Compost Grown Agaricus bisporus Lacks the Ability to Degrade and Consume Highly Substituted Xylan Fragments
Jurak, E. ; Patyshakuliyeva, A. ; Vries, R.P. de; Gruppen, H. ; Kabel, M.A. - \ 2015
PLoS ONE 10 (2015)8. - ISSN 1932-6203
wheat-flour arabinoxylan - h-1-nmr spectroscopy - aspergillus-awamori - enzyme-activities - button mushroom - mode - oligosaccharides - purification
The fungus Agaricus bisporus is commercially grown for the production of edible mushrooms. This cultivation occurs on compost, but not all of this substrate is consumed by the fungus. To determine why certain fractions remain unused, carbohydrate degrading enzymes, water-extracted from mushroom-grown compost at different stages of mycelium growth and fruiting body formation, were analyzed for their ability to degrade a range of polysaccharides. Mainly endo-xylanase, endo-glucanase, ß-xylosidase and ß-glucanase activities were determined in the compost extracts obtained during mushroom growth. Interestingly, arabinofuranosidase activity able to remove arabinosyl residues from doubly substituted xylose residues and a-glucuronidase activity were not detected in the compost enzyme extracts. This correlates with the observed accumulation of arabinosyl and glucuronic acid substituents on the xylan backbone in the compost towards the end of the cultivation. Hence, it was concluded that compost grown A. bisporus lacks the ability to degrade and consume highly substituted xylan fragments.
Effect of the DGAT1 K232A genotype of dairy cows on the milk metabolome and proteome
Lu, J. ; Boeren, S. ; Hooijdonk, A.C.M. van; Vervoort, J.J.M. ; Hettinga, K.A. - \ 2015
Journal of Dairy Science 98 (2015)5. - ISSN 0022-0302 - p. 3460 - 3469.
h-1-nmr spectroscopy - sample preparation - identification - stomatin - membrane - proteins - gene - cattle - yield
Diglyceride O-acyltransferase 1 (DGAT1) is the enzyme that catalyzes the synthesis of triglycerides from diglycerides and acyl-coenzyme A. The DGAT1 K232A polymorphism was previously shown to have a significant influence on bovine milk production characteristics (milk yield, protein content, fat content, and fatty acid composition). The mechanism of this influence has, however, not been elucidated. In this study, metabolomics (1H-nuclear magnetic resonance) and proteomics (laser chromatography-tandem mass spectrometry) were applied to determine the serum and lipid metabolite composition and milk fat globule membrane proteome of milk samples from cows with the DGAT1 KK and AA genotypes. The milk samples from cows with the DGAT1 KK genotype contained more stomatin, sphingomyelin, choline, and carnitine, and less citrate, creatine or phosphocreatine, glycerol-phosphocholine, mannose-like sugar, acetyl sugar phosphate, uridine diphosphate (UDP)-related sugar, and orotic acid compared with milk samples from cows with the DGAT1 AA genotype. Based on these results, we propose that the differences between the DGAT1 genotypes may be related to stomatin-sphingomyelin lipid rafts as well as structural (cell membrane) differences in epithelial cells of the mammary gland. In conclusion, our study shows that, in addition to previously described changes in triglyceride composition, cows differing in DGAT1 polymorphism differ in their milk proteome and metabolome, which may help in further understanding the effect of the DGAT1 K232A polymorphism on milk production characteristics.
Diversity in Production of Xylan-Degrading Enzymes Among Species Belonging to the Trichoderma Section Longibrachiatum
Toth, K. ; Gool, M.P. van; Schols, H.A. ; Samuels, G.J. ; Gruppen, H. ; Szakacs, G. - \ 2013
Bio Energy Research 6 (2013)2. - ISSN 1939-1234 - p. 631 - 643.
wheat-flour arabinoxylan - industrial applications - h-1-nmr spectroscopy - aspergillus-awamori - beta-glucosidase - biofuels - reesei - fermentation - hypocrea - endo-beta-1,4-xylanases
Xylan is an important part of plant biomass and represents a renewable raw material for biorefineries. Contrary to cellulose, the structure of hemicellulose is quite complex. Therefore, the biodegradation of xylan needs the cooperation of many enzymes. For industrial production of xylanase multienzyme complexes (cocktails) and selected monocomponent xylanases, different Trichoderma reesei mutants and recombinants are used. T. reesei QM 6a (wild-type parent of best existing mutants) was selected as a starting material in the 1960s when the modern in-depth analytical methods were not yet in use. Therefore, screening of fungi genetically close to T. reesei in biodegradation of xylan may have a scientific value. Fifteen different strains from Trichoderma section Longibrachiatum have been tested for extracellular xylan-degrading enzyme production on three carbon sources (wheat straw, corn fiber, and eucalyptus wood) in shake flask cultivation. The enzyme activities were evaluated by traditional colorimetric enzyme assays and by HPLC and matrix assisted laser desorption/ionization time-of-flight mass spectrometry. Degradation of xylan was studied on four different xylan-rich model substrates. T. reesei CPK 155, Trichoderma parareesei TUB F-2535, and Trichoderma gracile TUB F-2543 isolates were equally good or better in degradation of the wheat arabinoxylan (WAX) and corn fiber alcohol insoluble solids as hydolysis substrates than the well-known T. reesei QM 6a and RUT C30 strains. Though Trichoderma saturnisporum ATCC 18903 gave relatively low volumetric enzyme activities by traditional colorimetric assays, it could release quite large amount of hydrolysis products (mono- and oligosaccharides) from WAX. Therefore, these fungi may be potential candidates for further experiments. Enzyme production on wheat straw and corn fiber carbon sources was more effective than on eucalyptus wood
Enhanced NMR-based profiling of polyphenols in commercially available grape juices using solid-phase extraction
Savage, A.K. ; Duynhoven, J.P.M. van; Tucker, G. ; Daykin, C. - \ 2011
Magnetic Resonance in Chemistry 49 (2011)1. - ISSN 0749-1581 - p. S27 - S36.
nuclear-magnetic-resonance - fruit juices - h-1-nmr spectroscopy - liquid-extraction - by-products - green tea - wine - quality - identification - chromatography
Grapes and related products, such as juices, and in particular, their polyphenols, have previously been associated with many health benefits, such as protection against cardiovascular disease. Within grapes, a large range of structurally diverse polyphenols can be present, and their characterisation stands as a challenge. 1H NMR spectroscopy in principle would provide a rapid, nondestructive and straightforward method for profiling of polyphenols. However, polyphenol profiling and identification in grape juices is hindered because of signals of prevailing carbohydrates causing spectral overlap and compromising dynamic range. This study describes the development of an extraction method prior to analysis using 1H NMR spectroscopy, which can, potentially, significantly increase the number of detectable polyphenols and aid their identification, by reduction of signal overlap and selective removal of heavily dominating compounds such as sugars.
Comparison of two GM maize varieties with a near-isogenic non-GM variety using transcriptomics, proteomics and metabolomics
Barros, E. ; Lezar, S. ; Anttonen, M.J. ; Dijk, J.P. van; Rohlig, R.M. ; Kok, E.J. ; Engel, K.H. - \ 2010
Plant Biotechnology Journal 8 (2010)4. - ISSN 1467-7644 - p. 436 - 451.
gene-expression - h-1-nmr spectroscopy - safety assessment - food - tool - nmr - hybridization - microarrays - mutants - plants
P>The aim of this study was to evaluate the use of four nontargeted analytical methodologies in the detection of unintended effects that could be derived during genetic manipulation of crops. Three profiling technologies were used to compare the transcriptome, proteome and metabolome of two transgenic maize lines with the respective control line. By comparing the profiles of the two transgenic lines grown in the same location over three growing seasons, we could determine the extent of environmental variation, while the comparison with the control maize line allowed the investigation of effects caused by a difference in genotype. The effect of growing conditions as an additional environmental effect was also evaluated by comparing the Bt-maize line with the control line from plants grown in three different locations in one growing season. The environment was shown to play an important effect in the protein, gene expression and metabolite levels of the maize samples tested where 5 proteins, 65 genes and 15 metabolites were found to be differentially expressed. A distinct separation between the three growing seasons was also found for all the samples grown in one location. Together, these environmental factors caused more variation in the different transcript/protein/metabolite profiles than the different genotypes.
An inter-laboratory comparison demonstrates that [H-1]-NMR metabolite fingerprinting is a robust technique for collaborative plant metabolomic data collection.
Ward, J.L. ; Baker, J.M. ; Miller, S.J. ; Deborde, C. ; Maucourt, M. ; Biais, B. ; Rolin, D. ; Moing, A. ; Moco, S.I.A. ; Vervoort, J.J.M. ; Lommen, A. ; Schafer, H. ; Humpfer, E. ; Beale, M.H. - \ 2010
Metabolomics 6 (2010)2. - ISSN 1573-3882 - p. 263 - 273.
minimum reporting standards - nmr-spectroscopy - h-1-nmr spectroscopy - mass-spectrometry - metabonomics
In any metabolomics experiment, robustness and reproducibility of data collection is of vital importance. These become more important in collaborative studies where data is to be collected on multiple instruments. With minimisation of variance in sample preparation and instrument performance it is possible to elucidate even subtle differences in metabolite fingerprints due to genotype or biological treatment. In this paper we report on an inter laboratory comparison of plant derived samples by [1H]-NMR spectroscopy across five different sites and within those sites utilising instruments with different probes and magnetic field strengths of 9.4 T (400 MHz), 11.7 T (500 MHz) and 14.1 T (600 MHz). Whilst the focus of the study is on consistent data collection across laboratories, aspects of sample stability and the requirement for sample rotation within the NMR magnet are also discussed. Comparability of the datasets from participating laboratories was exceptionally good and the data were amenable to comparative analysis by multivariate statistics. Field strength differences can be adjusted for in the data pre-processing and multivariate analysis demonstrating that [1H]-NMR fingerprinting is the ideal technique for large scale plant metabolomics data collection requiring the participation of multiple laboratories.
Intra- and inter-metabolite correlation spectroscopy of tomato metabolomics data obtained by liquid chromatography-mass spectrometry and nuclear magnetic resonance
Moco, S.I.A. ; Forshed, J. ; Vos, C.H. de; Bino, R.J. ; Vervoort, J.J.M. - \ 2008
Metabolomics 4 (2008)3. - ISSN 1573-3882 - p. 202 - 215.
multivariate-analysis - h-1-nmr spectroscopy - fruit - nmr - identification - lc/ms - fusion
Nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LCMS) are frequently used as technological platforms for metabolomics applications. In this study, the metabolic profiles of ripe fruits from 50 different tomato cultivars, including beef, cherry and round types, were recorded by both 1H NMR and accurate mass LC-quadrupole time-of-flight (QTOF) MS. Different analytical selectivities were found for these both profiling techniques. In fact, NMR and LCMS provided complementary data, as the metabolites detected belong to essentially different metabolic pathways. Yet, upon unsupervised multivariate analysis, both NMR and LCMS datasets revealed a clear segregation of, on the one hand, the cherry tomatoes and, on the other hand, the beef and round tomatoes. Intra-method (NMR¿NMR, LCMS¿LCMS) and inter-method (NMR¿LCMS) correlation analyses were performed enabling the annotation of metabolites from highly correlating metabolite signals. Signals belonging to the same metabolite or to chemically related metabolites are among the highest correlations found. Inter-method correlation analysis produced highly informative and complementary information for the identification of metabolites, even in de case of low abundant NMR signals. The applied approach appears to be a promising strategy in extending the analytical capacities of these metabolomics techniques with regard to the discovery and identification of biomarkers and yet unknown metabolites.
Preparation of arabinoxylobiose from rye xylan using family 10 Aspergillus aculeatus endo-1,4-ß-d-xylanase
Rantanen, H. ; Virkki, L. ; Tuomainen, P. ; Kabel, M.A. ; Schols, H.A. ; Tenkanen, M. - \ 2007
Carbohydrate Polymers 68 (2007)2. - ISSN 0144-8617 - p. 350 - 359.
wheat-flour arabinoxylan - h-1-nmr spectroscopy - aspergillus-awamori - oligosaccharides - xylooligosaccharides - endoxylanases - hydrolysis - cellulase - endosperm - bacteria
Commercial xylanase preparation Shearzyme®, which contains the glycoside hydrolase family 10 endo-1,4-ß-d-xylanase from Aspergillus aculeatus, was used to prepare short-chain arabinoxylo-oligosaccharides (AXOS) from rye arabinoxylan (AX). A major AXOS was formed as a hydrolysis product. Longer AXOS were also produced as minor products. The pure GH10 xylanase from A. aculeatus was used as a comparison to ensure that the formed AXOS were consequence of the endoxylanase`s function instead of some side enzymes present in Shearzyme. The major AXOS was purified and the structure confirmed with various analysis methods (TLC, HPAEC-PAD, MALDI-TOF-MS, and one- and two-dimensional NMR spectroscopy with nano-probe) as ¿-l-Araf-(1 ¿ 3)-ß-d-Xylp-(1 ¿ 4)-d-Xylp (arabinoxylobiose). This is the first report on 13C NMR data of pure arabinoxylobiose. The yield of arabinoxylobiose was 12% from the quantified hydrolysis products. In conclusion, GH10 endoxylanase from A. aculeatus is thus able to cut efficiently the xylosidic linkage next to the arabinofuranosyl-substituted xylose unit which is not typical for all the GH10 endoxylanases. Interestingly, pure A. aculeatus xylanase showed notably activity towards p-nitrophenyl-ß-d-xylopyranose. In previously studies longer AXOS have been produced with Shearzyme but the formation of short-chain AXOS by A. aculeatus GH10 xylanase has not been studied before. Keywords: Arabinoxylan; Arabinoxylobiose; Aspergillus aculeatus; Glycoside hydrolase family 10; Shearzyme; Xylanase; Xylo-oligosaccharides
Tissue specialization at the metabolite level is perceived during the development of tomota fruit.
Moco, S.I.A. ; Capanoglu, E. ; Tikunov, Y.M. ; Bino, R.J. ; Boyacioglu, D. ; Hall, R.D. ; Vervoort, J.J.M. ; Vos, C.H. de - \ 2007
Journal of Experimental Botany 58 (2007)15-16. - ISSN 0022-0957 - p. 4131 - 4146.
mass-spectrometry - plant metabolomics - h-1-nmr spectroscopy - database - perspective - lycopene - growth - accumulation - expression - flavonoids
Fruit maturation and tissue differentiation are important topics in plant physiology. These biological phenomena are accompanied by specific alterations in the biological system, such as differences in the type and concentration of metabolites. The secondary metabolism of tomato (Solanum lycopersicum) fruit was monitored by using liquid chromatography (LC) coupled to photo-diode array (PDA) detection, fluorescence detection (FD), and mass spectrometry (MS). Through this integrated approach different classes of compounds were analysed: carotenoids, xanthophylls, chlorophylls, tocopherols, ascorbic acid, flavonoids, phenolic acids, glycoalkaloids, saponins, and other glycosylated derivatives. Related metabolite profiles of peel and flesh were found between several commercial tomato cultivars indicating similar metabolite trends despite the genetic background. For a single tomato cultivar, metabolite profiles of different fruit tissues (vascular attachment region, columella and placenta, epidermis, pericarp, and jelly parenchyma) were examined at the green, breaker, turning, pink, and red stages of fruit development. Unrelated to the chemical nature of the metabolites, behavioural patterns could be assigned to specific ripening stages or tissues. These findings suggest spatio-temporal specificity in the accumulation of endogenous metabolites from tomato fruit.
Metabolomics technologies and metabolite identification
Moco, S.I.A. ; Bino, R.J. ; Vos, C.H. de; Vervoort, J.J.M. - \ 2007
TrAC : Trends in Analytical Chemistry 26 (2007)9. - ISSN 0165-9936 - p. 855 - 866.
trap mass-spectrometry - h-1-nmr spectroscopy - nmr-spectroscopy - data sets - chromatography - database - probes - tool - acquisition - accuracy
Metabolomics studies rely on the analysis of the multitude of small molecules (metabolites) present in a biological system. Most commonly, metabolomics is heavily supported by mass spectrometry (MS) and nuclear magnetic resonance (NMR) as parallel technologies that provide an overview of the metabolome and high-power compound elucidation. Over and above large-scale analysis, a major effort is needed for unequivocal identification of metabolites. The combination of liquid chromatography (LC)-MS and NMR is a powerful methodology for identifying metabolites. Better chemical characterization of the metabolome will undoubtedly enlarge knowledge of any biological system.
Untargeted large-scale plant metabolomics using liquid chromatography coupled to mass spectrometry
Vos, C.H. de; Moco, S.I.A. ; Lommen, A. ; Keurentjes, J.J.B. ; Bino, R.J. ; Hall, R.D. - \ 2007
Nature protocols 2 (2007)4. - ISSN 1754-2189 - p. 778 - 791.
functional genomics - h-1-nmr spectroscopy - metabolites - tomato - identification - arabidopsis - ms - systems - combination - alignment
Untargeted metabolomics aims to gather information on as many metabolites as possible in biological systems by taking into account all information present in the data sets. Here we describe a detailed protocol for large-scale untargeted metabolomics of plant tissues, based on reversed phase liquid chromatography coupled to high-resolution mass spectrometry (LC-QTOF MS) of aqueous methanol extracts. Dedicated software, MetAlign, is used for automated baseline correction and alignment of all extracted mass peaks across all samples, producing detailed information on the relative abundance of thousands of mass signals representing hundreds of metabolites. Subsequent statistics and bioinformatics tools can be used to provide a detailed view on the differences and similarities between (groups of) samples or to link metabolomics data to other systems biology information, genetic markers and/or specific quality parameters. The complete procedure from metabolite extraction to assembly of a data matrix with aligned mass signal intensities takes about 6 days for 50 samples
Linking aboveground and belowground interactions via induced plant defenses
Bezemer, T.M. ; Dam, N.M. van - \ 2005
Trends in Ecology and Evolution 20 (2005)11. - ISSN 0169-5347 - p. 617 - 624.
arbuscular mycorrhizal fungi - herbivore-induced volatiles - root herbivory - entomopathogenic nematodes - h-1-nmr spectroscopy - arabidopsis-thaliana - induced resistance - chemical defenses - natural enemies - gene-expression
Plants have a variety of chemical defenses that often increase in concentration following attack by herbivores. Such induced plant responses can occur aboveground, in the leaves, and also belowground in the roots. We show here that belowground organisms can also induce defense responses aboveground and vice versa. Indirect defenses are particularly sensitive to interference by induced feeding activities in the other compartment, and this can disrupt multitrophic interactions. Unravelling the involvement of induced plant responses in the interactions between aboveground and belowground communities associated with plants is likely to benefit from comprehensive metabolomic analyses. Such analyses are likely to contribute to a better understanding of the costs and benefits involved in the selection for induced responses in plants
Identification of structural features of various (O-Acetylated) Xylo-Oligosaccharides from Xylan-Rich agricultural by-products: A review
Kabel, M.A. ; Schols, H.A. ; Voragen, A.G.J. - \ 2004
ACS symposium series 864 (2004). - ISSN 0097-6156 - p. 108 - 121.
anion-exchange chromatography - flight mass-spectrometry - wheat-flour arabinoxylan - aspergillus-awamori - h-1-nmr spectroscopy - side-chains - cell-walls - wood - nmr - glucuronoarabinoxylans
Hydrolysates obtained by hydrothermal treatment of four xylan rich by-products (wheat bran, brewery's spent grain, corn cobs and Eucalyptus wood) were characterised. Depending on the feedstock material studied, a wide variety of differently substituted xylo-oligosaccharides (XOS) and xylan-fragments were obtained. The structural features of the this way obtained arabinose, 4-O-methylglucuronic acid and O-acetyl substituted XOS are reviewed. High performance anion-exchange chromatography (HPAEC),reversed phase (RP)-high performance liquid chromatography (HPLC), mass spectrometry (MS), NMR spectroscopy, RP-HPLC-MS and RP-HPLC-NMR showed to be very useful for the separation and characterisation of the detailed structures of the substituted XOS
Location of O-acetyl substituents in xylo-oligosaccharides obtained from hydrothermally treated Eucalyptus wood
Kabel, M.A. ; Waard, P. de; Schols, H.A. ; Voragen, A.G.J. - \ 2003
Carbohydrate Research : an international journal 338 (2003)1. - ISSN 0008-6215 - p. 69 - 77.
anion-exchange chromatography - mimosa-scabrella bracatinga - acidic d-xylan - mass-spectrometry - liquid-chromatography - h-1-nmr spectroscopy - nmr - arabinoxylan - separation - wheat
A combination of techniques was used to localise the O-acetyl substituents in xylo-oligosaccharides, which are present in hydrolysates of hydrothermally treated Eucalyptus wood. Reversed-phase (RP)-high performance liquid chromatography (HPLC) coupled on-line to both a mass spectrometer and an evaporating light scattering (ELS) detector provided data about the order of elution of the various O-acetylated oligomers. The retention of the oligomers on the column depended on the number and position of the O-acetyl substituents within the xylo-oligosaccharides. One dimensional (1D)- and two dimensional (2D)-1H NMR spectroscopy was used to study the structural features of several xylotetramers separated by RP-HPLC, each having one O-acetyl substituent. O-Acetyl migration was proven to have occurred in these xylo-oligosaccharides. Mainly O-acetyl migration within the same xylosyl residue was observed. RP-HPLC–NMR was performed in order to study the structural features of the acetylated oligomers ‘on-line’ avoiding O-acetyl migration. Finally, the precise location of the 2-O- or 3-O-acetyl substituent in 6 xylotetramers and 4 xylotrimers separated by RP-HPLC was determined.