Enhancing itaconic acid production by Aspergillus terreus

Aspergillus terreus is successfully used for industrial production of itaconic acid. The acid is formed from cis-aconitate, an intermediate of the tricarboxylic (TCA) cycle, by catalytic action of cis-aconitate decarboxylase. It could be assumed that strong anaplerotic reactions that replenish the pool of the TCA cycle intermediates would enhance the synthesis and excretion rate of itaconic acid. In the phylogenetic close relative Aspergillus niger, upregulated metabolic flux through glycolysis has been described that acted as a strong anaplerotic reaction. Deregulated glycolytic flux was caused by posttranslational modification of 6-phosphofructo-1-kinase (PFK1) that resulted in formation of a highly active, citrate inhibition-resistant shorter form of the enzyme. In order to avoid complex posttranslational modification, the native A. niger pfkA gene has been modified to encode for an active shorter PFK1 fragment. By the insertion of the modified A. niger pfkA genes into the A. terreus strain, increased specific productivities of itaconic acid and final yields were documented by transformants in respect to the parental strain. On the other hand, growth rate of all transformants remained suppressed which is due to the low initial pH value of the medium, one of the prerequisites for the accumulation of itaconic acid by A. terreus mycelium. © 2010 Springer-Verlag.

Studies on Aspergillus niger glutamine synthetase: Regulation of enzyme levels by nitrogen sources and identification of active site residues

The specific activity of glutamine synthetase (L-glutamate: ammonia ligase, EC 6.3.1.2) in surface grown Aspergillus niger was increased 3-5 fold when grown on L-glutamate or potassium nitrate, compared to the activity obtained on ammonium chloride. The levels of glutamine synthetase was regulated by the availability of nitrogen source like NH4 + , and further, the enzyme is repressed by increasing concentrations of NH4 +. In contrast to other micro-organisms, the Aspergillus niger enzyme was neither specifically inactivated by NH4+ or L-glutamine nor regulated by covalent modification.Glutamine synthetase from Aspergillus niger was purified to homogenity. The native enzyme is octameric with a molecular weight of 385,000±25,000. The enzyme also catalyses Mn2+ or Mg2+-dependent synthetase and Mn2+-dependent transferase activity.Aspergillus niger glutamine synthetase was completely inactivated by two mol of phenylglyoxal and one mol of N-ethylmaleimide with second order rate constants of 3·8 M–1 min–1 and 760 M–1 min–1 respectively. Ligands like Mg. ATP, Mg. ADP, Mg. AMP, L-glutamate NH4+, Mn2+ protected the enzyme against inactivation. The pattern of inactivation and protection afforded by different ligands against N-ethylamaleimide and phenylglyoxal was remarkably similar. These results suggest that metal ATP complex acts as a substrate and interacts with an arginine ressidue at the active site. Further, the metal ion and the free nucleotide probably interact at other sites on the enzyme affecting the catalytic activity.

Statistical coupling analysis of aspartic proteinases based on crystal structures of the Trichoderma reesei enzyme and its complex with pepstatin A

The crystal structures of an aspartic proteinase from Trichoderma reesei (TrAsP) and of its complex with a competitive inhibitor, pepstatin A, were solved and refined to crystallographic R-factors of 17.9% (R(free)=21.2%) at 1.70 angstrom resolution and 15.81% (R(free) = 19.2%) at 1.85 angstrom resolution, respectively. The three-dimensional structure of TrAsP is similar to structures of other members of the pepsin-like family of aspartic proteinases. Each molecule is folded in a predominantly beta-sheet bilobal structure with the N-terminal and C-terminal domains of about the same size. Structural comparison of the native structure and the TrAsP-pepstatin complex reveals that the enzyme undergoes an induced-fit, rigid-body movement upon inhibitor binding, with the N-terminal and C-terminal lobes tightly enclosing the inhibitor. Upon recognition and binding of pepstatin A, amino acid residues of the enzyme active site form a number of short hydrogen bonds to the inhibitor that may play an important role in the mechanism of catalysis and inhibition. The structures of TrAsP were used as a template for performing statistical coupling analysis of the aspartic protease family. This approach permitted, for the first time, the identification of a network of structurally linked residues putatively mediating conformational changes relevant to the function of this family of enzymes. Statistical coupling analysis reveals coevolved continuous clusters of amino acid residues that extend from the active site into the hydrophobic cores of each of the two domains and include amino acid residues from the flap regions, highlighting the importance of these parts of the protein for its enzymatic activity. (C) 2008 Elsevier Ltd. All rights reserved.

Effects of Aspergillus spp. exogenous fibrolytic enzymes on in vitro fermentation of tropical forages

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Production, purification and characterization of a minor form of xylanase from Aspergillus versicolor

A strain of Aspergillus versicolor produces a xylanolytic complex containing two components, the minor component being designated xylanase II. The highest production of xylanase II was observed in cultures grown for 5 days in 1% wheat bran as carbon source, at pH 6.5. Xylanase II was purified 28-fold by DEAE-Sephadex and HPLC GF-5 10 gel filtration. Xylanase II was a monomeric glycoprotein, exhibiting a molecular mass of 32 kDa with 14.1% of carbohydrate content. Optimal pH and temperature values for the enzyme activity were about 6.0-7.0 and 55 degreesC, respectively. Xylanase II thermoinactivation at 50degreesC showed a biphasic curve. The ions Hg2+, Cu2+ and the detergent SDS were strong inhibitors, while Mn2+ ions and dithiothreitol were stimulators of the enzyme activity. The enzyme was specific for xylans, showing higher specific activity on birchwood xylan. The Michaelis-Menten constant (K-m) for birchwood xylan was estimated to be 2.3 mg ml(-1) while maximal velocity (V-max) was 233.1 mumol mg(-1) min(-1) of protein. The hydrolysis of oat spell xylan released only xylooligosaccharides. Published by Elsevier Ltd.

Fontes de carbono de baixo custo para a produção de xilanase termoestável por aspergillus niger

A strain of the flamentous fungus Aspergillus niger was isolated and shown to possess extracellular xylanolytic activity. These enzymes have biotechnological potential and can be employed in various industries. This fungus produced its highest xylanase activity in a medium made up of 0.1% CaCO3, 0.5% NaCl, 0.1% NH4Cl, 0.5% corn steep liquor and 1% carbon source, at pH 8.0. A low-cost hemicellulose residue (powdered corncob) proved to be an excellent inducer of the A. niger xylanolytic complex. Filtration of the crude culture medium with suspended kaolin was ideal for to clarify the extract and led to partial purifcation of the xylanolytic activity. The apparent molecular mass of the xylanase was about 32.3 kDa. Maximum enzyme activity occurred at pH 5.0 and 55-60oC. Apparent Km was 10.41 ± 0.282 mg/mL and Vmax was 3.32 ± 0.053 U/mg protein, with birchwood xylan as the substrate. Activation energy was 4.55 kcal/mol and half-life of the crude enzyme at 60oC was 30 minutes. Addition of 2% glucose to the culture medium supplemented with xylan repressed xylanase production, but in the presence of xylose the enzyme production was not affected.

Estudos bioquímicos e determinação da especificidade da peptidase produzida pelo fungo Aspergillus flavus

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Utilização de enzimas produzidas por Trichoderma reesei E Aspergillus niger na extração de óleos essenciais

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Effects of Aspergillus spp. exogenous fibrolytic enzymes on in vitro fermentation of tropical forages

BACKGROUND: Cellulose and hemicellulose are quantitatively the most important structural carbohydrates present in ruminant diets. Rumen micro-organisms produce enzymes that catalyse their hydrolysis, but the complex network formed by structural carbohydrates and lignin reduces their digestibility and restricts efficient utilisation of feeds by ruminants. This study aimed to produce two enzymatic extracts, apply them in ruminant diets to determine the best levels for ruminal digestibility and evaluate their effects on in vitro digestibility. RESULTS: In experiment 1 a two-stage in vitro technique was used to examine the effects of different enzymatic levels of Aspergillus japonicus and Aspergillus terricola on tropical forages. Enzyme addition had minor effects on corn silage at the highest enzymatic level. In experiment 2 an in vitro gas production (GP) technique was applied to determine apparent in vitro organic matter digestibility and metabolisable energy. The addition of enzymes in GP showed interesting results. Good data were obtained using sugar cane and Tifton-85 hay supplemented with extracts of A. japonicus and A. terricola respectively. CONCLUSION: Overall, the study suggests that addition of crude extracts containing exogenous fibrolytic enzymes to ruminant diets enhances the effective utilisation of ruminant feedstuffs such as forages. Copyright (c) 2012 Society of Chemical Industry

Xylanase and beta-Xylosidase Production by Aspergillus ochraceus: New Perspectives for the Application of Wheat Straw Autohydrolysis Liquor

The xylanase biosynthesis is induced by its substrate-xylan. The high xylan content in some wastes such as wheat residues (wheat bran and wheat straw) makes them accessible and cheap sources of inducers to be mainly applied in great volumes of fermentation, such as those of industrial bioreactors. Thus, in this work, the main proposal was incorporated in the nutrient medium wheat straw particles decomposed to soluble compounds (liquor) through treatment of lignocellulosic materials in autohydrolysis process, as a strategy to increase and undervalue xylanase production by Aspergillus ochraceus. The wheat straw autohydrolysis liquor produced in several conditions was used as a sole carbon source or with wheat bran. The best conditions for xylanase and beta-xylosidase production were observed when A. ochraceus was cultivated with 1% wheat bran added of 10% wheat straw liquor (produced after 15 min of hydrothermal treatment) as carbon source. This substrate was more favorable when compared with xylan, wheat bran, and wheat straw autohydrolysis liquor used separately. The application of this substrate mixture in a stirred tank bioreactor indicated the possibility of scaling up the process to commercial production.

Checkpoint Defects Leading to Premature Mitosis Also Cause Endoreplication of DNA in Aspergillus nidulans

The G2 DNA damage and slowing of S-phase checkpoints over mitosis function through tyrosine phosphorylation of NIMXcdc2 in Aspergillus nidulans. We demonstrate that breaking these checkpoints leads to a defective premature mitosis followed by dramatic rereplication of genomic DNA. Two additional checkpoint functions, uvsB and uvsD, also cause the rereplication phenotype after their mutation allows premature mitosis in the presence of low concentrations of hydroxyurea. uvsB is shown to encode a rad3/ATR homologue, whereas uvsD displays homology to rad26, which has only previously been identified in Schizosaccharomyces pombe. uvsBrad3 and uvsDrad26 have G2 checkpoint functions over mitosis and another function essential for surviving DNA damage. The rereplication phenotype is accompanied by lack of NIMEcyclinB, but ectopic expression of active nondegradable NIMEcyclinB does not arrest DNA rereplication. DNA rereplication can also be induced in cells that enter mitosis prematurely because of lack of tyrosine phosphorylation of NIMXcdc2 and impaired anaphase-promoting complex function. The data demonstrate that lack of checkpoint control over mitosis can secondarily cause defects in the checkpoint system that prevents DNA rereplication in the absence of mitosis. This defines a new mechanism by which endoreplication of DNA can be triggered and maintained in eukaryotic cells.

Aspergillazines A-E: novel heterocyclic dipeptides from an Australian strain of Aspergillus unilateralis

Biological and chemical pro ling of an Australian strain of the fungus Aspergillus unilateralis (MST-F8675), isolated from a soil sample collected near Mount Isa, Queensland, revealed a complex array of metabolites displaying broad chemotherapeutic properties. Noteworthy among these metabolites were a unique series of highly modified dipeptides aspergillazines A-E, incorporating a selection of unprecedented and yet biosynthetically related heterocyclic systems. Co-occurring with the aspergillazines was the recently described marine-derived fungal metabolite trichodermamide A (cf. penicillazine), whereas re-fermentation of A. unilateralis in NaCl (1%) enriched media resulted in co-production of the only other known example of this structure class, the marine-derived fungal metabolite trichodermamide B. Further investigation of A. unilateralis returned the known terrestrial fungal metabolite viridicatumtoxin as the cytotoxic and antibacterial principle, together with E-2-decenedioic acid, ferulic acid, (7E,7'E)-5,5'-diferulic acid and (7E,7'E)-8,5'-diferulic acid. The aromatic diacids have previously been reported from the chemical and enzymatic (esterase) treatment of plant cell wall material, with their isolation from A. unilateralis being their first apparent reported occurrence as natural products. Structures for all metabolites were determined by detailed spectroscopic analysis and, where appropriate, comparison to literature data and/or authentic samples.

Raman spectroscopy of some complex arsenate minerals—implications for soil remediation

The application of spectroscopy to the study of contaminants in soils is important. Among the many contaminants is arsenic, which is highly labile and may leach to non-contaminated areas. Minerals of arsenate may form depending upon the availability of specific cations for example calcium and iron. Such minerals include carminite, pharmacosiderite and talmessite. Each of these arsenate minerals can be identified by its characteristic Raman spectrum enabling identification.