Aspergillus Niger Var Research Articles

Synthesis of silver nanoparticles embedded on silica by gamma irradiation for utilization as antimicrobial agents

Silver nanoparticles deposited on silica (Ag nano/SiO2) have been prepared by Co-60 gamma irradiation of the mixture Ag+/SiO2/H2O/ethanol. The reduction of Ag+ in the suspension of SiO2 is brought by e-aq and H· generated during the radiolysis of water/ethanol solution. The presence of SiO2 prevents agglomeration of Ag clusters. The conversion dose (Ag+® Ag0) was determined by UV-Vis spectroscopy. Transmission electron microscopy (TEM) characterized the size of Ag nanoparticles. As a result, the particle sizes were determined to be in the range of 5-20 nm for an Ag+ concentration of 5 mM. The crystal structure of silver nanoparticles was also investigated by X-ray diffraction (XRD). In addition, the antifungal activity of Ag nano/SiO2 was tested against Aspergillus niger var Tieghn by plate count method. The results indicated that the antifungal efficiency of Ag nano/SiO2 was about 64, 71, 81, 82, and 96% at the concentrations of Ag nanoparticles of 30, 50, 70, 100, and 150 ppm, respectively.

The degradation of an aromatic organic compound by Aspergillus niger var tubingensis Ed8 produces metabolites that reduce Cr (VI)

Abstract Chromium Cr(VI) is a highly toxic environmental contaminant for any organism, its presence in the environment is mainly due to anthropogenic activities. The use of biotechnology has been implemented for the treatment of effluents contaminated with Cr(VI).Our working group has isolated several fungi and bacteria capable of removing Cr(VI) from the culture medium. Aspergillus niger var tubingensis Ed8 is a strain that can produce metabolites which reduce Cr (VI) to Cr (III). The objective of this work was to determine the effect of sodium salicylate on the growth of this strain and on the Cr(VI) reduction system, as well as to identify the metabolites that are produced from sodium salicylate. Our results show that the Culture medium containing sodium salicylate (20 mM) inhibits strain growth compared to the control condition (0 mM). However, it increases the specific reduction capacity of Cr (VI) red/mg Biomass in order of magnitude. Analysis of the culture medium corresponding to 48 h of incubation shows the presence of catechol and salicylate diminution. In addition, as a product of the enzymatic activity of a cell-free cellular extract, after 24 h of incubation, the consumption of salicylate is detected, as well as the presence of peaks corresponding to resorcinol and catechol. Our results show that it is possible to increase the Cr(VI) reducing capacity of the Ed8 strain, depending on the composition of the culture medium.

Safety evaluation of a food enzyme containing aspergillopepsin I and II from the Aspergillus niger var. macrosporus strain PTG8398.

The food enzyme with aspergillopepsin I (EC 3.4.23.18) and aspergillopepsin II (EC 3.4.23.19) activities is produced with a non‐genetically modified Aspergillus niger var. macrosporus strain PTG8398 by Meiji Seika Pharma Co., Ltd. The food enzyme was considered free from viable cells of the production organism. It is intended to be used in wine production. Based on the maximum use levels, dietary exposure to the food enzyme‐total organic solids (TOS) was estimated to be up to 0.14 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 919 mg TOS/kg bw per day, the highest dose tested which, when compared with the estimated dietary exposure, results in a margin of exposure above 6,700. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and four matches with respiratory allergens were found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is considered low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Effect of pH and temperature on phytase and biomass production by submerged fermentation with Aspergillus niger var. phoenicis URM 4924

The production of phytase and biomass (estimated by the ergosterol content) by submerged fermentation with Aspergillus niger var. phoenicis URM 4924 was studied. Experimental assays were carried under different conditions of pH (4.0 to 8.0) and temperature (25 to 35 ºC), and the influence of these variables on the responses was studied through a 22 central composite design and response surface methodology. Phytase and biomass production were strongly affected by the pH and temperature used during fermentation. Phytase activity was increased in up to 7.8-fold (from 1.04 to 8.09 U/mL) and the ergosterol content was increased in up to 38-fold (from 9.3 to 354.09 µg/mL). The maximum values of both responses were achieved when using pH 4.0 and 30 ºC. Good correlation (second-order fit, R2 = 0.9875) was found between the data obtained for phytase activity and ergosterol content, suggesting that the phytase production depends on the biomass formation. These results are of interest since they contribute for the development of an industrial process for phytase production with elevated yields by submerged fermentation.

Phytase production by Aspergillus niger var. phoenicis URM 4924 using factorial planning

Phytates represent a complex class of natural occurring compounds formed during the maturation process of seeds and grains, the main storage form of phosphorus in vegetables. The phytate molecule is a major anti-nutritional factor for monogastrics animals, possessing in their structure highly ionizable groups orthophosphate, which affect the availability of minerals in the gastrointestinal tract. Phytases form a group of enzymes with the ability to release phosphorus bound to phytate molecule, with the aim of increasing the utilization of phosphorus for absorption. The aim of this study was to evaluate parameters of the medium, such as the concentration of nitrogen and carbon source in the production of phytase by Aspergillus niger var. phoenicis URM 4924, using a factorial design. To evaluate the influence of parameters of the culture medium in the production of phytase was carried out factorial design and response surface methodology. The best conditions for production of phytase (8.80 U/mL) in submerged fermentation by A. niger var. phoenicis URM 4924 were 1.25% of the carbon source (rice bran) and 3.0% nitrogen source (corn steep liquor). A. niger var. phoenicis URM 4924 has potential for biotechnological production of phytase can be added to feed non-ruminant animals, thus contributing to the increased use of nutrients and production rates.

Diverse and bioactive endophytic Aspergilli inhabit Cupressaceae plant family.

Aspergilli are filamentous, cosmopolitan and ubiquitous fungi which have significant impact on human, animal and plant welfare worldwide. Due to their extraordinary metabolic diversity, Aspergillus species are used in biotechnology for the production of a vast array of biomolecules. However, little is known about Aspergillus species that are able to adapt an endophytic lifestyle in Cupressaceae plant family and are capable of producing cytotoxic, antifungal and antibacterial metabolites. In this work, we report a possible ecological niche for pathogenic fungi such as Aspergillus fumigatus and Aspergillus flavus. Indeed, our findings indicate that A. fumigatus, A. flavus, Aspergillus niger var. niger and A. niger var. awamori adapt an endophytic lifestyle inside the Cupressaceous plants including Cupressus arizonica, Cupressus sempervirens var. fastigiata, Cupressus semipervirens var. cereiformis, and Thuja orientalis. In addition, we found that extracts of endophytic Aspergilli showed significant growth inhibition and cytotoxicity against the model fungus Pyricularia oryzae and bacteria such as Bacillus sp., Erwinia amylovora and Pseudomonas syringae. These endophytic Aspergilli also showed in vitro antifungal effects on the cypress fungal phytopathogens including Diplodia seriata, Phaeobotryon cupressi and Spencermartinsia viticola. In conclusion, our findings clearly support the endophytic association of Aspergilli with Cupressaceae plants and their possible role in protection of host plants against biotic stresses. Observed bioactivities of such endophytic Aspergilli may represent a significant potential for bioindustry and biocontrol applications.

Mechanisms of interaction of chromium with Aspergillus niger var tubingensis strain Ed8

Experiments were conducted to determine the mechanisms of interaction with chromium of Aspergillus niger var tubingensis strain Ed8 in batch culture and in bioreactor experiments. Results obtained in this work showed that the interaction of A. niger var tubingensis Ed8 with Cr(VI) is based mainly in a reduction process and also, secondly, in a sorption process. Using electron microscopy techniques the ultrathin sections obtained from the mycelium biomass produced by the fungus in batch cultures showed the ability to incorporate Cr intracellulary, into low electron-dense inclusions, but not extracellularly. On the other hand, cultures without Cr(VI) of A. niger var tubingensis Ed8, grown in a bubble column bioreactor, reduced Cr(VI) immediately after repeated addition of this oxyanion; after six loads, 460 mg Cr(VI) was reduced to Cr(III) in 60 h, corresponding to a reduction rate of 2.62 mg Cr(VI)g(-1) dry biomass h(-1).

Effects of coagulating enzyme types (commercial calf rennet, Aspergillus niger var. awamori as recombinant chymosin and rhizomucor miehei as microbial rennet) on the chemical and sensory characteristics of white pickled cheese

The possibilities of using recombinant chymosin as an alternative coagulant to commercial calf rennet in the production of white pickled cheese was investigated. For this purpose, white pickled cheese produced by using commercial calf rennet, recombinant chymosin ( Aspergillus niger var. awamori ) and microbial rennet ( Rhizomucor miehei ) were compared in terms of their chemical and organoleptic properties. The cheese samples were stored in brine containing 12% salt at 4±1°C for 60 days. In the study, which was carried out in duplicate, pH, titratable acidity, dry matter, fat, fat-in-dry matter, protein, salt, nitrogeneous compounds (water sobuble nitrogen (WSN), ripening index (RI), non-protein nitrogen (NPN), electrophoretic and organoleptic properties of the cheese samples were determined at 1, 15, 30 and 60 days of storage. According to the results, the effects of enzyme type on the titratable acidity, dry matter, salt, nitrogeneous compounds and all sensory properties, except for the odour was significant (p<0.05). At the end of storage, the titratable acidity, salt, WSN, RI, NPN values and sensory scores of the cheeses increased, while the pH, fat, total nitrogen (TN), protein, b- and a s1 -casein contents of cheeses decreased compared to initial values. Keywords: White pickled cheese, calf rennet, recombinant chymosin, microbial rennet. African Journal of Biotechnology Vol. 12(37), pp. 5588-5594

The crystal structure of an intermediate dimer of aspergilloglutamic peptidase that mimics the enzyme-activation product complex produced upon autoproteolysis

Aspergilloglutamic peptidase from Aspergillus niger var. macrosporus (AGP) is one of the so-called pepstatin-insensitive acid endopeptidases, which are distinct from the well-studied aspartic peptidases. Among the known homologues of the glutamic peptidases, AGP is a unique two-chain enzyme with a light chain and a heavy chain bound non-covalently with each other, and thus is an interesting target for protein structure-function relationship studies. In this article, we report the crystal structure of a dimeric form of the enzyme at a resolution of 1.6 Å. This form has a unique structure in which the C-terminal region of the light chain of one of the molecules binds to the active site cleft of the other molecule like a part of a substrate. This form mimics the enzyme-activation product complex produced upon autoproteolysis, and provides a structural clue that could help to clarify the activation mechanism. This type of dimeric structure of a peptidase is here reported for the first time.

Changes and induction of aminopeptidase activities in response to pathogen infection during germination of pigeonpea (Cajanas cajan) seeds

Aminopeptidases play important role in the mobilization of storage proteins at the cotyledon during seed germination. It is often referred as inducible component of defense against herbivore attack. However the role of aminopeptidase in response to pathogen attack in germinating seeds is remained to be unknown. An attempt was made to analyze change in the aminopeptidase (EC 3.4.11.1) activity during germination of pigeonpea (Cajanus cajan L.) seeds by infecting the seeds with fungi. Two aminopeptidase activity bands (AP1 and AP2) were detected in control as well as infected pigeonpea seeds. During latter stages of germination in control seeds, AP1 activity was replaced by AP2 activity. However AP1 activity was significantly induced in germinating seeds infected with Fusarium oxysporum f.sp. ciceri and Aspergillus niger var. niger. The estimated molecular weights of AP1 and AP2 were ∼97 and 42.8kDa respectively. The induced enzyme was purified up to 30 fold by gel filtration chromatography. The purified enzyme was preferentially cleaved leucine p-nitroanilide than alanine p-nitroanilide. The enzyme was strongly inhibited by bestatin and 1,10-phenanthroline. Almost 50% of enzyme activity was inhibited by ethylene diamine tetra acetate. The purified enzyme showed broad pH optima ranging from pH 6.0 to 9.0 and optimum at pH 8.5. The induction of aminopeptidase activity during pigeonpea seed germination and in response to pathogen attack indicates significant involvement of these enzymes in primary as well as secondary metabolism of the seeds. These findings could be helpful to further dissect defensive role of aminopeptidases in seed germination which is an important event in plant's life.

A novel protease-resistant α-galactosidase with high hydrolytic activity from Gibberella sp. F75: gene cloning, expression, and enzymatic characterization

A novel alpha-galactosidase gene (aga-F75) from Gibberella sp. F75 was cloned and expressed in Escherichia coli. The gene codes for a protein of 744 amino acids with a 24-residue putative signal peptide and a calculated molecular mass of 82.94 kDa. The native structure of the recombinant Aga-F75 was estimated to be a trimer or tetramer. The deduced amino acid sequence showed highest identity (69%) with an alpha-galactosidase from Hypocrea jecorina (Trichoderma reesei), a member of the glycoside hydrolase family 36. Purified recombinant Aga-F75 was optimally active at 60 degrees C and pH 4.0 and was stable at pH 3.0-12.0. The enzyme exhibited broad substrate specificity and substantial resistance to neutral and alkaline proteases. The enzyme K (m) values using pNPG, melibiose, stachyose, and raffinose as substrates were 1.06, 1.75, 54.26, and 8.23 mM, respectively. Compared with the commercial alpha-galactosidase (Aga-A) from Aspergillus niger var. AETL and a protease-resistant alpha-galactosidase (Aga-F78) from Rhizopus sp. F78, Aga-F75 released 1.4- and 4.9-fold more galactose from soybean meal alone, respectively, and 292.5- and 8.6-fold more galactose from soybean meal in the presence of trypsin, respectively. The pH and thermal stability and hydrolytic activity of Aga-F75 make it potentially useful in the food and feed industries.

Probing the role of N-linked glycans in the stability and activity of fungal cellobiohydrolases by mutational analysis

The filamentous fungi Trichoderma reesei and Penicillium funiculosum produce highly effective enzyme mixtures that degrade the cellulose and hemicellulose components of plant cell walls. Many fungal species produce a glycoside hydrolase family 7 (Cel7A) cellobiohydrolase, a class of enzymes that catalytically process from the reducing end of cellulose. A direct amino acid comparison of these two enzymes shows that they not only have high amino acid homology, but also contain analogous N-linked glycosylation sites on the catalytic domain. We have previously shown (Jeoh et al. in Biotechnol Biofuels, 1:10, 2008) that expression of T. reesei cellobiohydrolase I in a commonly used industrial expression host, Aspergillus niger var. awamori, results in an increase in the amount of N-linked glycosylation of the enzyme, which negatively affects crystalline cellulose degradation activity as well as thermal stability. This complementary study examines the significance of individual N-linked glycans on the surface of the catalytic domain of Cel7A cellobiohydrolases from T. reesei and P. funiculosum by genetically adding or removing N-linked glycosylation motifs using site directed mutagenesis. Modified enzymes, expressed in A. niger var. awamori, were tested for activity and thermal stability. It was concluded that N-linked glycans in peptide loops that form part of the active site tunnel have the greatest impact on both thermal stability and enzymatic activity on crystalline cellulose for both the T. reesei and P. funiculosum Cel7A enzymes. Specifically, for the Cel7A T. reesei enzyme expressed in A. niger var. awamori, removal of the N384 glycosylation site yields a mutant with 70% greater activity after 120 h compared to the heterologously expressed wild type T. reesei enzyme. In addition, similar activity improvements were found to be associated with the addition of a new glycosylation motif at N194 in P. funiculosum. This mutant also exhibits 70% greater activity after 120 h compared to the wild type P. funiculosum enzyme expressed in A. niger var. awamori. Overall, this study demonstrates that “tuning” enzyme glycosylation for expression from heterologous expression hosts is essential for generating engineered enzymes with optimal stability and activity.

Aspergillus niger var. taxi, a new species variant of taxol-producing fungus isolated from Taxus cuspidata in China

To characterize and identify a new taxol-producing fungal strain HD86-9 isolated from Taxus cuspidata in China. Taxol extracted from strain HD86-9 was identified by HPLC and MS analyses. Strain HD86-9 was cultured and its morphology and phenotypes were described. HD86-9 displayed morphology most similar to that of Aspergillus niger but presented differences in the shape and size of the conidia. The growth evaluation showed that the maximal tolerable temperature of the new strain was 43 degrees C, higher than that of the model Aspergillus niger. The 18S rDNA and the internal transcribed spacer region including the 5.8S rDNA of HD86-9 were amplified by PCR; molecular analysis of these sequences revealed their high similarity of 98% to those of Aspergillus niger. The morphology and molecular analysis identified HD86-9 as a new variant of taxol-producing endophytic fungi, and it was named Aspergillus niger var. taxi D.P. Zhou, K. Zhao and W.X. Ping, var. nov. As the first report of a taxol-producing variant of Aspergillus niger species, this study offers important information and a new resource for the production of an important anticancer drug by endofungus fermentation.

Optium Conditions for the Production of Neutral Protease from local strain Aspergillus niger var cabonarius

The optimum conditions for the production of neutral protease from local strain Aspergillus niger var carbonarius by solid – state fermentation system (Wheat bran) moisted with 0.2 M phosphate buffer (PH7.0) . the hydration ratio was 1:5 (V:W) . the concentration of inoculum was 1×106 spores per 10 gram of solid materials , initial P H 6.5 and 96 hours of incubation period at 30? C .the enzyme activity was 1300 unit / ml and specific activity was 1550 unit / mg protein .

Optium Conditions for the Production of Neutral Protease from local strain Aspergillus niger var cabonarius

The optimum conditions for the production of neutral protease from local strain Aspergillus niger var carbonarius by solid – state fermentation system (Wheat bran) moisted with 0.2 M phosphate buffer (PH7.0) . the hydration ratio was 1:5 (V:W) . the concentration of inoculum was 1×106 spores per 10 gram of solid materials , initial P H 6.5 and 96 hours of incubation period at 30? C .the enzyme activity was 1300 unit / ml and specific activity was 1550 unit / mg protein .

Implications of cellobiohydrolase glycosylation for use in biomass conversion

The cellulase producing ascomycete, Trichoderma reesei (Hypocrea jecorina), is known to secrete a range of enzymes important for ethanol production from lignocellulosic biomass. It is also widely used for the commercial scale production of industrial enzymes because of its ability to produce high titers of heterologous proteins. During the secretion process, a number of post-translational events can occur, however, that impact protein function and stability. Another ascomycete, Aspergillus niger var. awamori, is also known to produce large quantities of heterologous proteins for industry. In this study, T. reesei Cel7A, a cellobiohydrolase, was expressed in A. niger var. awamori and subjected to detailed biophysical characterization. The purified recombinant enzyme contains six times the amount of N-linked glycan than the enzyme purified from a commercial T. reesei enzyme preparation. The activities of the two enzyme forms were compared using bacterial (microcrystalline) and phosphoric acid swollen (amorphous) cellulose as substrates. This comparison suggested that the increased level of N-glycosylation of the recombinant Cel7A (rCel7A) resulted in reduced activity and increased non-productive binding on cellulose. When treated with the N-glycosidase PNGaseF, the molecular weight of the recombinant enzyme approached that of the commercial enzyme and the activity on cellulose was improved.

Infection of the cones and seeds of Welwitschia mirabilis by Aspergillus niger var. phoenicis in the Namib-Naukluft Park

Welwitschia mirabilis Hook. fil. is a unique and rare dioecious desert gymnosperm endemic to the Namib Desert. The female plants bear 90–100 megasporophylls, of which 50–60% may be fertile, but up to 80% of those fertile seeds may be infected by Aspergillus niger var. phoenicis. This contamination results in seed and seedling death, potentially negatively affecting recruitment of plants into the population. The pattern of infection of the cones and seeds in the field was studied over a period of eight months at the Hope Mine in the Namib-Naukluft Park. Infection of the cones was found to peak coincident with the appearance of the pollination drops, and with high temperatures, winds and significant rainfall. W. mirabilis cones were microscopically examined and spores of A. niger var. phoenicis were found embedded in the dehydrating pollination drop at the tip of the micropyle, suggesting that this was one of the routes by which the seeds were infected. The heteropteran, Probergrothius sexpunctatis, was also implicated in the infection of the seeds, since it was found to be carrying spores of A. niger var. phoenicis and was observed feeding on mature seeds and immature cones. While most post-harvest treatments have proved ineffective in eradicating A. niger var. phoenicis from the seeds, promising results are presently reported using a fungicide containing tebuconazole as the active ingredient.

Activation profiles of the zymogen of aspergilloglutamic peptidase

Aspergilloglutamic peptidase produced by Aspergillus niger var. macrosporus belongs to the novel glutamic peptidase family. Its zymogen is autocatalytically activated under acidic conditions to the mature enzyme with a two-chain structure. Analyses by SDS-PAGE and mass spectrometry of the activation products of the recombinant zymogen showed that the major pathway of activation includes initial fast cleavage at Glu12-Ala13, followed by stepwise cleavages in the N-terminal and intervening propeptide regions. Essentially the same activation profile was obtained with the recombinant zymogen lacking the N-terminal 12-aa sequence. The missing region includes the most prominent cluster of basic residues of the propeptide, indicating low importance of this cluster for activation and refolding of the zymogen.

Evidence of RIP (repeat-induced point mutation) in transposase sequences of Aspergillus oryzae

A DNA methyl-binding column was used to isolate genomic fragments enriched for DNA-methylation from Aspergillus parasiticus. One of the isolated sequences presented 67% identity at the protein level with the transposase from the transposable element Tan1 of Aspergillus niger var. awamori, and was found to be present in at least 20 copies in the Aspergillus oryzae database. Analysis of four copies showed evidence of C:G to T:A transitions in at least 98.2% of the mutations found over a 1032–1180 bp region spanning a large part of the transposase sequence. Using copy specific primers three sequences were amplified from a different strain of A. oryzae and a similar pattern of C:G to T:A transitions was found. These transitions are similar to those observed in RIP, in Neurospora crassa, where cytosine-methylation is believed to be involved. Using methylation-sensitive Southern blotting, no evidence of methylation was found in the transposase sequences in these two A. oryzae strains as well as one A. parasiticus and one Aspergillus flavus strain.

Specific Inhibition and Stabilization of Aspergilloglutamic Peptidase by the Propeptide: IDENTIFICATION OF CRITICAL SEQUENCES AND RESIDUES IN THE PROPEPTIDE

Aspergilloglutamic peptidase (formerly called aspergillopepsin II) is an acid endopeptidase produced by Aspergillus niger var. macrosporus, with a novel catalytic dyad of a glutamic acid and a glutamine residue, thus belonging to a novel peptidase family G1. The mature enzyme is generated from its precursor by removal of the putative 41-residue propeptide and an 11-residue intervening peptide through autocatalytic activation. In the present study, the propeptide (Ala1-Asn41) and a series of its truncated peptides were chemically synthesized, and their effects on the enzyme activity and thermal stability were examined to identify the sequences and residues in the propeptide most critical to the inhibition and thermal stabilization. The synthetic propeptide was shown to be a potent competitive inhibitor of the enzyme (Ki = 27 nM at pH 4.0). Various shorter propeptide fragments derived from the central region of the propeptide had significant inhibitory effect, whereas their Ala scan-substituted peptides, especially R19A and H20A, showed only weak inhibition. Substitution of the Pro23-Pro24 sequence near His20 with an Ala-Ala sequence changed the peptide Lys18-Tyr25 to a substrate with His20 as the P1 residue. Furthermore, the propeptide was shown to be able to significantly protect the enzyme from thermal denaturation (DeltaTm = approximately 19 degrees C at pH 5.6). The protective potencies of the propeptide as well as truncated propeptides and their Ala scan-substituted peptides are parallel with their inhibitory potencies. These results indicate that the central part, and especially Arg19 and His20 therein, of the propeptide is most critical to the inhibition and thermal stabilization and that His20 interacts with the enzyme at or near the S1 site in a nonproductive fashion.