RESUMO
The application of pyrethroids and carbamates represents an environmental risk and may exert adverse effects on beneficial microorganisms such as Trichoderma, which contribute to the biocontrol of several fungal phytopathogens. This research evaluated the tolerance of several strains of Trichoderma to a selected culture medium contaminated with a commercial insecticide (H24®) composed of pyrethroids, permethrin and prallethrin, and carbamate propoxur, and determined the influence of this insecticide on the release of enzymes such as chitinases, peroxidases, and endoglucanases by a consortium of selected Trichoderma strains grown in liquid culture medium. Four out of 10 Trichoderma strains showed tolerance to 200ppm (â¼48.3% of growth) of the commercial insecticide after 96h of exposure to a contaminated solid medium. After eight days of growth in liquid culture, the insecticide enhanced extracellular protein content and peroxidase activities in the Trichoderma consortium but decreased both chitinase and glucanase activities. These fungal responses should be considered when implementing strategies that combine alternative pesticides and fungal biocontrollers for managing fungal phytopathogens.
Assuntos
Quitinases , Inseticidas , Piretrinas , Trichoderma , Trichoderma/metabolismo , Inseticidas/farmacologia , Piretrinas/farmacologia , Quitinases/metabolismo , Carbamatos , Meios de CulturaRESUMO
Lignocellulose is the most abundant natural biopolymer on earth and a potential raw material for the production of fuels and chemicals. However, only some organisms such as bacteria and fungi produce enzymes that metabolize this polymer. In this work we have demonstrated the presence of cellulolytic activity in the supernatant of Scenedesmus quadricauda cultures and we identified the presence of extracellular cellulases in the genome of five Scenedesmus species. Scenedesmus is a green alga which grows in both freshwater and saltwater regions as well as in soils, showing highly flexible metabolic properties. Sequence comparison of the different identified cellulases with hydrolytic enzymes from other organisms using multisequence alignments and phylogenetic trees showed that these proteins belong to the families of glycosyl hydrolases 1, 5, 9, and 10. In addition, most of the Scenedesmus cellulases showed greater sequence similarity with those from invertebrates, fungi, bacteria, and other microalgae than with the plant homologs. Furthermore, the data obtained from the three dimensional structure showed that both, their global structure and the main amino acid residues involved in catalysis and substrate binding are well conserved. Based on our results, we propose that different species of Scenedesmus could act as biocatalysts for the hydrolysis of cellulosic biomass produced from sunlight.
Assuntos
Celulases , Scenedesmus , Scenedesmus/metabolismo , Filogenia , Celulases/genética , Celulases/metabolismo , Bactérias/metabolismo , Hidrólise , Fungos/metabolismoRESUMO
Aspergillus niger KIJH was grown in solid and submerged fermentation using leaves and roots (with and without bark) of plants typically from Brazilian semiarid as substrate to produce a multienzymatic extract, which was characterised for its potential biotechnological applications. Solid-state fermentation (SSF) was applied to select the most promising plants biomass as induction substrates for the production of hydrolytic enzymes by fungus. The best biomasses were used as substrate in submerged fermentation (SmF) assays at two scales. Samples of up scale fermented culture were partially purified by ultrafiltration and activity and pH and temperature stability of CMCase and xylanase were evaluated. A. niger KIJH produced hydrolytic enzymes under SSF containing unconventional plants biomass from Brazilian semiarid. In SmF conditions, maximum CMCase (0.264 U mL-1) and xylanase (1.163 U mL-1) activities were induced by Jacaratia corumbensis. Scaling up the SmF to 500 mL of medium was able to maintain constant the production of CMCase (0.346 U mL-1) and xylanase (1.273 U mL-1) on the fermented culture. Ultrafiltered and concentrated extract presented CMCase activities practically constant in all temperature ranges (30-80°C) and pH (3.0-9.0), while xylanase optimum activity temperature was 50°C and pH in the range of 3.0 to 5.0. CMCase activity remained stable for 24 hours at 50°C(AU)
Assuntos
Biomassa , Aspergillus niger/crescimento & desenvolvimento , Fermentação , Substratos para Tratamento BiológicoRESUMO
Aspergillus niger KIJH was grown in solid and submerged fermentation using leaves and roots (with and without bark) of plants typically from Brazilian semiarid as substrate to produce a multienzymatic extract, which was characterised for its potential biotechnological applications. Solid-state fermentation (SSF) was applied to select the most promising plants biomass as induction substrates for the production of hydrolytic enzymes by fungus. The best biomasses were used as substrate in submerged fermentation (SmF) assays at two scales. Samples of up scale fermented culture were partially purified by ultrafiltration and activity and pH and temperature stability of CMCase and xylanase were evaluated. A. niger KIJH produced hydrolytic enzymes under SSF containing unconventional plants biomass from Brazilian semiarid. In SmF conditions, maximum CMCase (0.264 U mL-1) and xylanase (1.163 U mL-1) activities were induced by Jacaratia corumbensis. Scaling up the SmF to 500 mL of medium was able to maintain constant the production of CMCase (0.346 U mL-1) and xylanase (1.273 U mL-1) on the fermented culture. Ultrafiltered and concentrated extract presented CMCase activities practically constant in all temperature ranges (30-80°C) and pH (3.0-9.0), while xylanase optimum activity temperature was 50°C and pH in the range of 3.0 to 5.0. CMCase activity remained stable for 24 hours at 50°C
Assuntos
Aspergillus niger/crescimento & desenvolvimento , Biomassa , Fermentação , Substratos para Tratamento BiológicoRESUMO
Aspergillus niger KIJH was grown in solid and submerged fermentation using leaves and roots (with and without bark) of plants typically from Brazilian semiarid as substrate to produce a multienzymatic extract, which was characterised for its potential biotechnological applications. Solid-state fermentation (SSF) was applied to select the most promising plants biomass as induction substrates for the production of hydrolytic enzymes by fungus. The best biomasses were used as substrate in submerged fermentation (SmF) assays at two scales. Samples of up scale fermented culture were partially purified by ultrafiltration and activity and pH and temperature stability of CMCase and xylanase were evaluated. A. niger KIJH produced hydrolytic enzymes under SSF containing unconventional plants biomass from Brazilian semiarid. In SmF conditions, maximum CMCase (0.264 U mL-1) and xylanase (1.163 U mL-1) activities were induced by Jacaratia corumbensis. Scaling up the SmF to 500 mL of medium was able to maintain constant the production of CMCase (0.346 U mL-1) and xylanase (1.273 U mL-1) on the fermented culture. Ultrafiltered and concentrated extract presented CMCase activities practically constant in all temperature ranges (30-80°C) and pH (3.0-9.0), while xylanase optimum activity temperature was 50°C and pH in the range of 3.0 to 5.0. CMCase activity remained stable for 24 hours at 50°C a
RESUMO
Cello-oligosaccharides (COS) are oligomers with 2 to 6 ß-1,4-linked glucose units, with potential applications in the food/feed and bioenergy industrial sectors. In this study, the combination of five heterologous expressed endoglucanases varying the temperature and pH conditions were evaluated by design of experiments for COS production. Afterwards, the best combination was tested to produce COS from different pretreated sugarcane straws: ionic liquid, diluted acid, hydrothermal and steam-explosion. The results showed that steam explosion pretreated sugarcane straw treated with CtCel9R enzyme at 50 °C and pH 5.0 yielded 13.4 mg COS g biomass-1, 5-18-fold higher compared to the other pretreated straws. Under the conditions evaluated, the removal of hemicellulose and decrease in the cellulose crystallinity can benefits the enzymatic hydrolysis. This is the first study that combined the evaluation of different enzymes, conditions, and sugarcane straw pretreatments to optimize COS production in a single step without glucose formation.
Assuntos
Celulase , Saccharum , Celulose , Hidrólise , OligossacarídeosRESUMO
OBJECTIVES: To evaluate a strain of Fusarium verticillioides ITV03 isolated from wood residues in the Veracruz region of Mexico. Endoglucanase and ß-glucosidase production by submerged fermentation was optimized using a Box-Behnken design, where the independent variables were urea, ammonium sulfate and yeast extract. RESULTS: After optimization, an endoglucanase activity of 0.27 U/mL was achieved; subsequently, three carbon sources were evaluated (carboxymethyl cellulose, sweet sorghum bagasse cellulose and delignified sweet sorghum bagasse (DSSB). The results showed that DSSB yielded the greatest endoglucanase (0.28 U/mL) and ß-glucosidase (0.12 U/mL) activities. Both enzymatic activities were characterized for the effect of pH, temperature and thermostability. The optimal parameters of ß-glucosidase and endoglucanase activity were pH 5 and 4 respectively, the optimum temperature 60 °C. These enzymes were stable at 50 °C for 150.68 h and 8.54 h, with an activation energy (Ea(day)) of 265.55 kJ/mol and 44.40 kJ/mol respectively, for ß-glucosidase and endoglucanase. CONCLUSION: The present work shows that a native strain like F. verticillioides ITV03 using DSSB supplemented with nitrogen has a great potential as a producer of cellulase for lignocellulosic residue hydrolysis.
Assuntos
Celulose/química , Endo-1,4-beta-Xilanases/metabolismo , Fusarium/crescimento & desenvolvimento , Sorghum/química , beta-Glucosidase/metabolismo , Meios de Cultura/química , Estabilidade Enzimática , Fermentação , Proteínas Fúngicas/metabolismo , Fusarium/enzimologia , Fusarium/isolamento & purificação , Temperatura Alta , Concentração de Íons de Hidrogênio , México , Nitrogênio/química , Madeira/microbiologiaRESUMO
Glycoside hydrolase (GH) family 45 is one of the smallest and poorly studied endoglucanase family with a broad biotechnological application ranging from treatment of textiles to conversion of complex cell wall polysaccharides into simple oligo- and monosaccharides. In a present study, GH45 cellulase from Neurospora crassa OR74A (NcCel45A) was characterized both biochemically and structurally. HPLC analysis of the hydrolytic products confirmed the endo-ß(1,4) mode of action of the enzyme. Moreover, such pattern revealed that NcCel45A cannot hydrolyze efficiently oligosaccharides with a degree of polymerization smaller than six. The crystal structure of NcCel45A catalytic domain in the apo-form was determined at 1.9â¯Å resolution and the structure of the enzyme bound to cellobiose was solved and refined to 1.8â¯Å resolution. Comparative structural analyses and molecular dynamics simulations show that the enzyme dynamics is affected by substrate binding. Taken together, MD simulations and statistical coupling analysis revealed previously unknown correlation of a loop 6 with the breakdown of cellulose substrates by GH45.
Assuntos
Celulase/química , Celulose/metabolismo , Neurospora crassa/enzimologia , Domínio Catalítico , Cristalografia por Raios X , Hidrólise , Simulação de Dinâmica Molecular , Conformação Proteica , Especificidade por SubstratoRESUMO
Endophytes constitute plant-colonizing microorganisms in a mutualistic symbiosis relationship. They are found in most ecosystems reducing plant crops' biotic and abiotic stressors by stimulating immune responses, excluding plant pathogens by niche competition, and participating in antioxidant activities and phenylpropanoid metabolism, whose activation produces plant defense, structural support, and survival molecules. In fact, metabolomic studies have demonstrated that endophyte genes associated to specific metabolites are involved in plant growth promotion (PGP) by stimulating plant hormones production such as auxins and gibberellins or as plant protective agents against microbial pathogens, cancer, and insect pests, but eco-friendly and eco-safe. A number of metabolites of Gram-positive endophytes isolated from agriculture, forest, mangrove, and medicinal plants, mainly related to the Firmicutes phyla, possess distinctive biocontrol and plant growth-promoting activities. In general, Actinobacteria and Bacillus endophytes produce aromatic compounds, lipopeptides, plant hormones, polysaccharides, and several enzymes linked to phenylpropanoid metabolism, thus representing high potential for PGP and crop management strategies. Furthermore, Actinobacteria have been shown to produce metabolites with antimicrobial and antitumor activities, useful in agriculture, medicine, and veterinary areas. The great endophytes diversity, their metabolites production, and their adaptation to stress conditions make them a suitable and unlimited source of novel metabolites, whose application could reduce agrochemicals usage in food and drugs production.
RESUMO
The cellulases from Glycoside Hydrolyses family 12 (GH12) play an important role in cellulose degradation and plant cell wall deconstruction being widely used in a number of bioindustrial processes. Aiming to contribute toward better comprehension of these class of the enzymes, here we describe a high-yield secretion of a endoglucanase GH12 from Aspegillus terreus (AtGH12), which was cloned and expressed in Aspergillus nidulans strain A773. The purified protein was used for complete biochemical and functional characterization. The optimal temperature and pH of the enzyme were 55°C and 5.0 respectively, which has high activity against ß-glucan and xyloglucan and also is active toward glucomannan and CMC. The enzyme retained activity up to 60°C. AtGH12 is strongly inhibited by Cu2+, Fe2+, Cd2+, Mn2+, Ca2+, Zn2+ and EDTA, whereas K+, Tween, Cs+, DMSO, Triton X-100 and Mg2+ enhanced the enzyme activity. Furthermore, SAXS data reveal that the enzyme has a globular shape and CD analysis demonstrated a prevalence of a ß-strand structure corroborating with typical ß-sheets fold commonly found for other endoglucanases from GH12 family.
Assuntos
Aspergillus , Celulase , Clonagem Molecular , Proteínas Fúngicas , Expressão Gênica , Aspergillus/enzimologia , Aspergillus/genética , Celulase/biossíntese , Celulase/química , Celulase/genética , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas RecombinantesRESUMO
The giant snail Achatina fulica is considered an invasive species in most territories in which it was introduced, due to its ability to process a large amount of lignocellulose as a consequence of the presence of a cellulolytic-associated microflora. Streptomyces are well known as crucial agents in the decomposition of complex polymers in soil environments and also as cellulolytic symbionts commonly associated with herbivore insects. Here, we employed a combination of genomic and biochemical tools for a detailed evaluation of the cellulolytic potential of Streptomyces sp. I1.2, an aerobic bacterium isolated from the intestinal lumen of A. fulica in a screening for cellulolytic bacteria. Genomic analysis revealed that the ratio and diversity of CAZy domains and GH families coded by Streptomyces sp. I1.2 are comparable to those present in other highly cellulolytic bacteria. After growth on crystalline cellulose or sugarcane bagasse as sole carbon sources, the functionality of several genes encoding endoglucanases, cellobiohydrolases, xylanases, CBMs, and one ß-glucosidase were confirmed by the combination of enzymatic activity measurements, zymography, TLC, and cellulose-binding assays. The endoglucanases secreted by this isolate were stable at 50 °C and exhibited activity over a broad pH range between 4.0 and 8.0. The endoglucanases and cellobiohydrolases secreted by Streptomyces sp. I1.2 exhibited specific activities that were similar to the levels present in a commercial cellulase preparation from Trichoderma reesei, while I1.2 xylanase levels were even 350 % higher. The results presented here show that Streptomyces sp. I1.2 is promising for future biotechnological applications, since it is able to produce endoglucanases, cellobiohydrolases, and xylanases in appreciable amounts when grown on a low-cost residue such as sugarcane bagasse.
Assuntos
Celulose/metabolismo , Glicosídeo Hidrolases/análise , Streptomyces/enzimologia , Streptomyces/metabolismo , Animais , Carbono/metabolismo , Gastrópodes/microbiologia , Concentração de Íons de Hidrogênio , Hidrólise , Streptomyces/genética , Streptomyces/isolamento & purificação , TemperaturaRESUMO
Background: Cold-active endo-1, 4-β-glucanase (EglC) can decrease energy costs and prevent product denaturation in biotechnological processes. However, the nature EglC from C. farmeri A1 showed very low activity (800 U/L). In an attempt to increase its expression level, C. farmeri EglC was expressed in Escherichia coli as an N-terminal fusion to protein S (ProS) from Myxococcus xanthus. Results: A novel expression vector, pET(ProS-EglC), was successfully constructed for the expression of C. farmeri EglC in E. coli. SDS-PAGE showed that the recombinant protein (ProS-EglC) was approximately 60 kDa. The activity of ProS-EglC was 12,400 U/L, which was considerably higher than that of the nature EglC (800 U/L). ProS-EglC was active at pH 6.5-pH 8.0, with optimum activity at pH 7.0. The recombinant protein was stable at pH 3.5-pH 6.5 for 30 min. The optimal temperature for activity of ProS-EglC was 30°C-40°C. It showed greater than 50% of maximum activity even at 5°C, indicating that the ProS-EglC is a cold-active enzyme. Its activity was increased by Co2+ and Fe2+, but decreased by Cd2+, Zn2+, Li+, methanol, Triton-X-100, acetonitrile, Tween 80, and SDS. Conclusions: The ProS-EglC is promising in application of various biotechnological processes because of its cold-active characterizations. This study also suggests a useful strategy for the expression of foreign proteins in E. coli using a ProS tag.
Assuntos
Celulases/metabolismo , Citrobacter/enzimologia , Escherichia coli/enzimologia , Myxococcus xanthus/enzimologia , Temperatura Baixa , Vetores Genéticos , Proteínas RecombinantesRESUMO
Endoglucanases are the main cellulolytic enzymes secreted by the bacterium Xanthomonas campestris pv. campestris (Xcc). The major endoglucanase exported by this bacterium into an external milieu is an enzyme XccCel5A, which belongs to GH5 family subfamily 1 and is encoded by the gene engXCA. We purified XccCel5A using ammonium sulfate precipitation followed by size exclusion chromatography and identified it by zymogram analysis. Circular dichroism and fluorescence spectroscopy studies showed that XccCel5A is stable in a wide pH range and up to about 55°C and denatures at the higher temperatures. The optimal conditions for enzyme activity were identified as T=45°C and pH=7.0. Under the optimum conditions the catalytic efficiency (kcat/KM) of the enzyme was determined as 5.16×10(4)s(-1)M(-1) using carboxymethylcellulose (CMC) as a substrate. Our SAXS studies revealed extended tadpole-shape molecular assembly, typical for cellulases, and allowed to determine an overall shape of the enzyme and a relative position of the catalytic and cellulose binding domains.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Celulase/química , Celulase/metabolismo , Xanthomonas campestris/enzimologia , Proteínas de Bactérias/genética , Fenômenos Biofísicos , Domínio Catalítico , Celulase/genética , Dicroísmo Circular , Estabilidade Enzimática , Genes Bacterianos , Cinética , Modelos Moleculares , Conformação Proteica , Espalhamento a Baixo Ângulo , Espectrometria de Fluorescência , Difração de Raios X , Xanthomonas campestris/genéticaRESUMO
Neurospora crassa BGT-1 (NCU06381) and BGT-2 (NCU09175) are two putative glycoside hydrolases (GHs) with additional predicted glycosyltransferase activity and binding sites for a glycosyl phosphatidyl inositol (GPI) anchor that would facilitate their attachment to the plasma membrane (PM). To discern their role in key morphogenetic events during vegetative development of N. crassa, BGT-1 and BGT-2 were labeled with the green fluorescent protein (GFP). The gfp was inserted immediately after the signal peptide sequence, within the bgt-1 encoding sequence, or directly before the GPI-binding site in the case of bgt-2. Both BGT-1-GFP and BGT-2-GFP were observed at the PM of the hyphal apical dome, excluding the foremost apical region and the Spitzenkörper (Spk), where chitin and ß-1,3-glucan synthases have been previously found. These and previous studies suggest a division of labor of the cell wall synthesizing machinery at the hyphal dome: at the very tip, glucans are synthesized by enzymes that accumulate at the Spk, before getting incorporated into the PM, whereas at the subtending zone below the apex, glucans are presumably hydrolyzed, producing amenable ends for further branching and crosslinking with other cell wall polymers. Additionally, BGT-1-GFP and BGT-2-GFP were observed at the leading edge of new developing septa, at unreleased interconidial junctions, at conidial poles, at germling and hyphal fusion sites, and at sites of branch emergence, all of them processes that seemingly involve cell wall remodeling. Even though single and double mutant strains for the corresponding genes did not show a drastic reduction of growth rate, bgt-2Δ and bgt-1Δ::bgt-2Δ strains exhibited an increased resistance to the cell wall stressors calcofluor white (CW) and congo red (CR) than the reference strain, which suggests they present significant architectural changes in their cell wall. Furthermore, the conidiation defects observed in the mutants indicate a significant role of BGT-1 and BGT-2 on the re-arrangement of glucans needed at the conidiophore cell wall to allow conidial separation.
Assuntos
Parede Celular/metabolismo , Glicosídeo Hidrolases/metabolismo , Glicosiltransferases/metabolismo , Neurospora crassa/enzimologia , Membrana Celular/metabolismo , Quitina/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Reporter , Glicosídeo Hidrolases/genética , Glicosilfosfatidilinositóis/metabolismo , Glicosiltransferases/genética , Hifas , Neurospora crassa/citologia , Neurospora crassa/genética , Neurospora crassa/crescimento & desenvolvimento , Esporos Fúngicos , beta-Glucanas/metabolismoRESUMO
Enzymes that cleave the xyloglucan backbone at unbranched glucose residues have been identified in GH families 5, 7, 12, 16, 44, and 74. Fungi produce enzymes that populate 20 of 22 families that are considered critical for plant biomass deconstruction. We searched for GH12-encoding genes in 27 Eurotiomycetes genomes. After analyzing 50 GH12-related sequences, the conserved variations of the amino acid sequences were examined. Compared to the endoglucanases, the endo-xyloglucanase-associated YSG deletion at the negative subsites of the catalytic cleft with a SST insertion at the reducing end of the substrate-binding crevice is highly conserved. In addition, a highly conserved alanine residue was identified in all xyloglucan-specific enzymes, and this residue is substituted by arginine in more promiscuous glucanases. To understand the basis for the xyloglucan specificity displayed by certain GH12 enzymes, two fungal GH12 endoglucanases were chosen for mutagenesis and functional studies: an endo-xyloglucanase from Aspergillus clavatus (AclaXegA) and an endoglucanase from A. terreus (AtEglD). Comprehensive molecular docking studies and biochemical analyses were performed, revealing that mutations at the entrance of the catalytic cleft in AtEglD result in a wider binding cleft and the alteration of the substrate-cleavage pattern, implying that a trio of residues coordinates the interactions and binding to linear glycans. The loop insertion at the crevice-reducing end of AclaXegA is critical for catalytic efficiency to hydrolyze xyloglucan. The understanding of the structural elements governing endo-xyloglucanase activity on linear and branched glucans will facilitate future enzyme modifications with potential applications in industrial biotechnology.
Assuntos
Aspergillus/metabolismo , Celulase/metabolismo , Proteínas Fúngicas/metabolismo , Glucanos/metabolismo , Glicosídeo Hidrolases/metabolismo , Xilanos/metabolismo , Sequência de Aminoácidos , Aspergillus/química , Aspergillus/genética , Domínio Catalítico , Celulase/química , Celulase/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Filogenia , Dobramento de Proteína , Deleção de Sequência , Especificidade por SubstratoRESUMO
Cellulases, such as endoglucanases, exoglucanases and ß-glucosidases, are important enzymes used in the process of enzymatic hydrolysis of plant biomass. The bacteria Xanthomonas campestris pv. campestris expresses a large number of hydrolases and the major endoglucanase (XccEG), a member of glycoside hydrolase family 5 (GH5), is the most strongly secreted extracellularly. In this work, the native XccEG was purified from the extracellular extract and crystallization assays were performed on its catalytic domain. A complete data set was collected on an in-house X-ray source. The crystal diffracted to 2.7 Å resolution and belonged to space group C2, with unit-cell parameters a = 174.66, b = 141.53, c = 108.00 Å, ß = 110.49°. The Matthews coefficient suggests a solvent content of 70.1% and the presence of four protein subunits in the asymmetric unit.