 1422.2 U/l/h. Sa1-SSTrec purified from the culture supernatant was a monomeric glycoprotein optimally active at pH 5.0-6.0 and 45-50°C. The removal of N-linked oligosaccharides by Endo Hf treatment decreased the enzyme stability but had no effect on the substrate and product specificities. Sa1-SSTrec converted sucrose (600 g/l) into 1-kestose (GF₂) and nystose (GF₃) in a ratio 9:1 with their sum representing 55-60% (w/w) of the total carbohydrates in the reaction mixture. Variations in the sucrose (100-800 g/l) or enzyme (1.5-15 units per gram of substrate) concentrations kept unaltered the product profile. Sa1-SSTrec is an attractive candidate enzyme for the industrial production of short-chain fructooligosaccharides, most particularly 1-kestose.

Agave plants are members of the Agavaceae family and utilize crassulacean acid metabolism (CAM) for CO₂ fixation. Fructans are the main photosynthetic products produced by Agave plants, and are their principal source of storage carbohydrates. The aim of this work was to determine the chemical and molecular characterization of fructans from Agave durangensis. Fructans were extracted from 10 year old A. durangensis plants. Trimethylsilyl derivatization was employed to determine the monomer composition. The linkage types in these carbohydrates were determined by methylation followed by reduction and O-acetylation, and finally analysis by gas chromatography-mass spectrometry (GC-MS). Samples were shown to contain t-β-D-Fruf, t-α-D-Glup, i-α-D-6-Glup and 1,6-di-β-D-Fruf linkages. The analysis of the degree of polymerization (DP) was confirmed by MALDI-TOF-MS, showing a wide DP ranging from 2 to 29 units. The analyses performed revealed that fructans from A. durangensis are formed of 97.11% fructose and 2.89% glucose, and are a complex mixture of fructooligosaccharides of the neo-fructan type containing principally β-(2-1) and β-(2-6) linkages, with branch moieties.

Endo-inulinase is a member of glycosidase hydrolase family 32 (GH32) degrading fructans of the inulin type with an endo-cleavage mode and is an important class of industrial enzyme. In the present study, we report the first crystal structure of an endo-inulinase, INU2, from Aspergillus ficuum at 1.5 Å. It was solved by molecular replacement with the structure of exo-inulinase as search model. The 3D structure presents a bimodular arrangement common to other GH32 enzymes: a N-terminal 5-fold β-propeller catalytic domain with four β-sheets and a C-terminal β-sandwich domain organized in two β-sheets with five β-strands. The structural analysis and comparison with other GH32 enzymes reveal the presence of an extra pocket in the INU2 catalytic site, formed by two loops and the conserved motif W-M(I)-N-D(E)-P-N-G. This cavity would explain the endo-activity of the enzyme, the critical role of Trp40 and particularly the cleavage at the third unit of the inulin(-like) substrates. Crystal structure at 2.1 Å of INU2 complexed with fructosyl molecules, experimental digestion data and molecular modelling studies support these hypotheses.

Xanthomonas campestris pv phaseoli produced an extracellular endoinulinase (9.24 ± 0.03 U mL^-1) in an optimized medium comprising of 3% sucrose and 2.5% tryptone. X. campestris pv. phaseoli was further subjected to ethylmethanesulfonate mutagenesis and the resulting mutant, X. campestris pv. phaseoli KM 24 demonstrated inulinase production of 22.09 ± 0.03 U mL^-1 after 18 h, which was 2.4-fold higher than that of the wild type. Inulinase production by this mutant was scaled up using sucrose as a carbon source in a 5-L fermenter yielding maximum volumetric (21,865 U L^-1 h^-1) and specific (119,025 U g^-1 h^-1) productivities of inulinase after 18 h with an inulinase/invertase ratio of 2.6. A maximum FOS production of 11.9 g L^-1 h^-1 and specific productivity of 72 g g^-1 h^-1 FOS from inulin were observed in a fermenter, when the mutant was grown on medium containing 3% inulin and 2.5% tryptone. The detection of mono- and oligosaccharides in inulin hydrolysates by TLC analysis indicated the presence of an endoinulinase. This mutant has potential for large-scale production of inulinase and fructooligosaccharides.

Structure and health effects of inulin-type fructans have been extensively studied, while less is known about the properties of the graminan-type fructans in wheat. Arabinoxylan (AX) is another important indigestible component in cereal grains, which may have beneficial health effects. In this study, the fructan content in milling fractions of two wheat cultivars was determined and related to ash, dietary fibre and AX contents. The molecular weight distribution of the fructans was analysed with HPAEC-PAD and MALDI-TOF MS using ¹H NMR and enzymatic hydrolysis for identification of fructans. The fructan content (g/100 g) ranged from 1.5 ± 0.2 in flour to 3.6 ± 0.5 in shorts and 3.7 ± 0.3 in bran. A correlation was found between fructan content and dietary fibre content (r = 0.93, P < 0.001), but with a smaller variation in fructan content between inner and outer parts of the grain. About 50% of the dietary fibre consisted of AX in all fractions. The fructans were found to have a DP of up to 19 with a similar molecular weight distribution in the different fractions.

In traditional tequila production, the heads of Agave tequilana Weber var. azul are cooked in brick ovens to hydrolyze the fructan content and release fermentable sugars. The juice generated during cooking (known as “cooking honey”) was collected periodically in a tequila distillery and characterized to study the efficiency of fructan hydrolysis. The complex structure of fructans from A. tequilana was confirmed. The generation of 5-(hydroxymethyl)-furfural, an increase in absorbance and °Brix, and a decrease in pH and apparent average degree of polymerization of fructans during cooking were observed. The conversion of fructans in the flowing honey increased gradually from 20% at the onset of cooking to 98% after 25.5 h, where fructose represented more than 80% of the total carbohydrates. The proportion of non-hydrolyzed fructans in the cooking honey collected before this time resulted in a total ethanol loss of 6% in the tequila distillery investigated.

Bacterial fructosyltransferase (FTF) enzymes synthesize fructan polymers from sucrose. FTFs catalyse two different reactions, depending on the nature of the acceptor, resulting in: (i) transglycosylation, when the growing fructan chain (polymerization), or mono- and oligosaccharides (oligosaccharide synthesis), are used as the acceptor substrate; (ii) hydrolysis, when water is used as the acceptor. Lactobacillus reuteri 121 levansucrase (Lev) and inulosucrase (Inu) enzymes are closely related at the amino acid sequence level (86 % similarity). Also, the eight amino acid residues known to be involved in catalysis and/or sucrose binding are completely conserved. Nevertheless, these enzymes differ markedly in their reaction and product specificities, i.e. in β(2→6)- versus β(2→1)-glycosidic-bond specificity (resulting in levan and inulin synthesis, respectively), and in the ratio of hydrolysis versus transglycosylation activities [resulting in glucose and fructooligosaccharides (FOSs)/polymer synthesis, respectively]. The authors report a detailed characterization of the transglycosylation reaction products synthesized by the Lb. reuteri 121 Lev and Inu enzymes from sucrose and related oligosaccharide substrates. Lev mainly converted sucrose into a large levan polymer (processive reaction), whereas Inu synthesized mainly a broad range of FOSs of the inulin type (non-processive reaction). Interestingly, the two FTF enzymes were also able to utilize various inulin-type FOSs (1-kestose, 1,1-nystose and 1,1,1-kestopentaose) as substrates, catalysing a disproportionation reaction; to the best of our knowledge, this has not been reported for bacterial FTF enzymes. Based on these data, a model is proposed for the organization of the sugar-binding subsites in the two Lb. reuteri 121 FTF enzymes. This model also explains the catalytic mechanism of the enzymes, and differences in their product specificities.

Degradation of a sample of high-molecular (degree of polymerisation, DP, between 13 and 30) and low-molecular (DP below 12) inulin from Jerusalem artichoke during dry heating for 30 min at 165 and 195°C was analysed using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and thin layer chromatography. Dry heating at 195°C induced complete degradation of the fructan chains and the concomitant formation of low-molecular degradation products, most likely di-D-fructose dianhydrides. In vitro fermentation studies using mixed faecal samples of eight human volunteers for 24 h at 37°C showed significant stimulation of the growth of bifidobacteria and Enterobacteriaceae and a significant decrease of possibly pathogenic bacteria of the Clostridium histolyticum and C. lituseburense group by inulin samples heated at 195°C compared to unheated samples and samples heated at 165°C. This preliminary data may point to the hypothesis that heat-treated inulin or its degradation products may cause improvements of the gut microflora superior to native inulin.

Dry heating of inulin from chicory for up to 60 min at temperatures between 135 and 195°C resulted in a significant degradation of the fructan ranging from 20 to 100%. The choice of the analytical method has a significant influence on inulin quantification especially in heat-treated samples. The amount of inulin found after thermal treatment measured as fructose after acidic hydrolysis was significantly higher compared with corresponding data obtained with a method based on enzymatic hydrolysis. Using high-performance anion-exchange chromatography with pulsed amperometric detection as well as high-performance thin-layer chromatography, it was found that thermal treatment of inulin leads to a degradation of the long fructose chains and formation of new products, most likely di-D-fructose dianhydrides. These degradation products of inulin are cleavable by acid to fructose monomers, but their glycosidic bonds are no longer accessible for β-fructosidase, thus explaining the discrepancies in inulin quantification with respect to the method used. Inulin degradation must be taken into account when fructan is used as a prebiotic ingredient in thermally treated foods like bakery products.

The fructan content of Finnish rye grains (13 samples, seven cultivars, harvested in 1998-2000) varied at 4.6–6.6 g/100 g (db). Commercial whole grain rye flour and rye flakes had fructan content of 4 g/100 g, light refined rye flour had fructan content of 3 g/100 g, and rye bran had fructan content of 7 g/100 g. Fructan content as high as 23 g/100 g was detected in the water-extractable concentrate of rye bran. Finnish soft rye bread and rye crisp bread contained 2–3 g of fructan/100 g. According to the suggested new definition of dietary fiber, fructans are also classified as dietary fiber. This means that the dietary fiber content of some cereal foods such as rye products may be increased by as much as 20% due to the presence of fructans in the grain.

Agave plants utilize crassulacean acid metabolism (CAM) for CO₂ fixation. Fructans are the principal photosynthetic products generated by agave plants. These carbohydrates are fructose-bound polymers frequently with a single glucose moiety. Agave tequilana Weber var. azul is an economically important CAM species not only because it is the sole plant allowed for tequila production but because it is a potential source of prebiotics. Because of the large amounts of carbohydrates in A. tequilana, in this study the molecular structures of its fructans were determined by fructan derivatization for linkage analysis coupled with gas chromatography−mass spectrometry (GC−MS), nuclear magnetic resonance (NMR), and matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS). Fructans were extracted from 8-year-old A. tequilana plants. The linkage types present in fructans from A. tequilana were determined by permethylation followed by reductive cleavage, acetylation, and finally GC-MS analysis. Analysis of the degree of polymerization (DP) estimated by ¹H NMR integration and ¹³C NMR and confirmed by MALDI-TOF-MS showed a wide DP ranging from 3 to 29 units. All of the analyses performed demonstrated that fructans from A. tequilana consist of a complex mixture of fructooligosaccharides containing principally β(2 → 1) linkages, but also β(2 → 6) and branch moieties were observed. Finally, it can be stated that fructans from A. tequilana Weber var. azul are not an inulin type as previously thought.

We report a new and non-equipment demanding method of measuring the content of fructans as well as the contents of free glucose, free fructose and sucrose in foods and food products enzymatically. This method comprises hydrolysis of fructans into D-glucose and D-fructose enzymatically and measurement of the released sugars enzymatically. Sucrose is hydrolysed by α-glucosidase instead of β-fructosidase, which is normally used. In addition, sucrose is measured in the form of D-fructose instead of the typical D-glucose form, and the fructanase used to hydrolyse the fructans has fewer side effects than the fructanase reported as normally used. The method is tested on ten standard substances and five fructan products, and nine foods and food products are also analysed. The enzymatic measurement of the released sugars is confirmed by measurements done by high performance anion exchange chromatography with pulsed amperemetric detection.