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1.
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
2.
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
3.
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
4.
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
5.
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
6.
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
7.
Microbiol Res ; 223-225: 13-21, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31178046

RESUMO

Flavobacterium sp. AUG42 is a cellulase-producing bacterium isolated from the Antarctic oligochaete Grania sp. (Annelida). In this work, we report that AUG42 produces a glycoside hydrolase cocktail with CMCase, PASCase and cellobiase activities (optimum pHs and temperatures ranging from 5.5 to 6.5 and 40 to 50 °C, respectively). The time-course analyses of the bacterial growth and cellulase production showed that the cocktail has maximal activity at the stationary phase when growing at 16 °C with filter paper as a cellulosic carbon source, among the tested substrates. The analyses of the CAZome and the identification of secreted proteins by shotgun Mass Spectrometry analysis showed that five glycoside hydrolyses are present in the bacterial secretome, which probably cooperate in the degradation of the cellulosic substrates. Two of these glycoside hydrolyses may harbor putative carbohydrate binding modules, both with a cleft-like active site. The cellulolytic cocktail was assayed in saccharification experiments using carboxymethylcellulose as a substrate and results showed the release of glucose (a fermentable sugar) and other reducing-sugars, after 24 h incubation. The ecological relevance of producing cellulases in the Antarctic environment, as well as their potential use in the bio-refinery industry, are discussed.


Assuntos
Celulases/biossíntese , Celulases/química , Flavobacterium/enzimologia , Flavobacterium/metabolismo , Regiões Antárticas , Sequência de Bases , Carbono/metabolismo , Ciclo do Carbono , Carboximetilcelulose Sódica/metabolismo , Domínio Catalítico , Celulase , Celulases/genética , Celulose , Ensaios Enzimáticos , Fermentação , Flavobacterium/genética , Flavobacterium/crescimento & desenvolvimento , Glucose/metabolismo , Glicosídeo Hidrolases/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Especificidade por Substrato , Temperatura , beta-Glucosidase/metabolismo
8.
Microb Cell Fact ; 18(1): 97, 2019 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-31151435

RESUMO

BACKGROUND: Transglycosylation represents one of the most promising approaches for obtaining novel glycosides, and plant phenols and polyphenols are emerging as one of the best targets for creating new molecules with enhanced capacities. These compounds can be found in diet and exhibit a wide range of bioactivities, such as antioxidant, antihypertensive, antitumor, neuroprotective and anti-inflammatory, and the eco-friendly synthesis of glycosides from these molecules can be a suitable alternative for increasing their health benefits. RESULTS: Transglycosylation experiments were carried out using different GH3 ß-glucosidases from the fungus Talaromyces amestolkiae. After a first screening with a wide variety of potential transglycosylation acceptors, mono-glucosylated derivatives of hydroxytyrosol, vanillin alcohol, 4-hydroxybenzyl alcohol, and hydroquinone were detected. The reaction products were analyzed by thin-layer chromatography, high-pressure liquid chromatography, and mass spectrometry. Hydroxytyrosol and vanillyl alcohol were selected as the best options for transglycosylation optimization, with a final conversion yield of 13.8 and 19% of hydroxytyrosol and vanillin glucosides, respectively. NMR analysis confirmed the structures of these compounds. The evaluation of the biological effect of these glucosides using models of breast cancer cells, showed an enhancement in the anti-proliferative capacity of the vanillin derivative, and an improved safety profile of both glucosides. CONCLUSIONS: GH3 ß-glucosidases from T. amestolkiae expressed in P. pastoris were able to transglycosylate a wide variety of acceptors. Between them, phenolic molecules like hydroxytyrosol, vanillin alcohol, 4-hydroxybenzyl alcohol, and hydroquinone were the most suitable for its interesting biological properties. The glycosides of hydroxytyrosol and vanillin were tested, and they improved the biological activities of the original aglycons on breast cancer cells.


Assuntos
Neoplasias da Mama , Celulases/metabolismo , Glicosídeos/farmacologia , Talaromyces/enzimologia , Benzaldeídos/metabolismo , Álcoois Benzílicos/metabolismo , Celulases/química , Celulases/isolamento & purificação , Glicosídeos/química , Glicosídeos/isolamento & purificação , Glicosilação , Humanos , Hidroquinonas/metabolismo , Células MCF-7 , Álcool Feniletílico/análogos & derivados , Álcool Feniletílico/metabolismo , Especificidade por Substrato
9.
Z Naturforsch C J Biosci ; 74(3-4): 77-84, 2019 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-30710489

RESUMO

While the application of enzymes to synthetic and industrial problems continues to grow, the major development today is focused on multi-enzymatic cascades. Such systems are particularly attractive, because many commercially available enzymes operate under relatively similar operating conditions. This opens the possibility of one-pot operation with multiple enzymes in a single reactor. In this paper the concept of modules is introduced whereby groups of enzymes are combined in modules, each operating in a single reactor, but with the option of various operating strategies to avoid any complications of nonproductive interactions between the enzymes, substrates or products in a given reactor. In this paper the selection of modules is illustrated using the synthesis of the bulk chemical, gluconic acid, from lignocellulosic waste.


Assuntos
Catalase/química , Celulases/química , Gluconatos/síntese química , Glucose Oxidase/química , Lignina/química , Modelos Estatísticos , beta-Glucosidase/química , Biocatálise , Catalase/metabolismo , Celulases/metabolismo , Simulação por Computador , Fermentação , Gluconatos/química , Gluconatos/metabolismo , Glucose/química , Glucose/metabolismo , Glucose Oxidase/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Cinética , Lactonas/química , Lactonas/metabolismo , Lignina/metabolismo , Engenharia Metabólica/métodos , Temperatura , Resíduos , beta-Glucosidase/metabolismo
10.
Chembiochem ; 20(11): 1394-1399, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-30697892

RESUMO

Polymer-protein core-shell nanoparticles have been explored for enzyme immobilization. This work reports on the development of functional polymeric micelles for immobilizing His6 -tagged cellulases with controlled spatial orientation of enzymes, resulting in "artificial cellulosomes" for effective cellulose hydrolysis. Poly(styrene)-b-poly(styrene-alt-maleic anhydride) was prepared through one-pot reversible addition-fragmentation chain-transfer polymerization and modified with nitrilotriacetic acid (NTA) to afford an amphiphilic block copolymer. The self-assembled polymer was mixed with a solution of NiSO4 to form Ni-NTA-functionalized micelles, which could successfully capture His6 -tagged cellulases and form hierarchically structured core-shell nanoparticles with cellulases as the corona. Because the anchored enzymes are site-specifically oriented and in close proximity, synergistic catalysis that results in over twofold activity enhancement has been achieved.


Assuntos
Celulases/química , Celulossomas/química , Proteínas de Fluorescência Verde/química , Proteínas Luminescentes/química , Nanopartículas/química , Polímeros , Enzimas Imobilizadas , Micelas , Polimerização , Polímeros/síntese química , Polímeros/química
11.
Biomacromolecules ; 20(2): 769-777, 2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30657665

RESUMO

In this study, the interaction forces between different cellulosic nanomaterials and a protein domain belonging to cellulose binding modules family 1 (CBM1) were investigated at the molecular scale. Cellulose binding modules are protein domains found in carbohydrate active enzymes having an affinity toward cellulosic materials. Here, the binding force of a fusion protein containing a cellulose binding module (CBM1) produced recombinantly in E. coli was quantified on different cellulose nanocrystals immobilized on surfaces. Adhesion of the CBM on cellulose with different degrees of crystallinity as well as on chitin nanocrystals was examined. This study was carried out by single molecule force spectroscopy using an atomic force microscope, which enables the detection of binding force of individual molecules. The study contains a preliminary quantification of the interactions at the molecular level that sheds light on the development of new nanocellulose-based nanocomposites with improved strength and elasticity.


Assuntos
Celulases/metabolismo , Celulose/química , Nanoestruturas/química , Aderência Bacteriana , Celulases/química , Quitina/análogos & derivados , Escherichia coli , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Ligação Proteica , Domínios Proteicos
12.
Carbohydr Polym ; 207: 59-67, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30600044

RESUMO

A series of cellulosic substrates has been produced, treated with lytic polysaccharide monooxygenase (LPMO) from Streptomyces ambofaciens (SamLPMO10C), and analyzed by high performance anion exchange chromatography (HPAEC) with pulsed amperometric detection (PAD). The activity of the bacterial LPMO showed high variability depending on the origin and degree of crystallinity of the substrate. Additionally, we tested the effectiveness of SamLPMO10C in the nanofibrillation of flax, a high crystalline agricultural fiber, as a single pretreatment or in combination with cellulases. All pretreatments were followed by a mechanical defibrillation by high-pressure homogenization (HPH) to obtain cellulose nanofibrils (NFC). The combined LPMO-cellulase treatment showed higher fibrillation yield, optical transmittance and carboxylate content than control reactions. Therefore, it could be explored as a promising green alternative to reduce the energy consumption in the production of NFC. To our knowledge, this is the first study reporting the effect of a bacterial LPMO in nanocellulose production.


Assuntos
Celulose/química , Oxigenases de Função Mista/química , Nanofibras/química , Celulases/química , Cristalização , Ensaios Enzimáticos , Linho/química , Hidrólise , Paenibacillus/enzimologia , Streptomyces/enzimologia , Especificidade por Substrato , Têxteis
13.
Biotechnol Bioeng ; 116(3): 515-525, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30515756

RESUMO

The hydrolysis of cellulose by processive cellulases, such as exocellulase TrCel7A from Trichoderma reesei, is typically characterized by an initial burst of high activity followed by a slowdown, often leading to incomplete hydrolysis of the substrate. The origins of these limitations to cellulose hydrolysis are not yet fully understood. Here, we propose a new model for the initial phase of cellulose hydrolysis by processive cellulases, incorporating a bound but inactive enzyme state. The model, based on ordinary differential equations, accurately reproduces the activity burst and the subsequent slowdown of the cellulose hydrolysis and describes the experimental data equally well or better than the previously suggested model. We also derive steady-state expressions that can be used to describe the pseudo-steady state reached after the initial activity burst. Importantly, we show that the new model predicts the existence of an optimal enzyme-substrate affinity at which the pseudo-steady state hydrolysis rate is maximized. The model further allows the calculation of glucose production rate from the first cut in the processive run and reproduces the second activity burst commonly observed upon new enzyme addition. These results are expected to be applicable also to other processive enzymes.


Assuntos
Celulases , Celulose , Modelos Químicos , Celulases/química , Celulases/metabolismo , Celulose/química , Celulose/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Glucose/análise , Glucose/metabolismo , Hidrólise , Cinética , Trichoderma/enzimologia
14.
Trends Biotechnol ; 37(5): 518-531, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30477739

RESUMO

Lignin is central to overcoming recalcitrance in the enzyme hydrolysis of lignocellulose. While the term implies a physical barrier in the cell wall structure, there are also important biochemical components that direct interactions between lignin and the hydrolytic enzymes that attack cellulose in plant cell walls. Progress toward a deeper understanding of the lignin synthesis pathway - and the consistency between a range of observations over the past 40 years in the very extensive literature on cellulose hydrolysis - is resulting in advances in reducing a major impediment to cellulose conversion: the cost of enzymes. This review addresses lignin and its role in the hydrolysis of hardwood and other lignocellulosic residues.


Assuntos
Biomassa , Celulases , Lignina , Biocombustíveis , Celulases/química , Celulases/metabolismo , Etanol , Hidrólise , Lignina/química , Lignina/metabolismo , Madeira
15.
Braz. j. microbiol ; 49(4): 879-884, Oct.-Dec. 2018. tab, graf
Artigo em Inglês | LILACS | ID: biblio-1039268

RESUMO

ABSTRACT The multi-enzyme complex (crude extract) of white rot fungi Pleurotus ostreatus, Pleurotus eryngii, Trametes versicolor, Pycnosporus sanguineus and Phanerochaete chrysosporium were characterized, evaluated in the hydrolysis of pretreated pulps of sorghum straw and compared efficiency with commercial enzyme. Most fungi complexes had better hydrolysis rates compared with purified commercial enzyme.


Assuntos
Proteínas Fúngicas/química , Sorghum/química , Celulases/química , Fungos/enzimologia , Lignina/química , Proteínas Fúngicas/metabolismo , Caules de Planta/microbiologia , Caules de Planta/química , Sorghum/microbiologia , Celulases/metabolismo , Biocatálise , Fungos/química , Hidrólise , Lignina/metabolismo
16.
Adv Exp Med Biol ; 1104: 201-217, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30484250

RESUMO

Cellulose is the most abundant carbohydrate on earth and hydrolyzed by cellulases in nature. During catalysis, cellulase transfers protons to and from the oxygen atoms of the glycosidic bond and a water molecule. Since cellulose is an insoluble polymer, some kinds of cellulases, with high activity toward crystalline cellulose, move on the crystal surface with continuous hydrolysis of the molecular chain. In addition, binding and dissociation on/from the crystal surface are also important elementary steps of the reaction cycle. Recently, these interesting features of cellulases can be directly analyzed, due to the development of visualization techniques. In this chapter, we introduce (1) visualization of the protonation state of the catalytic residue by neutron crystallography, (2) visualization of processive movement on the crystal surface by high-speed atomic force microscopy , and (3) visualization of binding and dissociation events by single-molecule fluorescence microscopy.


Assuntos
Celulases/química , Celulose/química , Hidrólise
17.
Langmuir ; 34(45): 13514-13522, 2018 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-30372079

RESUMO

Effective regulation of cellulase adsorption is key to improving the efficiencies of the two major bottlenecks of lignocellulose hydrolysis and cellulase recovery. In this work, we investigated the effect of inhibitors, pH, and temperature on the adsorption of exo- and endoglucanases (Cel7A and Cel7B, respectively) on cellulose using quartz crystal microgravimetry with dissipation. The addition of glucose and cellobiose can both inhibit the hydrolysis activity of Cel7A, whereas only cellobiose can inhibit that of Cel7B. Notably, the adsorption was favored by acidic conditions (pH ≤ 4.8) and low temperature, whereas alkaline conditions (pH 9 and 10) facilitated enzyme desorption, which is useful to guide the process of cellulase recovery. The adsorption and hydrolysis activity of Cel7A and Cel7B were both higher at 45 °C than at 25 °C. These findings pave the way to effective regulation of cellulase adsorption and thus improve lignocellulose conversion and cellulase recovery.


Assuntos
Celulases/química , Celulose/química , Adsorção , Celobiose/química , Celulases/antagonistas & inibidores , Inibidores Enzimáticos/química , Glucose/química , Concentração de Íons de Hidrogênio , Hidrólise , Interações Hidrofóbicas e Hidrofílicas , Temperatura , Trichoderma/enzimologia
18.
Prep Biochem Biotechnol ; 48(9): 777-786, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30303453

RESUMO

The objective of the present study was to optimize parameters for the cultivation of Lichtheimia corymbifera (mesophilic) and Byssochlamys spectabilis (thermophilic) for the production of ß-glucosidases and to compare the catalytic and thermodynamic properties of the partially purified enzymes. The maximum amount of ß-glucosidase produced by L. corymbifera was 39 U/g dry substrate (or 3.9 U/mL), and that by B. spectabilis was 77 U/g (or 7.7 U/mL). The optimum pH and temperature were 4.5 and 55 °C and 4.0 and 50 °C for the enzyme from L. corymbifera and B. spectabilis, respectively. ß-Glucosidase produced by L. corymbifera was stable at pH 4.0-7.5, whereas the enzyme from B. spectabilis was stable at pH 4.0-6.0. Regarding the thermostability, ß-glucosidase produced by B. spectabilis remained stable for 1 h at 50 °C, and that from L. corymbifera was active for 1 h at 45 °C. Determination of thermodynamic parameters confirmed the greater thermostability of the enzyme produced by the thermophilic fungus B. spectabilis, which showed higher values of ΔH, activation energy for denaturation (Ea), and half-life t(1/2). The enzymes were stable in the presence of ethanol and were competitively inhibited by glucose. These characteristics contribute to their use in the simultaneous saccharification and fermentation of vegetable biomass.


Assuntos
Byssochlamys/enzimologia , Celulases/química , Proteínas Fúngicas/química , Mucorales/enzimologia , Byssochlamys/crescimento & desenvolvimento , Catálise , Celulases/antagonistas & inibidores , Celulases/isolamento & purificação , Técnicas de Cultura/métodos , Inibidores Enzimáticos/química , Etanol/química , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/isolamento & purificação , Glucose/química , Concentração de Íons de Hidrogênio , Cinética , Mucorales/crescimento & desenvolvimento , Temperatura , Termodinâmica
19.
Int J Biol Macromol ; 120(Pt A): 665-672, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30170053

RESUMO

The efficiency of enzymatic hydrolysis is reduced by the naturally recalcitrant complex polymers comprising lignocellulose. Increasing enzymatic conversion, which the complex macromolecules converted into simpler molecules, is still had to be overcome. High hydrostatic pressure (HHP), an emerging technology, is expected to ameliorate the situation. The effects of enzymatic hydrolysis after HHP pretreatment on pumpkin lignocellulose were studied and compared with hydrothermal and alkaline pretreatment. Further investigation was performed to evaluate the effect of enzymatic hydrolysis of pumpkin combined with HHP treatment. The samples underwent HHP treatment simultaneously exhibited overall better performance in enzymatic hydrolysis than the untreated. The highest glucose yield of 91.2% and xylose yield of 84.2% was achieved when 400 MPa HHP with high enzyme loading was applied. HHP exerted positive effects on enzyme-substrate interactions during the enzymatic hydrolysis of lignocellulose, which implied that HHP technology combined with enzymatic hydrolysis could be used to pretreat pumpkin pomace.


Assuntos
Celulases/química , Cucurbita/química , Glucose/química , Lignina/química , Xilose/química , Temperatura Alta , Concentração de Íons de Hidrogênio , Hidrólise , Pressão Hidrostática , Hidróxido de Sódio/química
20.
Braz J Microbiol ; 49(4): 879-884, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30150084

RESUMO

The multi-enzyme complex (crude extract) of white rot fungi Pleurotus ostreatus, Pleurotus eryngii, Trametes versicolor, Pycnosporus sanguineus and Phanerochaete chrysosporium were characterized, evaluated in the hydrolysis of pretreated pulps of sorghum straw and compared efficiency with commercial enzyme. Most fungi complexes had better hydrolysis rates compared with purified commercial enzyme.


Assuntos
Celulases/química , Proteínas Fúngicas/química , Fungos/enzimologia , Lignina/química , Sorghum/química , Biocatálise , Celulases/metabolismo , Proteínas Fúngicas/metabolismo , Fungos/química , Hidrólise , Lignina/metabolismo , Caules de Planta/química , Caules de Planta/microbiologia , Sorghum/microbiologia
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