1,1-Kestotetraose

Content: 100 mg
Shipping Temperature: Ambient
Storage Temperature: Ambient
Physical Form: Powder
Stability: > 2 years under recommended storage conditions
CAS Number: 13133-07-8
Molecular Formula: C24H42O21
Molecular Weight: 666.6
Purity: > 95%
Substrate For (Enzyme): endo-Inulinase

High purity 1,1-Kestotetraose for use in research, biochemical enzyme assays and in vitro diagnostic analysis.

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Megazyme publication

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.

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Sonochemical application reduces monosaccharide levels and improves cryoprotective effect of Jerusalem artichoke extract on Leuconostoc mesenteroides WiKim33 during freeze-drying.

Kim, Y. Y., Kim, H. M., Jeong, S. G., Yang, J. E., Kim, S. & Park, H. W. (2023). Ultrasonics Sonochemistry, 95, 106413.

Lactic acid bacteria (LAB) are being used for probiotic and starter cultures to prevent global damage to microbial cells. To retain the benefits of LAB in the commercially used powdered form, highly efficient cryoprotective agents are required during the manufacturing process. This study suggests a novel cryoprotective agent derived from Jerusalem artichoke (JA; Helianthus tuberous L.) and describes the mechanism of cryoprotective effect improvement by sonication treatment. The cryoprotective effect of JA extract was verified by examining the viability of Leuconostoc mesenteroides WiKim33 after freeze-drying (FD). Sonication of JA extract improved the cryoprotective effect. Sonication reduced fructose and glucose contents, which increased the induction of critical damage during FD by 15.84% and 46.81%, respectively. The cryoprotective effects of JA and sonication-treated JA extracts were determined using the viable cell count of Leu. mesenteroides WiKim33. Immediately after FD and storage for 24 weeks, the viability of Leu. mesenteroides WiKim33 with JA extract was 82.8% and 76.3%, respectively, while that of the sonication-treated JA extract was 95.2% and 88.8%, respectively. Our results show that reduction in specific monosaccharides was correlated with improved cryoprotective effect. This study adopted sonication as a novel treatment for improving the cryoprotective effect and verified its efficiency.

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High-yield production and purification of prebiotic inulin-type fructooligosaccharides.

Wienberg, F., Hövels, M. & Deppenmeier, U. (2022). AMB Express, 12(1), 1-16.

Due to the health-promoting effects and functional properties of inulin-type fructooligosaccharides (I-FOS), the global market for I-FOS is constantly growing. Hence, there is a continuing demand for new, efficient biotechnological approaches for I-FOS production. In this work, crude inulosucrase InuGB-V3 from Lactobacillus gasseri DSM 20604 was used to synthesize I-FOS from sucrose. Supplementation with 1 mM CaCl2, a pH of 3.5-5.5, and an incubation temperature of 40°C were found to be optimal production parameters at which crude inulosucrase showed high conversion rates, low sucrose hydrolysis, and excellent stability over 4 days. The optimal process conditions were employed in cell-free bioconversion reactions. By elevating the substrate concentration from 570 to 800 g L−1, the I-FOS concentration and the synthesis of products with a low degree of polymerization (DP) could be increased, while sucrose hydrolysis was decreased. Bioconversion of 800 g L−1 sucrose for 20 h resulted in an I-FOS-rich syrup with an I-FOS concentration of 401 ± 7 g L−1 and an I-FOS purity of 53 ± 1% [w/w]. I-FOS with a DP of 3-11 were synthesized, with 1,1-kestotetraose (DP4) being the predominant transfructosylation product. The high-calorie sugars glucose, sucrose, and fructose were removed from the generated I-FOS-rich syrup using activated charcoal. Thus, 81 ± 5% of the initially applied I-FOS were recovered with a purity of 89 ± 1%.

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Enzymatic degradation of FODMAPS via application of β-fructofuranosidases and α-galactosidases-A fundamental study.

Atzler, J. J., Ispiryan, L., Gallager, E., Sahin, A. W., Zannini, E. & Arendt, E. K. (2020).  Journal of Cereal Science, 102993.

Cereals and pulses often contribute to the intake of Fermentable Oligo-, Di-, Monosaccharides, and Polyols (FODMAPs) due to high amounts of fructans or galactooligosaccharides (GOS). FODMAPs can trigger symptoms of Irritable Bowel Syndrome (IBS) and therefore, the development of foods and beverages with a lower FODMAP-content are favourable for IBS patients. Enzyme technology is a promising tool to reduce the FODMAP-content in foods and to maintain product quality. This fundamental study investigates the efficiency of invertase, inulinase, and α-galactosidase as potential food additives to reduce the total FODMAP content of food ingredients. Extracts of high FODMAP ingredients, such as wheat and lentil, and standard solutions of various fructans and GOS were incubated with invertase, inulinase and α-galactosidase for 1 h and 2 h. Contents of oligosaccharides before and after treatment and related IBS-triggering reaction products were quantified using ion chromatography. Inulinase showed a high degradation yield (over 90% of degradation) for both GOS and fructans. For invertase only low degradation yields were measured. α-Galactosidase showed the highest efficiency in decomposing GOS (100% of degradation) and led to non-IBS triggering degradation products. This indicates a high potential for a combined inulinase/α-galactosidase treatment for products containing both fructans and GOS.

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Engineered thermostable β-fructosidase from Thermotoga maritima with enhanced fructooligosaccharides synthesis.

Menéndez, C., Martínez, D., Pérez, E. R., Musacchio, A., Ramírez, R., López-Munguía, A. & Hernández, L. (2019). Enzyme and Microbial Technology, 125, 53-62.

The thermostable β-fructosidase (BfrA) from the bacterium Thermotoga maritima converts sucrose into glucose, fructose, and low levels of short–chain fructooligosaccharides (FOS) at high substrate concentration (1.75 M) and elevated temperatures (60-70°C). In this research, FOS produced by BfrA were characterized by HPAE-PAD analysis as a mixture of 1–kestotriose, 6G-kestotriose (neokestose), and to a major extent 6-kestotriose. In order to increase the FOS yield, three BfrA mutants (W14Y, W14Y-N16S and W14Y-W256Y), designed from sequence divergence between hydrolases and transferases, were constructed and constitutively expressed in the non-saccharolytic yeast Pichia pastoris. The secreted recombinant glycoproteins were purified and characterized. The three mutants synthesized -kestotriose as the major component of a FOS mixture that includes minor amounts of tetra- and pentasaccharides. In all cases, sucrose hydrolysis was the predominant reaction. All mutants reached a similar overall FOS yield, with the average value 37.6% (w/w) being 3–fold higher than that of the wild–type enzyme (12.6%, w/w). None of the mutations altered the enzyme thermophilicity and thermostability. The single mutant W14Y, with specific activity of 841 U mg−1, represents an attractive candidate for the continuous production of FOS–containing invert syrup at pasteurization temperatures.

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Impact of grain sorghum polyphenols on microbiota of normal weight and overweight/obese subjects during in vitro fecal fermentation.

Ashley, D., Marasini, D., Brownmiller, C., Lee, J., Carbonero, F. & Lee, S. O. (2019). Nutrients, 11(2), 217.

The human gut microbiota is considered as a crucial mediator between diet and gut homeostasis and body weight. The unique polyphenolic profile of sorghum bran may promote gastrointestinal health by modulating the microbiota. This study evaluated gut microbiota and modulation of short-chain fatty acids (SCFA) by sorghum bran polyphenols in in vitro batch fermentation derived from normal weight (NW, n = 11) and overweight/obese (OO, n = 11) subjects’ fecal samples. Six separate treatments were applied on each batch fermentation: negative control (NC), fructooligosaccharides (FOS), black sorghum bran extract (BSE), sumac sorghum bran extract (SSE), FOS + BSE, or FOS + SSE; and samples were collected before and after 24 h. No significant differences in total and individual SCFA production were observed between NW and OO subjects. Differential responses to treatment according to weight class were observed in both phyla and genera. Sorghum bran polyphenols worked with FOS to enhance Bifidobacterium and Lactobacillus, and independently stimulated Roseburia and Prevotella (p < 0.05). Our results indicate that sorghum bran polyphenols have differential effects on gut health and may positively impact gut ecology, with responses varying depending on weight class.

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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 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.

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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 3.2.1.99) 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.

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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.

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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.

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