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1.
Bioresour Technol ; 313: 123616, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32563792

RESUMO

Trichoderma harzianum has attracting attention for its potential alternative use in biofuel production, due to a recognized competence for high diversity glycoside hydrolases (GH) enzyme complex, including higher ß-glucosidases and auxiliary proteins, using low-cost carbon sources. This strain constitutively overexpressed the global regulator putative methyltransferase - LAE1, in order to improve the GHs production. The recombinant strain achieved 79-fold increase in lae1 expression and high GHs productivity. The evaluation of the LAE1 impact to induce the GHs used soluble and lignocellulose inexpensive carbon sources in a stirred-tank bioreactor. Using sugarcane bagasse with sucrose, the overexpression of lae1 resulted in significantly increment of gh61b (31x), cel7a (25x), bgl1(20x) and xyn3 (20x) genes expression. Reducing sugar released from pretreated sugarcane bagasse, which hydrolyzed by recombinant crude enzyme cocktail, achieved 41% improvement. Therefore, lae1 overexpression effectively is a promising improving GHs target for biomass degradation by T. harzianum.


Assuntos
Celulases , Saccharum , Trichoderma , Biomassa , Metiltransferases
2.
Toxicon ; 180: 43-48, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32298663

RESUMO

Zearalenone, an oestogenic mycotoxin produced by Fusarium sp., occurs naturally in agricultural commodities. Economic losses and health concerns associated to mycotoxins has attracted research interest towards exploring novel approaches to detoxify mycotoxin-contaminated food and feed. The aim of the present work was to study the ability of 11 aflatoxin-degrading Bacillus strains to degrade ZEA. In addition, a qualitative assessment of protease, amylase and cellulase activity of the studied Bacillus strains was made. All strains were able to degrade 58-96.9% ZEA after 72 h. Toxicity towards Artemia salina was significantly reduced (P < 0.0001). Degradation extracts fluorescence decreased 50% indicating a probable cleavage of the lactone ring. Strains RC1A, RC3A and RC6A showed a remarkable enzymatic activity, showing potential to be used as feed additives.


Assuntos
Aflatoxinas/metabolismo , Amilases/metabolismo , Bacillus/metabolismo , Celulases/metabolismo , Peptídeo Hidrolases/metabolismo , Zearalenona/metabolismo , Agricultura , Inativação Metabólica
3.
Bioresour Technol ; 304: 122974, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32062498

RESUMO

Most additives that capable of enhancing enzymatic hydrolysis of lignocellulose are petroleum-based, which are not easy to recycle with poor biodegradability. In this work, highly recyclable and biodegradable sodium caseinate (SC) was used to enhance lignocellulosic hydrolysis with improved cellulase recyclability. When the pH decreased from 5.5 to 4.8, more than 96% SC could be precipitated from the solution and recovered. Adding SC increased enzymatic digestibility of dilute acid pretreated eucalyptus (Eu-DA) from 39.5% to 78.2% under Eu-DA loading of 10 wt% and pH = 5.5, and increase cellulase content in 72 h hydrolysate from only 15.2% of the original to 60.0%, which facilitated the recovery of cellulases through re-adsorption by fresh substrates. With multiple cycles of re-adsorption, application of SC not only increased the sugar yield of Eu-DA by 95.5%, but also reduced cellulase loading by 40%.


Assuntos
Celulase , Celulases , Caseínas , Hidrólise , Lignina
4.
Arch Microbiol ; 202(5): 935-951, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31927608

RESUMO

Considering the potential use of lignocellulosic biomass residues in microbial cultures to produce cellulases, the objective of this research was to investigate trends and discussions regarding scientific research conducted in this field through a bibliometric and scientometric analysis. Using the Elsevier Scopus database and VOSviewer software, scientific papers published between 2007 and 2018 were investigated. The results showed that the production of cellulases is related to obtaining xylanases and glucose. Obtaining of bioethanol and determining cellulolytic and xylanase activities were the relevant indicators for the use of these enzymes. China, India and Brazil are countries with a high number of publications in this field, most likely due to investments made between 2015 and 2017. This analysis showed that research on the use of lignocellulosic residues is focused on obtaining biofuels through enzymatic hydrolysis.


Assuntos
Biocombustíveis/microbiologia , Celulases/biossíntese , Lignina/metabolismo , Bactérias/enzimologia , Bactérias/metabolismo , Bibliometria , Biomassa , Brasil , China , Hidrólise , Índia
5.
ChemSusChem ; 13(1): 106-110, 2020 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-31593363

RESUMO

The activity of ß-glucosidases-the enzymes responsible for the final step in the enzymatic conversion of cellulose to glucose-can be maintained and manipulated under mechanochemical conditions in the absence of bulk solvent, either through an unexpected stabilization effect of inert surfaces, or by altering the enzymatic equilibrium. The reported results illustrate unique aspects of mechanoenzymatic reactions that are not observable in conventional aqueous solutions. They also represent the first reported strategies to enhance activity and favor either direction of the reaction under mechanochemical conditions.


Assuntos
Celulases/metabolismo , Catálise , Celulose/química , Cinética , Fenômenos Mecânicos , Polímeros/química , Propriedades de Superfície
6.
Biosci Biotechnol Biochem ; 84(2): 238-246, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31625450

RESUMO

The Cel genes from Bacillus licheniformis MSB03 were cloned and expressed to investigate binding ability on clay minerals and sea sand at pH ranging 3 to 9. FTIR analysis has been done to characterize bound enzymes on clay minerals. Subsequent, surveying of NCBI database for extracellular enzymes of soil bacteria was carried out. Among the five cloned Cel enzymes assayed for binding to clay minerals, only Cel5H enzyme had the binding ability. Enzyme Cel5H exhibited highest binding to montmorillonite followed by kaolinite and sea sand. Interestingly, Cel5H had higher pI value of 9.24 than other proteins (5.2-5.7). Cel5H binding to montmorillonite was shown to be negatively affected below pH 3 and above pH 9. Infrared absorption spectra of the Cel5H-montmorillonite complexes showed distinct peaks for clay minerals and bound proteins. Furthermore, database survey of soil bacterial extracellular enzymes revealed that Bacillus species enzymes had higher pI than other soil bacterial enzymes.


Assuntos
Bacillus licheniformis/enzimologia , Celulases/metabolismo , Argila , Bases de Dados de Proteínas , Ponto Isoelétrico , Minerais/metabolismo , Microbiologia do Solo , Celulases/genética , Clonagem Molecular , Hidrólise , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
7.
Insect Mol Biol ; 29(1): 124-135, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31449690

RESUMO

Many hemimetabolous insects produce their own cellulase enzymes from the glycoside hydrolase family 9, first observed in termites and cockroaches. Phasmatodea have multiple cellulases, some of which are multifunctional and can degrade xylan or xyloglucan. To discover when these abilities evolved, we identified cellulases from the Polyneoptera sampled by the 1000 Insect Transcriptome and Evolution (1KITE) project, including all cockroach and termite transcriptomes. We hoped to identify what role enzyme substrate specificities had in the evolution of dietary specification, such as leaf-feeding or wood-feeding. Putative cellulases were identified from the transcriptomes and analysed phylogenetically. All cellulases were amplified from an exemplar set of Polyneoptera species using rapid amplification of cDNA ends PCR and heterologously expressed in an insect cell line, then tested against different polysaccharides for their digestive abilities. We identified several multifunctional xyloglucanolytic enzymes across Polyneoptera, plus a large group of cellulase-like enzymes found in nearly all insect orders with no discernible digestive ability. Multifunctional xylanolytic cellulases remain unique to Phasmatodea. The presence or absence of multifunctional enzymes does not impact dietary specification, but rather having multiple, multifunctional cellulase genes is an ancestral state for Polyneoptera and possibly Insecta. The prevalence of multifunctional cellulases in other animals demands further investigation.


Assuntos
Celulases/genética , Insetos/enzimologia , Insetos/genética , Polissacarídeos/metabolismo , Animais , Evolução Biológica , Celulases/química , Dieta , Insetos/classificação , Insetos/metabolismo , Enzimas Multifuncionais , Filogenia , Transcriptoma
8.
Appl Biochem Biotechnol ; 190(2): 448-463, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31378843

RESUMO

Cellulases, as environmentally appropriate catalysts specifically acting on cellulosic substrates, are important for the industrial conversion of lignocellulose and modification of cellulose products. After decades of research, a fundamental understanding of cellulase-mediated hydrolysis of cellulose is that its ability to processively act as a key for the complete enzymatic hydrolysis of natural crystalline cellulose. Two types of processive cellulases are known: exoglucanases and processive endoglucanases. Exoglucanases are typical processive enzymes, and they have been studied in detail so that their modes of action and mechanisms are reasonably well characterized. Conversely, endoglucanases are less well characterized because of the non-universality and structural diversity. However, processive endoglucanases have certain characteristics that exoglucanases lack such as hydrolysis product diversity and independent hydrolyze natural crystalline cellulose. Therefore, besides the conversion of cellulose to monosaccharide, they might be useful for modification of fibrous substrates and preparation of cellulose oligosaccharides. Herein, we review in detail the sources, hydrolysis products, application, and possible hydrolysis mechanisms of processive endoglucanases.


Assuntos
Celulase/metabolismo , Bactérias/enzimologia , Catálise , Celulases/metabolismo , Celulose/metabolismo , Fungos/enzimologia
9.
Biochim Biophys Acta Proteins Proteom ; 1868(1): 140297, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31672609

RESUMO

Two forms of C1/C4-oxidizing lytic polysaccharide monooxygenase (PvLPMO9A) from Penicillium verruculosum (Talaromyces verruculosus) homologously expressed in P. verruculosum B1-537 auxotrophic strain were isolated in a homogeneous state using two-stage chromatography. The PvLPMO9A-hm form represented a full-size enzyme encoded by the intact lpmo1 gene, while the PvLPMO9A-lm was a truncated enzyme variant consisting of a conserved catalytic core of AA9 family LPMOs and lacking a C-terminal extra peptide sequence that is present in PvLPMO9A-hm. The N-terminal histidine was partially methylated in both enzymes. Most of properties of PvLPMO9A-hm and PvLPMO9A-lm, such as specific activities determined using the 2,6-dimethoxyphenol/H2O2 assay, pH-optima of activity observed at pH 7.5, synergistic effects exhibited with purified cellobiohydrolase I (Cel7A) and/or endoglucanase II (Cel5A) from P. verruculosum in hydrolysis of Avicel and milled aspen wood, were also very similar, except for the higher PvLPMO9A-hm thermostability studied using differential scanning calorimetry (DSC). The DSC profile for the PvLPMO9A-hm holoenzyme demonstrated two overlapping peaks (with maxima at 56.3 and 59.6 °C) due to the presence of two unfolding protein domains, while the PvLPMO9A-lm DSC profile represented one peak with maximum at 48.1 °C. After removing the active site copper with EDTA, the PvLPMO9A-hm and PvLPMO9A-lm melting temperatures decreased by ~10-11 and ~1 °C, respectively. These data show that both active site copper and C-terminal domain present in the PvLPMO9A-hm protect the enzyme from thermal unfolding, while the stabilizing effect of metal is much less pronounced in the truncated PvLPMO9A-lm form.


Assuntos
Proteínas Fúngicas/química , Oxigenases de Função Mista/química , Penicillium/enzimologia , Sequência de Aminoácidos , Domínio Catalítico , Celulases/química , Celulose/química , Cobre/química , Estabilidade Enzimática , Proteínas Fúngicas/genética , Oxigenases de Função Mista/genética , Domínios Proteicos
10.
Proc Natl Acad Sci U S A ; 116(46): 23061-23067, 2019 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-31666327

RESUMO

Cellulase enzymes deconstruct recalcitrant cellulose into soluble sugars, making them a biocatalyst of biotechnological interest for use in the nascent lignocellulosic bioeconomy. Cellobiohydrolases (CBHs) are cellulases capable of liberating many sugar molecules in a processive manner without dissociating from the substrate. Within the complete processive cycle of CBHs, dissociation from the cellulose substrate is rate limiting, but the molecular mechanism of this step is unknown. Here, we present a direct comparison of potential molecular mechanisms for dissociation via Hamiltonian replica exchange molecular dynamics of the model fungal CBH, Trichoderma reesei Cel7A. Computational rate estimates indicate that stepwise cellulose dethreading from the binding tunnel is 4 orders of magnitude faster than a clamshell mechanism, in which the substrate-enclosing loops open and release the substrate without reversing. We also present the crystal structure of a disulfide variant that covalently links substrate-enclosing loops on either side of the substrate-binding tunnel, which constitutes a CBH that can only dissociate via stepwise dethreading. Biochemical measurements indicate that this variant has a dissociation rate constant essentially equivalent to the wild type, implying that dethreading is likely the predominant mechanism for dissociation.


Assuntos
Celulases/química , Proteínas Fúngicas/química , Trichoderma/enzimologia , Sítios de Ligação , Domínio Catalítico , Celulases/metabolismo , Celulose/química , Celulose/metabolismo , Proteínas Fúngicas/metabolismo , Cinética , Simulação de Dinâmica Molecular , Trichoderma/química
11.
PLoS One ; 14(11): e0224803, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31730665

RESUMO

Talaromyces leycettanus JCM12802 is a great producer of thermophilic glycoside hydrolases (GHs). In this study, two cellulases (TlCel5A and TlCel6A) belonging to GH5 and GH6 respectively were expressed in Pichia pastoris and functionally characterized. The enzymes had acidic and thermophilic properties, showing optimal activities at pH 3.5-4.5 and 75-80°C, and retained stable at temperatures up to 60°C and over a broad pH range of 2.0-8.0. TlCel5A and TlCel6A acted against several cellulose substrates with varied activities (3,101.1 vs. 92.9 U/mg to barley ß-glucan, 3,905.6 U/mg vs. 109.0 U/mg to lichenan, and 840.3 and 0.09 U/mg to CMC-Na). When using Avicel, phosphoric acid swollen cellulose (PASC) or steam-exploded corn straw (SECS) as the substrate, combination of TlCel5A and TlCel6A showed significant synergistic action, releasing more reduced sugars (1.08-2.87 mM) than the individual enzymes. These two cellulases may represent potential enzyme additives for the efficient biomass conversion and bioethanol production.


Assuntos
Celulases/metabolismo , Celulose/metabolismo , Talaromyces/enzimologia , Temperatura , Sequência de Aminoácidos , Celulases/química , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
12.
Proc Natl Acad Sci U S A ; 116(49): 24729-24737, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31740605

RESUMO

The order Coleoptera (beetles) is arguably the most speciose group of animals, but the evolutionary history of beetles, including the impacts of plant feeding (herbivory) on beetle diversification, remain poorly understood. We inferred the phylogeny of beetles using 4,818 genes for 146 species, estimated timing and rates of beetle diversification using 89 genes for 521 species representing all major lineages and traced the evolution of beetle genes enabling symbiont-independent digestion of lignocellulose using 154 genomes or transcriptomes. Phylogenomic analyses of these uniquely comprehensive datasets resolved previously controversial beetle relationships, dated the origin of Coleoptera to the Carboniferous, and supported the codiversification of beetles and angiosperms. Moreover, plant cell wall-degrading enzymes (PCWDEs) obtained from bacteria and fungi via horizontal gene transfers may have been key to the Mesozoic diversification of herbivorous beetles-remarkably, both major independent origins of specialized herbivory in beetles coincide with the first appearances of an arsenal of PCWDEs encoded in their genomes. Furthermore, corresponding (Jurassic) diversification rate increases suggest that these novel genes triggered adaptive radiations that resulted in nearly half of all living beetle species. We propose that PCWDEs enabled efficient digestion of plant tissues, including lignocellulose in cell walls, facilitating the evolution of uniquely specialized plant-feeding habits, such as leaf mining and stem and wood boring. Beetle diversity thus appears to have resulted from multiple factors, including low extinction rates over a long evolutionary history, codiversification with angiosperms, and adaptive radiations of specialized herbivorous beetles following convergent horizontal transfers of microbial genes encoding PCWDEs.


Assuntos
Biodiversidade , Evolução Biológica , Besouros/genética , Transferência Genética Horizontal , Genoma de Inseto , Animais , Bactérias/enzimologia , Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Parede Celular/química , Parede Celular/metabolismo , Celulases/genética , Celulases/metabolismo , Besouros/enzimologia , Besouros/microbiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fungos/enzimologia , Fungos/genética , Herbivoria/genética , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Lignina/química , Lignina/metabolismo , Filogenia , Plantas/química , Polissacarídeo-Liase/genética , Polissacarídeo-Liase/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo
13.
Proc Natl Acad Sci U S A ; 116(45): 22545-22551, 2019 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-31636211

RESUMO

Two fluorescence-tagged carbohydrate-binding modules (CBMs), which specifically bind to crystalline (CBM2a-RRedX) and paracrystalline (CBM17-FITC) cellulose, were used to differentiate the supramolecular cellulose structures in bleached softwood Kraft fibers during enzyme-mediated hydrolysis. Differences in CBM adsorption were elucidated using confocal laser scanning microscopy (CLSM), and the structural changes occurring during enzyme-mediated deconstruction were quantified via the relative fluorescence intensities of the respective probes. It was apparent that a high degree of order (i.e., crystalline cellulose) occurred at the cellulose fiber surface, which was interspersed by zones of lower structural organization and increased cellulose accessibility. Quantitative image analysis, supported by 13C NMR, scanning electron microscopy (SEM) imaging, and fiber length distribution analysis, showed that enzymatic degradation predominates at these zones during the initial phase of the reaction, resulting in rapid fiber fragmentation and an increase in cellulose surface crystallinity. By applying this method to elucidate the differences in the enzyme-mediated deconstruction mechanisms, this work further demonstrated that drying decreased the accessibility of enzymes to these disorganized zones, resulting in a delayed onset of degradation and fragmentation. The use of fluorescence-tagged CBMs with specific recognition sites provided a quantitative way to elucidate supramolecular substructures of cellulose and their impact on enzyme accessibility. By designing a quantitative method to analyze the cellulose ultrastructure and accessibility, this study gives insights into the degradation mechanism of cellulosic substrates.


Assuntos
Proteínas de Bactérias/química , Celulases/química , Cellulomonas/enzimologia , Celulose/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Celulases/genética , Celulases/metabolismo , Cellulomonas/química , Cellulomonas/genética , Celulose/metabolismo , Fluorescência , Hidrólise , Cinética , Microscopia Confocal
14.
Appl Microbiol Biotechnol ; 103(21-22): 8711-8724, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31628521

RESUMO

Biocatalysts provide a major advantage to bio-based economy over chemical catalysts by catalyzing various useful transformations in an environment friendly manner along with other major benefits of selectivity, specificity, and low energy consumption. Since last decade, cellulase is the 3rd highest used enzyme in industry in various processes. Xylanase is also one amongst the widely used enzymes, and many industrial applications require synergistic action of both of these enzymes. These applications predominantly include bioethanol production, deinking of waste paper, animal feed processing, food processing, paper and pulp production, removal of fine fibers from textile material (biostoning), and pharmaceuticals. These enzymes are produced by microorganisms (fungi and bacteria), and hence, the microorganisms producing both cellulases and xylanases are in high demand by these industries. This review focuses on the synergistic applications of cellulase and xylanase enzymes across various industrial sectors. It also discusses the potential applications and the need of the microbial systems (fungi and bacteria) secreting both of these enzymes and the future prospects of their development into an integral part of various industrial processes.


Assuntos
Bactérias/enzimologia , Reatores Biológicos/microbiologia , Celulases/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Fungos/enzimologia , Bactérias/metabolismo , Biocombustíveis/microbiologia , Fungos/metabolismo , Indústrias
15.
J Environ Manage ; 252: 109496, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31605913

RESUMO

Currently, the increasing demand of biopesticides production to replace chemical pesticides which are excessively used has made solid state fermentation (SSF) technology the need of the hour. In spite of advantages, true potential of SSF process has not been fully realized at industrial scale. A fermentation process for 6-pentyl-a-pyrone (6 PP), conidia, and lytic enzymes (cellulases, lipase, amylase) production by Trichoderma asperellum TF1 was scaled-up from 250 mL flasks and glass Raimbault column packed with 20 g of solid substrates (dry weight) to 5 Kg of solid substrate by using a new plastic single used bioreactor. For column and single used bioreactor, the fermentation was done with the application of humid air during all the process however flasks are not hermetically closed that some oxygen could flow by diffusion. T. asperellum growth was investigated using a mixture of vine shoots, jatropha cake, olive pomace and olive oil as substrate in all systems in parallel at 25 °C during 7 days. Overall, the conditions applied on the single used bioreactor resulted in the optimum 6-PP production (7.36 ±â€¯0.37 mg g DM-1), lipases (38.73 ±â€¯0.21U/g DM), amylases (15.22 ±â€¯0.13 U/g DM), and conidia production (8.55 ±â€¯0.04 × 109 conidia/g DM).


Assuntos
Celulases , Trichoderma , Reatores Biológicos , Fermentação , Esporos Fúngicos
16.
Microb Pathog ; 137: 103740, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31513898

RESUMO

Mastitis is one of the most important diseases that are threatening modern dairy farms. Biofilms of mastitic teat canal have serious clinical implications because of colonized pathogens having the ability to construct an extracellular polymeric substance (EPS) with increased tolerance to antimicrobials leads to difficulty in eradicating the infection. In this study, we investigated the synergistic biofilm disruptive effect of a combination of carbohydrate hydrolases targeting extracellular polysaccharides of biofilm matrix and we termed it as 'Biofilm Clippers (BC)'. Our findings demonstrate that the BC formulation exhibits intense biofilm-disrupting activity against Staphylococcus aureus biofilms. The results of the study showed that BC enables activity equivalent to physiologically achievable concentrations in disrupting biofilms of S. aureus in vitro. The synergistic anti-biofilm activities of BC on S. aureus biofilms demonstrated that the biofilm matrix is predominant of complex polysaccharides. Further, the confocal microscopic analysis demonstrates that the BC formulation is highly effective compared to the single treatment of either of the enzymes in disrupting the biofilm. To the best of our knowledge, this is the first report on the synergistic anti-biofilm activity of a class of enzyme formulation against mastitic biofilm mass. Even though a small study showed a promising effect on mastitic teat canal, further extensive investigation on a large number of bovines for mastitis therapeutic potential of this BC-derived product is now warranted.


Assuntos
Biofilmes/efeitos dos fármacos , Matriz Extracelular de Substâncias Poliméricas/efeitos dos fármacos , Mastite Bovina/tratamento farmacológico , Amilases/farmacologia , Animais , Antibacterianos/farmacologia , Bovinos , Celulases/farmacologia , Esterases/farmacologia , Feminino , Glândulas Mamárias Animais , Manosidases/farmacologia , Mastite Bovina/microbiologia , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/isolamento & purificação
17.
Phys Chem Chem Phys ; 21(38): 21485-21496, 2019 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-31535114

RESUMO

As a non-catalytic domain, carbohydrate binding modules (CBMs) are often considered to play some key roles in the degradation and recognition of polysaccharides catalyzed by cellulases. In this work, we investigated the recognition dynamics of cello- or xylo-saccharides by two typical CBMs (CBM16-1 and CBM22-2), which are grouped into Type B CBMs. By combining extensive molecular dynamics, principle component analysis, and binding free energy calculations, we constructed several complex models of the two CBMs in both complex cello- and xylo-oligosaccharides. The corresponding substrate recognition affinity and critical residues having significant contributions were systematically investigated. The residues containing aromatic side chain groups were shown to contribute significantly to substrate binding. The calculated binding free energies were in fairly good agreement with the experimental measurements with the absolute mean error of 0.69 kcal mol-1. The overall electrostatic interactions were shown to have negative effects on substrate recognition. Further metadynamics simulations revealed the substrate dissociation process.


Assuntos
Celulases/química , Simulação de Dinâmica Molecular , Oligossacarídeos/química , Sítios de Ligação , Análise de Componente Principal , Ligação Proteica , Conformação Proteica , Eletricidade Estática , Termodinâmica
18.
Carbohydr Res ; 485: 107811, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31526927

RESUMO

The use of Zophobas morio extracts in the aspect of cellulose hydrolysis is presented for the first time. The aim of this study was to investigate the action of enzymes obtained from Z. morio on cellulose hydrolysis and to determine their influence on the structural properties of cellulose with use the Fourier transform infrared spectroscopy (FTIR) and gel permeation chromatography (GPC). Cellulose hydrolysis products were analyzed by high performance liquid chromatography (HPLC). This analysis indicated that microcrystalline cellulose with smaller particle size was more susceptible to enzymatically treatment. Moreover, our investigation of cellulase activity showed a different profile of the used enzyme during particular developmental stages of Z. morio. Midgut extracts obtained from adult insects are more effective in degrading cellulose than extracts from larvae. The analysis of cellulose hydrolysis confirms that the efficiency of this reaction also depends on the structure of cellulosic materials and internal conditions of enzymatic reaction. In this study the cellulolytic activity of Z. morio midgut extracts showed that these insects could be valuable sources of cellulases.


Assuntos
Celulases/metabolismo , Celulose/química , Besouros/enzimologia , Animais , Hidrólise
19.
Enzyme Microb Technol ; 130: 109370, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31421724

RESUMO

Secretome evaluations of lignocellulose-decay basidiomycetes can reveal new enzymes in selected fungal species that degrade specific substrates. Proteins discovered in such studies can support biorefinery development. Brown-rot (Gloeophyllum trabeum) and white-rot (Pleurotus ostreatus) fungi growing in sugarcane bagasse solid-state cultures produced 119 and 63 different extracellular proteins, respectively. Several of the identified enzymes are suitable for in vitro biomass conversion, including a range of cellulases (endoglucanases, cellobiohydrolases and ß-glucosidases), hemicellulases (endoxylanases, α-arabinofuranosidases, α-glucuronidases and acetylxylan esterases) and carbohydrate-active auxiliary proteins, such as AA9 lytic polysaccharide monooxygenase, AA1 laccase and AA2 versatile peroxidase. Extracellular oxalate decarboxylase was also detected in both fungal species, exclusively in media containing sugarcane bagasse. Interestingly, intracellular AA6 quinone oxidoreductases were also exclusively produced under sugarcane bagasse induction in both fungi. These enzymes promote quinone redox cycling, which is used to produce Fenton's reagents by lignocellulose-decay fungi. Hitherto undiscovered hypothetical proteins that are predicted in lignocellulose-decay fungi genomes appeared in high relative abundance in the cultures containing sugarcane bagasse, which suggests undisclosed, new biochemical mechanisms that are used by lignocellulose-decay fungi to degrade sugarcane biomass. In general, lignocellulose-decay fungi produce a number of canonical hydrolases, as well as some newly observed enzymes, that are suitable for in vitro biomass digestion in a biorefinery context.


Assuntos
Basidiomycota/metabolismo , Celulose/metabolismo , Lignina/metabolismo , Metaboloma , Pleurotus/metabolismo , Saccharum/metabolismo , Biomassa , Celulases/metabolismo , Celulose 1,4-beta-Celobiosidase/metabolismo , Proteínas Fúngicas/metabolismo , Glucose/metabolismo , Madeira/metabolismo , Madeira/microbiologia
20.
An Acad Bras Cienc ; 91(3): e20180583, 2019 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-31365652

RESUMO

Isolation and screening of new fungal strains from extreme and understudied environments, such as caves, is a promising approach to find higher yields enzyme producers. Cellulolytic fungal strains isolated from a Brazilian cave were evaluated for their enzymatic production after submerged (SmF) and solid-state fermentation (SSF). After SmF, three strains were selected for their high enzymatic activities: Aspergillus ustus for endoglucanase (4.76 U/mg), Talaromyces bruneus for ß-glucosidase (11.71 U/mg) and Aspergillus sp. (CBMAI 1926) for total cellulase (1.70 U/mg). After SSF, these strains, showed better yields compared to the reference strain Aspergillus niger 3T5B8. Aspergillus sp. (CBMAI 1926) stood out as a new species that expressed activity of total cellulases (0.10 U/mg) and low protein concentration (0.44 mg/mL). In conclusion, these isolated strains have a more efficient and promising cellulolytic enzyme complex that can be used in fermentation and saccharification processes with a lower protein concentration and a higher enzymatic activity than the reference strain. Therefore, beside the new genetic material characterized, our study highlights the benefits of cave extreme environments exploitation to find new potentially valuable strains.


Assuntos
Cavernas , Celulases/metabolismo , Ambientes Extremos , Fungos/metabolismo , Brasil , Fungos/classificação
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