Versatile high resolution oligosaccharide microarrays for plant glycobiology and cell wall research.
Pedersen, H. L., Fangel, J. U., McCleary, B., Ruzanski, C., Rydahl, M. G., Ralet, M. C., Farkas, V., Von Schantz, L., Marcus, S. E., Andersen, M.C. F., Field, R., Ohlin, M., Knox, J. P., Clausen, M. H. & Willats, W. G. T. (2012). Journal of Biological Chemistry, 287(47), 39429-39438.
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.
Fructan content of rye and rye products.
Karppinen, S., Myllymäki, O., Forssell, P. & Poutanen, K. (2003). Cereal Chemistry, 80(2), 168-171.
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.
Heat-induced degradation of inulin.
Böhm, A., Kaiser, I., Trebstein, A. & Henle, T. (2005). European Food Research and Technology, 220(5-6), 466-471.
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.
Distribution and characterisation of fructan in wheat milling fractions.
Haskå, L., Nyman, M. & Andersson, R. (2008). Journal of Cereal Science, 48(3), 768-774.
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.
Structure of fructans from excised leaves of New Zealand flax.
Sims, I. M., Cairns, A. J. & Furneaux, R. H. (2001). Phytochemistry, 57(5), 661-668.
The accumulation of total water-soluble carbohydrate, and specifically sucrose and fructan, by excised leaves of Phormium tenax and P. cookianum (family Phormiaceae J. G. Agardh, order Asparagales) was investigated. Total water-soluble carbohydrate content of excised leaves of P. tenax and P. cookianum increased during 48 h of continuous illumination at an average rate of 1.3 and 0.9 mg g-1 fresh weight leaf per hour, respectively. The sucrose content of excised leaves increased throughout the experimental period. The fructan content of excised leaves of P. tenax increased slightly throughout the experimental period, whilst that of P. cookianum was variable and showed no overall change. Chemical and spectroscopic analysis of the fructans obtained from the two Phormium species showed that they were similar to each other and contained mostly 1-linked and terminal fructofuranosyl (Fruf) residues, together with smaller amounts of 6-linked Fruf, 1,6-branched Fruf, terminal and 6-linked glucopyranosyl residues. Separation of the fructans by thin-layer and high-performance anion-exchange chromatography revealed the presence of a complex mixture of fructo-oligosaccharides and higher molecular weight fructan. The branched structure of the fructans isolated from excised leaves of Phormium resembles that of fructans and fructo-oligosaccharides isolated from some related species within the order Asparagales (Agave vera cruz, Cordyline australis and Urginea maritima), but is distinct from the linear structure of fructans from others (Allium cepa and Asparagus officinalis). The structural heterogeniety of fructans within both the order Asparagales and superorder Liliiflorae may be a useful chemotaxonomic aid.
Effect of dry heated inulin on selected intestinal bacteria.
Böhm, A., Kleessen, B. & Henle, T. (2006). European Food Research and Technology, 222(5), 737-740.
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.
Fructans from oat and rye: Composition and effects on membrane stability during drying.
Hincha, D. K., Livingston III, D. P., Premakumar, R., Zuther, E., Obel, N., Cacela, C. & Heyer, A. G. (2007). Biochimica et Biophysica Acta (BBA)-Biomembranes, 1768(6), 1611-1619.
Fructans have been implicated in the abiotic stress tolerance of many plant species, including grasses and cereals. To elucidate the possibility that cereal fructans may stabilize cellular membranes during dehydration, we used liposomes as a model system and isolated fructans from oat (Avena sativa) and rye (Secale cereale). Fructans were fractionated by preparative size exclusion chromatography into five defined size classes (degree of polymerization (DP) 3 to 7) and two size classes containing high DP fructans (DP > 7 short and long). They were characterized by high performance liquid chromatography (HPLC) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The effects of the fructans on liposome stability during drying and rehydration were assessed as the ability of the sugars to prevent leakage of a soluble marker from liposomes and liposome fusion. Both species contain highly complex mixtures of fructans, with a DP up to 17. The two DP > 7 fractions from both species were unable to protect liposomes, while the fractions containing smaller fructans were protective to different degrees. Protection showed an optimum at DP 4 and the DP 3, 4, and 5 fractions from oat were more protective than all other fractions from both species. In addition, we found evidence for synergistic effects in membrane stabilization in mixtures of low DP with DP > 7 fructans. The data indicate that cereal fructans have the ability to stabilize membranes under stress conditions and that there are size and species dependent differences between the fructans. In addition, mixtures of fructans, as they occur in living cells may have protective properties that differ significantly from those of the purified fractions.
Linear Ion Trap MSn of Enzymatically Synthesized 13C-Labeled Fructans Revealing Differentiating Fragmentation Patterns of β (1-2) and β (1-6) Fructans and Providing a Tool for Oligosaccharide Identification in Complex Mixtures.
Harrison, S., Xue, H., Lane, G., Villas-Boas, S. & Rasmussen, S. (2012). Analytical Chemistry, 84(3), 1540-1548.
Fructans are polymeric carbohydrates, which play important roles as plant reserve carbohydrates and stress protectants, and are beneficial for human health and animal production. Fructans are formed by the addition of β-D-fructofuranosyl units to sucrose, leading to very complex mixtures of 1-kestose based inulins, 6-kestose linked levans, and 6G-kestose derived neoseries inulins and levans in cool season grasses such as Lolium perenne. The identification of isomeric fructan oligomers in chromatographic analysis of crude plant extracts is often hampered by the lack of authentic standards, and unambiguous peak assignment usually requires time-consuming analyses of purified fructan oligomers. We have developed a LC-MSn method for the separation and detection of fructan isomers and present here evidence for specific MSn fragmentation patterns associated with β 1-2 (inulins) and β 2-6 (levans) fructans. LC-MSn analysis of 13C labeled fructan oligomers produced by L. perenne fructosyltransferases expressed in yeast has enabled us to account for the observed fragmentation patterns in terms of preferential cleavage of the glycosidic bond between O- and fructose C2 in both inulins and levans and to differentiate reducing-end from nonreducing end cross ring cleavages in levans. We propose that higher order MS fragmentation patterns can be used to distinguish between the two major classes of fructan, i.e., inulins and levans, without the need for authentic standards.
Enhanced fructooligosaccharides and inulinase production by a Xanthomonas campestris pv. phaseoli KM 24 mutant.
Naidoo, K., Ayyachamy, M., Permaul, K. & Singh, S. (2009). Bioprocess and Biosystems Engineering, 32(5), 689-695.
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.
Towards a better understanding of the generation of fructan structure diversity in plants: molecular and functional characterization of a sucrose: fructan 6-fructosyltransferase (6-SFT) cDNA from perennial ryegrass (Lolium perenne).
Lasseur, B., Lothier, J., Wiemken, A., Van Laere, A., Morvan-Bertrand, A., Van den Ende, W. & Prud'homme, M. P. (2011). Journal of Experimental Botany, 62(6), 1871-1885.
The main storage compounds in Lolium perenne are fructans with prevailing β(2–6) linkages. A cDNA library of L. perenne was screened using Poa secunda sucrose:fructan 6-fructosyltransferase (6-SFT) as a probe. A full-length Lp6-SFT clone was isolated as shown by heterologous expression in Pichia pastoris. High levels of Lp6-SFT transcription were found in the growth zone of elongating leaves and in mature leaf sheaths where fructans are synthesized. Upon fructan synthesis induction, Lp6-SFT transcription was high in mature leaf blades but with no concomitant accumulation of fructans. In vitro studies with the recombinant Lp6-SFT protein showed that both 1-kestotriose and 6G-kestotriose acted as fructosyl acceptors, producing 1- and 6-kestotetraose (bifurcose) and 6G,6-kestotetraose, respectively. Interestingly, bifurcose formation ceased and 6G,6-kestotetraose was formed instead, when recombinant fructan:fructan 6G-fructosyltransferase (6G-FFT) of L. perenne was introduced in the enzyme assay with sucrose and 1-kestotriose as substrates. The remarkable absence of bifurcose in L. perenne tissues might be explained by a higher affinity of 6G-FFT, as compared with 6-SFT, for 1-kestotriose, which is the first fructan formed. Surprisingly, recombinant 6-SFT from Hordeum vulgare, a plant devoid of fructans with internal glucosyl residues, also produced 6G,6-kestotetraose from sucrose and 6G-kestotriose. In the presence of recombinant L. perenne 6G-FFT, it produced 6G,6-kestotetraose from 1-kestotriose and sucrose, like L. perenne 6-SFT. Thus, we demonstrate that the two 6-SFTs have close catalytic properties and that the distinct fructans formed in L. perenne and H. vulgare can be explained by the presence of 6G-FFT activity in L. perenne and its absence in H. vulgare.
Molecular structures of fructans from Agave tequilana Weber var. azul.
Lopez, M. G., Mancilla-Margalli, N. A. & Mendoza-Diaz, G. (2003). Journal of Agricultural and Food Chemistry, 51(27), 7835-7840.
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.
Heterologous expression of a Penicillium purpurogenum exo-arabinanase in Pichia pastoris and its biochemical characterization.
Mardones, W., Callegari, E. & Eyzaguirre, J. (2015). Fungal Biology, 119(12), 1267-1278.
Arabinan is a component of pectin, which is one of the polysaccharides present in lignocelluose. The enzymes degrading the main chain of arabinan are the endo- (EC 22.214.171.124) and exo-arabinanases (3.2.1.-). Only three exo-arabinanases have been biochemically characterized; they belong to glycosyl hydrolase family 93. In this work, the cDNA of an exo-arabinanase (Arap2) from Penicillium purpurogenum has been heterologously expressed in Pichia pastoris. The gene is 1310 bp long, has three introns and codes for a protein of 380 amino acid residues; the mature protein has a calculated molecular mass of 39 823 Da. The heterologously expressed Arap2 has a molecular mass in the range of 60–80 kDa due to heterogeneous glycosylation. The enzyme is active on debranched arabinan with optimum pH of 4-5.5 and optimal temperature of 40°C, and has an exo-type action mode, releasing arabinobiose from its substrates. The expression profile of arap2 in corncob and sugar beet pulp follows a different pattern and is not related to the presence of arabinan. This is the first exo-arabinanase studied from P. purpurogenum and the first expressed in yeast. The availability of heterologous Arap2 may be useful for biotechnological applications requiring acidic conditions.
Fructooligosaccharides production by Schedonorus arundinaceus sucrose: sucrose 1-fructosyltransferase constitutively expressed to high levels in Pichia pastoris.
Hernández, L., Menéndez, C., Pérez, E. R., Martínez, D., Alfonso, D., Trujillo, L. E., Ramírez, R., Sobrino, A., Mazola, Y., Musacchio, A. & Pimentel, E. (2017). Journal of biotechnology, 266, 59-71.
The non-saccharolytic yeast Pichia pastoris was engineered to express constitutively the mature region of sucrose:sucrose 1-fructosyltransferase (1-SST, EC 126.96.36.199) 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.