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.
Induction and partial characterization of intracellular β from Thermoascus aurantiacus and its application in the synthesis of 6-kestose.
Katapodis, P. & Christakopoulos, P. (2004). World Journal of Microbiology and Biotechnology, 20(7), 667-672.
Production of β-fructofuranosidase from the thermophilic fungus Thermoascus aurantiacus was enhanced by optimization of the type of nitrogen source as well as the type and concentration of carbon source. Submerged batch cultivation in a laboratory bioreactor (7 l) using the optimized medium allowed the production of 85 mU/ml of β-fructofuranosidase. The enzyme showed both transfructosylating and hydrolytic activities and was optimally active at 60°C and pH 5.0. The enzyme showed the ability to catalyse the synthesis of 1-kestose and the reaction was maximized at 30% (w/v) initial sucrose concentration.
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.
Biosynthesis of fructo-oligosaccharides by Sporotrichum thermophile during submerged batch cultivation in high sucrose media.
Katapodis, P., Kalogeris, E., Kekos, D., Macris, B. J. & Christakopoulos, P. (2004). Applied Microbiology and Biotechnology, 63(4), 378-382.
Biosynthesis of fructo-oligosaccharides (FOS) was observed during growth of the thermophilic fungus Sporotrichum thermophile on media containing high sucrose concentrations. Submerged batch cultivation with the optimum initial sucrose concentration of 250 g/l allowed the production of 12.5 g FOS/l. The FOS mixture obtained was composed of three sugars, which were isolated by size-exclusion chromatography. They were characterized by acid hydrolysis and HPLC as 1-kestose, 6-kestose and neokestose. The mechanism of osmotic adaptation of S. thermophile was investigated and sugars and amino acids were found to be the predominant compatible solutes. The fungus accumulated glutamic acid, arginine, alanine, leucine and lysine, in order to balance the outer osmotic pressure. Fatty acid analysis of the membrane lipids showed a relatively high percentage of unsaturated lipids, which is known to be associated with high membrane fluidity.
A reverse-phase liquid chromatography/mass spectrometry method for the analysis of high-molecular-weight fructooligosaccharides.
Harrison, S. J., Fraser, K., Lane, G. A., Villas-Boas, S. & Rasmussen, S. (2009). Analytical Biochemistry, 395(1), 113-115.
Many important crop and forage plants accumulate polymeric water-soluble carbohydrates as fructooligosaccharides (or fructans). We have developed an improved method for the analysis of the full fructan complement in plant extracts based on porous graphitized carbon chromatography coupled to negative electrospray ionization mass spectrometry. By the use of profile data collection and multiple charge state ions, the effective mass range of the ion trap was extended to allow for the analysis of very high-molecular-weight oligosaccharides. This method allows the separation and quantification of isomeric fructan oligomers ranging from degree of polymerization (DP) 3 to DP 49.
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.
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.
Physico-chemical and transglucosylation properties of recombinant sucrose phosphorylase from Bifidobacterium adolescentis DSM20083.
van den Broek, L. A. M., van Boxtel, E. L., Kievit, R. P., Verhoef, R., Beldman, G. & Voragen, A. G. J. (2004). Applied Microbiology and Biotechnology, 65(2), 219-227.
Clones of a genomic library of Bifidobacterium adolescentis were grown in minimal medium with sucrose as sole carbon source. An enzymatic fructose dehydrogenase assay was used to identify sucrose-degrading enzymes. Plasmids were isolated from the positive colonies and sequence analysis revealed that two types of insert were present, which only differed with respect to their orientation in the plasmid. An open reading frame of 1,515 nucleotides with high homology for sucrose phosphorylases was detected on these inserts. The gene was designated SucP and encoded a protein of 56,189 Da. SucP was heterologously expressed in Escherichia coli, purified, and characterized. The molecular mass of SucP was 58 kDa, as estimated by SDS-PAGE, while 129 kDa was found with gel permeation, suggesting that the native enzyme was a dimer. The enzyme showed high activity towards sucrose and a lower extent towards α-glucose-1-phosphate. The transglucosylation properties were investigated using a broad range of monomeric sugars as acceptor substrate for the recombinant enzyme, while α-glucose-1-phosphate served as donor. D- and L-arabinose, D- and L-arabitol, and xylitol showed the highest production of transglucosylation products. The investigated disaccharides and trisaccharides were not suitable as acceptors. The structure of the transglucosylation product obtained with D-arabinose as acceptor was elucidated by NMR. The structure of the synthesized non-reducing dimer was α-Glcp(1→1) β-Araf.
Expression and activity analysis of sucrose:sucrose 1-fructosyltransferase from onion.
Han, Y., Chen, L., Mao, D., Tang, L. & Guan, L. (2010). New Biotechnology, 27(4), 324-329.
This study was designed to express the onion fructosyltransferase by Escherichia coli DH5α, and obtain the optimal conditions of FST-1 activity. Thereby, fructosyltransferase gene was obtained by RT-PCR from onion in this experiment, and named FST-1. The expressed proteins were analyzed by SDS-PAGE. FST-1 activity was identified by the high performance liquid chromatography (HPLC). The optimal conditions of FST-1 were analyzed by the dinonylnaphthalene sulfonic acid (DNS) and orthogonal test. Results revealed that FST-1 was identified to 98% similarity with fructosyltransferase mRNA of onion (accession number: AJ006066). FST-1 was successfully expressed in E. coli DH5α. HPLC results indicated that the expressed protein from FST-1 had a good transferring activity for fructose. The optimal conditions of FST-1 in catalyzing reaction were the pH 5.0, 45°C and 60% sucrose substrate. The results in this experiment would lay the foundation for the large-scale of kestose by bio-catalysis method.
Enzymatic determination of inulin and fructooligosaccharides in food.
Korakli, M., Hinrichs, C., Ehrmann, M. A. & Vogel, R. F. (2003). European Research and Technology, 217(6), 530-534.
Inulin and fructooligosaccharides (FOS) are widely distributed throughout the plant kingdom. They have been increasingly used in various foods due to their beneficial nutritional attributes. An enzymatic method was developed for rapid and accurate determination of inulin and/or FOS in food. β-Fructofuranosidase heterologously expressed in Escherichia coli was used as a hydrolyzing enzyme. After extraction with water, filtration and appropriate dilution, inulin was hydrolyzed using β-fructofuranosidase and the liberated sugars were determined enzymatically. The average recovery of inulin and/or FOS in food matrixes was 97% with a coefficient of variation of 5%. The method provided an inexpensive technique requiring only standard laboratory equipment for determining inulin and/or FOS with high accuracy and precision.
An improved method for quantitative analysis of total fructans in plant tissues.
Liu, Z., Mouradov, A., Smith, K. F. & Spangenberg, G. (2011). Analytical Biochemistry, 418(2), 253-259.
Current methods for measuring fructan levels in plant tissues are time-consuming and costly. They often involve multiple or sequential extractions, enzymatic or acid hydrolysis of fructan polymers, and multiple HPLC runs to quantify fructan-derived hexoses. Here we describe a new method that requires a single extraction step, followed by selective precipitation of fructans by acetone, acid hydrolysis of the precipitate, and a short (10 min) HPLC run to complete the procedure. We used perennial ryegrass samples to show that the new method has similar sensitivity, but better reproducibility, than a more complex method that is widely used. We have used the new method to study developmentally related changes in fructan levels in glasshouse-grown perennial ryegrass plants.
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.