Mapping sugar beet pectin acetylation pattern

Homogalacturonan-derived partly methylated and/or acetylated oligogalacturonates were recovered after enzymatic hydrolysis (endo-polygalacturonase+pectin methyl esterase+side-chain degrading enzymes) of sugar beet pectin followed by anion-exchange and size exclusion chromatography. Around 90% of the GalA and 75% of the acetyl groups present in the initial sugar beet pectin were recovered as homogalacturonan-derived oligogalacturonates, the remaining GalA and acetyl belonging to rhamnogalacturonic regions. Around 50% of the acetyl groups present in sugar beet homogalacturonans were recovered as partly methylated and/or acetylated oligogalacturonates of degree of polymerisation 5 whose structures were determined by electrospray ionization ion trap mass spectrometry (ESI-IT-MSn). 2-O-acetyl- and 3-O-acetyl-GalA were detected in roughly similar amounts but 2,3-di-O-acetylation was absent. Methyl-esterified GalA residues occurred mainly upstream 2-O-acetyl GalA. Oligogalacturonates containing GalA residues that are at once methyl- and acetyl-esterified were recovered in very limited amounts. A tentative mapping of the distribution of acetyl and methyl esters within sugar beet homogalacturonans is proposed. Unsubstituted GalA residues are likely to be present in limited amounts (approximately 10% of total GalA residues), due to the fact that methyl and acetyl groups are assumed to be most often not carried by the same residues.     

Endopolygalacturonases reveal molecular features for processivity pattern and tolerance towards acetylated pectin

Endopolygalacturonases (EndoPGs) hydrolyse the 1-4 linkages between two alpha-d-galacturonic acids (GalA) of the smooth homogalacturonan regions of pectin. GalA may be methyl-esterified on the carboxylic group and acetyl-esterified on the hydroxylic groups. EndoPG activity most often decreases with such increasing degree of substitution. In this paper, we used bioinformatics and molecular modelling technics to explain the tolerance profile at the molecular scale and processivity scheme of three endoPGs with respect to acetylated pectin substrate; the first two enzymes originate from Aspergillus niger (AnPGI and AnPGII) and the third from Fusarium moniliforme (FmPG). Partly acetylated and methylated homogalacturonan fragments in complex with the three PGs were successively modelled in silico. The amino acid residues involved in substrate binding were identified for each enzyme. Similarly, the docking pattern of the differently decorated oligomers in the catalytic groove was individually characterized for each enzyme. This work shows full agreement with our previous extensive mass spectrometry analysis of the hydrolytic products that established distinct tolerance profiles for the three endoPGs and earlier work that ascertained processivity, specifically for AnPGI. In our previous work, AnPGI was shown to be the most powerful enzyme among the three enzymes with an enhanced tolerance towards O2- and O3-acetylated substrates. We report here amino acids of AnPGI that are unique in binding the pectin backbone and that are identified as possibly crucial for its specificity, namely S191(An)(PGI)/D240(An)(PGI). Similarly, topologically equivalent residues in AnPGII and FmPG were identified that could impede such binding; S234(An)(PGII)/S91(An)(PGII) and S245(Fm)(PG)/V89(Fm)(PG). In addition, we report here, from normal mode analysis computed on AnPG1, a shear bending motion of 15 A of amplitude that fully accredits the processive action pattern for this enzyme, with D240(An)(PGI) and R96(An)(PGI) working as crampons to favour the sliding of the substrate. Conversely, the same method clearly evidences a hinge binding motion for AnPGII and FmPG that should only authorize one hydrolytic event per enzyme/substrate encounter.     

Mode of action of Fusarium moniliforme endopolygalacturonase towards acetylated pectin

Endopolygalacturonase from Fusarium moniliforme was used to degrade acetylated homogalacturonan previously prepared from sugar beet pulp. The initial velocity and the final percentage of hydrolysis decreased very rapidly with increasing degree of acetylation, showing that acetyl substitution markedly affected the enzymatic activity. MALDI-TOF mass spectrometry was used to analyse the reaction products and to show acetyl groups on the oligogalacturonates. The results demonstrated that the enzyme was able to accommodate acetyl groups in its active site cleft. The influence of acetyl groups on the mode of action of the enzyme was discussed and compared to the influence of methyl groups.     

The faeA genes from Aspergillus niger and Aspergillus tubingensis encode ferulic acid esterases involved in degradation of complex cell wall polysaccharides.

We report the cloning and characterization of a gene encoding a ferulic acid esterase, faeA, from Aspergillus niger and Aspergillus tubingensis. The A. niger and A. tubingensis genes have a high degree of sequence identity and contain one conserved intron. The gene product, FAEA, was overexpressed in wild-type A. tubingensis and a protease-deficient A. niger mutant. Overexpression of both genes in wild-type A. tubingensis and an A. niger protease-deficient mutant showed that the A. tubingensis gene product is more sensitive to degradation than the equivalent gene product from A. niger. FAEA from A. niger was identical to A. niger FAE-III (C. B. Faulds and G. Williamson, Microbiology 140:779-787, 1994), as assessed by molecular mass, pH and temperature optima, pI, N-terminal sequence, and activity on methyl ferulate. The faeA gene was induced by growth on wheat arabinoxylan and sugar beet pectin, and its gene product (FAEA) released ferulic acid from wheat arabinoxylan. The rate of release was enhanced by the presence of a xylanase. FAEA also hydrolyzed smaller amounts of ferulic acid from sugar beet pectin, but the rate was hardly affected by addition of an endo-pectin lyase.     

Glycon specificity profiling of alpha-glucosidases using monodeoxy and mono-O-methyl derivatives of p-nitrophenyl alpha-D-glucopyranoside

Hydrolysis of probe substrates, eight possible monodeoxy and mono-O-methyl analogs of p-nitrophenyl alpha-D-glucopyranoside (pNP alpha-D-Glc), modified at the C-2, C-3, C-4, and C-6 positions, was studied as part of investigations into the glycon specificities of seven alpha-glucosidases (EC 3.2.1.20) isolated from Saccharomyces cerevisiae, Bacillus stearothermophilus, honeybee (two enzymes), sugar beet, flint corn, and Aspergillus niger. The glucosidases from sugar beet, flint corn, and A. niger were found to hydrolyze the 2-deoxy analogs with substantially higher activities than against pNP alpha-D-Glc. Moreover, the flint corn and A. niger enzymes showed hydrolyzing activities, although low, for the 3-deoxy analog. The other four alpha-glucosidases did not exhibit any activities for either the 2- or the 3-deoxy analogs. None of the seven enzymes exhibited any activities toward the 4-deoxy, 6-deoxy, or any of the methoxy analogs. The hydrolysis results, with the deoxy substrate analogs, demonstrated that alpha-glucosidases having remarkably different glycon specificities exist in nature. Further insight into the hydrolysis of deoxyglycosides was obtained by determining the kinetic parameters (k(cat) and K(m)) for the reactions of sugar beet, flint corn, and A. niger enzymes.     

Aspergillus niger I-1472 and Pycnoporus cinnabarinus MUCL39533, selected for the biotransformation of ferulic acid to vanillin, are also able to produce cell wall polysaccharide-degrading enzymes and feruloyl esterases

The filamentous fungal strains Aspergillus niger I-1472 and Pycnoporus cinnabarinus MUCL39533, previously selected for the bioconversion of ferulic acid to vanillic acid and vanillin respectively, were grown on sugar beet pulp. A large spectrum of polysaccharide-degrading enzymes was produced by A. niger and very few levels of feruloyl esterases were found. In contrast, P. cinnabarinus culture filtrate contained low amount of polysaccharide-degrading enzymes and no feruloyl esterases. In order to enhance feruloyl esterases in A. niger cultures, feruloylated oligosaccharide-rich fractions were prepared from sugar beet pulp or cereal bran and used as carbon sources. Number of polysaccharide-degrading enzymes were induced. Feruloyl esterases were much higher in maize bran-based medium than in sugar beet pulp-based medium, demonstrating the ability of carbon sources originating from maize to induce the synthesis of feruloyl esterases. Thus, A. niger I-1472 could be interesting to release ferulic acid from sugar beet pulp or maize bran.     

Expression profiling of pectinolytic genes from Aspergillus niger

The expression of 26 pectinolytic genes from Aspergillus niger was studied in a wild type strain and a CreA derepressed strain, under 16 different growth conditions, to obtain an expression profile for each gene. These expression profiles were then submitted to cluster analysis to identify subsets of genes with similar expression profiles. With the exception of the feruloyl esterase encoding genes, all genes were expressed in the presence of D-galacturonic acid, polygalacturonate, and/or sugar beet pectin. Despite this general observation five distinct groups of genes were identified. The major group consisted of 12 genes of which the corresponding enzymes act on the pectin backbone and for which the expression, in general, is higher after 8 and 24 h of incubation, than after 2 or 4 h. Two other groups of genes encoding pectin main chain acting enzymes were detected. Two additional groups contained genes encoding L-arabinose and D-galactose releasing enzymes, and ferulic acid releasing enzymes, respectively. The genes encoding beta-galactosidase and the L-arabinose releasing enzymes were not only expressed in the presence of D-galacturonic acid, but also in the presence of L-arabinose, suggesting that they are under the control of two regulatory systems. Similarly, the rhamnogalacturonan acetylesterase encoding gene was not only expressed in the presence of D-galacturonic acid, polygalacturonate and sugar beet pectin, but also in the presence of L-rhamnose. The data presented provides indications for a general pectinolytic regulatory system responding to D-galacturonic acid or a metabolite derived from it. In addition, subsets of pectinolytic genes are expressed in response to the presence of L-arabinose, L-rhamnose or ferulic acid.     

Rhamnogalacturonan acetylesterase: a novel enzyme from Aspergillus aculeatus,specific for the deacetylation of hairy (ramified) regions of pectins

Rhamnogalacturonan acetylesterase, able to specifically hydrolyse the acetyl asters present in modified hairy (ramified) regions (MHR) of apple pectin, was identified. The enzyme removed about 70% of the total acetyl groups in MHR. This acetylesterase did not cause the release of acetyl groups from a range of other acetylated substrates, either synthetic or extracted from plants, including the acetylated smooth regions present in beet pectin. Pretreatment of pectic polysaccharides in order to remove arabinose side chains had no effect on the acetyl release, wor was an effect found on the rate or degree of acetyl release, when the purified acetylesterase was combined with pectolytic enzymes, pectin methylesterase or arabinanases. Correspondence to: A. G. J. Voragen     

New polygalacturonases from Trichoderma reesei: characterization and their specificities to partially methylated and acetylated pectins

Two extracellular isoenzymes of polygalacturonases PG1 and PG2 were isolated from 3-day-old culture filtrates of Trichoderma reesei. The two enzymes were purified to homogeneity by ion-exchange, gel filtration and hydrophobic interaction chromatographies. PG1 and PG2 exhibit similar molecular weights from gel filtration and SDS-PAGE. Their properties, including optimal pH and temperature, thermal stability and Km were compared. Characterization of substrate specificity showed that the two enzymes had higher affinity toward PGA (B0100) derived from sugar beet pectin (SBP) than PGA from lime pectin. A series of SBPs with different distribution patterns of methyl and acetyl groups, produced by treatment with either plant pectin methylesterase (P-series) or fungal pectin methylesterase (F-series) or base catalysis (B-series), was used as substrates for PG1 and PG2. Substrates with a low degree of esterification were preferred substrates. The activities of PG1 and PG2 were strongly correlated to the degree of methylation and very little effect from acetylation. The products generated by digestion of selected lime and SBPs were analysed using matrix assisted laser desorption ionisation time of flight (MALDI TOF) MS. A mode of action revealed a random cleavage pattern for PG1 and PG2, confirming that these enzymes are endopolygalacturonases.     

Purification and characterisation of two exo-polygalacturonases from Aspergillus niger able to degrade xylogalacturonan and acetylated homogalacturonan

Two exo-polygalacturonases (EC 3.2.1.67) were purified from a commercial Aspergillus niger enzyme preparation by ammonium sulfate precipitation, preparative electrofocusing, anion-exchange and size-exclusion chromatographies. The enzymes had molar masses of 82 kDa (exo-PG1) and 56 kDa (exo-PG2). Exo-PG1 was stable over wider pH and temperature ranges than exo-PG2. Addition of 0.01 mM HgCl(2) increased the exo-PG2 activity 3.4 times but did not affect exo-PG1. Analysis of the reaction products of (reduced) pentagalacturonate by high-performance anion-exchange chromatography revealed that both enzymes split the substrate from the non-reducing end in a multi-chain attack mode. Exo-PG1 had a broad specificity towards oligogalacturonates with different degrees of polymerisation, while digalacturonate was the most favorable substrate for exo-PG2. Both enzymes degraded xylogalacturonan from pea hull in an exo manner to produce galacturonic acid and Xyl-GalA disaccharide, as identified by electrospray ionization-ion trap mass spectrometry (ESI-ITMS). Moreover, exo-PGs split acetylated homogalacturonan in an exo manner, producing galacturonic acid and acetylated galacturonic acid, as shown by ESI-ITMS.     

Cloning and characterization of two rhamnogalacturonan hydrolase genes from Aspergillus niger.

A rhamnogalacturonan hydrolase gene of Aspergillus aculeatus was used as a probe for the cloning of two rhamnogalacturonan hydrolase genes of Aspergillus niger. The corresponding proteins, rhamnogalacturonan hydrolases A and B, are 78 and 72% identical, respectively, with the A. aculeatus enzyme. In A. niger cultures which were shifted from growth on sucrose to growth on apple pectin as a carbon source, the expression of the rhamnogalacturonan hydrolase A gene (rhgA) was transiently induced after 3 h of growth on apple pectin. The rhamnogalacturonan hydrolase B gene was not induced by apple pectin, but the rhgB gene was derepressed after 18 h of growth on either apple pectin or sucrose. Gene fusions of the A. niger rhgA and rhgB coding regions with the strong and inducible Aspergillus awamori exlA promoter were used to obtain high-producing A. awamori transformants which were then used for the purification of the two A. niger rhamnogalacturonan hydrolases. High-performance anion-exchange chromatography of oligomeric degradation products showed that optimal degradation of an isolated highly branched pectin fraction by A. niger rhamnogalacturonan hydrolases A and B occurred at pH 3.6 and 4.1, respectively. The specific activities of rhamnogalacturonan hydrolases A and B were then 0.9 and 0.4 U/mg, respectively, which is significantly lower than the specific activity of A. aculeatus rhamnogalacturonan hydrolase (2.5 U/mg at an optimal pH of 4.5). Compared to the A enzymes, the A. niger B enzyme appears to have a different substrate specificity, since additional oligomers are formed.     

Control of dehydrodiferulate cross-linking in pectins from sugar-beet tissues

Pectins were extracted from roots, petioles and leaves of sugar beet, and cross-linked using hydrogen peroxide and peroxidase. The effects on dehydrodiferulate formation were monitored by HPLC and TLC. Dehydrodimers were formed in different proportions to those found in vivo. There was a net loss of around 50% of the phenolic groups (monomers plus dimers) during dimerisation. Gel filtration showed that root and petiole pectin, but not leaf pectin, increased in molecular weight during cross-linking. The effects of varying the cross-linking conditions were investigated, and it was found that hydrogen peroxide concentration was the most important factor in controlling both the type and amount of dehydrodiferulate formed.     

Production of feruloyl/ρ-coumaroyl esterase activity by Penicillium expansum, Penicillium brevicompactum and Aspergillus niger

Extracellular esterase production by Penicillium expansum, Penicillium brevicompactum and Aspergillus niger was determined in both liquid and solid-state culture. Methyl ferulate was used as the main carbon source in liquid culture whereas wheat bran and sugar beet pulp were used in solid-state culture. Extracted enzyme for each fungus showed activity in the presence of ONP butyrate, methyl ferulate, methyl coumarate and two 'natural'feruloylated carbohydrate esters. Higher enzyme recoveries were obtained using wheat bran in solid-state culture. Higher levels of feruloyl esterase activity were recovered from P. expansum on all feruloylated substrates than from P. brevicompactum or A. niger. Using ONP butyrate as substrate the pH and temperature optima for the esterases of both Penicillium spp. were 6.0 and 25–30°C. Aspergillus niger esterase activity showed a broader temperature range with an optimum at 40°C.     

Feruloyl esterases as a tool for the release of phenolic compounds from agro-industrial by-products

Agro-industrial by-products are a potential source of added-value phenolic acids with promising applications in the food and pharmaceutical industries. Here two purified feruloyl esterases from Aspergillus niger, FAEA and FAEB were tested for their ability to release phenolic acids such as caffeic acid, p-coumaric acid and ferulic acid from coffee pulp, apple marc and wheat straw. Their hydrolysis activity was evaluated and compared with their action on maize bran and sugar beet pulp. The specificity of both enzymes against natural and synthetic substrates was evaluated; particular attention was paid to quinic esters and lignin monomers. The efficiency of both enzymes on model substrates was studied. We show the ability of these enzymes to hydrolyze quinic esters and ester linkages between phenolic acids and lignin monomer.     

Effect of carbon sources on the synthesis of pectinase by Aspergilli

The synthesis of pectinase is investigated using six species of Aspergillus, with five media differing either in their carbon sources or level of carbon source(s). Five of the six species used, synthesized appreciable amounts of pectinase in the media containing sugars. Pectinase synthesis was highest for A. niger, NCIM 548, with all the sugar containing media. A. foetidus, NCIM 510, was the only one among the organisms studied, that responded well to the medium containing pectin in the absence of additional sugars supplied in the medium.     

Exploration of members of Aspergillus sections Nigri, Flavi, and Terrei for feruloyl esterase production

The ability of members of Aspergillus sections Nigri, Flavi, and Terrei to produce feruloyl esterases was studied according to their substrate specificity against synthetic methyl esters of hydroxycinnamic acids. Type A feruloyl esterases (FAEA), induced during growth on cereal-derived products, show a preference for the phenolic moiety of substrates that contain methoxy substitutions, as found in methyl sinapinate, whereas type B feruloyl esterases (FAEB) show a preference for the phenolic moiety of substrates that contain hydroxyl substitutions, as occurs in methyl caffeate. All the strains of Aspergillus section Nigri (e.g., A. niger and A. foetidus) were able to produce feruloyl esterases with activity profiles similar to those reported for FAEA and FAEB of A. niger when grown on oat-spelt xylan and sugar beet pulp, respectively. The two genes encoding these proteins, faeA and faeB, were identified by Southern blot analysis. The strains of Aspergillus sections Flavi (e.g., A. flavus, A. flavo-furcatus, and A. tamarii) and Terrei (e.g., A. terreus) were able to produce type A and type B enzymes. faeA was revealed in genomic DNA of these strains, and FAEA was determined by immunodetection in cultures grown in oat-spelt xylan. In addition, type B enzymes, not related to faeB, were efficiently induced by oat-spelt xylan and exhibited very original activity profiles on sugar beet pulp. This work confirms that the members of the genus Aspergillus are good feruloyl esterase producers.     

果胶酶亲和吸附剂的制备及其性能比较

以甜菜渣、果胶、海藻酸钠为原料 ,与表氯醇在碱性条件下交联 ,制备了果胶酶亲和吸附剂并应用这几种亲和吸附剂对草酸青霉果胶酶和黑曲霉果胶酶进行亲和层析实验。结果表明 :用小于 60目的甜菜渣粉末和海藻酸钠制备的果胶酶亲和吸附剂 (分别简写为SBP吸附剂和SA吸附剂 )具有较好的纯化果胶酶性能 ,其中SBP吸附剂性能稳定 ,而SA吸附剂膨胀系数较大 ,在流动相改变时引起流速不稳。     

The beta-1,4-endogalactanase A gene from Aspergillus niger is specifically induced on arabinose and galacturonic acid and plays an important role in the degradation of pectic hairy regions.

The Aspergillus nigerbeta-1,4-endogalactanase encoding gene (galA) was cloned and characterized. The expression of galA in A. niger was only detected in the presence of sugar beet pectin, d-galacturonic acid and l-arabinose, suggesting that galA is coregulated with both the pectinolytic genes as well as the arabinanolytic genes. The corresponding enzyme, endogalactanase A (GALA), contains both active site residues identified previously for the Pseudomonas fluorescensbeta-1,4-endogalactanase. The galA gene was overexpressed to facilitate purification of GALA. The enzyme has a molecular mass of 48.5 kDa and a pH optimum between 4 and 4.5. Incubations of arabinogalactans of potato, onion and soy with GALA resulted initially in the release of d-galactotriose and d-galactotetraose, whereas prolonged incubation resulted in d-galactose and d-galactobiose, predominantly. MALDI-TOF analysis revealed the release of l-arabinose substituted d-galacto-oligosaccharides from soy arabinogalactan. This is the first report of the ability of a beta-1,4-endogalactanase to release substituted d-galacto-oligosaccharides. GALA was not active towards d-galacto-oligosaccharides that were substituted with d-glucose at the reducing end.     

Sequence analysis and overexpression of a pectin lyase gene (pel1) from Aspergillus oryzae KBN616

A gene (pel1) encoding pectin lyase (Pel1) was isolated from a shoyu koji mold, Aspergillus oryzae KBN616, and characterized. The structural gene comprised 1,196 bp with a single intron. The ORF encoded 381 amino acids with a signal peptide of 20 amino acids. The deduced amino acid sequence showed high similarity to those of Aspergillus niger pectin lyases and Glomerella cingulata PnlA. The pel1 gene was successfully overexpressed under the promoter of the A. oryzae TEF1 gene. The molecular mass of the recombinant pectin lyase substantially coincided with that calculated based on nucleotide sequence.