The Aspergillus niger faeB gene encodes a second feruloyl esterase involved in pectin and xylan degradation and is specifically induced in the presence of aromatic compounds

The faeB gene encoding a second feruloyl esterase from Aspergillus niger has been cloned and characterized. It consists of an open reading frame of 1644 bp containing one intron. The gene encodes a protein of 521 amino acids that has sequence similarity to that of an Aspergillus oryzae tannase. However, the encoded enzyme, feruloyl esterase B (FAEB), does not have tannase activity. Comparison of the physical characteristics and substrate specificity of FAEB with those of a cinnamoyl esterase from A. niger [Kroon, Faulds and Williamson (1996) Biotechnol. Appl. Biochem. 23, 255-262] suggests that they are in fact the same enzyme. The expression of faeB is specifically induced in the presence of certain aromatic compounds, but not in the presence of other constituents present in plant-cell-wall polysaccharides such as arabinoxylan or pectin. The expression profile of faeB in the presence of aromatic compounds was compared with the expression of A. niger faeA, encoding feruloyl esterase A (FAEA), and A. niger bphA, the gene encoding a benzoate-p-hydroxylase. All three genes have different subsets of aromatic compounds that induce their expression, indicating the presence of different transcription activating systems in A. niger that respond to aromatic compounds. Comparison of the activity of FAEA and FAEB on sugar-beet pectin and wheat arabinoxylan demonstrated that they are both involved in the degradation of both polysaccharides, but have opposite preferences for these substrates. FAEA is more active than FAEB towards wheat arabinoxylan, whereas FAEB is more active than FAEA towards sugar-beet pectin

Accessory enzymes from Aspergillus involved in xylan and pectin degradation

The xylanolytic and pectinolytic enzyme systems from Aspergillus have been the subject of study for many years. Although the main chain cleaving enzymes and their encoding genes have been studied in detail, little information is available about most of the accessory enzymes and their corresponding genes. This thesis describes the purification and characterisation of two accessory enzymes from Aspergillus , feruloyl esterase A (FaeA) andα-glucuronidase A (AguA), and the activities of these enzymes on polymeric substrates in relation to other accessory enzymes. Furthermore, the characterisation and regulation of the FaeA and AguA encoding genes ( fae A and agu A), and some genes encoding other accessory enzymes is studied.FaeA is the major feruloyl esterase produced when Aspergillus niger is grown on xylan or crude substrates such as wheat bran or sugar beet pulp. Addition of ferulic acid, the product of FaeA, to media containing xylan increases the production of this enzyme. FaeA is able to release ferulic acid from xylan and pectin oligosaccharides, as well as from synthetic substrates such as methylferulate. The fae A gene was cloned from A. niger and Aspergillus tubingensis . A blast of the deduced amino acid sequence of FaeA revealed no significant homology to other proteins, except for a small region of FaeA which was highly similar to the active site of lipases. Based on this homology, a 3-dimensional model for FaeA was proposed by Pickersgill et al. Although only 16 amino acid differences were observed between FaeA from A. niger and A. tubingensis , the latter enzyme was found to be much more sensitive to proteolytic degradation.AguA from A. tubingensis was able to release (4-O-methyl-) glucuronic acid from xylan derived oligosaccharides, but had very little activity towards the intact polysaccharide. The agu A gene was cloned, and database analysis of the deduced amino acid sequence revealed homology to theα-glucuronidases from Trichoderma reesei and Thermotoga maritima .Regulation of fae A and agu A expression was studied in A. niger and compared to other xylanolytic genes. Both genes were found to be under the control of the xylanolytic transcriptional activator protein XlnR, which also regulates endoxylanase,β-xylosidase, acetylxylan esterase, arabinoxylan arabinofuranohydrolase, and endoglucanase gene expression. In a XlnR negative mutant no expression of fae A and agu A was observed on xylose or xylan. Expression of fae A in this mutant was observed in the presence of ferulic acid, indicating the presence of a second system for the induction of this gene. This system seems to be specific for fae A, since no expression of agu A or other xylanolytic genes was observed under these conditions. In a wild-type A. niger strain, expression levels of fae A were higher on a combination of xylose and ferulic acid than the sum of the expression levels on xylose and ferulic acid alone, suggesting a synergistic effect of these two inducing systems.The carbon catabolite repressor protein CreA is involved in the repression of xylanolytic gene expression in the presence of easy metabolisable carbon sources, such as glucose or fructose. Expression of agu A and fae A on xylose and xylan, as well as expression of fae A on ferulic acid was repressed in the presence of glucose. Depending on the concentration of xylose present in the medium, this sugar also triggers CreA mediated repression of xylanolytic gene expression. Using a concentration range from 1 to 100 mM, it was shown that expression levels of fae A, agu A, and genes encoding endoxylanase B andβ-xylosidase decreased with increasing xylose concentrations in an A. niger wild type strain. In a CreA derepressed mutant constant levels of XlnR induced gene expression were observed indicating that the xylose concentration has a modulating effect via CreA.A gene ( agl B) encoding anα-galactosidase, which was produced when A. niger was grown on crude wheat arabinoxylan, was cloned and the expression of this gene was compared with the expression of two otherα-galactosidase encoding genes ( agl A and agl C) and aβ-galactosidase encoding gene ( lac A) from A. niger . All four genes had specific expression profiles with respect to monomeric sugars, galacto-oligosaccharides and polymeric substrates. High expression on xylan was only observed for agl B and lac A, suggesting that these genes may be part of the xylanolytic system from A. niger . This was confirmed using a XlnR negative mutant, which showed no ( lac A) or reduced ( agl B) expression of these genes on xylose.Synergy was studied between the accessory enzymes from Aspergillus involved in xylan degradation and two main chain cleaving enzymes, endoxylanase A (XlnA) andβ-xylosidase (XlnD). Except forα-L-arabinofuranosidase B (AbfB), the activity of all accessory enzymes on xylan was increased in the presence of XlnA. Similarly, the presence of accessory enzymes increased the activity of XlnA on xylan, indicating synergy between these enzymes. Synergy was also observed between the accessory enzymes, resulting in more efficient degradation of xylan. These results confirm that the gene products of XlnR regulated genes are in fact all part of the xylanolytic enzyme system of Aspergillus .Similarly, the synergy of enzymes involved in the degradation of the hairy regions in sugar beet pectin was studied. Degradation of the pectin backbone did not influence the activity of the arabinose releasing enzymes, AbfB and endoarabinase (AbnA), but had a strong effect on the release of ferulic acid by FaeA and the release of galactose by endogalactanase (GalA) andβ-galactosidase (LacA). Synergy was also observed between galactose- and ferulic acid- releasing enzymes.The accessory enzymes from Aspergillus involved in the degradation of xylan and pectin form a diverse group of enzymes which actively co-operate in polysaccharide degradation. Common factors have been identified in the regulation of the genes encoding these enzymes, but the expression patterns of the different genes also indicate the presence of other factors influencing specific genes. This most likely enables Aspergillus to modulate the production of these enzymes to obtain an efficient mixture for the degradation of the variety of substrates it encounters.</p

Analysis of regulation of pentose utilisation in Aspergillus niger reveals evolutionary adaptations in Eurotiales

Aspergilli are commonly found in soil and on decaying plant material. D-xylose and L-arabinose are highly abundant components of plant biomass. They are released from polysaccharides by fungi using a set of extracellular enzymes and subsequently converted intracellularly through the pentose catabolic pathway (PCP)

Growth and hydrolase profiles can be used as characteristics to distinguish Aspergillus niger and other black aspergilli

Wild type Aspergillus niger isolates from different biotopes from all over the world were compared to each other and to the type strains of other black Aspergillus species with respect to growth and extracellular enzyme profiles. The origin of the A. niger isolate did not result in differences in growth profile with respect to monomeric or polymeric carbon sources. Differences were observed in the growth rate of the A. niger isolates, but these were observed on all carbon sources and not specific for a particular carbon source. In contrast, carbon source specific differences were observed between the different species. Aspergillus brasiliensis is the only species able to grow on D-galactose, and A. aculeatus had significantly better growth on Locus Bean gum than the other species. Only small differences were found in the extracellular enzyme profile of the A. niger isolates during growth on wheat bran, while large differences were observed in the profiles of the different black aspergilli. In addition, differences were observed in temperature profiles between the black Aspergillus species, but not between the A. niger isolates, demonstrating no isolate-specific adaptations to the environment

Isolation and characterization of two specific regulatory Aspergillus niger mutants shows antagonistic regulation of arabinan and xylan metabolism

This paper describes two Aspergillus niger mutants (araA and araB) specifically disturbed in the regulation of the arabinanase system in response to the presence of L-arabinose. Expression of the three known L-arabinose-induced arabinanolytic genes, abfA, abfB and abnA, was 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 activities Of L-arabitol dehydrogenase and L-arabinose reductase, involved in L-arabinose catabolism, were decreased in the araA and araB strains. Finally, the data show that the gene encoding D-xylulose kinase, xkiA, is also under control of the arabinanolytic regulatory system. L-Arabitol, most likely the true inducer of the arabinanolytic and L-arabinose catabolic genes, accumulated to a high intracellular concentration in the araA and araB mutants. This indicates that the decrease of expression of the arabinanolytic genes was not due to lack of inducer accumulation. Therefore, it is proposed that the araA and araB mutations are localized in positive-acting components of the regulatory system involved in the expression of the arabinanase-encoding genes and the genes encoding the L-arabinose catabolic pathway

Genomic and exoproteomic diversity in plant biomass degradation approaches among Aspergilli

We classified the genes encoding carbohydrate-active enzymes (CAZymes) in 17 sequenced genomes representing 16 evolutionarily diverse Aspergillus species. We performed a phylogenetic analysis of the encoding enzymes, along with experimentally characterized CAZymes, to assign molecular function to the Aspergilli CAZyme families and subfamilies. Genome content analysis revealed that the numbers of CAZy genes per CAZy family related to plant biomass degradation follow closely the taxonomic distance between the species. On the other hand, growth analysis showed almost no correlation between the number of CAZyme genes and the efficiency in polysaccharide utilization. The exception is A. clavatus where a reduced number of pectinolytic enzymes can be correlated with poor growth on pectin. To gain detailed information on the enzymes used by Aspergilli to breakdown complex biomass, we conducted exoproteome analysis by mass spectrometry. These results showed that Aspergilli produce many different enzymes mixtures in the presence of sugar beet pulp and wheat bran. Despite the diverse enzyme mixtures produced, species of section Nigri, A. aculeatus, A. nidulans and A. terreus, produce mixtures of enzymes with activities that are capable of digesting all the major polysaccharides in the available substrates, suggesting that they are capable of degrading all the polysaccharides present simultaneously. For the other Aspergilli, typically the enzymes produced are targeted to a subset of polysaccharides present, suggesting that they can digest only a subset of polysaccharides at a given time.Peer reviewe

Теплофизические модели слоисто-неоднородных горных массивов

Стисло розглянуто математичні моделі процесів переносу тепла в шаруватонеоднорідних гірничих масивах. Запропоновано загальний метод моделювання теплопереносу в шаруватих системах різної геометрії. Знайдено рівняння «склеювання», за допомогою якого розглянуто асимптотичні випадки.Mathematical models of heat transfer in layered inhomogeneous rock media are summarized. A general method of modeling the heat transfer in layered systems of a different geometry is proposed. A “matching” equation for different asymptotic cases has been found