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.     

Overexpression of Aspergillus tubingensis faeA in protease-deficient Aspergillus niger enables ferulic acid production from plant material

The production of ferulic acid esterase involved in the release of ferulic acid side groups from xylan was investigated in strains of Aspergillus tubingensis, Aspergillus carneus, Aspergillus niger and Rhizopus oryzae. The highest activity on triticale bran as sole carbon source was observed with the A. tubingensis T8.4 strain, which produced a type A ferulic acid esterase active against methyl p-coumarate, methyl ferulate and methyl sinapate. The activity of the A. tubingensis ferulic acid esterase (AtFAEA) was inhibited twofold by glucose and induced twofold in the presence of maize bran. An initial accumulation of endoglucanase was followed by the production of endoxylanase, suggesting a combined action with ferulic acid esterase on maize bran. A genomic copy of the A. tubingensis faeA gene was cloned and expressed in A. niger D15#26 under the control of the A. niger gpd promoter. The recombinant strain has reduced protease activity and does not acidify the media, therefore promoting high-level expression of recombinant enzymes. It produced 13.5 U/ml FAEA after 5 days on autoclaved maize bran as sole carbon source, which was threefold higher than for the A. tubingensis donor strain. The recombinant AtFAEA was able to extract 50 % of the available ferulic acid from non-pretreated maize bran, making this enzyme suitable for the biological production of ferulic acid from lignocellulosic plant material.     

AFM studies of water-soluble wheat arabinoxylans--effects of esterase treatment

The degradation products of water-soluble wheat arabinoxylans treated with Aspergillus niger ferulic acid esterase (FAEA-able to cleave 5,5'- and 8-O-4'-ferulic acid dimers) have been characterised by atomic force microscopy (AFM) and size exclusion chromatography. The AFM images of arabinoxylans confirmed that a small proportion ( approximately 15%) of the population of arabinoxylan molecules contain xylan-based branches attached to the xylan-based backbone. Treatment with FAEA reduced the contour length of the molecules suggesting that certain dimeric ferulic acid linkages may play a previously unconfirmed role in the elongation of arabinoxylans. Overnight treatment with FAEA led to a reduction in the density of branches suggesting that they may also be linked to the backbone through phenolic linkages.     

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.     

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.     

Synergy between enzymes from Aspergillus involved in the degradation of plant cell wall polysaccharides

Synergy in the degradation of two plant cell wall polysaccharides, water insoluble pentosan from wheat flour (an arabinoxylan) and sugar beet pectin, was studied using several main-chain cleaving and accessory enzymes. Synergy was observed between most enzymes tested, although not always to the same extent. Degradation of the xylan backbone by endo-xylanase and beta-xylosidase was influenced most strongly by the action of alpha-L-arabinofuranosidase and arabinoxylan arabinofuranohydrolase resulting in a 2.5-fold and twofold increase in release of xylose, respectively. Ferulic acid release by feruloyl esterase A and 4-O-methyl glucuronic acid release by alpha-glucuronidase depended largely on the degradation of the xylan backbone by endo-xylanase but were also influenced by other enzymes. Degradation of the backbone of the pectin hairy regions resulted in a twofold increase in the release of galactose by beta-galactosidase and endo-galactanase but did not significantly influence the arabinose release by arabinofuranosidase and endo-arabinase. Ferulic acid release from sugar beet pectin by feruloyl esterase A was affected most strongly by the presence of other accessory 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.     

Construction of engineered bifunctional enzymes and their overproduction in Aspergillus niger for improved enzymatic tools to degrade agricultural by-products

Two chimeric enzymes, FLX and FLXLC, were designed and successfully overproduced in Aspergillus niger. FLX construct is composed of the sequences encoding the feruloyl esterase A (FAEA) fused to the endoxylanase B (XYNB) of A. niger. A C-terminal carbohydrate-binding module (CBM family 1) was grafted to FLX, generating the second hybrid enzyme, FLXLC. Between each partner, a hyperglycosylated linker was included to stabilize the constructs. Hybrid proteins were purified to homogeneity, and molecular masses were estimated to be 72 and 97 kDa for FLX and FLXLC, respectively. Integrity of hybrid enzymes was checked by immunodetection that showed a single form by using antibodies raised against FAEA and polyhistidine tag. Physicochemical properties of each catalytic module of the bifunctional enzymes corresponded to those of the free enzymes. In addition, we verified that FLXLC exhibited an affinity for microcrystalline cellulose (Avicel) with binding parameters corresponding to a Kd of 9.9 x 10(-8) M for the dissociation constant and 0.98 micromol/g Avicel for the binding capacity. Both bifunctional enzymes were investigated for their capacity to release ferulic acid from natural substrates: corn and wheat brans. Compared to free enzymes FAEA and XYNB, a higher synergistic effect was obtained by using FLX and FLXLC for both substrates. Moreover, the release of ferulic acid from corn bran was increased by using FLXLC rather than FLX. This result confirms a positive role of the CBM. In conclusion, these results demonstrated that the fusion of naturally free cell wall hydrolases and an A. niger-derived CBM onto bifunctional enzymes enables the increase of the synergistic effect on the degradation of complex substrates.     

Heterogeneity of Aspergillus niger microcolonies in liquid shaken cultures

The fungus Aspergillus niger forms (sub)millimeter microcolonies within a liquid shaken culture. Here, we show that such microcolonies are heterogeneous with respect to size and gene expression. Microcolonies of strains expressing green fluorescent protein (GFP) from the promoter of the glucoamlyase gene glaA or the ferulic acid esterase gene faeA were sorted on the basis of diameter and fluorescence using the Complex Object Parametric Analyzer and Sorter (COPAS) technology. Statistical analysis revealed that the liquid shaken culture consisted of two populations of microcolonies that differ by 90 μm in diameter. The population of small microcolonies of strains expressing GFP from the glaA or faeA promoter comprised 39% and 25% of the culture, respectively. Two populations of microcolonies could also be distinguished when the expression of GFP in these strains was analyzed. The population expressing a low level of GFP consisted of 68% and 44% of the culture, respectively. We also show that mRNA accumulation is heterogeneous within microcolonies of A. niger. Central and peripheral parts of the mycelium were isolated with laser microdissection and pressure catapulting (LMPC), and RNA from these samples was used for quantitative PCR analysis. This analysis showed that the RNA content per hypha was about 45 times higher at the periphery than in the center of the microcolony. Our data imply that the protein production of A. niger can be improved in industrial fermentations by reducing the heterogeneity within the culture.     

Evidence for a blockwise distribution of acetyl groups onto homogalacturonans from a commercial sugar beet (Beta vulgaris) pectin

Commercial acid-extracted sugar beet pectin was extensively hydrolysed using an endo-polygalacturonase (AnPGI from Aspergillus niger or AnPGII from A. niger or FmPG from Fusarium moniliforme) in combination with Aspergillus aculeatus pectin methyl-esterase (AaPME). The homogalacturonan-derived oligogalacturonates released were quantified by high-performance anion-exchange chromatography and their structure determined by mass spectrometry. The different endo-polygalacturonases exhibited variable tolerance towards acetyl groups. AnPGI was the most active and FmPG the less. A hypothetical homogalacturonan was constructed using the AnPGI-recovered oligogalacturonates as building blocks and the validity of the model was checked taking into account FmPG observed requirements and hydrolysis products. A blockwise repartition of the acetyl groups onto sugar beet pectin homogalacturonan is proposed.     

Production of a chimeric enzyme tool associating the Trichoderma reesei swollenin with the Aspergillus niger feruloyl esterase A for release of ferulic acid

The main goals of this work were to produce the fusion protein of the Trichoderma reesei swollenin I (SWOI) and Aspergillus niger feruloyl esterase A (FAEA) and to study the effect of the physical association of the fusion partners on the efficiency of the enzyme. The fusion protein was produced up to 25 mg l⁻¹ in the T. reesei strains Rut-C30 and CL847. In parallel, FAEA alone was produced for use as a control protein in application tests. Recombinant FAEA and SWOI–FAEA were purified to homogeneity and characterized. The biochemical and kinetic characteristics of the two recombinant proteins were found to be similar to those of native FAEA, except for the temperature stability and specific activity of the SWOI–FAEA. Finally, the SWOI–FAEA protein was tested for release of ferulic acid from wheat bran. A period of 24 h of enzymatic hydrolysis with the SWOI–FAEA improved the efficiency of ferulic acid release by 50% compared with the results obtained using the free FAEA and SWOI. Ferulic acid is used as an antioxidant and flavor precursor in the food and pharmaceutical industries. This is the first report of a potential application of the SWOI protein fused with an enzyme of industrial interest.     

Probing the determinants of substrate specificity of a feruloyl esterase, AnFaeA, from Aspergillus niger

Feruloyl esterases hydrolyse phenolic groups involved in the cross-linking of arabinoxylan to other polymeric structures. This is important for opening the cell wall structure making material more accessible to glycoside hydrolases. Here we describe the crystal structure of inactive S133A mutant of type-A feruloyl esterase from Aspergillus niger (AnFaeA) in complex with a feruloylated trisaccharide substrate. Only the ferulic acid moiety of the substrate is visible in the electron density map, showing interactions through its OH and OCH(3) groups with the hydroxyl groups of Tyr80. The importance of aromatic and polar residues in the activity of AnFaeA was also evaluated using site-directed mutagenesis. Four mutant proteins were heterologously expressed in Pichia pastoris, and their kinetic properties determined against methyl esters of ferulic, sinapic, caffeic and p-coumaric acid. The k(cat) of Y80S, Y80V, W260S and W260V was drastically reduced compared to that of the wild-type enzyme. However, the replacement of Tyr80 and Trp260 with smaller residues broadened the substrate specificity of the enzyme, allowing the hydrolysis of methyl caffeate. The role of Tyr80 and Trp260 in AnFaeA are discussed in light of the three-dimensional structure.     

Overproduction of the Aspergillus niger feruloyl esterase for pulp bleaching application

A well-known industrial fungus for enzyme production, Aspergillus niger, was selected to produce the feruloyl esterase FAEA by homologous overexpression for pulp bleaching application. The gpd gene promoter was used to drive FAEA expression. Changing the nature and concentration of the carbon source nature (maltose to glucose; from 2.5 to 60 g l(-1)), improved FAEA activity 24.5-fold and a yield of 1 g l(-1) of the corresponding protein in the culture medium was achieved. The secreted FAEA was purified 3.5-fold to homogeneity in a two-step purification procedure with a recovery of 69%. The overproduced protein was characterised and presented properties in good agreement with those of native FAEA. The recombinant FAEA was tested for wheat straw pulp bleaching, with or without a laccase mediator system and xylanase. Best results were obtained using a bi-sequential process with a sequence including xylanase, FAEA and laccase, and yielded very efficient delignification--close to 75%--and a kappa number of 3.9. This is the first report on the potential application of recombinant FAEA in the pulp and paper sector.     

Structural features of a polygalacturonase gene cloned from Aspergillus oryzae KBN616

Abstract A genomic gene encoding a polygalacturonase from Aspergillus oryzae , used in soy sauce production, was cloned and sequenced. The structural gene comprises 1227 bp coding for 363 amino acids with a putative prepropeptide of 28 amino acids and the open reading frame is disrupted by two short introns of 57 bp and 81 bp. The deduced amino acid sequence of the mature protein showed 63, 63, 63 and 64% homology with those of Aspergillus niger polygalacturonase I, Aspergillus niger polygalacturonase II, Aspergillus tubingensis polygalacturonase II and Cochliobolus carbonum polygalacturonase, respectively. There is, however, little homology among fungal, plant and bacterial polygalacturonases.     

Comparative analysis of the complete mitochondrial genomes of Aspergillus niger mtDNA type 1a and Aspergillus tubingensis mtDNA type 2b.

To understand the differences in the organization of mitochondrial genomes of the very closely related Aspergillus niger and Aspergillus tubingensis species, we determined the complete genome sequence of the 1a mtDNA type of A. niger and 2b mtDNA type of A. tubingensis and now we provide a comparative analysis of the two mtDNAs. We found that (1) the organization (gene content and order) of the two genomes is almost identical and (2) the size difference between them is principally attributed to the different intron content of their cox1, atp9 and ndh4L genes.     

Heterologous gene expression in Aspergillus niger: a glucoamylase-porcine pancreatic prophospholipase A2 fusion protein is secreted and processed to yield mature enzyme.

The cDNA gene encoding porcine pancreatic prophospholipase A2 (proPLA2) was cloned into an Aspergillus niger expression vector downstream of the glucoamylase (glaA) gene promoter region. When this construct was transformed into A. niger, no detectable PLA2 was produced. Evidence was obtained showing that the PLA2 gene was transcribed and that PLA2 is extremely susceptible to both intracellular and extracellular proteases of A. niger, thus indicating that translation products would be rapidly degraded. By fusing the proPLA2-encoding sequence to the entire glaA gene, secreted yields of PLA2 up to 10 micrograms/ml were obtained from a transformed protease-deficient strain of A. niger. PLA2 was secreted in young cultures as a fusion protein, but in older cultures, it was processed from the glucoamylase carrier protein. Secreted PLA2 was shown to be enzymatically active and to have the correct N-terminal amino acid (aa) sequence, although another form of processed PLA2 was also produced. This form included two aa of the proregion from PLA2. The potential for improving yields of secreted heterologous proteins from A. niger still further is discussed.     

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.