Influence of pectin and glucose on growth and polygalacturonase production by Aspergillus niger in solid-state cultivation

The solid-state production of endo- and exo-polygalacturonases (PG) by Aspergillus niger was studied in a media containing wheat bran, salts, and different citric pectin and/or glucose concentrations. Kinetic analysis of the process indicated that the formation of PG and the growth of A. niger are associated processes. By increasing citric pectin from 0 to 16% (w/w), the maximum A. niger concentration (X _m) was raised from 94 to 121 mg/g dry medium suggesting that pectin can be used by A. niger as a growth substrate besides its role as an inducer. With 16% (w/w) pectin, 281 U exo-PG/gdm and 152 U endo-PG/gdm were obtained. Otherwise, pectin concentrations from 20 to 30% (w/w) hindered both production and growth. A. niger concentrations of 108–113 mg/gdm were achieved in runs with glucose from 5 to 12% (w/w), whereas at 16 and 20% (w/w) glucose, lower X _m values (ca. 100 mg/gdm) were measured. The addition of glucose to the wheat bran medium, up to 10% (w/w) led to maximum endo-PG titers slightly lower than those found in the absence of glucose. Nevertheless, exo-PG formation in these media was strongly increased and activities over 370 U/gdm were achieved. The results suggest that in experiments with pectin concentrations until 16% (w/w), exo-PG production was repressed by pectin-degradation products although these same substances had favored biomass growth. When glucose concentrations over 10% (w/w) were added to the media, the maximum activities of both enzymes decreased drastically, suggesting that glucose at high concentrations also exerts a repressive effect on PG production.     

Advances in citric acid fermentation by Aspergillus niger: biochemical aspects, membrane transport and modeling

Citric acid is regarded as a metabolite of energy metabolism, of which the concentration will rise to appreciable amounts only under conditions of substantive metabolic imbalances. Citric acid fermentation conditions were established during the 1930s and 1940s, when the effects of various medium components were evaluated. The biochemical mechanism by which Aspergillus niger accumulates citric acid has continued to attract interest even though its commercial production by fermentation has been established for decades. Although extensive basic biochemical research has been carried out with A. niger, the understanding of the events relevant for citric acid accumulation is not completely understood. This review is focused on citric acid fermentation by A. niger. Emphasis is given to aspects of fermentation biochemistry, membrane transport in A. niger and modeling of the production process.     

Comprehensive glycan analysis of recombinant Aspergillus niger endo-polygalacturonase C

The enzyme PGC is produced by the fungus Aspergillus niger during invasion of plant cell walls. The enzyme has been homologously overexpressed to provide sufficient quantities of purified enzyme for biological studies. We have characterized this enzyme in terms of its posttranslational modifications (PTMs) and found it to be both N- and O-glycosylated. The glycosyl moieties have also been characterized. This has involved a combination of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), liquid chromatography (LC)-ion trap, and LC-electrospray ionization (ESI) mass spectrometries in conjunction with trypsin degradation and beta-elimination, followed by Michael addition with dithiothreitol (BEMAD). This is the first demonstration of the ability of BEMAD to map glycosylation sites other than O-GlcNAc sites. The complete characterization of all PTMs on PGC allows us to model them on the peptide backbone, revealing potential roles played by the glycans in modulating the interaction of the enzyme with other macromolecules.     

Comparative sequence analysis of citrate synthase and 18S ribosomal DNA from a wild and mutant strains of Aspergillus niger with various fungi

A mutation was induced in Aspergillus niger wild strain using ethidium bromide resulting in enhanced expression of citric acid by three folds and 112.42 mg/mL citric acid was produced under optimum conditions with 121.84 mg/mL of sugar utilization. Dendograms of 18S rDNA and citrate synthase from different fungi including sample strains were made to assess homology among different fungi and to study the correlation of citrate synthase gene with evolution of fungi. Subsequent comparative sequence analysis revealed strangeness between the citrate synthase and 18S rDNA phylogenetic trees. Furthermore, the citrate synthase movement suggests that the use of traditional marker molecule of 18S rDNA gives misleading information about the evolution of citrate synthase in different fungi as it has shown that citrate synthase gene transferred independently among different fungi having no evolutionary relationships. Random amplified polymorphic DNA (RAPD-PCR) analysis was also employed to study genetic variation between wild and mutant strains of A. niger and only 71.43% similarity was found between both the genomes. Keeping in view the importance of citric acid as a necessary constituent of various food preparations, synthetic biodegradable detergents and pharmaceuticals the enhanced production of citric acid by mutant derivative might provide significant boost in commercial scale viability of this useful product.

Abbreviations

CS - Citrate synthase, CA - Citric acid, RAPD - Random amplified polymorphic DNA, TAF - Total amplified fragments, PAF - Polymorphic amplified fragments, CAF - Common amplified fragments.     

Structural insights into the processivity of endopolygalacturonase I from Aspergillus niger

Endopolygalacturonase I is a processive enzyme, while the 60% sequence identical endopolygalacturonase II is not. The 1.70 A resolution crystal structure of endopolygalacturonase I reveals a narrowed substrate binding cleft. In addition, Arg96, a residue in this cleft previously shown to be critical for processivity, interacts with the substrate mimics glycerol and sulfate in several well-defined conformations in the six molecules in the asymmetric unit. From this we conclude that both Arg96 and the narrowed substrate binding cleft contribute to retaining the substrate while it moves through the active site after a cleavage event has occurred.     

Atypical regioselective biohydrolysis on steroidal oxiranes by Aspergillus niger whole cells: some stereochemical features

5,6-Epoxycholestan-3beta-ol derivatives were hydrolyzed in a diastereoconvergent manner by growing and resting cells of several strains of Aspergillus niger, particularly A. niger ATCC 11394. These strains displayed opposite regioselectivity toward each isomer in an alpha and beta epoxide mixture, thus, the nucleophilic attack took place at the less substituted and the most substituted carbon atom on each diasteromer, respectively. These biocatalysts opened trisubstituted oxiranes but were unable to hydrolyze the disubstituted oxiranes in the tested sterol derivatives. These findings suggest that A. niger strains possess another hydrolytic ability different from the commercial A. niger epoxide hydrolase (EH) that did not accept this kind of steroidal oxiranes as substrates.     

Treatment of APMP pulping effluent based on aerobic fermentation with Aspergillus niger and post-coagulation/flocculation

A novel two-stage biological/flocculation process was developed for treating the pulping effluent from the alkaline peroxide mechanical pulping (APMP) process. In the first biological stage, the aerobic fermentation by using Aspergillus niger can decrease the chemical oxygen demand (COD) by about 60% while producing about 7 g/l of solid biomass. In the second stage (post-coagulation/flocculation), the residual COD, turbidity and color, can be further decreased by using alum and polyacrylamide (PAM). The overall removal efficiencies of COD, color and turbidity from the APMP pulping effluent by the above two-stage biological-coagulation/flocculation process were 93%, 92% and 99%, respectively, under the conditions studied.     

High efficient expression of cellobiase gene from Aspergillus niger in the cells of Trichoderma reesei

The cellobiase gene from Aspergillus niger was cloned and connected with the strong promoter Pcbh1 from Trichoderma reesei to construct a recombinant plasmid pHB9 with the hygromycin B resistance marker. The plasmid was transformed into conidia of T. reesei using the modified PEG-CaCl₂ method. Main factors effecting the transformation were discussed and about 99-113 transformants/μg DNA could be obtained under optimal conditions. It was found that the molecular mass of the recombinant cellobiase was about 120 kDa by SDS-PAGE analysis. The activity of cellobiase could reach 5.3 IU/ml after 48 h fermentation, which was as high as 106 times compared with that of the host strain. Meanwhile, the filter paper activity of recombinant T. reesei was 1.44-fold of the host strain. Saccharification of corncob residue with the crude enzyme showed that the hydrolysis yield (84.2%) of recombinant T. reesei was 21% higher than that (69.5%) of the host strain.     

Pollen-specific pectin methylesterase involved in pollen tube growth

Pollen tube elongation in the pistil is a crucial step in the sexual reproduction of plants. Because the wall of the pollen tube tip is composed of a single layer of pectin and, unlike most other plant cell walls, does not contain cellulose or callose, pectin methylesterases (PMEs) likely play a central role in the pollen tube growth and determination of pollen tube morphology. Thus, the functional studies of pollen-specific PMEs, which are still in their infancy, are important for understanding the pollen development. We identified a new Arabidopsis pollen-specific PME, AtPPME1, characterized its native expression pattern, and used reverse genetics to demonstrate its involvement in determination of the shape of the pollen tube and the rate of its elongation.     

Utility of Aspergillus niger citrate synthase promoter for heterologous expression

Citrate synthase is a central player in the acidogenic metabolism of Aspergillus niger. The 5′ upstream sequence (0.9 kb DNA) of citrate synthase gene (citA) from A. niger NCIM 565 was analyzed and its promoter function demonstrated through the heterologous expression of two proteins. The cloned citrate synthase promoter (PcitA) sequence was able to express bar coding sequence thereby conferring phosphinothricin resistance. This sequence was further analyzed by systematic deletions to define an effective but compact functional promoter. The PcitA driven egfp expression showed that PcitA was active in all differentiation cell-stages of A. niger. EGFP expression was highest on non-repressible carbon sources like acetate and glycerol. Mycelial EGFP levels increased during acidogenic growth suggesting that PcitA is functional throughout this cultivation. A. niger PcitA is the first Krebs cycle gene promoter used to express heterologous proteins in filamentous fungi.     

Mathematical modelling and assessment of the pH homeostasis mechanisms in Aspergillus niger while in citric acid producing conditions

In this work we introduce an extended model of the Aspergillus niger metabolism while in citrate production conditions. The model includes many recent findings related to various transport processes. It now considers a new information about the fructose uptake system and the proton and amino acids carriers between cytoplasm and the external medium. It also accounts for recent information about both the malate-citrate antiport between mitochondria and cytoplasm and the dihydrogen citrate ion excretion symport with protons. Finally, the model also accounts for new information about the glycerol-3-phosphate shuttle and pH buffering systems. Provided with this updated representation and after having assessed its quality and dynamic behaviour, we were able to explain the observed pH homoeostasis found in A. niger while in citrate producing conditions. The model also serves to enhance our comprehension of the molecular mechanisms operating in order to keep homoeostasis of pH in A. niger and other fungi, bacteria and yeast of biotechnological relevance.     

Oxalic acid production from lipids by a mutant of Aspergillus niger at different pH

Crude rapeseed oil and post-refining fatty acids were used as substrates for oxalic acid production by a mutant of Aspergillus niger. Both the final concentration and the yield of the product were highest at pH 4 to 5. With a medium containing 50 g lipids l^–1, production reached a maximum of 68 g oxalic acid l^–1 after 7 d. A high yield of the product (up to 1.4 g oxalic acid g^–1 lipids consumed) was achieved with oil and fatty acids combined.     

Identification of pollen-expressed pectin methylesterase inhibitors in Arabidopsis

Pectin methylesterases (PMEs) play an essential role during plant development by affecting the mechanical properties of the plant cell wall. Previous work indicated that plant PMEs may be subject to post-translational regulation. Here, we report the analysis of two proteinaceous inhibitors of PME in Arabidopsis thaliana (AtPMEI1 and 2). The functional analysis of recombinant AtPMEI1 and 2 proteins revealed that both proteins are able to inhibit PME activity from flowers and siliques. Quantitative RT-PCR analysis indicated that expression of AtPMEI1 and 2 mRNAs is tightly regulated during plant development with highest mRNA levels in flowers. Promotor::GUS fusions demonstrated that expression is mostly restricted to pollen.     

An Experimental Study on Citric Acid Production by Aspergillus niger Using Gelidiella acerosa as a Substrate

Citric acid (CA) is the most important commercial product which is produced by using various sugar substrates in the terrestrial environment. The present study made an attempt to produce citric acid by the fungal strain Aspergillus niger from red seaweed Gelidiella acerosa is the best alternative to sugar substrate in the marine environment. In this study three types of production media were prepared including control (sucrose) by following standard fermentation conditions. The acid production was indicated by the reduction of pH levels. The control medium gave the highest yield of 80 g/l at pH 1.5 and the medium containing crude seaweed powder and other compositions gave the yield of 30 g/l at pH 3.5 whereas the medium containing crude seaweed and 10% sucrose gave the yield of 50 g/l at pH 3.0. When calculating the benefit cost ratio, crude seaweed powder and 10% sucrose yielded 50 g of citric acid at the lower cost of Rs. 35, whereas the other two media gave the yield of 80 and 30 g respectively with the cost of Rs. 77 and 28. In economic point of view, the medium containing seaweed and 10% sucrose showed more benefit with lower cost.     

Treatment of poplar alkaline peroxide mechanical pulping (APMP) effluent with Aspergillus niger

Although the moderate load (COD of 5000-10,000 mg/L) and biodegradability of the APMP pulping effluent should allow biological treatment, toxic compounds in the effluent can interfere with this type of treatment. Studies were conducted to determine if treatment of the effluent with Aspergillus niger S13 was feasible. Under the optimized conditions (3% inoculum, pH 6, shaking at 160 rpm, 60-72 h, and 30 °C), this fungus was able to remove about 97% of the methyl tertiary butyl ether (MTBE) extractives, and 60%, 77% and 43% of the chemical oxygen demand, turbidity and color even without a pre-flocculation step. These results are of practical interest in China because the APMP process has become popular, and efficient and cost-effective effluent treatment technologies are in high demand.     

Immobilization of the epoxide hydrolase from Aspergillus niger

Three methods for the immobilization of the epoxide hydrolase from the fungus Aspergillus niger were tested. The highest immobilization yield (90%) and retention of activity (65%) were obtained by adsorption onto DEAE-cellulose compared to adsorption onto hydrophobic porous polypropylene and covalent linkage using Eupergit resin. The enzymatic properties of the immobilized enzyme were similar to those of the free enzyme with respect to the effect of temperature and pH on both activity and stability as well as the effect of solvent (DMF) on activity. The kinetic parameters were affected leading to lower K _M(app) and higher Vm _(app).     

Ultrasound effects on invertase from Aspergillus niger

Ultrasound effects on the release and activity of invertase from Aspergillus niger cultivated in a medium containing sucrose and peptone and in another with sugar-cane molasses and peptone were investigated. Irradiation was conducted for periods of 2–10 min. with waves of amplitude 20 and 40 using an ultrasound processor of 20 kHz. Product formation was determined as reducing equivalents formed by time units using 3,5-dinitrosalicylic acid. Total and specific activities of the culture supernatants were compared in the presence and absence of sonication. Both amplitudes promoted a significant increase of total invertase activity in the time periods investigated and the highest values were obtained with an amplitude of 20. Ultrasound irradiation caused cell disruption, thus releasing invertase and, after 4 min, activation of the enzyme also occurred. The best conditions for production, extraction and activation of invertase were in molasses medium containing peptone and irradiation with ultrasound waves at 20 for 8 min. This method showed high efficiency for the extraction and activation of invertase from A. niger as well as a great potential for use in industrial processes.     

Site-directed mutagenesis of Aspergillus niger NRRL 3135 phytase at residue 300 to enhance catalysis at pH 4.0

Increased phytase activity for Aspergillus niger NRRL 3135 phytaseA (phyA) at intermediate pH levels (3.0-5.0) was achieved by site-directed mutagenesis of its gene at amino acid residue 300. A single mutation, K300E, resulted in an increase of the hydrolysis of phytic acid of 56% and 19% at pH 4.0 and 5.0, respectively, at 37 degrees C. This amino acid residue has previously been identified as part of the substrate specificity site for phyA and a comparison of the amino acid sequences of other cloned fungal phytases indicated a correlation between a charged residue at this position and high specific activity for phytic acid hydrolysis. The substitution at this residue by either another basic (R), uncharged (T), or acidic amino acid (D) did not yield a recombinant enzyme with the same favorable properties. Therefore, we conclude that this residue is not only important for the catalytic function of phyA, but also essential for imparting a favorable pH environment for catalysis.     

Cloning, Expression, and Characterization of an GHF 11 Xylanase from Aspergillus niger XZ-3S

A xylanase gene (xynZF-2) from the Aspergillus niger XZ-3S was cloned and expressed in Escherichia coli. The coding region of the gene was separated by only one intron with the 68 bp in length. It encoded 225 amino acid residues of a protein with a calculated molecular weight of 24.04 kDa plus a signal peptide of 18 amino acids. The amino acid sequence of the xynZF-2 gene had a high similarity with those of family 11 of glycosyl hydrolases reported from other microorganisms. The mature peptide encoding cDNA was subcloned into pET-28a(+) expression vector. The resultant recombinant plasmid pET-28a-xynZF-2 was transformed into E. coli BL21(DE3), and finally the recombinant strain BL21/xynZF-2 was obtained. A maximum activity of 42.33 U/mg was gained from cellular of E. coli BL21/xynZF-2 induced by IPTG. The optimum temperature and pH for recombinant enzyme which has a good stability in alkaline conditions were 40 °C and 5.0, respectively. Fe³⁺ had an active effect on the enzyme obviously.     

Biotransformation of terpenes from Stemodia maritima by Aspergillus niger ATCC 9142.

Incubation of stemodin (1) in cultures of Aspergillus niger ATCC 9142 resulted in the production of 2alpha,3beta,13-trihydroxystemodane (2), 2alpha,7beta,13-trihydroxystemodane (3) and 2alpha,13,16beta-trihydroxystemodane (4), while stemodinone (5) afforded 13,18-dihydroxystemodan-2-one (6) and 13,16beta-dihydroxystemodan-2-one (7). Four novel metabolites were obtained from the bioconversion of stemarin (8) by the fungus, namely 18-hydroxystemaran-19-oic acid (9), 7beta,18-dihydroxystemaran-19-oic acid (10), 7alpha,18,19-trihydroxystemarane (11) and 1beta-hydroxystemaran-19-oic acid (12). 19-N,N-Dimethylcarbamoxy-13-hydroxystemarane (13) was also transformed to afford 19-N,N-dimethylcarbamoxy-13,17xi,18-trihydroxystemarane (14).