Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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    From Beet Pulp To Building Blocks and Polymers Developing Value Added Materials From GalX
    Leeuwen, M.B. van; Gootjes, L. ; Vogelzang, W. ; Knoop, J.R.I. ; Haveren, J. van; Es, D.S. van - \ 2016
    biobased economy - materialen uit biologische grondstoffen - reststromen - agrarische afvalstoffen - biopolymeren - galacturonzuur - bietenpulp - bioplastics - biobased economy - biobased materials - residual streams - agricultural wastes - biopolymers - galacturonic acid - beet pulp - bioplastics
    Poster of the Carbohydrate Competence Center. The increasing demand for 2nd generation bio-based performance materials presents an excellent opportunity for the development of agricultural residues like sugar beet pulp (SBP) as feedstock for renewable polymers. Refining SBP yields various industrially interesting components, such as galacturonic acid. This sugar acid can be transformed into a novel family of bio-based building blocks called GalX. Here we report on the use of GalX building blocks in bio-based polymers.
    Identification of novel isomeric pectic oligosaccharides using hydrophilic interaction chromatography coupled to traveling-wave ion mobility mass spectrometry
    Leijdekkers, A.G.M. ; Huang, J.H. ; Bakx, E.J. ; Gruppen, H. ; Schols, H.A. - \ 2015
    Carbohydrate Research : an international journal 404 (2015). - ISSN 0008-6215 - p. 1 - 8.
    interaction liquid-chromatography - fluorescent labels - galacturonic acid - cell walls - separation
    Separation and characterization of complex mixtures of pectic oligosaccharides still remains challenging and often requires the use of multiple analytical techniques, especially when isomeric structures are present. In this work, it is demonstrated that the coupling of hydrophilic interaction chromatography (HILIC) to traveling-wave ion mobility mass spectrometry (TWIMMS) enabled the simultaneous separation and characterization of complex mixtures of various isomeric pectic oligosaccharides. Labeling of oligosaccharides with 3-aminoquinoline (3-AQ) improved MS-ionization efficiency of the oligosaccharides and reduced the complexity of the product ion mass spectra, without losing resolution of the HILIC separation. In addition, labeling enabled quantification of oligosaccharides on molar basis using in-line fluorescence detection. Isomeric structures were distinguished using TWIMMS. The 3-AQ-HILIC–TWIMMS method was used to characterize a series of isomeric sugar beet rhamnogalacturonan I derived oligosaccharides carrying a glucuronic acid substituent. Thereby, some novel structural features were identified for the first time: glucuronic acid was attached to O-3 or to O-2 of galacturonic acid residues and a single galacturonic acid residue within an oligomer could contain both an acetyl group and a glucuronic acid substituent.
    Pectin degradation by Botrytis cinerea: recognition of endopolygalacturonases by an Arabidopsis receptor and utilization of Dgalacturonic acid
    Lisha Zhang, Lisha - \ 2013
    Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Jan van Kan. - S.l. : s.n. - ISBN 9789461735409 - 188
    botrytis cinerea - plantenziekteverwekkende schimmels - pectinen - degradatie - celwanden - arabidopsis - receptoren - polygalacturonase - galacturonzuur - botrytis cinerea - plant pathogenic fungi - pectins - degradation - cell walls - arabidopsis - receptors - polygalacturonase - galacturonic acid

    The necrotrophic fungal plant pathogenBotrytis cinerea is able to infect over 200 host plants and cause severe damage to crops, both pre- and post-harvest. B. cinerea often penetrates host leaf tissue at the anticlinal cell wall and subsequently grows into and through the middle lamella, which consists mostly of low-methylesterified pectin. Effective pectin degradation thus is important for virulence of B. cinerea. Chapter 1 describes the chemical structures of plant cell wall polysaccharides, the cell wall-associated mechanisms that confer resistance against pathogens, and the microbial enzymes involved in cell wall decomposition. It then discusses the plant cell wall degrading enzymes of pathogenic fungi and illustrates with case studies the process of pectin decomposition by B. cinerea.

    Chapter 2describes the molecular identification and functional characterization of a novel MAMP receptor RBPG1, a Leucine-Rich Repeat Receptor-Like Protein (LRR-RLP), that recognizes fungal endo-polygalacturonases (endo-PGs), in particular the B. cinerea protein BcPG3. Infiltration of the BcPG3 protein into Arabidopsis thaliana accession Col-0 induced a necrotic response. Heat-inactivated protein and a catalytically inactive mutant protein retained the ability to induce necrosis. An 11-amino acid peptide stretch was identified that is conserved among many fungal but not plant endo-PGs. A synthetic peptide comprising this sequence was unable to induce necrosis. A map-based cloning strategy, combined with comparative and functional genomics, led to the identification of the RBPG1 gene, which is required for responsiveness of A. thaliana to the BcPG3 protein. Co-immunoprecipitation experiments demonstrated that RBPG1 and BcPG3 form a complex inNicotiana benthamiana, which also involves the A. thaliana LRR-RLK SOBIR1. The sobir1 mutant plants no longer respond to BcPG3. Furthermore, overexpression of RBPG1 in the BcPG3-non-responsive accession Br-0 did not enhance resistance to a number of microbial pathogens.

    Chapter 3describes the functional, biochemical and genetic characterization of the D-galacturonic acid catabolic pathway in B. cinerea. The B. cinerea genome contains two non-homologous galacturonate reductase genes (Bcgar1 and Bcgar2), a galactonate dehydratase gene (Bclgd1), and a 2-keto-3-deoxy-L-galactonate aldolase gene (Bclga1). Targeted gene replacement of all four genes in B. cinerea, either separately or in combinations, yielded mutants that were affected in growth on D-galacturonic acid, pectate, or pectin as the sole carbon source. The extent of growth reduction of the mutants on pectic substrates was positively correlated to the proportion of D-galacturonic acid present in the pectic substrate. The virulence of these mutants on different host plants is discussed in Chapter 4. These mutants showed reduced virulence on N. benthamiana and A. thaliana leaves, but not on tomato leaves. The cell walls of N. benthamiana and A. thaliana leaves have a higher D-galacturonic acid content as compared to tomato. Additional in vitro growth assays with the knockout mutants suggested that the reduced virulence of D-galacturonic acid catabolism-deficient mutants on N. benthamiana and A. thaliana is not only due to the inability of the mutants to utilize an abundant carbon source as nutrient, but also due to the growth inhibition by catabolic intermediates.

    In Chapter 5, the functional characterization of two putative D-galacturonic acid transporters is presented. Bchxt15 is highly and specifically induced by D-galacturonic acid, whereas Bchxt13 is highly expressed in the presence of all carbon sources tested except for glucose. Subcellular localization of BcHXT13-GFP and BcHXT15-GFP fusion proteins expressed under their native promoter suggests that the fusion proteins are localized in plasma membranes and intracellular vesicles. Knockout mutants in the Bchxt13 and Bchxt15 genes, respectively, were neither affected in their growth on D-galacturonic acid as the sole carbon source, nor in their virulence on tomato and N. benthamiana leaves.

    Chapter 6describes the genome-wide transcriptome analysis in B. cinerea grown in media containing glucose and pectate as sole carbon sources. Genes were identified that are significantly altered in their expression during growth on these two carbon sources. Conserved sequence motifs were identified in the promoters of genes involved in pectate decomposition and D-galacturonic acid utilization. The role of these motifs in regulating D-galacturonic acid-induced expression was functionally analysed in thepromoter of the Bclga1 gene, which encodes one of the key enzymes in the D-galacturonic acid catabolic pathway. The regulation by D-galacturonic acid required the presence of sequences encompassing the GAE1 motif and a binding motif for the pH-dependent transcriptional regulator PacC.

    Chapter 7 provides a general discussion of the results presented in this thesis. A model of the concerted action of pectin degradation and subsequent monosaccharide consumption and co-regulation of gene expression is proposed.

    Okra pectin contains an unusual substitution of its rhamnosyl residues with acetyl and alpha-linked galactosyl groups
    Sengkhamparn, N. ; Bakx, E.J. ; Verhoef, R.P. ; Schols, H.A. ; Sajjaanantakul, T. ; Voragen, A.G.J. - \ 2009
    Carbohydrate Research : an international journal 344 (2009)14. - ISSN 0008-6215 - p. 1842 - 1851.
    hairy ramified regions - fat ingredient substitute - rhamnogalacturonan oligomers - structural-characterization - galacturonic acid - oligosaccharides - polysaccharides - degradation - substances - products
    The okra plant, Abelmoschus esculentus (L.) Moench, a native plant from Africa, is now cultivated in many other areas such as Asia, Africa, Middle East, and the southern states of the USA. Okra pods are used as vegetables and as traditional medicines. Sequential extraction showed that the Hot Buffer Soluble Solids (HBSS) extract of okra consists of highly branched rhamnogalacturonan (RG) I containing high levels of acetyl groups and short galactose side chains. In contrast, the CHelating agent Soluble Solids (CHSS) extract contained pectin with less RG I regions and slightly longer galactose side chains. Both pectic populations were incubated with homogeneous and well characterized rhamnogalacturonan hydrolase (RGH), endo-polygalacturonase (PG), and endo-galactanase (endo-Gal), monitoring both high and low molecular weight fragments. RGH is able to degrade saponified HBSS and, to some extent, also non-saponified HBSS, while PG and endo-Gal are hardly able to degrade either HBSS or saponified HBSS. In contrast, PG is successful in degrading CHSS, while RGH and endo-Gal are hardly able to degrade the CHSS structure. These results point to a much higher homogalacturonan (HG) ratio for CHSS when compared to HBSS. In addition, the CHSS contained slightly longer galactan side chains within its RG I region than HBSS. Matrix-assisted laser desorption ionization-time of flight mass spectrometry indicated the presence of acetylated RG oligomers in the HBSS and CHSS enzyme digests and electron spray ionization-ion trap-mass spectrum showed that not only galacturonosyl residues but also rhamnosyl residues in RG I oligomers were O-acetylated. NMR spectroscopy showed that all rhamnose residues in a 20 kDa HBSS population were O-acetylated at position O-3. Surprisingly, the NMR data also showed that terminal a-linked galactosyl groups were present as neutral side chain substituents. Taken together, these results demonstrate that okra contained RG I structures which have not been reported before for pectic RG I.
    Fingerprinting complex pectins by chromatographic separation combined with ELISA detection
    Verhoef, R.P. ; Lu, Y. ; Knox, J.P. ; Voragen, A.G.J. ; Schols, H.A. - \ 2009
    Carbohydrate Research : an international journal 344 (2009)14. - ISSN 0008-6215 - p. 1808 - 1817.
    polysaccharide rhamnogalacturonan-ii - enzymatically-tailored pectins - hairy ramified regions - cell-walls - plant-cell - arabinogalactan-proteins - extracellular polysaccharides - monoclonal-antibodies - structural features - galacturonic acid
    Enzyme-resistant pectin or modified hairy regions were subjected to size exclusion (HPSEC) and weak anion exchange (WAX) chromatography. Fractions collected after separation were tested for the presence of different pectic epitopes using the monoclonal antibodies LM2, LM5, LM6, and JIM7. Separation by HPSEC showed that based on molecular weight the different epitopes were restricted to distinct molecular weight populations. WAX chromatography resulted in an even better separation of the different pectic epitopes present. A clear separation between arabino galactan type II epitopes and the RG I side chains, (1,5)-a-l-arabinan and (1,4)-ß-d-galactan, could be established. Arabinogalactan type II was found in the first populations eluting off the WAX column. The observations made within the ELISA assays of the collected fractions could be confirmed by determination of the sugar composition of the individual populations obtained. The sugar composition of the AGII positive populations eluting off the WAX column shows the presence of significant amounts of rhamnose and galacturonic acid. Together with the delay on an anion exchanger, this observation indicates a possible linkage between RGI and AGII. The volume of the individual fractions collected provides enough material for a maximum of 20 different antibodies to be tested from one analytical separation.
    CE-MSn of complex pectin-derived oligomers
    Coenen, G.J. ; Kabel, M.A. ; Schols, H.A. ; Voragen, A.G.J. - \ 2008
    Electrophoresis 29 (2008)10. - ISSN 0173-0835 - p. 2101 - 2111.
    anion-exchange chromatography - capillary zone electrophoresis - trap mass-spectrometry - hairy ramified regions - maldi-tof ms - rhamnogalacturonan-i - 8-aminonaphthalene-1,3,6-trisulfonic acid - galacturonic acid - apple pectin - oligosaccharides
    As pectin molecules are too large and heterogeneous to analyze as a whole, the polymer is usually degraded to smaller oligomers, which are often analyzed by high-performance anion exchange chromatography (HPAEC). However, the high salt concentration necessary to elute pectin oligomers by HPAEC is incompatible with online mass detection. To overcome such a disadvantage, a CE-IT-MS system was set up to further elucidate the fine structure of charged oligosaccharides. An effective separation of differently substituted galacturonic acid containing oligomers was obtained by low-pH CE-LIF analysis. By adapting the buffer and capillary online MS detection was enabled. Moreover, with MS/MS it was possible to localize sugar residues' substitutions. With this combined CE-MS approach LIF electropherograms of xylogalacturonan and rhamnogalacturonan I digests could be annotated. The method was further exemplified by a complex oligomer mixture of acid hydrolyzed apple pectin, which was separated and characterized by CE-MSn. Oligomers present in low amounts could be localized by their corresponding m/z, as was demonstrated by selected mass range representation.
    Assessment of the pectinolytic network of Aspergillus niger by functional genomics : insights from the transcriptome
    Martens-Uzunova, E.S. - \ 2008
    Wageningen University. Promotor(en): Willem de Vos; C.A.M.J.J. van den Hondel, co-promotor(en): Peter Schaap. - Wageningen : Wageningen University - ISBN 9789085048879 - 208
    aspergillus niger - polygalacturonase - galacturonzuur - metabolisme - biochemische omzettingen - functionele genomica - transcriptomics - aspergillus niger - polygalacturonase - galacturonic acid - metabolism - biochemical pathways - functional genomics - transcriptomics
    More than a century ago, in 1889, A. Fernbach presented a detailed report about the invertase of Aspergillus niger in the third edition of “Annales De L'institut Pasteur”. Since then, many of the enzymes secreted by A. niger have found a broad range of applications, and today they are produced on an industrial scale. This filamentous fungus is also used as a primary source for the production of organic acids and other economically important metabolites. Although, many of these fermentation processes are well established, the underlying genetics are still not well understood. The recent determination of the genome sequence of A. niger illustrated the versatile metabolic capacities of this fungus and created the opportunity for challenging research aiming to unravel the fine tuned metabolic network and the full enzymatic potential of A. niger.
    The work described in this thesis is focused on the genome mining and transcriptional profiling of the enzymatic network of A. niger involved in pectin degradation and utilization. Pectin was chosen because of the broad application of it and its derivates in food, feed, medicines, and cosmetics and the economical importance of this polymer in several technological processes. This study addresses several issues concerning pectin degradation by A. niger:
    i) the complete inventory of the known and potential pectinolytic activities encoded in the genome of this fungus; ii) the understanding of dynamics of their gene-expression on (complex) carbon sources in order to unravel underling metabolic networks; iii) the evolutionary aspects of the pectinolytic system of A. niger and other filamentous fungi.
    Chapter 1 of this thesis discusses the importance of fungi and A. niger for the biotech industry, and gives a brief introduction to the structural elements of pectin, the types of enzymatic activities involved in its degradation, and the present knowledge of the metabolism of its major constituent – galacturonic acid. The current advances in fungal ‘omics research are pointed out. Chapter 2 presents the construction of pectin specific cDNA libraries from A. niger, and the annotation of more than 200 of the obtained expressed sequence tags. Chapter 3 focuses on the roles of and the interactions between the 21 genes within glycoside hydrolase family 28 - the largest group of A. niger pectinases. A special emphasis is given to a new, previously unanticipated, group of exo-acting enzymes. Chapter 4 describes the survey for all genes encoding pectinolytic activities in A. niger and their transcriptional profiles. In this chapter, a hypothetical model of the pectinolytic network of A. niger is proposed. Chapter 5 focuses on the identification of the genes involved in galacturonic acid catabolism in A. niger, the verification of some of the enzymatic activities encoded by them, and elaborates on the presence of an evolutionary conserved galacturonic acid pathway operating only in filamentous fungi capable of plant cell wall degradation. In Chapter 6, the evolutionary conservation of transcriptional response of the pectinolytic system of A. niger is compared to that of the model organism A. nidulans. This chapter further describes the identification of the evolutionary conserved, regulatory core of functional orthologs involved in galacturonic acid utilization and metabolism. In Chapter 7, the results of the work described in this thesis are summarized and discussed.
    Elucidation of the chemical fine structure of polysaccharides from soybean and maize kernel cell walls
    Huisman, M.M.H. - \ 2000
    Agricultural University. Promotor(en): A.G.J. Voragen; H.A. Schols. - S.l. : S.n. - ISBN 9789058081872 - 159
    celwanden - polysacchariden - pectinen - galactanen - galacturonzuur - zea mays - glycine max - cell walls - polysaccharides - pectins - galactans - galacturonic acid - zea mays - glycine max

    The subject of this thesis was the elucidation of the chemical fine structure of polysaccharides from cell walls of soybean and maize kernel. The two species investigated represent different taxonomic groups, soybean belonging to the dicotyledonous and maize to the monocotyledonous plants. Besides representing the most important structures present in cell wall material, these raw materials are of great importance in food and feed industry.

    The characterisation of the soybean cell wall polysaccharides started with the isolation of the cell wall material as Water-Unextractable Solids (WUS) from soybean meal (chapter 2). The isolation procedure yielded a WUS fraction containing almost all polysaccharides present in the meal and only few other components. WUS was sequentially extracted with chelating agent (Chelating agent Soluble Solids, ChSS), dilute alkali (Dilute Alkali Soluble Solids, DASS), 1 m alkali (1 m Alkali Soluble Solids, 1 MASS) and 4 m alkali (4 m Alkali Soluble Solids, 4 MASS) to leave a cellulose-rich residue (RES). The pectin-rich extracts (ChSS and DASS) were found to have identical sugar compositions and contained predominantly galactose, arabinose, and uronic acid residues. The 1 MASS fraction contained xylose in addition to the former three sugars. The hemicellulose-rich fraction (4 MASS) contained mainly xylose and glucose. No indications were found that ChSS and DASS were structurally different, although it is obvious that their arrangement in the cell wall was not identical.

    The intact cell wall polysaccharides in the meal and WUS were hardly degradable by enzymes. Once extracted, the polysaccharides from WUS were degraded more easily (chapter 3). The arabinogalactan side chains in the pectin-rich ChSS fraction could to a large extent be removed by the combined action of endo-galactanase, exo-galactanase, endo-arabinanase, and arabinofuranosidase B. The remaining polymer (fraction P) was isolated and represented 30% of the polysaccharides in the ChSS fraction (12% of the polysaccharides in the WUS). This polymer still contained some remaining arabinose and galactose residues, which could not be removed by the enzyme mixture used.

    The pectic backbone (fraction P) appeared to be resistant to enzymatic degradation by both established (like polygalacturonase) and novel pectic enzymes (like RG-hydrolase). After partial acid hydrolysis of the isolated pectic backbone, one oligomeric and two polymeric populations were obtained by size-exclusion chromatography. Monosaccharide and linkage analyses, enzymatic degradation, and NMR spectroscopy of these two polymeric populations showed that the pectic substances in the original extract (ChSS) contained both rhamnogalacturonan and xylogalacturonan regions, while homogalacturonan was absent (chapter 4). The absence of homogalacturonan distinguishes the pectic substances from soybean from pectic polysaccharides extracted from other sources, which contain homogalacturonan and rhamnogalacturonan regions and can be degraded with polygalacturonase and RG-hydrolase, respectively. Acid hydrolysis of fraction P improves the susceptibility of the remaining polymers for RG hydrolase and exo-galacturonase.

    The xylogalacturonan present in the ChSS fraction distinguishes itself from xylogalacturonan from other sources known so far. A part of the xylose residues in the xylogalacturonan is substituted with fucose and the xylogalacturonan is resistant to degradation with XGH.

    The arabinogalactan side chains, which were removed from the ChSS fraction to obtain fraction P, were the subjects of investigation in chapter 5. Fractionation, monosaccharide and linkage analyses, enzymatic degradation, and mass spectrometry of the oligosaccharides in the digest of ChSS after enzymatic digestion with arabinogalactan degrading enzymes indicated the presence of common linear (1,4)-linked galacto-oligosaccharides, and both linear and branched arabino-oligosaccharides. In addition, the results unambiguously showed the presence of oligosaccharides containing (1,4)-linked galactose residues bearing an arabino pyranose residue at the non-reducing terminus, and a mixture of linear oligosaccharides constructed of (1,4)-linked galactose residues interspersed with one internal (1,5)-linked arabinofuranose residue. The presence of an internal arabinofuranose residue in a pectic arabinogalactan chain in cell wall polysacchairdes has not been reported previously, not in soybean, nor in other fruit or vegetable cell walls. Another uncommon feature is the presence of arabinopyranose residues in pectic arabinogalactan.

    The pectic substances form only one network of the plant cell wall, the other is the cellulose/hemicellulose network. The hemicelluloses were solubilised from the residue with 1 and 4 m KOH solutions (chapter 6). The polysaccharides extracted with 1 m KOH were fractionated by ion-exchange chromatography, yielding a neutral and a pectic population. The sugar composition of the neutral population indicated the presence of xyloglucans and possibly xylans. Enzymatic degradation with endo-xylanases and endo-glucanases showed the presence of xyloglucan fragments only. Analysis of the digest formed after incubation of the neutral population with endo-glucanase V showed the formation of the characteristic poly-XXXG xyloglucan oligomers (XXG, XXXG, XXFG, XLXG, and XLFG), so three out of four glucose residues carry a side chain.

    In chapter 7, the structural features of glucuronoarabinoxylans from maize kernels are described. First of all, maize kernel cell wall material was isolated as Water-Unextractable Solids (WUS). As expected the non-starch polysaccharides (NSP) had concentrated in the WUS (57%). These NSP were composed mainly of glucose, xylose, arabinose, and glucuronic acid. Sequential extractions with a saturated Ba(OH) 2 -solution (BE1 extract), and distilled water (BE2 extract) were used to solubilise glucuronoarabinoxylans from maize WUS. The glycosidic linkage composition of the extracts and their resistance to endo-xylanase treatment indicated that the extracted glucuronoarabinoxylans were highly substituted. In the maize BE1 extract 25% of the xylose was unsubstituted, 38% was monosubstituted and 15% was disubstituted. The glucuronoarabinoxylans in maize BE1 appeared to be resistant to endo-xylanase treatment, but could be degraded by a sub-fraction of Ultraflo, a commercial enzyme preparation from Humicola insolens . The digest contained a number of series of oligomers: pentose n , pentose n GlcA, pentose n hexose, and pentose n GlcA 2 . The presence of these glucuronic acid-containing series of oligomers showed that the glucuronic acids in the glucuronoarabinoxylancan can be very close to each other, but are not distributed blockwise. Finally, a new measure for the degree of substitution of glucuronoarabinoxylans was defined. It turned out that the degree of substitution in maize BE1 is much higher (87%) than in sorghum (70%) and wheat flour BE1 (56%). This indicates that the glucuronoarabinoxylans in maize BE1 are more complex than those in sorghum BE1 and explains their resistance to endo-xylanase treatment.

    From this research, it can be concluded that both soybean and maize kernel cell wall polysaccharides distinguish themselves in a number of respects from other plant cell walls polysaccharides. The absence of homogalacturonan, but also the presence of internal (1,5)-linked arabinofuranose and terminal arabinopyranose in the pectic arabinogalactan side chains from soybean cell walls and the complexity of the glucuronoarabinoxylan from maize kernel cell walls are discussed in chapter 8. In addition, it was shown that techniques like mass spectrometry and NMR spectroscopy are powerfull techniques to be used after (enzymatic) fragmentation, for chemical characterisation of the original polysaccharides.

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