Improvement of Laccase Production by Thielavia terrestris Co3Bag1. Enhancing the Bio-Catalytic Performance of the Native Thermophilic TtLacA via Immobilization in Copper Alginate Gel Beads.

A 32-fold increase in laccase activity production by the thermophilic biomass-degrading fungus T. terrestris Co3Bag1 was achieved when the microorganism was grown on a modified medium containing fructose, sodium nitrate, and copper. A 70 kDa laccase (TtLacA), produced under the above conditions, was purified, immobilized in copper alginate gel beads, and characterized. TtLacA, both free and immobilized enzymes, exhibited optimal activity at pH 3.0, at a temperature of 65 and 70 °C, respectively, although both displayed 70% of activity from 40 to 70 °C. Free and immobilized enzymes retained at least 80% of relative activity in the pH range from 3 to 4.6. Immobilized TtLacA manifested a 2.3-fold higher thermal stability than the free form of the enzyme at 60 and 70 °C. Immobilized TtLacA retained 95% initial activity for six consecutive reuse cycles at 60 °C, and also retained 86% of initial activity after 12 days of storage at 4 °C. Based on the biochemical features, thermophilic TtLacA may be an efficient enzyme for dye decolorization and other industrial applications at high temperatures or acidic conditions. This work represents the first report about the immobilization and biochemical characterization of a thermophilic laccase from a member of the genus Thielavia.

Identification of α-L-fucosidase (ALFuc) of Blastocystis sp. subtypes ST1, ST2 and ST3.

Blastocystis sp. is a common intestinal microorganism. The α-L-fucosidase (ALFuc) is an enzyme long associated with the colonization of the gut microbiota. However, this enzyme has not been experimentally identified in Blastocystis cultures. The objective of the present study was to identify ALFuc in supernatants of axenic cultures of Blastocystis subtype (ST)1 ATCC-50177 and ATCC-50610 and to compare predicted ALFuc proteins of alfuc genes in sequenced STs1-3 isolates in human Blastocystis carriers. Excretion/secretion (Es/p) and cell lysate proteins were obtained by processing Blastocystis ATCC cultures and submitting them to SDS-PAGE and immunoblotting. In addition, 18 fecal samples from symptomatic Blastocystis human carriers were analyzed by sequencing of amplification products for subtyping. A complete identification of the alfuc gene and phylogenetic analysis were performed. Immunoblotting showed that the amplified band corresponding to ALFuc (~51 kDa) was recognized only in the ES/p. Furthermore, prediction analysis of ALFuc 3D structures revealed that the domain α-L-fucosidase and the GH29 family's catalytic sites were conserved; interestingly, the galactose-binding domain was recognized only in ST1 and ST2. The phylogenetic inferences of ALFuc showed that STs1-3 were clearly identifiable and grouped into specific clusters. Our results show, for the first time through experimental data that ALFuc is a secretion product of Blastocystis sp., which could have a relevant role during intestinal colonization; however, further studies are required to clarify this condition. Furthermore, the alfuc gene is a promising candidate for a phylogenetic marker, as it shows a conserved classification with the SSU-rDNA gene.

Identification of α-L-fucosidase (ALFuc) of Blastocystis sp. subtypes ST1, ST2 and ST3

ABSTRACT Blastocystis sp. is a common intestinal microorganism. The α-L-fucosidase (ALFuc) is an enzyme long associated with the colonization of the gut microbiota. However, this enzyme has not been experimentally identified in Blastocystis cultures. The objective of the present study was to identify ALFuc in supernatants of axenic cultures of Blastocystis subtype (ST)1 ATCC-50177 and ATCC-50610 and to compare predicted ALFuc proteins of alfuc genes in sequenced STs1-3 isolates in human Blastocystis carriers. Excretion/secretion (Es/p) and cell lysate proteins were obtained by processing Blastocystis ATCC cultures and submitting them to SDS-PAGE and immunoblotting. In addition, 18 fecal samples from symptomatic Blastocystis human carriers were analyzed by sequencing of amplification products for subtyping. A complete identification of the alfuc gene and phylogenetic analysis were performed. Immunoblotting showed that the amplified band corresponding to ALFuc (~51 kDa) was recognized only in the ES/p. Furthermore, prediction analysis of ALFuc 3D structures revealed that the domain α-L-fucosidase and the GH29 family's catalytic sites were conserved; interestingly, the galactose-binding domain was recognized only in ST1 and ST2. The phylogenetic inferences of ALFuc showed that STs1-3 were clearly identifiable and grouped into specific clusters. Our results show, for the first time through experimental data that ALFuc is a secretion product of Blastocystis sp., which could have a relevant role during intestinal colonization; however, further studies are required to clarify this condition. Furthermore, the alfuc gene is a promising candidate for a phylogenetic marker, as it shows a conserved classification with the SSU-rDNA gene.

Characterization of polyphenol oxidase from purple sweet potato (Ipomoea batatas L. Lam) and its affinity towards acylated anthocyanins and caffeoylquinic acid derivatives.

Biochemical characterization of polyphenol oxidase (PPO) present in purple sweet potato (PSP) is a key step in developing efficient methodologies to control oxidative damage caused by this enzyme to the valuable components of PSP, such as caffeoylquinic acid derivatives and acylated anthocyanins. Thus, this work focused on the assessment of the effects of pH, temperature, and chemical agents on the PPO activity as well as characterization of the PPO substrate specificity towards major phenolic compounds found in PSP. The optimum conditions of enzyme activity were pH 7 and a temperature range of 20-30 °C at which phenolic substrates were oxidized with 72.5-99.8% yield. Zn2+ ions remarkably reduced PPO activity while Cu2+ ions improved enzyme performance. The highest substrate preference was shown for 3,4,5-tri-caffeoylquinic and 3,5-di-caffeoylquinic acid, followed by 5-caffeoylquinic and caffeic acid, 3,4- and 4,5-di-caffeoylquinic acids, peonidin-3-caffeoyl-p-hydroxybenzoyl-sophoroside-5-glucoside. The highest Km values were found for 4,5-feruloyl-caffeoylquinic acid and catechol.

Effect of different carbon sources on the growth and enzyme production of a toxigenic and a non-toxigenic strain of Aspergillus flavus.

Two strains of A. flavus one toxigenic (CECT 2687) and the other non-toxigenic (NRRL 6541) were studied for their genomic potential, growth capacity, and the production of enzymes on simple sugars, polysaccharides, and complex substrates under solid-state fermentation (SSF). According to the genome analysis, this fungus has many genes to degrade different types of polysaccharides and therefore it would be able to grow on different substrates. Both strains grow in all the carbon sources, but visibly CECT2687 grows slower than NRRL6541. However, we propose the growth index (GI) to establish a dry weight-diameter relationship as a more reliable measure that truly shows the growth preferences of the fungus. Considering this, the NRRL6541 shows less growth in 11 of the 16 evaluated carbon sources than CECT2687. Complex substrates were the best carbon source for the growth of both strains. Corncob (CC) induced the production of xylanases, pectinases, and almost all the accessory enzymes evaluated (except for α-xylosidase) this could make it an agricultural waste of interest to produce hemicellulolytic enzymes. Both strains produce a great variety of xylanases and pectinases (pathogenicity factors) making A. flavus a good potential candidate for the degradation of polysaccharides with a high content of xylan and pectin.

Analysis of polysaccharide hydrolases secreted by Aspergillus flavipes FP-500 on corn cobs and wheat bran as complex carbon sources.

Aspergillus flavipes FP-500 is a Mexican native strain that has been reported as a good producer of xylanases and pectinases; therefore, it promises a strong impact on biotechnology. To provide an overview of protein secretion by A. flavipes, we carried out a comparative proteome analysis of extracellular proteins in liquid cultures with two heterogeneous agro-industrial residues; corn cob (CC) and wheat bran (WB), as carbon sources. Extracellular proteins obtained from both cultures were identified using MS/MS spectrometry. We identified 134 proteins, which were classified into four groups: glycosyl hydrolases (GH), esterases/proteases, miscellaneous proteins, and unidentified proteins. Around 50% of the total proteins identified were GH such as xylanases, ß-xylosidases, ß-galactosidases, cellulolytic enzymes like ß-glucosidase, endoglucanases, and cellobiohydrolases. From this family, a core of 22 (16%) of the proteins identified were found in both substrates, CC and WB, whereas 30% and 54% were unique for CC and WB, respectively. In the esterases/proteases group, proteases, lipases and esterases like feruloylesterases, and acetyl-xylanesterase were identified. Proteins with diverse functions such as monophosphate dehydrogenase or N-acetylglucosaminidase were present. Here, we present strong evidences indicating that the composition and heterogeneity of the used carbon source determine the specific set of protein secreted by the fungus.

Purification and biochemical characterization of a novel thermophilic exo-ß-1, 3-glucanase from the thermophile biomass-degrading fungus Thielavia terrestris Co3Bag1

Background: The aim of this work was to purify and characterize exo-ß-1,3-glucanase, namely, TtBgnA, from the thermophilic fungus Thielavia terrestris Co3Bag1 and to identify the purified enzyme. Results: The thermophilic biomass-degrading fungus T. terrestris Co3Bag1 displayed ß-1,3-glucanase activity when grown on 1% glucose. An exo-ß-1,3-glucanase, with an estimated molecular mass of 129 kDa, named TtBgnA, was purified from culture filtrates from T. terrestris Co3Bag1. The enzyme exhibited optimum activity at pH 6.0 and 70°C and half-lives (t1/2) of 54 and 37 min at 50 and 60°C, respectively. Substrate specificity analysis showed that laminarin was the best substrate studied for TtBgnA. When laminarin was used as the substrate, the apparent KM and Vmax values were determined to be 2.2 mg mL-1 and 10.8 U/mg, respectively. Analysis of hydrolysis products by thin-layer chromatography (TLC) revealed that TtBgnA displays an exo mode of action. Additionally, the enzyme was partially sequenced by tandem mass spectrometry (MS/MS), and the results suggested that TtBgnA from T. terrestris Co3Bag1 could be classified as a member of the GH-31 family. Conclusions: This report thus describes the purification and characterization of TtBgnA, a novel exo-ß-1,3-glucanase of the GH-31 family from the thermophilic fungus T. terrestris Co3Bag1. Based on the biochemical properties displayed by TtBgnA, the enzyme could be considered as a candidate for potential biotechnological applications.

Extraction and Identification of Anthocyanins in Corn Cob and Corn Husk from Cacahuacintle Maize.

Pigmented maize has been extensively studied due to its high anthocyanin content. This study has been focused mainly on kernel, although the whole plant of purple corn is a potential source of anthocyanins. First, general parameters of extraction (solvent system, solvent-to-solid ratio, number of extractions, and acid type) were established depending on the total anthocyanins content. Then, three extraction methods to access anthocyanins were compared: maceration extraction (ME), ultrasound-assisted extraction (UAE), and microwave-assisted extraction (MAE). Since the residual material still possessed an intense color, a further treatment was performed by application of enzymatic-assisted extraction (EAE). Three enzymatic cocktails (Xylanases, Celluclast, and Depol), pH, and temperature were evaluated to establish optimal reaction conditions. Subsequent analysis and identification of the anthocyanins obtained by four different extraction techniques were performed using HPLC and HPLC-mass spectrometry, respectively. The most efficient method was UAE using 20 min of ultrasound (100 W) preceded by sample treatment in the following conditions: ethanol/water/lactic acid mixture (80:19:1), two extractions, 1:10 solvent-to-solid ratio. As a result, anthocyanins from corn cob and corn husk were extracted at concentrations of 24.32 and 25.80 mg/gDW, respectively. No difference in the anthocyanins profile for samples extracted by three different methods was observed. However, an enhanced presence of cyanidin-3-(6''malonyl)glucoside was detected in the sample corresponding to the EAE method. Therefore, the Cahuacintle corn husk can be considered as a competitive source of anthocyanins with the available commercial sources. PRACTICAL APPLICATION: The by-products obtained from Cacahuacintle purple corn can be potentially used as natural colorants thanks to their anthocyanins content. In this work, we established the most efficient extraction method of anthocyanins from corn husk and corn cob, and demonstrated that their anthocyanins profile is comparable to other Peruvian purple corns, which are currently used as natural colorants. Therefore, the extraction procedure described in this study might be scaled-up in an industrial process to get access to anthocyanins from undervalued wastes.

Lactic Acid Fermentation of Arabinoxylan From Nejayote by Streptococcus infantarius ssp. infantarius 25124 Isolated From Pozol.

Streptococcus infantarius ssp. infantarius 25124 (Sii-25124) is a lactic acid bacterium (LAB) isolated from pozol, a refreshing beverage prepared by suspending fermented nixtamal (a thermal and alkali-treated maize dough) in water. Although Lactobacillus are the predominant strains in fermented doughs, such as sourdoughs, and non-nixtamalized fermented maize foods, the pozol microbiota is markedly different. This may be the result of the nixtamalization process, which could act as a selective force of some strains. Sii-25124 has been reported as the main amylolytic LAB in pozol; starch is the primary carbon source on nixtamal since monosaccharides and disaccharides are lost during nixtamalization; however, non-amylolytic LAB counts are higher than amylolytic LAB in pozol after 24-h fermentation suggesting that another carbon source is being used by the former bacteria. Hemicellulose (arabinoxylan in maize) becomes available via nixtamalization and is subsequently metabolized by LAB. The aim of this work was to determine whether this bacterium is able to use arabinoxylan as the only carbon source in a defined medium containing arabinoxylan extracted from either nejayote (wash water produced during nixtamal preparation), or beechwood xylan. Xylanase activity in the presence of nejayote arabinoxylan (135.8 ± 48.7 IU/mg protein) was higher than that of beechwood (62.5 ± 19.8 IU/mg protein). Other enzymatic activities, such as arabinofuranosidase and acetyl esterase, were also detected, suggesting the adaptation of the bacterium studied to nixtamal dough. It was concluded that Streptococcus infantarius 25124 isolated from pozol was able to use arabinoxylans, which are present in nixtamal dough, so fermentation does not depend exclusively on free sugars and starch.

Comparative genomics reveals high biological diversity and specific adaptations in the industrially and medically important fungal genus Aspergillus.

BACKGROUND: The fungal genus Aspergillus is of critical importance to humankind. Species include those with industrial applications, important pathogens of humans, animals and crops, a source of potent carcinogenic contaminants of food, and an important genetic model. The genome sequences of eight aspergilli have already been explored to investigate aspects of fungal biology, raising questions about evolution and specialization within this genus. RESULTS: We have generated genome sequences for ten novel, highly diverse Aspergillus species and compared these in detail to sister and more distant genera. Comparative studies of key aspects of fungal biology, including primary and secondary metabolism, stress response, biomass degradation, and signal transduction, revealed both conservation and diversity among the species. Observed genomic differences were validated with experimental studies. This revealed several highlights, such as the potential for sex in asexual species, organic acid production genes being a key feature of black aspergilli, alternative approaches for degrading plant biomass, and indications for the genetic basis of stress response. A genome-wide phylogenetic analysis demonstrated in detail the relationship of the newly genome sequenced species with other aspergilli. CONCLUSIONS: Many aspects of biological differences between fungal species cannot be explained by current knowledge obtained from genome sequences. The comparative genomics and experimental study, presented here, allows for the first time a genus-wide view of the biological diversity of the aspergilli and in many, but not all, cases linked genome differences to phenotype. Insights gained could be exploited for biotechnological and medical applications of fungi.

Scaling-up and ionic liquid-based extraction of pectinases from Aspergillus flavipes cultures.

The viability of the scaling-up of pectinases production by Aspergillus flavipes at 5L-bioreactor scale has been demonstrated by keeping constant the power input, and a drastic increase in the endo- and exopectinolytic enzyme production was recorded (7- and 40-fold, respectively). The main process variables were modelled by means of logistic and Gompertz equations. In order to overcome the limitations of the conventional downstream strategies, a novel extraction strategy was proposed on the basis of the adequate salting-out potential of two biocompatible cholinium-based ionic liquids (N1112OHCl and N1112OHH2PO4) in aqueous solutions of Tergitol, reaching more than 90% of extraction.

The thermophilic biomass-degrading fungus Thielavia terrestris Co3Bag1 produces a hyperthermophilic and thermostable ß-1,4-xylanase with exo- and endo-activity.

A hyperthermophilic and thermostable xylanase of 82 kDa (TtXynA) was purified from the culture supernatant of T. terrestris Co3Bag1, grown on carboxymethyl cellulose (CMC), and characterized biochemically. TtXynA showed optimal xylanolytic activity at pH 5.5 and at 85 °C, and retained more than 90% of its activity at a broad pH range (4.5-10). The enzyme is highly thermostable with a half-life of 23.1 days at 65 °C, and active in the presence of several metal ions. Circular dichroism spectra strongly suggest the enzyme gains secondary structures when temperature increases. TtXynA displayed higher substrate affinity and higher catalytic efficiency towards beechwood xylan than towards birchwood xylan, oat-spelt xylan, and CMC. According to its final hydrolysis products, TtXynA displays endo-/exo-activity, yielded xylobiose, an unknown oligosaccharide containing about five residues of xylose and a small amount of xylose on beechwood xylan. Finally, this report represents the description of the first fungal hyperthermophilic xylanase which is produced by T. terrestris Co3Bag1. Since TtXynA displays relevant biochemical properties, it may be a suitable candidate for biotechnological applications carried out at high temperatures, like the enzymatic pretreatment of plant biomass for the production of bioethanol.

Biodegradative Activities of Selected Environmental Fungi on a Polyester Polyurethane Varnish and Polyether Polyurethane Foams.

UNLABELLED: Polyurethane (PU) is widely used in many aspects of modern life because of its versatility and resistance. However, PU waste disposal generates large problems, since it is slowly degraded, there are limited recycling processes, and its destruction may generate toxic compounds. In this work, we isolated fungal strains able to grow in mineral medium with a polyester PU (PS-PU; Impranil DLN) or a polyether PU (PE-PU; Poly Lack) varnish as the only carbon source. Of the eight best Impranil-degrading strains, the six best degraders belonged to the Cladosporium cladosporioides complex, including the species C. pseudocladosporioides, C. tenuissimum, C. asperulatum, and C. montecillanum, and the two others were identified as Aspergillus fumigatus and Penicillium chrysogenum The best Impranil degrader, C. pseudocladosporioides strain T1.PL.1, degraded up to 87% after 14 days of incubation. Fourier transform infrared (FTIR) spectroscopy analysis of Impranil degradation by this strain showed a loss of carbonyl groups (1,729 cm(-1)) and N-H bonds (1,540 and 1,261 cm(-1)), and gas chromatography-mass spectrometry (GC-MS) analysis showed a decrease in ester compounds and increase in alcohols and hexane diisocyanate, indicating the hydrolysis of ester and urethane bonds. Extracellular esterase and low urease, but not protease activities were detected at 7 and 14 days of culture in Impranil. The best eight Impranil-degrading fungi were also able to degrade solid foams of the highly recalcitrant PE-PU type to different extents, with the highest levels generating up to 65% of dry-weight losses not previously reported. Scanning electron microscopy (SEM) analysis of fungus-treated foams showed melted and thinner cell wall structures than the non-fungus-treated ones, demonstrating fungal biodegradative action on PE-PU. IMPORTANCE: Polyurethane waste disposal has become a serious problem. In this work, fungal strains able to efficiently degrade different types of polyurethanes are reported, and their biodegradative activity was studied by different experimental approaches. Varnish biodegradation analyses showed that fungi were able to break down the polymer in some of their precursors, offering the possibility that they may be recovered and used for new polyurethane synthesis. Also, the levels of degradation of solid polyether polyurethane foams reported in this work have never been observed previously. Isolation of efficient polyurethane-degrading microorganisms and delving into the mechanisms they used to degrade the polymer provide the basis for the development of biotechnological processes for polyurethane biodegradation and recycling.

Closely related fungi employ diverse enzymatic strategies to degrade plant biomass.

BACKGROUND: Plant biomass is the major substrate for the production of biofuels and biochemicals, as well as food, textiles and other products. It is also the major carbon source for many fungi and enzymes of these fungi are essential for the depolymerization of plant polysaccharides in industrial processes. This is a highly complex process that involves a large number of extracellular enzymes as well as non-hydrolytic proteins, whose production in fungi is controlled by a set of transcriptional regulators. Aspergillus species form one of the best studied fungal genera in this field, and several species are used for the production of commercial enzyme cocktails. RESULTS: It is often assumed that related fungi use similar enzymatic approaches to degrade plant polysaccharides. In this study we have compared the genomic content and the enzymes produced by eight Aspergilli for the degradation of plant biomass. All tested Aspergilli have a similar genomic potential to degrade plant biomass, with the exception of A. clavatus that has a strongly reduced pectinolytic ability. Despite this similar genomic potential their approaches to degrade plant biomass differ markedly in the overall activities as well as the specific enzymes they employ. While many of the genes have orthologs in (nearly) all tested species, only very few of the corresponding enzymes are produced by all species during growth on wheat bran or sugar beet pulp. In addition, significant differences were observed between the enzyme sets produced on these feedstocks, largely correlating with their polysaccharide composition. CONCLUSIONS: These data demonstrate that Aspergillus species and possibly also other related fungi employ significantly different approaches to degrade plant biomass. This makes sense from an ecological perspective where mixed populations of fungi together degrade plant biomass. The results of this study indicate that combining the approaches from different species could result in improved enzyme mixtures for industrial applications, in particular saccharification of plant biomass for biofuel production. Such an approach may result in a much better improvement of saccharification efficiency than adding specific enzymes to the mixture of a single fungus, which is currently the most common approach used in biotechnology.

Glucose kinases from Streptomyces peucetius var. caesius.

Glucose kinases (Glks) are enzymes of the glycolytic pathway involved in glucose phosphorylation. These enzymes can use various phosphoryl donors such as ATP, ADP, and polyphosphate. In several streptomycetes, ATP-glucose kinase (ATP-Glk) has been widely studied and regarded as the main glucose phosphorylating enzyme and is likely a regulatory protein in carbon catabolite repression. In cell extracts from the doxorubicin overproducing strain Streptomyces peucetius var. caesius, grown in glucose, a polyphosphate-dependent Glk (Pp-Glk) was detected by zymogram. Maximum activity was observed during the stationary growth phase (48 h) of cells grown in 100 mM glucose. No activity was detected when 20 mM glutamate was used as the only carbon source, supporting a role for glucose in inducing this enzyme. Contrary to wild-type strains of Streptomyces coelicolor, Streptomyces lividans, and Streptomyces thermocarboxydus K-155, S. peucetius var. caesius produced 1.8 times more Pp-Glk than ATP-Glk. In addition, this microorganism produced five and four times more Pp-Glk and anthracyclines, respectively, than its wild-type S. peucetius parent strain, supporting a role for this enzyme in antibiotic production in the overproducer strain. A cloned 726-bp DNA fragment from S. peucetius var. caesius encoded a putative Pp-Glk, with amino acid identities between 83 and 87 % to orthologous sequences from the above-cited streptomycetes. The cloned fragment showed the polyphosphate-binding sequences GXDIGGXXIK, TXGTGIGSA, and KEX(4)SWXXWA. Sequences for the Zn-binding motif were not detected in this fragment, suggesting that Pp-Glk is not related to the Glk ROK family of proteins.

Spatial and developmental differentiation of mannitol dehydrogenase and mannitol-1-phosphate dehydrogenase in Aspergillus niger.

The presence of a mannitol cycle in fungi has been subject to discussion for many years. Recent studies have found no evidence for the presence of this cycle and its putative role in regenerating NADPH. However, all enzymes of the cycle could be measured in cultures of Aspergillus niger. In this study we have analyzed the localization of two enzymes from the pathway, mannitol dehydrogenase and mannitol-1-phosphate dehydrogenase, and the expression of their encoding genes in nonsporulating and sporulating cultures of A. niger. Northern analysis demonstrated that mpdA was expressed in both sporulating and nonsporulating mycelia, while expression of mtdA was expressed only in sporulating mycelium. More detailed studies using green fluorescent protein and dTomato fused to the promoters of mtdA and mpdA, respectively, demonstrated that expression of mpdA occurs in vegetative hyphae while mtdA expression occurs in conidiospores. Activity assays for MtdA and MpdA confirmed the expression data, indicating that streaming of these proteins is not likely to occur. These results confirm the absence of the putative mannitol cycle in A. niger as two of the enzymes of the cycle are not present in the same part of A. niger colonies. The results also demonstrate the existence of spore-specific genes and enzymes in A. niger.

Post-genomic insights into the plant polysaccharide degradation potential of Aspergillus nidulans and comparison to Aspergillus niger and Aspergillus oryzae.

The plant polysaccharide degradative potential of Aspergillus nidulans was analysed in detail and compared to that of Aspergillus niger and Aspergillus oryzae using a combination of bioinformatics, physiology and transcriptomics. Manual verification indicated that 28.4% of the A. nidulans ORFs analysed in this study do not contain a secretion signal, of which 40% may be secreted through a non-classical method.While significant differences were found between the species in the numbers of ORFs assigned to the relevant CAZy families, no significant difference was observed in growth on polysaccharides. Growth differences were observed between the Aspergilli and Podospora anserina, which has a more different genomic potential for polysaccharide degradation, suggesting that large genomic differences are required to cause growth differences on polysaccharides. Differences were also detected between the Aspergilli in the presence of putative regulatory sequences in the promoters of the ORFs of this study and correlation of the presence of putative XlnR binding sites to induction by xylose was detected for A. niger. These data demonstrate differences at genome content, substrate specificity of the enzymes and gene regulation in these three Aspergilli, which likely reflect their individual adaptation to their natural biotope.

Constitutive and inducible pectinolytic enzymes from Aspergillus flavipes FP-500 and their modulation by pH and carbon source / Enzimas pectinolíticas constitutivas e indutíveis de Aspergillus flavipes FP-500 e sua modulação pelo pH e fonte de carbono

Growth and enzymes production by Aspergillus flavipes FP-500 were evaluated on pectin, polygalacturonic acid, galacturonic acid, arabinose, rhamnose, xylose, glycerol and glucose at different initial pH values. We found that the strain produced exopectinases, endopectinases and pectin lyases. Exopectinases and pectin lyase were found to be produced at basal levels as constitutive enzymes and their production was modulated by the available carbon source and pH of culture medium and stimulated by the presence of inducer in the culture medium. Endo-pectinase was basically inducible and was only produced when pectin was used as carbon source. Our results suggest that pectinases in A. flavipes FP-500 are produced in a concerted way. The first enzyme to be produced was exopectinase followed by Pectin Lyase and Endo-pectinase.

Avaliou-se o crescimento e a produção de enzimas por Aspergillus flavipes FP-500 em pectina, ácido poligalacturônico, ácido galacturônico, arabinose, ramnose, xilose, glicerol e glicose, em diferentes valores de pH inicial. Verificamos que a cepa produziu exopectinases, endopectinases e pectina liases. Exopectinases e pectina liases foram produzidas em níveis basais como enzimas constitutivas e sua produção foi modulada pela fonte de carbono disponível e pelo pH do meio de cultura e estimulada pela presença de indutores no meio de cultura. Endopectinase foi indutível e produzida somente quando pectina foi utilizada como fonte de carbono. Nossos resultados sugerem que as pectinases de A. flavipes FP-500 são produzidas de forma planejada. A primeira enzima a ser produzida foi expopectinase, seguida por pectina liase e endopectinase.

Constitutive and inducible pectinolytic enzymes from Aspergillus flavipes FP-500 and their modulation by pH and carbon source.

Growth and enzymes production by Aspergillus flavipes FP-500 were evaluated on pectin, polygalacturonic acid, galacturonic acid, arabinose, rhamnose, xylose, glycerol and glucose at different initial pH values. We found that the strain produced exopectinases, endopectinases and pectin lyases. Exopectinases and pectin lyase were found to be produced at basal levels as constitutive enzymes and their production was modulated by the available carbon source and pH of culture medium and stimulated by the presence of inducer in the culture medium. Endo-pectinase was basically inducible and was only produced when pectin was used as carbon source. Our results suggest that pectinases in A. flavipes FP-500 are produced in a concerted way. The first enzyme to be produced was exopectinase followed by Pectin Lyase and Endo-pectinase.

Kinetic study on inducibility of polygalacturonases from Aspergillus flavipes FP-500

The aim of this work was to describe growth dynamics, substrate depletion and polygalacturonases production by Aspergillus flavipes FP-500 in batch cultures by means of unstructured models. The microorganism was cultivated on several mono- di- and poly- saccharides, and then the culture development modeled with Monod and Leudeking-Piret equations. The kinetic parameters related to the models (µmax, ãx/s, alpha and beta) were obtained by minimizing the quadratic residuals function with a simplex algorithm. An accurate description of experimental data was attained with the proposed models. Besides, modeling provided significant kinetic information on microbial degradation of complex substrates, such as the correlation between specific growth rate µmax and production yield á, suggesting that A. flavipes FP-500 polygalacturonases are actually constitutive, but also that there is a certain degree of induciblility in these enzymatic activities.