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PubMed1998

Recombinant glucoamylase production by Aspergillus niger B1 in chemostat and pH auxostat cultures.

The production of glucoamylase (GAM) by Aspergillus niger B1, a genetic transformant containing an additional 20 copies of the homologous glucoamylase gene (glaA) was studied in nutrient (maltodextrin)-limited chemostat and nutrient-excess pH auxostat cultures. In these culture systems the specific production rate of GAM increased with dilution rate and reached a maximum (up to 15.0 mg GAM [g biomass]-1 h-1) when A. niger B1 was grown at its maximum specific growth rate in pH auxostat culture, indicating that GAM is a growth-associated product. The appearance of spontaneous morphological mutants was observed in all continuous flow cultures grown at pH 5.4, with a light brown mutant always displacing the parental strain. However, no morphological mutants were observed in cultures grown at pH 4.0. Further, when A. niger B1 was grown in pH auxostat culture, the specific production rate of GAM was 31% higher at pH 4.0 than at pH 5.4. Southern blot analyses showed that some morphological mutants (including the light brown mutant) isolated from a pH auxostat culture had lost copies of the glaA genes.

Aspergillus niger↗
PubMed1996

Three-dimensional structure of Endo-1,4-beta-xylanase I from Aspergillus niger: molecular basis for its low pH optimum.

The crystal structure of endo-1,4-beta-xylanase I from Aspergillus niger has been solved by molecular replacement and was refined to 2.4 A resolution. The final R-factor for all data from 6 to 2.4 A is 17.9%. The A. niger xylanase has a characteristic fold which is unique for family G xylanases (root-mean-square deviation = 1.1 A to Trichoderma reesei xylanase I, which has 53% sequence identity). It consists of a single domain composed predominantly of beta-strands. Two beta-sheets are twisted around a deep, long cleft, which is lined with many aromatic amino acid residues and is large enough to accommodate at least four xylose residues. The two conserved glutamate residues, Glu79 and Glu170, which are likely to be involved in catalysis, reach into this cleft from opposite sides. A niger xylanase I is of particular commercial interest because of its low pH optimum. A model is proposed which explains this low pH optimum compared to other members of xylanase family G.

Amino Acid Sequence↗
PubMed1991

Induction, purification and characterisation of arabinases produced by Aspergillus niger.

The induction of arabinases in Aspergillus niger N400 was studied on different simple and complex carbon sources. Sugar beet pulp was found to be an inducer of three arabinan degrading enzymes (alpha-L-arabinofuranosidase A, alpha-L-arabinofuranosidase B and endoarabinase). These enzymes were purified from A. niger culture fluid after growth of the fungus in medium employing sugar beet pulp as the carbon source and were characterised both physico-chemically (Mw 83,000, 67,000, 43,000 Da and, pI 3.3, 3.5 and 3.0 for alpha-L-arabinofuranosidases A and B and endo-arabinase, respectively) and kinetically (Km on p-nitrophenyl-alpha-L-arabinofuranoside 0.68 and 0.52 mM for alpha-L-arabinofuranosidases A and B, resp.; Km on sugar beet arabinan 0.24 and 3.7 g/l for alpha-L-arabinofuranosidase B and endoarabinase, resp.). The amino acid compositions of the three enzymes were determined also. The enzymic properties were compared with those of arabinases purified from a commercial A. niger enzyme preparation. Differences were found though the kinetic data suggest considerable similarity between the enzymes from the different sources. Antibodies raised in mice against the three enzymes were found to be highly specific and no crossreactivity with other proteins present in culture filtrates was observed. A mixture of these antibodies has been used to analyze specific induction of these individual enzymes on simple and complex substrates by Western blotting.

Amino Acids↗
PubMed1990

Genetic analysis of Aspergillus niger: isolation of chlorate resistance mutants, their use in mitotic mapping and evidence for an eighth linkage group.

This paper describes the use of chlorate resistant mutants in genetic analysis of Aspergillus niger. The isolated mutants could be divided into three phenotypic classes on the basis of nitrogen utilization. These were designated nia, nir and cnx as for Aspergillus nidulans. All mutations were recessive to their wild-type allele in heterokaryons as well as in heterozygous diploids. The mutations belong to nine different complementation groups. In addition a complex overlapping complementation group was found. Evidence for the existence of eight linkage groups was obtained. Two linked chlorate resistance mutations and two tryptophan auxotrophic markers, which were unlinked to any of the known markers, form linkage group VIII. We used the chlorate resistance mutations as genetic markers for the improvement of the mitotic linkage map of A. niger. We determined the linear order of three markers in linkage group VI as well as the position of the centromere by means of direct selection of homozygous cnxA1 recombinants. In heterozygous diploid cultures diploid chlorate resistant segregants appeared among conidiospores with a frequency of 3.9 x 10(-5) (cnxG13 in linkage group I) to 2.1 x 10(-2) (cnxD6 in linkage group III). The mean frequency of haploid chlorate resistant segregants was 1.3 x 10(-3). The niaD1 and niaD2 mutations were also complemented by transformation with the A. niger niaD+ gene cloned by Unkles et al. (1989). Mitotic stability of ten Nia+ transformants was determined. Two distinct stability classes were found, showing revertant frequencies of 5.0 x 10(-3) and 2.0 x 10(-5) respectively.

Aspergillus niger↗
PubMed1992

Cloning and expression of a member of the Aspergillus niger gene family encoding alpha-galactosidase.

An enzyme with alpha-galactosidase activity and an apparent molecular weight of 82 kDa was purified from culture medium of Aspergillus niger. The N-terminal amino acid sequence of the purified protein shows similarity to the N-terminal amino acid sequence of alpha-galactosidases from several other organisms. Oligonucleotides, based on the N-terminal amino acid sequence, were used as probes to clone the corresponding gene from a lambda EMBL3 gene library of A. niger. The cloned gene (aglA) was shown to be functional by demonstrating that the 82 kDa alpha-galactosidase is absent from a strain with a disruption of the aglA gene, and is over-produced in strains containing multiple copies of the aglA gene. Enzyme activity assays revealed that the 82 kDa alpha-galactosidase A represents a minor extracellular alpha-galactosidase activity in A. niger.

Amino Acid Sequence↗
PubMed1988

Nucleotide sequence of the Aspergillus niger trpC gene: structural relationship with analogous genes of other organisms.

The nucleotide sequence of the Aspergillus niger tryptophan C (trpC) gene was determined. Northern hybridization and S1-mapping experiments showed the presence of a 2.6 kb trpC poly(A)+ RNA with two very short (5 and 6 nucleotides) noncoding 5'-regions. Comparison of the predicted amino acid sequence with that of trp gene proteins of pro- and eukaryotic organisms revealed three functional domains (G, C, F) in the A. niger TrpC protein which catalyse the glutamine amidotransferase reaction (GAT), the indole-3-glycerol phosphate synthase reaction (IGPS) and the N-(5'-phosphoribosyl) anthranilate isomerase reaction (PRAI), respectively. These domains are highly conserved and bordered by short areas showing less homology. Within the F domain of the trpC gene in A. niger, A. nidulans and Neurospora crassa, a region encoding 30 amino acids was found which is absent in the analogous genes of Saccharomyces cerevisiae and prokaryotic organisms. This region has features of a mutated in-phase intron.

Amino Acid Sequence↗
PubMed1992

Arginine and proline genes of Aspergillus niger.

Aspergillus niger mutants defective in arginine or proline biosynthesis have been isolated and 12 genetic loci were identified. Mutation was induced by low doses UV, and mutants were isolated after filtration enrichment. The mutants were classified according to their phenotype in growth tests and were further characterized in complementation tests. The arginine auxotrophic mutants represent nine complementation groups. Three additional complementation groups were found for mutants that could grow on proline (two of them on arginine too). Linkage group analysis was done in somatic diploids obtained from a mutant and a master strain with genetic markers on six chromosomes. The arg genes belong to six different linkage groups and the pro genes to two. One arg-mutant could be complemented by transformation with the A. nidulans argB+ gene, and this A. niger gene thus appeared to be homologous to the A. nidulans argB. We isolated an A. niger strain with the argB gene tightly linked with the nicA1 marker. This strain is very suitable as acceptor for transformation with an argB-plasmid, because transformants with inserts on the homologous site can be recognized and analyzed genetically using the nicA1 marker gene.

Arginine↗
PubMed1989

Amino acid sequence of cytochrome c from Aspergillus niger.

Cytochrome c from Aspergillus niger consists of two forms, a major one (80%) with 111 amino acid residues and a minor one (20%) with 108 residues, missing the three N-terminal residues of the major one. The primary sequence of A. niger cytochrome c was determined by standard spinning-cup Edman degradation of purified peptides and of pairs of peptides, from which the desired sequence was readily deduced by subtraction of common sequencies. Except for the extension and some variability at the N-terminal sequence, the A. niger protein conforms well with other cytochrome c structures.

Amino Acid Sequence↗
PubMed1996

Cloning and characterisation of genes (pkc1 and pkcA) encoding protein kinase C homologues from Trichoderma reesei and Aspergillus niger.

Oligonucleotides, designed on the basis of conserved flanking amino acid sequence segments within the catalytic domain of eukaryotic protein kinase C (PKC) proteins, were used as primers for polymerase chain reactions to amplify a 427-bp chromosomal DNA fragment from the filamentous fungus Trichoderma reesei. This fragment was then used to isolate genes encoding PKC homologues of T. reesei and Aspergillus niger (pkc1 and pkcA, respectively). The genes contain six (T. reesei) and eight (A. niger) introns, which exhibit notable conservation in position with those found in the corresponding Schizosaccharomyces pombe pkc1+ and Drosophila melanogaster dPKC53Ebr genes. A single 4.2-kb transcript was detected in Northern analyses. The deduced PKC1 (T.reesei, 126 kDa) and PKCA (A. niger, 122 kDa) amino acid sequences reveal domains homologous to the C1 and C3/C4 domains of PKC-related proteins, but lack typical Ca(2+)-binding (C2) domains. Both contain a large, extended N-terminus, which shares a high degree of similarity with the corresponding regions of Saccharomyces cerevisiae PKC1 and S. pombe pkc1+ and pkc2+ proteins, but which is not present in PKCs of Dictyostelium or higher eukaryotes. This extended region can be divided into three subdomains; the N-terminal one contains a hydrophobic helix-turn-helix motif, whereas the C-terminal one contains potential targets for proteolytic processing. A polyclonal antiserum raised against the pseudosubstrate-binding domain of PKC1 recognizes in T. reesei a 115-120 kDa protein in Western blots. Expression of pkc1 cDNA in insect cells directs the synthesis of a PKC1 protein of similar size. The T. reesei PKC1 protein was partially purified and some of its properties examined: it is stimulated about twofold by phospholipids or phorbol esters but is not stimulated by Ca2+. We conclude that these PKC proteins from filamentous fungi represent the Ca(2+)-insensitive fungal homologues of the nPKC family.

Amino Acid Sequence↗
PubMed1997

Synthesis of isopropyl-1-thio-beta-D-glucopyranoside (IPTGlc), an inducer of Aspergillus niger B1 beta-glucosidase production.

Production of beta-glucosidase in Aspergillus niger B1 is subjected to catabolic repression by glucose. Aspergillus niger B1 grown on bran as a carbon source secreted beta-glucosidase. The maximum level of the enzyme was reached after 7 d of fermentation. Addition of 1% glucose to the medium suppressed beta-glucosidase production to undetectable levels. In this study, the organic synthesis of a potential inducer of beta-glucosidase production by A. niger B1's reported. Isopropyl-1-thio-beta-D-glucopyranoside (IPTGlc) was synthesized using a two-step organic synthesis protocol. The H-NMR data agreed with those reported previously for the galactoside analog. When IPTGlc was added 24 h after inoculation at a final concentration of 0.4 mM, similar levels of beta-glucosidase were reached 3 to 4 d earlier as compared to fermentation without IPTGlc induction. In practice, this may translate to a more efficient method of producing beta-glucosidase from this fungus.

Aspergillus niger↗
PubMed1992

Protection of Aspergillus niger cellulases by urea during growth on glucose or glycerol supplemented media.

The cellulase enzymes of Aspergillus niger were found to undergo catabolite repression in the presence of glucose and glycerol accompanied by sudden drop in pH of the fermentation medium below 2.0. This sudden drop in pH caused inactivation of cellulolytic enzymes produced by Aspergillus niger. The supplementation of nitrogen sources, especially urea, protects A. niger cellulases from inactivation caused by a sudden drop in pH, since urea helped to maintain the pH of the fermentation medium between 3.5 and 4.5. The role of urea in the protection of cellulase was more prominent when it was used in combination with glycerol (5%).

Aspergillus niger↗
PubMed1996

Detection of endogenous beta-glucuronidase activity in Aspergillus niger.

An endogenous beta-glucuronidase that hydrolyses the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide (X-gluc) in Aspergillus niger is reported. The activity was induced when the fungus was grown in media containing xylan, but was either very low, or absent, when grown on glucose. Endogenous beta-glucuronidase was primarily located in newly formed hyphae, and was apparent at pH values between 3 and 6. Hydrolysis of X-gluc was sensitive to the inhibitor D-saccharic acid 1,4-lactone and was irreversibly inactivated by heating. The bacterial uid A beta-glucuronidase reporter gene was strongly expressed in the hyphae of transformed A. niger but, in contrast to the endogenous activity, the enzyme was also active at pH 7-8.5. Histochemical localization of uidA expression in A. niger, without interference from the endogenous beta-glucuronidase activity, was achieved by staining at this pH.

Aspergillus niger↗
PubMed1998

Secretion, purification, and characterisation of barley alpha-amylase produced by heterologous gene expression in Aspergillus niger.

Efficient production of recombinant barley alpha-amylase has been achieved in Aspergillus niger. The cDNA encoding alpha-amylase isozyme 1 (AMY1) and its signal peptide was placed under the control of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter and the A. nidulans trpC gene terminator. Secretion yields up to 60 mg/l were obtained in media optimised for alpha-amylase activity and low protease activity. The recombinant AMY1 (reAMY1) was purified to homogeneity and found to be identical to native barley AMY1 with respect to size, pI, and immunoreactivity. N-terminal sequence analysis of the recombinant protein indicated that the endogenous plant signal peptide is correctly processed in A. niger. Electrospray ionisation/mass spectrometry gave a molecular mass for the dominant form of 44,960 Da, in accordance with the loss of the LQRS C-terminal residues; glycosylation apparently did not occur. The activities of recombinant and native barley alpha-amylases are very similar towards insoluble and soluble starch as well as 2-chloro-4-nitrophenol beta-D-maltoheptaoside and amylose (degree of polymerisation = 17). Barley alpha-amylase is the first plant protein efficiently secreted and correctly processed by A. niger using its own signal sequence.

Aspergillus niger↗
PubMed2006

Biosorption potency of Aspergillus niger for removal of chromium (VI).

Aspergillus niger isolated from soil and effluent of leather tanning mills had higher activity to remove chromium. The potency of Aspergillus niger was evaluated in shake flask culture by absorption of chromium at pH 6 and temperature 30 degrees C. The results of the study indicated removal of more than 75% chromium by Aspergillus niger determined by diphenylcarbazide colorimetric assay and atomic absorption spectrophotometry after 7 days. Study of microbial Cr(VI) reduction and identification of reduction intermediates has been hindered by the lack of analytical techniques that can identify the oxidation state with subcellular spatial resolution. Therefore, removal of chromium was further substantiated by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDX), which indicated an accumulation of chromium in the fungal mycelium.

Aspergillus niger↗
PubMed1996

Isolation by genetic complementation of two differentially expressed genes for beta-isopropylmalate dehydrogenase from Aspergillus niger.

We have constructed an Aspergillus niger cDNA library with a yeast expression vector. The library DNA complemented a leucine auxotroph of Saccharomyces cerevisiae (strain BWG1-7a) at a frequency of 4x10(-4). Plasmids rescued from the yeast prototrophs also complemented Escherichia coli (strain MC1066) deficient in leucine biosynthesis. Sequence determination of the rescued plasmids revealed two genes for beta-isopropylmalate dehydrogenase, which we called leu2A and leu2B. Genomic-blot analysis suggested that both leu2A and leu2B were derived from single-copy genes. Northern-blot hybridization showed that in nutrient-rich medium a leu2A transcript accumulated during germination and log-phase growth while the leu2B transcript appeared late in the growth phase. In minimal medium, only leu2A expression was greatly stimulated. We examined the codon preference of these two genes. Whereas leu2A shows a bias in codon usage typical of A. niger genes, leu2B does not. These results indicate the presence in A. niger of two highly divergent, differentially regulated, isozymes for beta-isopropylmalate dehydrogenase.

3-Isopropylmalate Dehydrogenase↗
PubMed1997

Arabinoxylan degradation by fungi: characterization of the arabinoxylan-arabinofuranohydrolase encoding genes from Aspergillus niger and Aspergillus tubingensis.

The genes encoding the enzyme arabinoxylan arabinofuranohydrolase, which releases L-arabinose from arabinoxylan, have been cloned from the closely related fungi Aspergillus niger and Aspergillus tubingensis and were shown to be functional in A. niger. Integration of multiple copies in the genome resulted in over-expression of the enzymes. The arabinofuranohydrolases encoded comprise 332 amino acids and have 94% amino acid identity. Their primary structure is not related to those of other alpha-L-arabinofuranosidases, except for a low similarity with XYLC, a bacterial alpha-L-arabinofuranosidase from Pseudomonas fluorescens which acts on oat spelt xylan. The axhA expression pattern in A. niger differed from that of abfB, since it was strongly induced by birchwood xylan and much less by L-arabitol or L-arabinose. Furthermore, Northern analysis revealed that axhA expression was de repressed in creAd mutants and carbon catabolite repressed by D-glucose.

Amino Acid Sequence↗
PubMed1999

Cloning and expression of the cDNA encoding an alternative oxidase gene from Aspergillus niger WU-2223L.

A cDNA fragment encoding the mitochondrial alternative oxidase, the enzyme responsible for cyanide-insensitive and salicylhydroxamic acid (SHAM)-sensitive respiration, from the citric acid-producing fungus Aspergillus niger WU-2223L was cloned and expressed in Escherichia coli as a host strain. Synthetic primers were designed from the conserved nucleotide sequences of the alternative oxidase genes from higher plants and a yeast. The 210-bp DNA fragment was amplified by PCR with these primers using chromosomal DNA of WU-2223L as a template, and was employed to screen a cDNA library of A. niger. One full-length cDNA clone of 1.2 kb was obtained, and was sequenced to reveal that the clone contained an open reading frame (ORF-AOX1) encoding a polypeptide of 351 amino acids. The predicted amino-acid sequence exhibited 50%, 55%, and 52% homology to the alternative oxidases of Hansenula anomala, Neurospora crassa and Sauromatum guttatum, respectively. In the 5'-terminus region of the ORF-AOX1, a mitochondrial targeting motif was found. The whole open reading frame of ORF-AOX1 was ligated to plasmid pKK223-3 to construct the expression vector pKAOX1. The E. coli transformant harboring pKAOX1 showed cyanide-insensitive and SHAM-sensitive respiration, and expression was increased approximately two-fold by the addition of IPTG. These results indicated that the ORF-AOX1 encodes an alternative oxidase of A. niger.

Amino Acid Sequence↗
PubMed2002

Regulation of the alpha-glucuronidase-encoding gene ( aguA) from Aspergillus niger.

The alpha-glucuronidase gene aguA from Aspergillus niger was cloned and characterised. Analysis of the promoter region of aguA revealed the presence of four putative binding sites for the major carbon catabolite repressor protein CREA and one putative binding site for the transcriptional activator XLNR. In addition, a sequence motif was detected which differed only in the last nucleotide from the XLNR consensus site. A construct in which part of the aguA coding region was deleted still resulted in production of a stable mRNA upon transformation of A. niger. The putative XLNR binding sites and two of the putative CREA binding sites were mutated individually in this construct and the effects on expression were examined in A. niger transformants. Northern analysis of the transformants revealed that the consensus XLNR site is not actually functional in the aguA promoter, whereas the sequence that diverges from the consensus at a single position is functional. This indicates that XLNR is also able to bind to the sequence GGCTAG, and the XLNR binding site consensus should therefore be changed to GGCTAR. Both CREA sites are functional, indicating that CREA has a strong influence on aguA expression. A detailed expression analysis of aguA in four genetic backgrounds revealed a second regulatory system involved in activation of aguA gene expression. This system responds to the presence of glucuronic and galacturonic acids, and is not dependent on XLNR.

Aspergillus niger↗