Mode of action of Bacillus licheniformis pectin methylesterase on highly methylesterified and acetylated pectins
Remoroza, C.A. ; Wagenknecht, M. ; Buchholt, H.C. ; Moerschbacher, B.M. ; Gruppen, H. ; Schols, H.A. - \ 2015
Carbohydrate Polymers 115 (2015). - ISSN 0144-8617 - p. 540 - 550.
sugar-beet pectins - aspergillus-niger - esterified oligogalacturonides - environmental-conditions - structural features - de-esterification - escherichia-coli - methyl esterase - degradation - calcium
A gene encoding a putative pectinesterase from Bacillus licheniformis DSM13 was cloned and expressed in Escherichia coli. The resulting recombinant enzyme (BliPME) was purified and characterized as a pectin methylesterase. The enzyme showed maximum activity at pH 8.0 and 50 °C. BliPME is able to release up to 100% of the methylesters from lime pectin (DM 34–76 ¿ DM 0) and up to 73% of all methylesters from SBPs (DM 30–73 ¿ DM 14). BliPME efficiently de-methylesterifies lemon pectins and SBPs in a blockwise manner and is quite tolerant towards the acetyl groups present within the SBPs. Detailed analysis of the BliPME-modified pectins using HILIC–MSn and the classical calcium reactivity measurement showed that the enzyme generates pectins with low methylesterification (lime and SBP) and high acetyl content (SBP) while creating blocks of nonmethylesterified galacturonic acid residues. The high activity of BliPME towards highly methylesterified and acetylated pectins makes this novel esterase more efficient in removing methylesters from highly esterified beet pectin compared to other PMEs, e.g. Aspergillus niger PME.
Biosynthetic genes and activity spectrum of antifungal polyynes from Collimonas fungivorans Ter331
Fritsche, K. ; Berg, M. van den; Boer, W. de; Beek, T.A. van; Raaijmakers, J.M. ; Veen, J.A. van; Leveau, J.H.J. - \ 2014
Environmental Microbiology 16 (2014)5. - ISSN 1462-2912 - p. 1334 - 1345.
pseudomonas-fluorescens pf-5 - aspergillus-niger - sp nov. - identification - caryoynencins - antibiotics - mycophagy - bacteria
The antifungal activity of bacteria from the genus Collimonas has been well documented, but the chemistry and gene functions that underlie this phenotype are still poorly understood. Screening of a random plasposon insertion library of Collimonas fungivorans Ter331 for loss-of-function mutants revealed the importance of gene cluster K, which is annotated to code for the biosynthesis of a secondary metabolite and which features genes for fatty acid desaturases and polyketide synthases. Mutants in gene cluster K had lost the ability to inhibit hyphal growth of the fungus Aspergillus niger and were no longer able to produce and secrete several metabolites that after extraction and partial purification from wildtype strain Ter331 were shown to share a putative ene-triyne moiety. Some but not all of these metabolites were able to inhibit growth of A.¿niger, indicating functional variation within this group of Collimonas-produced polyyne-like 'collimomycins'. Polymerase chain reaction analysis of isolates representing different Collimonas species indicated that the possession of cluster K genes correlated positively with antifungal ability, further strengthening the notion that this cluster is involved in collimomycin production. We discuss our findings in the context of other bacterially produced polyynes and the potential use of collimomycins for the control of harmful fungi.
Two-step enzymatic fingerprinting of sugar beet pectin
Remoroza, C.A. ; Broxterman, S.E. ; Gruppen, H. ; Schols, H.A. - \ 2014
Carbohydrate Polymers 108 (2014). - ISSN 0144-8617 - p. 338 - 347.
galacturonic acid distribution - aspergillus-niger - esterified oligogalacturonides - endopolygalacturonase - quantification - degradation - oligomers - enzymes - lyase
A two-step enzymatic fingerprinting method was introduced to analyze a highly methylesterified and acetylated sugar beet pectin having a degree of methylesterification (DM) of 62 and acetylation of 30. A cocktail of pectolytic enzymes, including endo-polygalacturonase II (endo-PGII) and pectin lyase (PL), was used for the first digestion. The endo-PGII and PL resistant pectin fragments were isolated and subjected to a second digestion using fungal pectin methylesterase and endo-PGII. After the two sequential digestions, 78% of the total GalA residues present in the parental pectin were recovered as mono- and oligomers, which were used to quantitatively describe the parental SBP. For this reason, the descriptive parameters degree of blockiness (DBabs), degree of hydrolysis by PG (DHPG) and degree of hydrolysis by PL (DHPL) were established for both digestions. The first digestion revealed the presence of short blocks of nonesterified GalA residues and blocks of partly methylesterified and acetylated GalA residues in the parental SBP, in addition to blocks of highly methylesterified and acetylated GalA residues. The second digestion revealed the presence of blocks of methylesterified, partly methylesterified and/or acetylated GalA residues in a sequence not to be degradable by neither endo-PGII nor by PL. The acetyl groups were present in an blockwise manner. Application of the method to two differently prepared DM 50 SBPs showed that the two pectins differ in the ratio of blocks of nonesterified and blocks of partly methylesterified and acetylated GalA residues.
Descriptive parameters for revealing substitution patterns of sugar beet pectins using pectolytic enzymes
Remoroza, C.A. ; Buchholt, H.C. ; Gruppen, H. ; Schols, H.A. - \ 2014
Carbohydrate Polymers 101 (2014). - ISSN 0144-8617 - p. 1205 - 1215.
aspergillus-niger - mass-spectrometry - acetyl groups - endopolygalacturonase - homogalacturonans - quantification - esterification - degradation - oligomers - methyl
Enzymatic fingerprinting was applied to sugar beet pectins (SBPs) modified by either plant or fungal pectin methyl esterases and alkali catalyzed de-esterification to reveal the ester distributions over the pectin backbone. A simultaneous pectin lyase (PL) treatment to the commonly used endo-polygalacturonase (endo-PG) degradation showed to be effective in degrading both high and low methylesterified and/or acetylated homogalaturonan regions of SBP simultaneously. Using LC-HILIC–MS/ELSD, we studied in detail all the diagnostic oligomers present, enabling us to discriminate between differently prepared sugar beet pectins having various levels of methylesterification and acetylation. Furthermore, distinction between commercially extracted and de-esterified sugar beet pectin having different patterns of substitution was achieved by using novel descriptive pectin parameters. In addition to DBabs approach for nonmethylesterified sequences degradable by endo-PG, the “degree of hydrolysis” (DHPG) representing all partially saturated methylesterified and/or acetylated galacturonic acid (GalA) moieties was introduced as a new parameter. Consequently, the description DHPL has been introduced to quantify all esterified unsaturated GalA oligomers.
Pathway transfer in fungi: Transporters are the key to success
Straat, L. van der; Graaff, L.H. de - \ 2014
Bioengineered 5 (2014)5. - ISSN 2165-5979 - p. 335 - 339.
itaconic acid production - saccharomyces-cerevisiae - lactococcus-lactis - aspergillus-niger - cephalosporin production - penicillium-chrysogenum - functional expression - membrane-proteins - ceft gene - overproduction
Itaconic acid is an important building block for the chemical industry. Currently, Aspergillus terreus is the main organism used for itaconic acid production. Due to the enormous citric acid production capacity of Aspergillus niger, this host is investigated as a potential itaconic acid production host. Several strategies have been tried so far: fermentation optimization, expression of cis-aconitate decarboxylase (cadA) alone and in combination with aconitase targeted to the same compartment, chassis optimization, and the heterologous expression of two transporters flanking the cadA gene. We showed that the heterologous expression of these two transporters were key to improving itaconic acid production in an A. niger strain that was unable to produce oxalic acid and gluconic acid. The expression of transporters has increased the production levels of other industrially relevant processes as well, such as ß-lactam antibiotics and bioethanol. Thus far, the role of transporters in production process optimization is a bit overlooked.
Fungal endopolygalacturonases are recognized as MAMPs by the Arabidopsis Receptor-Like Protein RBPG1
Zhang, L. ; Kars, I. ; Essenstam, B. ; Liebrand, T.W.H. ; Wagemakers, L. ; Elberse, J. ; Tagkalaki, P. ; Tjoitang, D. ; Ackerveken, G. van den; Kan, J.A.L. van - \ 2014
Plant Physiology 164 (2014)1. - ISSN 0032-0889 - p. 352 - 364.
ethylene-inducing xylanase - agrobacterium-mediated transformation - innate immunity - nicotiana-benthamiana - necrotizing activity - enzymatic-activity - aspergillus-niger - plasma-membrane - plant immunity - active-site
Plants perceive microbial invaders using pattern recognition receptors that recognize microbe-associated molecular patterns. In this study, we identified RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES1 (RBPG1), an Arabidopsis (Arabidopsis thaliana) leucine-rich repeat receptor-like protein, AtRLP42, that recognizes fungal endopolygalacturonases (PGs) and acts as a novel microbe-associated molecular pattern receptor. RBPG1 recognizes several PGs from the plant pathogen Botrytis cinerea as well as one from the saprotroph Aspergillus niger. Infiltration of B. cinerea PGs into Arabidopsis accession Columbia induced a necrotic response, whereas accession Brno (Br-0) showed no symptoms. A map-based cloning strategy, combined with comparative and functional genomics, led to the identification of the Columbia RBPG1 gene and showed that this gene is essential for the responsiveness of Arabidopsis to the PGs. Transformation of RBPG1 into accession Br-0 resulted in a gain of PG responsiveness. Transgenic Br-0 plants expressing RBPG1 were equally susceptible as the recipient Br-0 to the necrotroph B. cinerea and to the biotroph Hyaloperonospora arabidopsidis. Pretreating leaves of the transgenic plants with a PG resulted in increased resistance to H. arabidopsidis. Coimmunoprecipitation experiments demonstrated that RBPG1 and PG form a complex in Nicotiana benthamiana, which also involves the Arabidopsis leucine-rich repeat receptor-like protein SOBIR1 (for SUPPRESSOR OF BIR1). sobir1 mutant plants did not induce necrosis in response to PGs and were compromised in PG-induced resistance to H. arabidopsidis.
Carbohydrate utilization and metabolism is highly differentiated in Agaricus bisporus
Patyshakuliyeva, A. ; Jurak, E. ; Kohler, A. ; Baker, A. ; Battaglia, E. ; Bruijn, W. de; Burton, K.S. ; Challen, M.P. ; Cuotinho, P.M. ; Eastwood, D.C. ; Gruben, B.S. ; Makela, M.R. ; Martin, F. ; Nadal, M. ; Brink, J. van den; Wiebenga, A. ; Zhou, M. ; Henrissat, B. ; Kabel, M.A. ; Gruppen, H. ; Vries, R.P. de - \ 2013
BMC Genomics 14 (2013). - ISSN 1471-2164 - 14 p.
cell-wall polysaccharides - aspergillus-niger - wheat-straw - d-galactose - trehalose phosphorylase - mannitol dehydrogenase - sporophore development - enzyme-activities - button mushroom - fruit bodies
Background - Agaricus bisporus is commercially grown on compost, in which the available carbon sources consist mainly of plant-derived polysaccharides that are built out of various different constituent monosaccharides. The major constituent monosaccharides of these polysaccharides are glucose, xylose, and arabinose, while smaller amounts of galactose, glucuronic acid, rhamnose and mannose are also present. Results - In this study, genes encoding putative enzymes from carbon metabolism were identified and their expression was studied in different growth stages of A. bisporus. We correlated the expression of genes encoding plant and fungal polysaccharide modifying enzymes identified in the A. bisporus genome to the soluble carbohydrates and the composition of mycelium grown compost, casing layer and fruiting bodies. Conclusions - The compost grown vegetative mycelium of A. bisporus consumes a wide variety of monosaccharides. However, in fruiting bodies only hexose catabolism occurs, and no accumulation of other sugars was observed. This suggests that only hexoses or their conversion products are transported from the vegetative mycelium to the fruiting body, while the other sugars likely provide energy for growth and maintenance of the vegetative mycelium. Clear correlations were found between expression of the genes and composition of carbohydrates. Genes encoding plant cell wall polysaccharide degrading enzymes were mainly expressed in compost-grown mycelium, and largely absent in fruiting bodies. In contrast, genes encoding fungal cell wall polysaccharide modifying enzymes were expressed in both fruiting bodies and vegetative mycelium, but different gene sets were expressed in these samples
Degradation of different pectins by fungi:correlations and contrasts between the pectinolytic enzyme sets identified in genomes and the growth on pectins of different origin
Benoit, I. ; Coutinho, P.M. ; Schols, H.A. ; Gerlach, G.F. ; Henrissat, B. ; Vries, R.P. de - \ 2012
BMC Genomics 13 (2012). - ISSN 1471-2164
aspergillus-niger - rhizopus-oryzae - podospora-anserina - trichoderma-reesei - sequence - protein - nidulans - acid - homogalacturonan - tryptoquivaline
Background: Pectins are diverse and very complex biomolecules and their structure depends on the plant speciesand tissue. It was previously shown that derivatives of pectic polymers and oligosaccharides from pectins havepositive effects on human health. To obtain specific pectic oligosaccharides, highly defined enzymatic mixes arerequired. Filamentous fungi are specialized in plant cell wall degradation and some produce a broad range ofpectinases. They may therefore shed light on the enzyme mixes needed for partial hydrolysis.Results: The growth profiles of 12 fungi on four pectins and four structural elements of pectins show that thepresence/absence of pectinolytic genes in the fungal genome clearly correlates with their ability to degradepectins. However, this correlation is less clear when we zoom in to the pectic structural elements.Conclusions: This study highlights the complexity of the mechanisms involved in fungal degradation of complexcarbon sources such as pectins. Mining genomes and comparative genomics are promising first steps towards theproduction of specific pectinolytic fractions.
Combined HILIC-ELSD/ESI-MSn enables the separation, identification and quantification of sugar beet pectin derived oligomers
Remoroza, C.A. ; Cord-Landwehr, S. ; Leijdekkers, A.G.M. ; Moerschbacher, B.M. ; Schols, H.A. ; Gruppen, H. - \ 2012
Carbohydrate Polymers 90 (2012)1. - ISSN 0144-8617 - p. 41 - 48.
performance liquid-chromatography - aspergillus-niger - mass-spectrometry - acetyl groups - acid - methyl - oligogalacturonides - lyase - esterification - degradation
The combined action of endo-polygalacturonase (endo-PGII), pectin lyase (PL), pectin methyl esterase (fungal PME) and RG-I degrading enzymes enabled the extended degradation of methylesterified and acetylated sugar beet pectins (SBPs). The released oligomers were separated, identified and quantified using hydrophilic interaction liquid chromatography (HILIC) with online electrospray ionization ion trap mass spectrometry (ESI-IT-MSn) and evaporative light scattering detection (ELSD). By MSn, the structures of galacturonic acid (GalA) oligomers having an acetyl group in the O-2 and/or O-3 positions eluting from the HILIC column were elucidated. The presence of methylesterified and/or acetylated galacturonic acid units within an oligomer reduced the interaction with the HILIC column significantly compared to the unsubstituted GalA oligomers. The HILIC column enables a good separation of most oligomers present in the digest. The use of ELSD to quantify oligogalacturonides was validated using pure GalA standards and the signal was found to be independent of the chemical structure of the oligomer being detected. The combination of chromatographic and enzymatic strategies enables to distinguish SBPs having different methylesters and acetyl group distribution.
Effects of temperature, pH, incubation time and pepsin concentration on the in vitro stability of intrinsic phytase of wheat, barley and rye
Esmaeilipour, O. ; Krimpen, M.M. van; Jongbloed, A.W. ; Jonge, L.H. de; Bikker, P. - \ 2012
Animal Feed Science and Technology 175 (2012)3-4. - ISSN 0377-8401 - p. 168 - 174.
aspergillus-niger - phosphorus utilization - pigs - feedstuffs - plant - feed - acid
Three experiments were conducted to evaluate the effects of temperature (T), pH, time of incubation and pepsin on the stability of intrinsic phytase of different cereals. Experiment one was conducted as a 3×4×3×3 factorial arrangement with three feed ingredients (rye, wheat and barley), four T (20, 38, 55 and 80 °C), three pH levels (3.0, 5.5 and 8.0) and three incubation times (30, 60 and 120 min), with two replicates per treatment. Stability was calculated as the ratio of the residual phytase activity of a treated sample to the non-treated (reference sample) times 100. Phytase activity of the reference samples of rye, wheat and barley was 3.14, 1.77 and 0.66 FTU/g, respectively. Overall, rye showed the highest and barley the lowest stability (0.58 vs. 0.47, P0.90). Wheat and rye phytases were stable at pH 3.0 and 5.5 (20 and 38 °C). In experiment two, the effect of shorter period of time (10 and 30 min) was assessed on the stability of cereal phytases. Barley phytase showed the highest residual phytase activity among cereals at 65 °C. In experiment three, stability of intrinsic phytase of these feed ingredients was determined after incubation in different concentrations of pepsin (0, 5 and 10 mg/mL) at pH 2.0. Intrinsic phytases of wheat and rye were resistant to pepsin, but barley phytase was susceptible to pepsin and its stability decreased to 0.57 after pre-incubation for 60 min in 5 mg pepsin/mL. This knowledge regarding the effect of T, incubation time and pH on stability of cereal intrinsic phytases may contribute to optimize available P contents of diets, thereby reducing phosphorus excretion of monogastrics.
The ferulic acid esterases of Chrysosporium lucknowense C1: Purification, characterization and their potential application in biorefinery
Kuhnel, S. ; Pouvreau, L.A.M. ; Appeldoorn, M.M. ; Hinz, S.W.A. ; Schols, H.A. ; Gruppen, H. - \ 2012
Enzyme and Microbial Technology 50 (2012)1. - ISSN 0141-0229 - p. 77 - 85.
sugar-beet pulp - plant-cell walls - aspergillus-niger - maize bran - feruloylated oligosaccharides - structural-characterization - wheat bran - degradation - classification - arabinoxylans
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.
Characterization and mode of action of two acetyl xylan esterases from Chrysosporium lucknowense C1 active towards acetylated xylans
Pouvreau, L.A.M. ; Jonathan, M.C. ; Kabel, M.A. ; Hinz, S.W.A. ; Gruppen, H. ; Schols, H.A. - \ 2011
Enzyme and Microbial Technology 49 (2011). - ISSN 0141-0229 - p. 312 - 320.
alpha-l-arabinofuranosidases - penicillium-purpurogenum - trichoderma-reesei - aspergillus-niger - feruloyl esterase - genome sequence - wheat bran - glucuronidase - gene - oligosaccharides
Two novel acetyl xylan esterases, Axe2 and Axe3, from Chrysosporium lucknowense (C1), belonging to the carbohydrate esterase families 5 and 1, respectively, were purified and biochemically characterized. Axe2 and Axe3 are able to hydrolyze acetyl groups both from simple acetylated xylo-oligosaccharides and complex non-soluble acetylglucuronoxylan. Both enzymes performed optimally at pH 7.0 and 40 °C. Axe2 has a clear preference for acetylated xylo-oligosaccharides (AcXOS) with a high degree of substitution and Axe3 does not show such preference. Axe3 has a preference for large AcXOS (DP 9–12) when compared to smaller AcXOS (especially DP 4–7) while for Axe2 the size of the oligomer is irrelevant. Even though there is difference in substrate affinity towards acetylated xylooligosaccharides from Eucalyptus wood, the final hydrolysis products are the same for Axe2 and Axe3: xylo-oligosaccharides containing one acetyl group located at the non-reducing xylose residue remain as examined using MALDI-TOF MS, CE-LIF and the application of an endo-xylanase (GH 10).
Mode of action of Chrysosporium lucknowense C1 a-l-arabinohydrolases
Kuhnel, S. ; Westphal, Y. ; Hinz, S.W.A. ; Schols, H.A. ; Gruppen, H. - \ 2011
Bioresource Technology 102 (2011)2. - ISSN 0960-8524 - p. 1636 - 1643.
glycoside hydrolase family - aspergillus-niger - geobacillus-stearothermophilus - streptomyces-avermitilis - l-arabinofuranosidases - degradation - substrate - arabinan - oligosaccharides - fermentation
The mode of action of four Chrysosporium lucknowense C1 a-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.
Chrysosporium lucknowense arabinohydrolases effectively degrade sugar beet arabinan
Kuhnel, S. ; Hinz, S.W.A. ; Pouvreau, L.A.M. ; Wery, J. ; Schols, H.A. ; Gruppen, H. - \ 2010
Bioresource Technology 101 (2010)21. - ISSN 0960-8524 - p. 8300 - 8307.
alpha-l-arabinofuranosidases - cell wall polysaccharides - aspergillus-niger - purification - enzymes - cloning - expression - biofuels - gene
The filamentous fungus Chrysosporium lucknowense (C1) is a rich source of cell wall degrading enzymes. In the present paper four arabinose releasing enzymes from C1 were characterized, among them one endoarabinanase, two arabinofuranosidases and one exoarabinanase. Combinations of these enzymes released up to 80% of the arabinose present in sugar beet arabinan to fermentable monosugars. Besides the main product arabinobiose, unknown arabinose oligomers are produced from highly branched arabinan when endoarabinanase was combined with exoarabinanase and/or arabinofuranosidase. All described arabinose releasing enzymes are temperature stable up to 50 °C and have a broad pH stability. This makes C1 arabinohydrolases suitable for many biotechnical applications, like co-fermentation bioethanol production.
First characterization of bioactive components in soybean tempe that protect human and animal intestinal cells against enterotoxigenic Escherichia coli (ETEC) infection
Roubos-van den Hil, P.J. ; Schols, H.A. ; Nout, M.J.R. ; Zwietering, M.H. ; Gruppen, H. - \ 2010
Journal of Agricultural and Food Chemistry 58 (2010)13. - ISSN 0021-8561 - p. 7649 - 7656.
soya bean tempe - aspergillus-niger - adhesion - fermentation - inhibition - diarrhea - piglets - k88 - polysaccharides - arabinanases
Tempe extracts can inhibit the adhesion of enterotoxigenic Escherichia coli (ETEC) to intestinal cells and thereby can play a role in controlling ETEC-induced diarrhea. The component responsible for this adhesion inhibition activity is still unknown. This research describes the purification and partial characterization of this bioactive component of tempe. After heating, defatting, and protease treatment, the extracts were found to remain active. However, after treatment with polysaccharide-degrading enzyme mixtures the bioactivity was lost. Ultrafiltration revealed the active component to be >30 kDa. Further purification of the bioactive tempe extracts yielded an active fraction with an increased carbohydrate content of higher arabinose content than the nonactive fractions. In conclusion, the bioactive component contains arabinose and originates from the arabinan or arabinogalactan side chain of the pectic cell wall polysaccharides of the soybeans, which is probably released or formed during fermentation by enzymatic modifications
Hemicellulase production in Chrysosporium lucknowense C1
Hinz, S.W.A. ; Pouvreau, L.A.M. ; Joosten, R. ; Bartels, J. ; Jonathan, M.C. ; Wery, J. ; Schols, H.A. - \ 2009
Journal of Cereal Science 50 (2009). - ISSN 0733-5210 - p. 318 - 323.
recombinant saccharomyces-cerevisiae - fungus trichoderma-reesei - alpha-glucuronidase - aspergillus-niger - wheat arabinoxylan - ferulic acid - feruloylated oligosaccharides - degrading enzymes - maize bran - xylose
Filamentous fungi are widely used for enzyme production for the biofuel industry. The ascomycetous fungus Chrysosporium lucknowense C1 was isolated as a natural producer of neutral cellulases. It is at present an attractive alternative to well known fungi like Aspergillus sp. and Trichoderma reesei for protein production on a commercial scale. Besides many cellulases, a large number of hemicellulases (particularly xylanases and arabinofuranosidases) and esterases (acetyl xylan esterases and ferulic acid esterases) encoding genes have also been identified in the C1 genome. Many of these extracellular enzymes have been selectively expressed in C1 and then purified and characterized. Four arabinofuranosidases, two acetyl xylan esterases, two ferulic acid esterases, an a-glucuronidase and four xylanases have been purified and characterized. All these enzymes were found to be active towards arabinoxylans, demonstrating the high potential of C1 as a producer of hemicellulolytic enzymes.
Analysis of variance components reveals the contribution of sample processing to transcript variation
Veen, D. van der; Oliveira, J.M. ; Berg, W.A.M. van den; Graaff, L.H. de - \ 2009
Applied and Environmental Microbiology 75 (2009)8. - ISSN 0099-2240 - p. 2414 - 2422.
time rt-pcr - aspergillus-niger - gene-expression - microarray experiments - quantification - information - standards - nidulans - cloning - growth
The proper design of DNA microarray experiments requires knowledge of biological and technical variation of the studied biological model. For the filamentous fungus Aspergillus niger, a fast, quantitative real-time PCR (qPCR)-based hierarchical experimental design was used to determine this variation. Analysis of variance components determined the contribution of each processing step to total variation: 68% is due to differences in day-to-day handling and processing, while the fermentor vessel, cDNA synthesis, and qPCR measurement each contributed equally to the remainder of variation. The global transcriptional response to d-xylose was analyzed using Affymetrix microarrays. Twenty-four statistically differentially expressed genes were identified. These encode enzymes required to degrade and metabolize D-xylose-containing polysaccharides, as well as complementary enzymes required to metabolize complex polymers likely present in the vicinity of D-xylose-containing substrates. These results confirm previous findings that the d-xylose signal is interpreted by the fungus as the availability of a multitude of complex polysaccharides. Measurement of a limited number of transcripts in a defined experimental setup followed by analysis of variance components is a fast and reliable method to determine biological and technical variation present in qPCR and microarray studies. This approach provides important parameters for the experimental design of batch-grown filamentous cultures and facilitates the evaluation and interpretation of microarray data
Structure and function of 2,3-dimethylmalate lyase, a PEP mutase/isocitrate lyase superfamily member
Narayanan, B. ; Niu, W. ; Joosten, H.J. ; Li, Z. ; Kuipers, R.K. ; Schaap, P.J. ; Dunaway-Mariano, D. ; Herzberg, O. - \ 2009
Journal of Molecular Biology 386 (2009)2. - ISSN 0022-2836 - p. 486 - 503.
nicotinic-acid metabolism - petal death protein - crystal-structure - 2-methylisocitrate lyase - isocitrate lyase - aspergillus-niger - mycobacterium-tuberculosis - enzyme superfamily - escherichia-coli - catalysis
The Aspergillus niger genome contains four genes that encode proteins exhibiting greater than 30% amino acid sequence identity to the confirmed oxaloacetate acetyl hydrolase (OAH), an enzyme that belongs to the phosphoenolpyruvate mutase/isocitrate lyase superfamily. Previous studies have shown that a mutant A. niger strain lacking the OAH gene does not produce oxalate. To identify the function of the protein sharing the highest amino acid sequence identity with the OAH (An07g08390, Swiss-Prot entry Q2L887, 57% identity), we produced the protein in Escherichia coli and purified it for structural and functional studies. A focused substrate screen was used to determine the catalytic function of An07g08390 as (2R,3S)-dimethylmalate lyase (DMML): k(cat)=19.2 s(-1) and K(m)=220 microM. DMML also possesses significant OAH activity (k(cat)=0.5 s(-1) and K(m) =220 microM). DNA array analysis showed that unlike the A. niger oah gene, the DMML encoding gene is subject to catabolite repression. DMML is a key enzyme in bacterial nicotinate catabolism, catalyzing the last of nine enzymatic steps. This pathway does not have a known fungal counterpart. BLAST analysis of the A. niger genome for the presence of a similar pathway revealed the presence of homologs to only some of the pathway enzymes. This and the finding that A. niger does not thrive on nicotinamide as a sole carbon source suggest that the fungal DMML functions in a presently unknown metabolic pathway. The crystal structure of A. niger DMML (in complex with Mg(2+) and in complex with Mg(2+) and a substrate analog: the gem-diol of 3,3-difluoro-oxaloacetate) was determined for the purpose of identifying structural determinants of substrate recognition and catalysis. Structure-guided site-directed mutants were prepared and evaluated to test the contributions made by key active-site residues. In this article, we report the results in the broader context of the lyase branch of the phosphoenolpyruvate mutase/isocitrate lyase superfamily to provide insight into the evolution of functional diversity
Identification of fungal oxaloacetate hydrolyase within the isocitrate lyase/PEP mutase enzyme superfamily using a sequence marker-based method
Joosten, H.J. ; Han, Y. ; Niu, W. ; Vervoort, J.J.M. ; Dunaway-Mariano, D. ; Schaap, P.J. - \ 2008
Proteins : Structure, Function, and Bioinformatics 70 (2008)1. - ISSN 0887-3585 - p. 157 - 166.
oxalic-acid biosynthesis - petal death protein - sclerotinia-sclerotiorum - oxalate production - aspergillus-niger - pathogenicity - palustris - rolfsii
Aspergillus niger produces oxalic acid through the hydrolysis of oxaloacetate, catalyzed by the cytoplasmic enzyme oxaloacetate acetylhydrolase (OAH). The A. niger genome encodes four additional open reading frames with strong sequence similarity to OAH yet only the oahA gene encodes OAH activity. OAH and OAH-like proteins form subclass of the isocitrate lyase/PEP mutase enzyme superfamily, which is ubiquitous present filamentous fungi. Analysis of function-specific residues using a superfamily-based approach revealed an active site serine as a possible sequence marker for OAH activity. We propose that presence of this serine in family members correlates with presence of OAH activity whereas its absence correlates with absence of OAH. This hypothesis was tested by carrying out a serine mutagenesis study with the OAH from the fungal oxalic acid producer Botrytis cinerea and the OAH active plant petal death protein as test systems
pH-gradient ion-exchange chromatography: An analytical tool for design and optimization of protein separations
Ahamed, T. ; Nfor, B. ; Verhaert, P. ; Deden, G. van; Wielen, L. van der - \ 2007
Journal of Chromatography. A, Including electrophoresis and other separation methods 1164 (2007)1-2. - ISSN 0021-9673 - p. 181 - 188.
performance liquid-chromatography - aspergillus-niger - rapid separation - purification - isoforms - columns
This work demonstrates that a highly linear, controllable and wide-ranged pH-gradient can be generated through an ion-exchange chromatography (IEC) column. Such a pH-gradient anion-exchange chromatography was evaluated with 17 model proteins and found that acidic (pI <6) and basic (pI > 8) proteins elute roughly at their pI, whereas neutral proteins (pI 6–8) elute at pH 8–9 regardless their pI values. Because of the flat nature of protein titration curves from pH 6 to 9, neutral proteins indeed exhibit nearly zero net charge at pH 9. The elution-pH in pH-gradient IEC or the titration curve, but not the pI, was identified as the key parameter for pH optimization of preparative IEC in a fast and rational way. The pH-gradient IEC was also applied and found to be an excellent analytical tool for the fractionation of crude protein mixtures.