Showing papers on "Aspergillus niger published in 1991"

Localization of growth and secretion of proteins in aspergillus-niger

Abstract: Hyphal growth and secretion of proteins in Aspergillus niger were studied using a new method of culturing the fungus between perforated membranes which allows visualization of both parameters. At the colony level the sites of occurrence of growth and general protein secretion were correlated. In 4-d-old colonies both growth and secretion were localized at the periphery of the colony, whereas in a 5-d-old colony growth and secretion also occurred in a more central zone of the colony where conidiophore differentiation was observed. However, in both cases glucoamylase secretion was mainly detected at the periphery of the colonies. At the hyphal level immunogold labelling showed glucoamylase secretion at the tips of leading hyphae only. Microautoradiography after labelling with N-acetylglucosamine showed that these hyphae were probably all growing. Glucoamlyase secretion could not be demonstrated immediately after a temperature shock which stopped growth. These results indicate that glucoamylase secretion is located at the tips of growing hyphae only.

Structure of the Aspergillus niger pelA gene and its expression in Aspergillus niger and Aspergillus nidulans.

Abstract: Aspergillus niger pectin lyases are encoded by a multigene family. The complete nucleotide sequence of the pectin lyase PLA-encoding gene pelA has been determined. Comparison of the deduced amino acid sequence with the deduced amino acid sequence of the other characterized pectin lyase, PLD, shows that the proteins share 69% amino acid identity. When grown on media with pectin as the sole carbon source, A. niger transformants containing multiple copies of the pelA gene show raised mRNA levels and overexpression of the gene product PLA compared with the wild-type strain. PLA was purified and characterized. In A. nidulans transformants PLA is also produced in medium containing a high concentration of glucose and no pectin.

Commercial levels of chymosin production by Aspergillus.

Abstract: We have increased the production of bovine chymosin in Aspergillus niger var. awamori to more than one gram per liter of secreted authentic enzyme by combining a mutagenesis protocol with a novel robotic screening program. Analysis of the superior chymosin producing strains indicated that they have enhanced capabilities to secrete extracellular proteins.

Biosorption of zinc by fungal mycelial wastes

Abstract: Waste mycelia from several industrial fermentation plants (Aspergillus niger, Penicillium chrysogenum, Claviceps paspali) were used as a biosorbent for zinc ions from aqueous environments, both batchwise as well as in a column mode. With all mycelia testes, biosorption per biomass dry weight was a function of pH (increasing with increasing pH between 1.0 and 9.0), biomass concentration (decreasing at high biomass concentrations) and the zinc concentration. Under optimized conditions, A. niger and C. paspali were superior to P. chrysogenum. Treatment of A. niger biomass with NaOH further increased its biosorbent capacity. Desorption of biosorbed zinc was achieved by elution with 0.1 m HCl, best results being obtained with NaOH-treated A. niger. Such treatment did not affect the capacity for biosorption in repeated experiments. NaOH-treated A. niger mycelia were also successfully used in removal of zinc from polluted waters in Austria, thereby showing that the simultaneous presence of other naturally occurring ions does not affect biosorption.

Efficient KEX2-Like Processing of a Glucoamylase-Interleukin-6 Fusion Protein by Aspergillus Nidulans and Secretion of Mature Interleukin-6

Abstract: We have designed an expression vector for the secretion of human interleukin-6 (hIL-6) in which the mature protein is fused through a spacer peptide, containing a KEX-2 like protein processing signal, to the entire Aspergillus niger glucoamylase (glaA) gene. Transformation of Aspergillus nidulans with this vector results in fungal strains secreting equimolar amounts of the glucoamylase and IL-6 proteins. The KEX2-type processing signal, Lys-Arg, is recognized and cleaved efficiently by an enzyme present in A. nidulans resulting in the secretion of an authentic mature hIL-6 protein at levels of up to 5 mg/l.

The use of RFLP analysis in classification of the black Aspergilli: reinterpretation of the Aspergillus niger aggregate

Abstract: By studying ribosomal banding patterns in ethidium bromide-stained gels of chromosomal digests we are able to provide a rapid and reliable classification of a number of taxonomically important isolates of the black Aspergilli. This classification is supported by Southern blots using several pectin lyase genes, isolated from A. niger CBS 120.49, as probes. Taxonomy on the basis of RFLP analysis leads to a classification which is completely different from, but more reliable than, the current one which is mainly based on morphological characteristics. The 23 Aspergillus niger isolates investigated could be divided into two distinct groups on the basis of our results. We propose that these groups represent two different species: A. niger and A. tubigensis. This is supported by preliminary results showing failure of heterokaryon formation between typical representatives of both groups.

Expression and sequence comparison of the Aspergillus niger and Aspergillus tubigensis genes encoding polygalacturonase II.

Abstract: The structure and expression of the polygalacturonase-encoding pgaII genes of two recently recognized species, Aspergillus niger and Aspergillus tubigensis, was investigated. While the structure of the pgaII genes is very similar, showing 83% DNA sequence identity and 94% identity at the amino acid level, they have diverged significantly. The NH2-terminal sequence suggests that these PGs are made as pre pro-proteins and the secretory propeptide of the PGII precursors shows sequence homology with some other fungal pro-peptides. The expression of the pgaII genes is strongly regulated by the carbon source and the A. tubigensis gene is expressed and regulated in A. niger transformants. The low similarity of the fungal PGs with those of bacterial and plant origin is discussed in relation to the possible functional role of specific amino acids.

Leaching with Penicillium simplicissimum: Influence of Metals and Buffers on Proton Extrusion and Citric Acid Production.

Abstract: In the presence of insoluble metal oxides (industrial filter dust, zinc oxide, synthetic mixture of metal oxides), Penicillium simplicissimum developed the ability to excrete considerable amounts of citric acid (>100 mM). Parallel with the increase of citric acid concentration in the culture broth, zinc was solubilized from zinc oxide. The adsorption of filter dust onto the mycelium (the pellets formed were less than 1 mm in diameter) was required for not only the citric acid excretion but also the leaching of zinc. When the filter dust was replaced with a synthetic mixture of metal oxides or with zinc oxide in combination with trace elements, levels of adsorption and citric acid production were observed to be similar to those in experiments where industrial filter dust was used. The two most important properties of the filter dust were its heavy-metal content and its buffering capacity. These properties were simulated by adding heavy metals in soluble form (as chlorides, sulfates, or nitrates) or soluble buffers to the medium. Both heavy metals and buffers were not able to induce a citric acid efflux. As with citric acid production by Aspergillus niger, the addition of manganese lowered citric acid excretion (by 40% with metal oxide-induced citric acid efflux and by 100% with urea-induced citric acid efflux). Copper antagonized the effect of manganese. The mechanism for the bulk of citric acid excretion by P. simplicissimum, however, seemed to be different from that described for citric acid accumulation by A. niger. Because of the inefficiency of metals in solubilized form and of soluble buffers to induce a strong citric acid efflux, adsorption of an insoluble metal compound (zinc oxide) turned out to be essential. Surface phenomena possibly involving the plasma membrane H+-ATPase are thought to participate in the induction of citric acid excretion by P. simplicissimum in the presence of industrial filter dust.

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

Abstract: 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.

The use of Aspergillus niger for the bioconversion of olive mill waste-waters

Abstract: Olive mill waste-water was used for protein production in small-scale experiments, using non-sterilized medium without pH control. A 14 g/1 concentration of proteins, 61% chemical oxygen demand removal and a 58% reduction in total phenolic compounds were obtained using an Aspergillus niger strain. The removal of phenolic compounds resulted in a change in the colour of the waste-water from black to brown.

Identification and characterization of a second polygalacturonase gene of Aspergillus niger

Abstract: The filamentous fungus Aspergillus niger produces several endopolygalacturonases that are involved in the degradation of pectin. PGI, the enzyme representing the second most abundant activity in a commercial enzyme preparation, was further characterized and the corresponding gene was isolated. The nucleotide sequence of the pgaI gene was determined and the protein coding region was found to be interrupted by two short introns, one of which has a unusual donor splice site. The deduced 368 amino acids long protein with a putative prepropeptide of 31 amino acids shows 60% sequence identity to PGII in the mature protein. PGI overproducing A. niger strains were obtained by cotransformation with the cloned gene.

Intracellular location of enzymes involved in citrate production by Aspergillus niger

Abstract: The intracellular distribution and maximal activities of nine enzymes involved in the biosynthesis and degradation of citric acid in Aspergillus niger were determined under conditions of growth and of citric acid production. Under these conditions the intracellular location of the enzymes in most cases resembled that described for other filamentous fungi. Pyruvate carboxylase was found predominantly or exclusively in the cytosol. A single isoenzyme of NADP–isocitrate dehydrogenase was present, which appeared to be localised in the mitochondrion. No significant differences in maximal enzyme activities were observed except for NADP–isocitrate dehydrogenase, which showed decreased activity in production-phase mycelia. The results obtained support the scheme proposed by C. P. Kubicek for the intracellular organisation of citric acid formation but provide little evidence that this process is controlled at the level of the biosynthesis of any of the enzymes examined here. Key words: pyruvate carboxylase, citric...

Fermentation of a yeast producing A. niger glucose oxidase: scale-up, purification and characterization of the recombinant enzyme.

Abstract: We have developed a fermentation process to produce up to 3 grams per liter of active, secreted glucose oxidase from a recombinant Saccharomyces cerevisiae. Real-time size-exclusion HPLC analysis is used to monitor enzyme production during fermentation, and purification to more than 95 percent is obtained using only filtration methods. The recombinant enzyme is stable to higher temperatures and a wider pH range than the native Aspergillus niger enzyme, and is free of contaminating amylase, cellulase and catalase.

Optimization of cellulase production by Aspergillus niger NCIM 1207.

Abstract: Aspergillus niger NCIM 1207 produces high levels of extracellular β-glucosidase and xylanase activities in submerged fermentation Among the nitrogen sources, ammonium sulfate, ammonium dihy-drogen orthophosphate, and corn-steep liquor were the best for the production of cellulolytic enzymes by A niger The optimum pH and temperature for cellulase production were 30-55 and 28°C, respectively The cellulase complex of this strain was found to undergo catabolite repression in the presence of high concentrations of glucose Glycerol at all concentrations caused catabolite repression of cellulase production The addition of glucose (up to 1% concentration) enhanced the production of cellulolytic enzymes, but a higher concentration of glucose effected the pronounced repression of enzymes Generally the growth on glucose- or glycerol-containing medium was accompanied by a sudden drop in the pH of the fermentation medium to 20

Optimization of the fermentation of olive mill waste-waters by Aspergillus niger

Abstract: The fermentation of olive mill waste-waters (OMW) by Aspergillus niger was studied. On the basis of factorial design experiments, suspended solids and concentration of OMW, nitrogen source, sulphate and size of inocula were all found to be significant by affecting mycelium growth and chemical oxygen demand (COD) removal. Neither the absence of yeast extract, magnesium, sodium, potassium nor of calcium limited the growth of A. niger. With media lacking additional nitrogen and sulphate, the growth was limited. The optimal inoculum obtained was between 106 and 107 spores/g COD. The highest biomass and the greatest COD removal were obtained with removed COD to N:SO inf4 sup¨- ratios averaging 100 to 3:1.5.

Production of fructooligosaccharides from sucrose by a transfructosylase from Aspergillus niger

Abstract: A strain of Aspergillus niger isolated from sugarcane fields, produced an extracellular transfructosylase in the culture medium. Sucrose and raffinose induced the production to the enzyme, which was purified by 138-fold. The optimum pH for activity and stability were 5.5 and 6.5, respectively. Its optimum temperature was 55°C. The enzyme hydrolysed sucrose rapidly and simultaneously formed fructooligosaccharides by transfructosylation.

Submerged citric acid fermentation: rheological properties ofAspergillus niger broth in a stirred tank reactor

Abstract: A large number of submerged citric acid fermentations in a beet molasses substrate was studied. The development of Aspergillus niger from conidia to pellets was followed. Rheological characteristics of the fermentation broth including the pellets were determined. The results obtained confirm the fact that the non-Newtonian pseudoplastic behaviour of the fermentation broth was due to the presence of mycelial pellets. The most significant changes in rheological properties occurred during the period of maximal citric acid production and increase in biomass.

Concomitant loss of respiratory chain NADH: ubiquinone reductase (complex I) and citric acid accumulation in Aspergillus niger

Abstract: Growth, citric acid production and enzymatic activity of the mitochondrial respiratory enzymes of a wild-type and a citric-acid-producing mutant of Aspergillus niger have been compared during fermentation under citric-acid-accumulating and non-accumulating conditions. Under non-accumulating conditions, both strains showed standard growth and no citric acid production. The mutant strain was characterized by delayed onset of growth and lowered cell yield. Under citric-acid-accumulating conditions the wild-type strain exhibited decelerated growth and a maximal citric acid concentration of 12 g l−1. Reduced, but continuing growth and citric acid production of 32 g l−1 was observed for the mutant strain. In general, the mutant strain exhibited reduced activity for the proton-pumping respiratory complexes and enhanced activity for the alternative respiratory enzymes. In contrast to the stable activity of complex I in the wild-type strain, this complex was selectively lost in the mutant strain at the onset of citric acid production, while the alternative NADH dehydrogenases were kept at enhanced and constant activity. A possible causal connection between the loss of complex I and citric acid accumulation is discussed.

Characterization of a polygalacturonase gene of Aspergillus niger RH5344.

Abstract: Summary We have cloned a gene encoding a polygalacturonase (PG) in the filamentous fungus Aspergillus niger RH5344. The structural gene comprises 1141 bp codIng for 362 amino acids and the open reading frame is disrupted by one intron of 52 bp. Eukaryotic consensus sequences for transcription regulation are found only in deviated forms. The biological functionality of the isolated PG gene was established by retransformation in A. niger and Aspergillus awamori. In addition, we have found that the PG protein of A. niger shares significant similarities with PG proteins from tomato and Erwinia carotovora. Comparison of the three enzymes revealed a highly conserved region in their C-terminal region probably comprising the elements of substrate binding and the catalytic centre.

Purification and characterization of an acid-resistant triacylglycerol lipase from Aspergillus niger

Abstract: An acid-resistant lipase from Aspergillus niger was purified from a crude commercial preparation using alcohol precipitation, acid treatment, size exclusion and ion-exchange chromatography, and polyacrylamide gel electrophoresis followed by electroelution. The commercial lipase was activated 3.7-fold by the alcohol precipitation and acid treatment steps. Overall purification was 40-fold with a yield of 36%. The N-terminal sequence of the electroeluted protein was found to be XVSTSTLDELQFALQ. The amino acid composition of the enzyme was also determined. The activity of the purified preparation toward triolein was determined using an emulsion hydrolysis assay and was found to be 170 units/mg protein

Phytic acid content reduction in canola meal by various microorganisms in a solid state fermentation process

Abstract: Solid state fermentation of canola meal has been carried out for the reduction of its phytic acid content using the following microorganisms: Rhizopus oligosporus NRRL 2990, Aspergillus niger NRC 5765 and NRC 401 121, Aspergillus ficuum NRRL 3135 and a wild Saccharomyces cerevisiae strain. The results showed that all these microorganisms can be used for the reduction of the phytic acid content in the tested material. A. ficuum which completely hydrolyzed the phytic acid in 48 hours was the most efficient. Buffered systems, aeration and an increase in inoculum concentration caused faster and higher reduction of phytic acid content in canola meal.