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Wageningen, Netherlands

Kuhnel S.,Wageningen University | Westphal Y.,Wageningen University | Hinz S.W.A.,Dyadic Netherlands | Schols H.A.,Wageningen University | Gruppen H.,Wageningen University
Bioresource Technology | Year: 2011

The mode of action of four Chrysosporium lucknowense C1 α- l-arabinohydrolases was determined to enable controlled and effective degradation of arabinan. The active site of endoarabinanase Abn1 has at least six subsites, of which the subsites -1 to +2 have to be occupied for hydrolysis. Abn1 was able to hydrolyze a branched arabinohexaose with a double substituted arabinose at subsite -2. The exo acting enzymes Abn2, Abn4 and Abf3 release arabinobiose (Abn2) and arabinose (Abn4 and Abf3) from the non-reducing end of reduced arabinose oligomers. Abn2 binds the two arabinose units only at the subsites -1 and -2. Abf3 prefers small oligomers over large oligomers. It is able to hydrolyze all linkages present in beet arabinan, including the linkages of double substituted residues. Abn4 is more active towards polymeric substrate and releases arabinose monomers from single substituted arabinose residues. Depending on the combination of the enzymes, the C1 arabinohydrolases can be used to effectively release branched arabinose oligomers and/or arabinose monomers. © 2010 Elsevier Ltd. Source

Kool M.M.,Wageningen University | Schols H.A.,Wageningen University | Wagenknecht M.,University of Munster | Hinz S.W.A.,Dyadic Netherlands | And 2 more authors.
Carbohydrate Polymers | Year: 2014

Screening of eight carbohydrate acetyl esterases for their activity towards xanthan resulted in the recognition of one active esterase. AXE3, a CAZy family CE1 acetyl xylan esterase originating from Myceliophthora thermophila C1, removed 31% of all acetyl groups present in xanthan after a 48 h incubation. AXE3 activity towards xanthan was only observed when xanthan molecules were in the disordered conformation. Optimal performance towards xanthan was observed at 53 °C in the complete absence of salt, a condition favouring the disordered conformation. AXE3-deacetylated xanthan was hydrolyzed using cellulases and analyzed for its repeating units using UPLC-HILIC-ELSD/ESI-MS. This showed that AXE3 specifically removes the acetyl groups positioned on the inner mannose and that acetyl groups positioned on the outer mannose are not removed at all. After a prolonged incubation at optimal conditions, 57% of all acetyl groups, representing 70% of all acetyl groups on the inner mannose units, were hydrolyzed. © 2014 Elsevier Ltd. Source

Westphal Y.,Wageningen University | Kuhnel S.,Wageningen University | de Waard P.,Wageningen Center | Hinz S.W.A.,Dyadic Netherlands | And 3 more authors.
Carbohydrate Research | Year: 2010

Sugar beet arabinan consists of an α-(1,5)-linked backbone of l-arabinosyl residues, which can be either single or double substituted with α-(1,2)- and/or α-(1,3)-linked l-arabinosyl residues. Neutral branched arabino-oligosaccharides were isolated from sugar beet arabinan by enzymatic degradation with mixtures of pure and well-defined arabinohydrolases from Chrysosporium lucknowense followed by fractionation based on size and analysis by MALDI-TOF MS and HPAEC. Using NMR analysis, two main series of branched arabino-oligosaccharides have been identified, both having an α-(1,5)-linked backbone of l-arabinosyl residues. One series carries single substituted α-(1,3)-linked l-arabinosyl residues at the backbone, whereas the other series consists of a double substituted α-(1,2,3,5)-linked arabinan structure within the molecule. The structures of eight such branched arabino-oligosaccharides were established. © 2010 Elsevier Ltd. All rights reserved. Source

Kuhnel S.,Wageningen University | Pouvreau L.,NIZO food research | Appeldoorn M.M.,Wageningen University | Hinz S.W.A.,Dyadic Netherlands | And 2 more authors.
Enzyme and Microbial Technology | Year: 2012

Three ferulic acid esterases from the filamentous fungus Chrysosporium lucknowense C1 were purified and characterized. The enzymes were most active at neutral pH and temperatures up to 45°C. All enzymes released ferulic acid and p-coumaric acid from a soluble corn fibre fraction. Ferulic acid esterases FaeA1 and FaeA2 could also release complex dehydrodiferulic acids and dehydrotriferulic acids from corn fibre oligomers, but released only 20% of all ferulic acid present in sugar beet pectin oligomers. Ferulic acid esterase FaeB2 released almost no complex ferulic acid oligomers from corn fibre oligomers, but 60% of all ferulic acid from sugar beet pectin oligomers. The ferulic acid esterases were classified based on both, sequence similarity and their activities toward synthetic substrates. The type A ferulic acid esterases FaeA1 and FaeA2 are the first members of the phylogenetic subfamily 5 to be biochemically characterized. Type B ferulic acid esterase FaeB2 is a member of subfamily 6. © 2011 Elsevier Inc. Source

Visser H.,Dyadic Netherlands | Joosten V.,Dyadic Netherlands | Punt P.J.,TNO | Punt P.J.,Leiden University | And 9 more authors.
Industrial Biotechnology | Year: 2011

The filamentous fungus C1 was developed into an expression platform for screening and production of diverse industrial enzymes. C1 shows a lowviscosity morphology in submerged culture, enabling the use of complex growth and production media. This morphology furthermore allowed C1 to be used as a host for high-throughput robotic screening of gene libraries. A C1-genetic toolbox was developed, which enabled the generation of a large collection of dedicated C1 host strains and gene-expression strategies. The 38 Mbp genome was sequenced and found to be rich in biomass-hydrolyzing-enzyme-encoding genes. C1 production strains have been developed that produce large quantities of these enzyme mixtures (up to 100 g/L total protein). Recombinant C1 strains were constructed that produce single enzymes in a relatively pure form, facilitating enzyme purification and characterization, as well as for commercial applications. Molecular phylogenetic studies revealed that C1, previously classified as Chrysosporium lucknowense based on morphological characteristics, is actually a Myceliophthora thermophila isolate. In addition, C1 has proven to be a source of novel industrial enzymes, and the C1-technology platform developed has been applied as a tool for research on and production of industrial enzymes for various industrial applications, such as biofuels and biorefineries. © 2011 Mary Ann Liebert, Inc. Source

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