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.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Bioactive components of fermented soya beans effective against diarrhoea-associated bacteria
    Roubos-van den Hil, P.J. - \ 2010
    Wageningen University. Promotor(en): Marcel Zwietering; Harry Gruppen, co-promotor(en): Rob Nout. - [S.l. : S.n. - ISBN 9789085857136 - 152
    tempé - sojabonen - diarree - enzymactiviteit - arabinose - fermentatie - bioactieve verbindingen - tempeh - soyabeans - diarrhoea - enzyme activity - arabinose - fermentation - bioactive compounds
    Tempe is a fermented food, obtained by fungal fermentation of soya beans. During the processing specific flavour, texture and nutritional properties are achieved. Previous research has indicated that tempe reduced the incidence and severity of diarrhoea. In this thesis the bioactive effects of tempe on diarrhoea-associated bacteria are described.
    Tempe appeared to be antibacterial against Bacillus cereus cells and spores, but not against enterotoxigenic Escherichia coli (ETEC). Nevertheless, tempe was found to inhibit the adhesion of ETEC to intestinal epithelial cells.
    Experiments showed that the inhibition of adhesion was caused by an interaction between ETEC and tempe extracts. A range of ETEC strains was shown to be sensitive for the anti-adhesive component, making the bioactivity of broader interest for applications in feed matrices.
    Furthermore, several substrates (legumes and cereals) were fermented and all fermented legumes tested were equally bioactive as the fermented soya beans, whereas the fermented cereals were not active. The use of different starter cultures showed that Bacillus spp., moulds and some yeasts were capable to release or form the bioactive component during fermentation, thus the bioactivity was not specific for one microbial species.
    After heating, defatting and protease treatment of the bioactive tempe extracts, they remained bioactive. On the contrary, after treatment with polysaccharide degrading enzyme mixtures the bioactivity was lost. This suggests that the bioactive component contains carbohydrates, and explains the interaction between ETEC and tempe extracts, which could indeed be established by carbohydrates of the tempe extract. Ultra-filtration revealed the bioactive component to have molecular masses >30 kDa. Further purification yielded an active fraction with an increased carbohydrate content. Monosaccharide analysis showed the importance of arabinose in the bioactive components.
    In conclusion, the bioactive component is released or formed during fermentation by enzymatic degradation of legumes. The bioactive component is of carbohydrate nature and contains arabinose, which originates from arabinan or arabinogalactan chains of the pectic cell wall polysaccharides of legumes.
    Integrated molecular analysis of sugar metabolism of Sulfolobus solfataricus
    Brouns, S.J.J. - \ 2007
    Wageningen University. Promotor(en): Willem de Vos; John van der Oost. - [S.l.] : s.n. - ISBN 9789085047131 - 166
    arabinose - oxidoreductasen - kristaleiwitten - thermofiele bacteriën - arabinose - oxidoreductases - crystal proteins - thermophilic bacteria - cum laude
    cum laude graduation (with distinction)
    Regulation and control of L-arabinose catabolism in Aspergillus niger
    Groot, M.J.L. de - \ 2005
    Wageningen University. Promotor(en): A.J.J. van Ooyen, co-promotor(en): G.J.G. Ruijter. - [S.l.] : S.n. - ISBN 9789085042518 - 131
    arabinose - katabolisme - aspergillus niger - genexpressieanalyse - eiwitexpressieanalyse - arabinose - catabolism - aspergillus niger - genomics - proteomics
    This thesis describes studies on the biochemical properties and regulation of L-arabinose metabolism and arabinan degrading enzymes of Aspergillus niger. We focused on the investigation of the catabolic pathway, firstly by isolating pathway specific regulatory mutants using a newly developed selection system and, secondly, by purifying the enzymes and characterising their kinetics for use in metabolic control analysis. Finally, by cloning genes encoding these enzymes we were able to analyse expression of these genes.

    Using a D-xylulose kinase deficient strain we developed a mutant selection system that identified genes involved in pentose catabolism and their regulation. The A.niger strain carrying the xkiA1 mutation lacks D-xylulose kinase and cannot grow on pentoses, such as D-xylose and L-arabinose, but these sugars still repress the use of other carbon sources such as D-gluconate. We used this genetic background to select for pentose-derepressed mutants on media containing combinations of gluconate with xylitol, L-arabinose or D-xylose. A subset of these mutants was further analysed and turned out to have rather interesting properties as described in chapters two and three.

    One of the mutants isolated using this method carried a mutation called xtlA36 and is described in chapter two. This mutation results in a severe decrease in xylitol consumption, suggesting that xtlA36 inactivates a xylitol transporter and opens the way of isolation of genes encoding the corresponding genes. Two other A.niger mutants, carrying araA and araB are specifically disturbed in the regulation of the arabinanase system in the presence of L-arabinose. Expression of three arabinanolytic genes, abfA, abfB and abnA , is substantially decreased or absent in the araA and araB , strains compared to the wild-type when incubated in the presence of L-arabinose or L-arabitol. In addition, the intracellular enzyme activities of L-arabitol dehydrogenase and L-arabinose reductase, involved in L-arabinose catabolism, were decreased in the araA and araB strains.

    L-arabitol, most likely the true inducer of the arabinanolytic and L-arabinose catabolic genes, accumulates to a high intracellular concentration in the araA and araB mutants. This indicates that the decreased expression of the arabinolytic genes is not due to lack of inducer accumulation. Therefore, we propose that araA and araB are mutations in positively acting components of the regulatory system involved in the expression of the arabinanase encoding genes and the genes encoding the L-arabinose catabolic pathway.

    Chapter four describes cloning of the A.niger D-xylulose kinase encoding gene ( xkiA ) by direct complementation of the strain deficient in D-xylulose kinase activity. This enabled us to investigate the expression of xkiA in the presence of L-arabinose, L-arabitol, and D-xylose. Although XKI is part of the D-xylose catabolic pathway, expression of xkiA appeared not to be mediated by XLNR, the xylose-dependent positively acting xylanolytic regulator. Expression of xkiA is subject to carbon catabolite repression (ccr) but the wide domain regulator CREA is not directly involved. Using the araA and araB strains described in chapter three, we showed that xkiA is under control of the arabinanolytic regulatory system.

    Overexpression of xkiA enabled us to purify the encoded D-xylulose kinase enzyme. The molecular mass, determined using Electrospray Ionization Mass Spectrometry (ESI-MS) concurred with the calculated molecular mass of 62816.6 Da. The activity of D-xylulose kinase is highly specific for D-xylulose. Kinetic parameters were determined, including Km (D-xylulose), 0.76 mM and Km (ATP), 0.061 mM.

    In a D-xylulose kinase deficient strain a higher accumulation of intracellular arabitol and xylitol correlated to increased transcript levels of the genes encoding arabinan and xylan degrading enzymes, respectively. This supports the suggestion that L-arabitol may be the specific low molecular weight inducer of the genes involved in arabinan degradation. It also suggests a possible role for xylitol in the induction of xylanolytic genes. Overproduction of XKIA did not reduce the size of the intracellular arabitol and xylitol pools, and had no effect on expression of genes encoding xylan and arabinan degrading enzymes. This suggests that the enzymes preceding D-xylulose kinase in the L-arabinose/D-xylose catabolic pathway probably have more control on the flux through this pathway than D-xylulose kinase itself.

    In chapter five, we cloned the genes encodingA.nigerL-arabitol dehydrogenase ( ladA ) and xylitol dehydrogenase ( xdhA ), and produced the enzymes in Escherichia coli . Analysis of the substrate specificity showed that LADA is most active on L-arabitol and also has significant activity on xylitol, but only low activity on D-sorbitol and galactitol. XDHA has the highest activity on xylitol, significant activity on D-sorbitol, but very low activity on L-arabitol. The higher activity on sorbitol for XDHA is in agreement with the amino acid similarity of the different enzyme classes, since a phylogenetic tree of L-arabitol dehydrogenases, xylitol dehydrogenases and sorbitol dehydrogenases (SDH) suggests that xylitol dehydrogenases are more similar to sorbitol dehydrogenases than L-arabitol dehydrogenases. Expression analysis of the pentose catabolic pathway genes confirmed the model in which an arabinose specific regulator activates the expression of all genes required for the conversion of L-arabinose to D-xylulose-5-phosphate. In addition, XLNR regulates the first step and, to a lesser extent, the other steps of the conversion of D-xylose into D-xylulose-5-phosphate.

    Using dye-affinity chromatography we isolated enzymes of the L-arabinose and D-xylose catabolic pathways that had not been described previously. Using the complete set of kinetic parameters a metabolic model was constructed which we used to perform steady state metabolic control analysis (chapter six). The metabolic model was used to analyse flux and metabolite concentration control of the L-arabinose catabolic pathway. The model predicts that flux control does not only reside at the enzyme following the intermediate with the highest concentration, L-arabitol, but is distributed over the first three steps in the pathway, preceding and following L-arabitol. Flux control appeared to be strongly dependent on the intracellular L-arabinose concentration. At 5 mM intracellular L-arabinose, a level that resulted in realistic intermediate concentrations in the model, flux control coefficients for L-arabinose reductase, L-arabitol dehydrogenase and L-xylulose reductase were 0.68, 0.17 and 0.14 respectively. This analysis can be used as a guide to identify targets for metabolic engineering aiming at either flux or metabolite level optimisation of the L-arabinose catabolic pathway of A.niger .

    In chapter seven the results from chapters two through six are discussed in light of possible engineering applications and recent scientific developments such as genomics.
    Effect of roasting on the carbohydrate composition of Coffea arabica beans.
    Oosterveld, A. ; Voragen, A.G.J. ; Schols, H.A. - \ 2003
    Carbohydrate Polymers 54 (2003)2. - ISSN 0144-8617 - p. 183 - 192.
    rich pectic polysaccharides - sugar-beet pulp - chemical characterization - hot-water - arabinose - green
    Coffee beans (arabica) with different degrees of roast were sequentially extracted with water (90 °C, 1 h), water (170 °C, 30 min), and 0.05 M NaOH (0 °C, 1 h). The amount and composition of polysaccharides, oligosaccharides and monosaccharides in the extracts and residues were analyzed. The results were compared with the composition of the same batch of green arabica coffee beans. Although part of our results were already reported in rather fragmented studies, this study gives a more complete overview of the amount and composition of unextractable polymers, extractable polymers, oligomers, monomers, and their conversion into (non-sugar) degradation products as a function of their degree of roast. It was found that most carbohydrates in the roasted coffee bean were present as polysaccharides (extractable or unextractable). The fact that only a small part of the carbohydrates in the extracts were recovered as oligomer and even less as monomers, showed that oligomers and especially monomers were converted very rapidly into Maillard and pyrolysis products. Cellulose remains unextractable and its solubility was not affected by the degree of roast. Galactomannans were also mainly present as unextractable polymers in green beans, but were solubilized to a large extent with increasing degrees of roast. The arabinogalactans in the roasted bean were highly soluble at the extraction conditions used. The arabinose as present as side-chains in the arabinogalactans were found to be more susceptible to degradation at more severe roasting conditions than the galactans. Also evidence was found that populations of arabinogalactans with very different ara:gal ratios exist in the roasted beans as well as in the green beans.
    Extraction and characterization of polysaccharides from green and roasted Coffea arabica beans.
    Oosterveld, A. ; Harmsen, H. ; Voragen, A.G.J. ; Schols, H.A. - \ 2003
    Carbohydrate Polymers 52 (2003)3. - ISSN 0144-8617 - p. 285 - 296.
    rich pectic polysaccharides - sugar-beet pulp - hot-water - arabinose
    Polysaccharides were sequentially extracted from green and roasted Coffea arabica beans with water (90 °C), EDTA, 0.05, 1, and 4 M NaOH and characterized chemically. Additionally, the beans were subjected to a single extraction with water at 170 °C. Green arabica coffee beans contained large proportions of 1¿4-linked mannans, of which on average 1 in every 23 mannopyranose residues was branched with single unit galactose side-chains at O-6. A part of these galactomannans could be extracted relatively easy with water and EDTA. These galactomannans were found to have a relatively high degree of branching (gal:man1:8) and a relatively low molecular weight in comparison to the remaining galactomannans (gal:man1:15–24). Additionally, 1¿3-linked galactans, heavily branched at O-6 with side-chains containing arabinose and galactose residues, were present in the green coffee beans, as well as smaller amounts of pectins, cellulose, and xyloglucans. Roasting resulted in a loss of 8% of the dry weight. This could be partly explained by the relatively high percentage of sugars which was lost during the roasting process, most probably as a result of conversion into, e.g. Maillard and pyrolysis products. After roasting the extractability of polysaccharides was increased significantly. A decrease in the degree of branching as well as a decrease in molecular weight of arabinogalactans, galactomannans, and xyloglucans was observed after roasting.
    A molecular analysis of (hemi-)cellulose degradation by Aspergilli
    Gielkens, M.M.C. - \ 1999
    Agricultural University. Promotor(en): A.J.J. van Ooyen; L.H. de Graaff. - S.l. : S.n. - ISBN 9789058081384 - 136
    aspergillus - biodegradatie - cellulose - arabinose - aspergillus - biodegradation - cellulose - arabinose

    Glycosylhydrolases like cellulases and xylanases are of great importance for the ecological recycling of biomass. The saprophytic fungi, e.g Aspergillus niger , are capable of degrading plant cell wall material by secreting these enzymes. Because of their properties, a whole range of commercial enzyme preparations containing fungal polysaccharidases is used in industrial applications. For example, xylanases and cellulases are used in food and feed applications, and in pulp and paper applications, whereas cellulases are also used the textile industry in biostoning applications. The aim research described in this thesis is to clone genes encoding novel activities capable of degrading (hemi-)cellulose. Many of these novel activities are minor activities, which are difficult to detect and identify using standard conditions for growth. Several strategies will therefore be exploited to find these novel activities in Aspergilli. Firstly, the conditions of induction need to be optimal. This involves both the carbon source as well as the duration of growth (Chapter 4). Secondly, accumulation of the inducer often results in higher levels of expression (Chapter 3).

    Also the use of derepressed creAdstrains can result in elevated levels of expression as is shown in Chapters 2 and 3. Chapter 3 also shows that gene disruption of majorα-L-arabinofuranosidase can reveal minor activities which are otherwise difficult to identify. Finally, increasing the gene dosage of a specific transcriptional activator can increase the transcription levels of genes controlled by that activator (Chapters 5 and 6). Despite the fact that many glycosylhydrolases and their encoding genes have been isolated from a wide variety of microorganisms, little is known at the molecular level about the factors that are involved in the expression of these genes. The research described in this thesis gives a better understanding of the mechanisms underlying the regulation of expression of genes encoding cellulose- and hemicellulose-degrading enzymes produced by Aspergillus .

    Chapter 2 describes the isolation of A. niger creAdmutants relieved of carbon repression and the effects of the mutations on the expression of arabinanases and L-arabinose catabolic enzymes. Carbon repression is a global regulatory mechanism by which in the presence of D-glucose or other rapidly metabolisable carbon sources the expression of genes involved in the utilisation of less-favoured carbon sources is repressed. The arabinanolytic system was selected to illustrate that the expression of genes involved in the utilisation of less-favoured carbon sources could be enhanced in derepressed creAdmutant strains.

    Chapters 3 and 4 focus on genes encoding enzymes that are able to release L-arabinose from arabinoxylan. Chapter 3 describes the cloning and characterisation of the A. nidulans abfB gene, as well as the analysis of expression of the L-arabinofuranosidase encoding gene in an A. nidulans wild-type strain and several mutant strains. Super-induction of the abfB gene can be accomplished by combining a mutation leading to the intracellular accumulation of an inducer with a creAdmutation, as is shown by expression analyses. This chapter also describes the identification of minor L-arabinose releasing activities when the majorα-L-arabinofuranosidase activity was disrupted in a derepressed creAdgenetic background.

    The cloning, characterisation and analysis of expression of the axhA genes from the closely related fungi A. niger and A. tubingensis is described in Chapter 4. These genes encode an arabinoxylan-arabinofuranohydrolase A enzyme which specifically releases L-arabinose substituents from arabinoxylan. This chapter also describes the transcriptional analysis of the axhA and abfB genes in A. niger . It demonstrates that the regulation of transcription of the two genes differs significantly, although both genes encode L-arabinose releasing activities.

    The transcription of genes encoding enzymes involved in xylan degradation and two endoglucanases involved in the degradation of cellulose in A. niger is studied in Chapter 5. In particular, the role of the xylanolytic transcriptional activator XlnR in the regulation of transcription of these genes was investigated. This analysis is extended in Chapter 6, which describes the cloning and characterisation of two cellobiohydrolase encoding genes in A. niger , cbhA and cbhB , which are also involved in the degradation of cellulose. The results described in Chapters 5 and 6 illustrate that the range of genes transcriptionally regulated by a specific activator is not necessarily limited to genes encoding pathway specific enzymes but that it also can include genes encoding non-pathway specific activities.

    Furthermore, the data described in these chapters give evidence that increasing the gene dosage of a specific transcriptional activator could elevate the expression of a broad range of genes controlled by that activator. Based on data described in this thesis a working model is proposed in which XlnR plays a central role in the degradation of plant cell walls.

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