RESUMO
Nanocellulose are promising Pickering emulsion stabilizers for being sustainable and non-toxic. In this work, semicrystalline cellulose oligomers (SCCO), which were synthesized from maltodextrin using cellobiose as primer by in vitro enzymatic biosystem, were exploited as stabilizers for oil-in-water Pickering emulsions. At first, the morphology, structure, thermal and rheological properties of SCCO suspensions were characterized, showing that SCCO had a sheet morphology and typical cellulose-â ¡ structure with 56 % crystallinity. Then the kinetic stabilities of emulsions containing various amounts of SCCO were evaluated against external stress such as pH, ionic strength, and temperature. Noting that SCCO-Pickering emulsions exhibited excellent stabilities against changes in centrifugation, pH, ionic strengths, and temperatures, and it was also kinetically stable for up to 6 months. Both SCCO suspensions and their emulsions exhibited gel-like structures and shear-thinning behaviors. These results demonstrated great potential of SCCO to be applied as nanocellulosic emulsifiers in food, cosmetic and pharmaceutical industries.
Assuntos
Celobiose/química , Celulose/química , Emulsificantes/química , Polissacarídeos/química , Celulose/ultraestrutura , Cosméticos/química , Cristalização , Emulsões , Tecnologia de Alimentos/métodos , Humanos , Concentração de Íons de Hidrogênio , Cinética , Concentração Osmolar , Temperatura , Água/químicaRESUMO
Β-glucosidases are key enzymes used in second-generation biofuel production. They act in the last step of the lignocellulose saccharification, converting cellobiose in glucose. However, most of the ß-glucosidases are inhibited by high glucose concentrations, which turns it a limiting step for industrial production. Thus, ß-glucosidases have been targeted by several studies aiming to understand the mechanism of glucose tolerance, pH and thermal resistance for constructing more efficient enzymes. In this paper, we present a database of ß-glucosidase structures, called Glutantßase. Our database includes 3842 GH1 ß-glucosidase sequences collected from UniProt. We modeled the sequences by comparison and predicted important features in the 3D-structure of each enzyme. Glutantßase provides information about catalytic and conserved amino acids, residues of the coevolution network, protein secondary structure, and residues located in the channel that guides to the active site. We also analyzed the impact of beneficial mutations reported in the literature, predicted in analogous positions, for similar enzymes. We suggested these mutations based on six previously described mutants that showed high catalytic activity, glucose tolerance, or thermostability (A404V, E96K, H184F, H228T, L441F, and V174C). Then, we used molecular docking to verify the impact of the suggested mutations in the affinity of protein and ligands (substrate and product). Our results suggest that only mutations based on the H228T mutant can reduce the affinity for glucose (product) and increase affinity for cellobiose (substrate), which indicates an increment in the resistance to product inhibition and agrees with computational and experimental results previously reported in the literature. More resistant ß-glucosidases are essential to saccharification in industrial applications. However, thermostable and glucose-tolerant ß-glucosidases are rare, and their glucose tolerance mechanisms appear to be related to multiple and complex factors. We gather here, a set of information, and made predictions aiming to provide a tool for supporting the rational design of more efficient ß-glucosidases. We hope that Glutantßase can help improve second-generation biofuel production. Glutantßase is available at http://bioinfo.dcc.ufmg.br/glutantbase .
Assuntos
Biocombustíveis/microbiologia , Bases de Dados de Compostos Químicos , beta-Glucosidase , Sequência de Aminoácidos , Bactérias/genética , Bactérias/metabolismo , Celobiose/química , Genes Bacterianos , Glucose/efeitos adversos , Glucose/química , Lignina/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Mutação , Paenibacillus polymyxa/genética , Paenibacillus polymyxa/metabolismo , Conformação Proteica , Streptomyces/genética , Streptomyces/metabolismo , beta-Glucosidase/síntese química , beta-Glucosidase/química , beta-Glucosidase/genéticaRESUMO
Many industrial enzymes can be highly glycosylated, including the ß-glucosidase enzymes. Although glycosylation plays an important role in many biological processes, such chains can cause problems in the multipoint immobilization techniques of the enzymes, since the glycosylated chains can cover the reactive groups of the protein (e.g., Lys) and do not allow those groups to react with reactive groups of the support (e.g., aldehyde and epoxy groups). Nevertheless, the activated glycosylated chains can be used as excellent crosslinking agents. The glycosylated chains when oxidized with periodate can generate aldehyde groups capable of reacting with the amino groups of the protein itself. Such intramolecular crosslinks may have significant stabilizing effects. In this study, we investigated if the intramolecular crosslinking occurs in the oxidized ß-glucosidase and its effect on the stability of the enzyme. For this, the oxidation of glycosidic chains of ß-glucosidase was carried out, allowing to demonstrate the formation of aldehyde groups and subsequent interaction with the amine groups and to verify the stability of the different forms of free enzyme (glycosylated and oxidized). Furthermore, we verified the influence of the glycosidic chains on the immobilization of ß-glucosidase from Aspergillus niger and on the consequent stabilization. The results suggest that intramolecular crosslinking occurred and consequently the oxidized enzyme showed a much greater stabilization than the native enzyme (glycosylated). When the multipoint immobilization was performed in amino-epoxy-agarose supports, the stabilization of the oxidized enzyme increases by a 6-fold factor. The overall stabilization strategy was capable to promote an enzyme stabilization of 120-fold regarding to the soluble unmodified enzyme.
Assuntos
Lisina/química , Oxigênio/química , beta-Glucosidase/química , Aspergillus niger/enzimologia , Biomassa , Celobiose/química , DEAE-Celulose/química , Estabilidade Enzimática , Enzimas Imobilizadas/química , Fermentação , Glicólise , Glicosídeos , Glicosilação , Concentração de Íons de Hidrogênio , Hidrólise , Sefarose/química , Temperatura , Fatores de TempoRESUMO
The present study demonstrated the preparation of three different acid-functionalised magnetic nanoparticles (MNPs) and evaluation for their catalytic efficacy in hydrolysis of cellobiose. Initially, iron oxide (Fe3O4)MNPs were synthesised, which further modified by applying silica coating (Fe3O4-MNPs@Si) and functionalised with alkylsulfonic acid (Fe3O4-MNPs@Si@AS), butylcarboxylic acid (Fe3O4-MNPs@Si@BCOOH) and sulphonic acid (Fe3O4-MNPs@Si@SO3H) groups. The Fourier transform infrared analysis confirmed the presence of above-mentioned acid functional groups on MNPs. Similarly, X-ray diffraction pattern and energy dispersive X-ray spectroscopy analysis confirmed the crystalline nature and elemental composition of MNPs, respectively. TEM micrographs showed the synthesis of spherical and polydispersed nanoparticles having diameter size in the range of 20-80â nm. Cellobiose hydrolysis was used as a model reaction to evaluate the catalytic efficacy of acid-functionalised nanoparticles. A maximum 74.8% cellobiose conversion was reported in case of Fe3O4-MNPs@Si@SO3H in first cycle of hydrolysis. Moreover, thus used acid-functionalised MNPs were magnetically separated and reused. In second cycle of hydrolysis, Fe3O4-MNPs@Si@SO3H showed 49.8% cellobiose conversion followed by Fe3O4-MNPs@Si@AS (45%) and Fe3O4-MNPs@Si@BCOOH (18.3%). However, similar pattern was reported in case of third cycle of hydrolysis. The proposed approach is considered as rapid and convenient. Moreover, reuse of acid-functionalised MNPs makes the process economically viable.
Assuntos
Celobiose/química , Nanopartículas de Magnetita/química , Ácidos Sulfônicos/química , Ácidos Carboxílicos/química , Celobiose/análise , Hidrólise , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
The tertiary structure of proteins has been represented as a network, in which residues are nodes and their contacts are edges. Protein structure networks contain residues, called hubs or central, which are essential to form short connection pathways between any pair of nodes. Hence hub residues may effectively spread structural perturbations through the protein. To test whether modifications nearby to hub residues could affect the enzyme active site, mutations were introduced in the ß-glycosidase Sfßgly (PDB-ID: 5CG0) directed to residues that form an α-helix (260-265) and a ß-strand (335-337) close to one of its main hub residues, F251, which is approximately 14 Å from the Sfßgly active site. Replacement of residues A263 and A264, which side-chains project from the α-helix towards F251, decreased the rate of substrate hydrolysis. Mutation A263F was shown to weaken noncovalent interactions involved in transition state stabilization within the Sfßgly active site. Mutations placed on the opposite side of the same α-helix did not show these effects. Consistently, replacement of V336, which side-chain protrudes from a ß-strand face towards F251, inactivated Sfßgly. Next to V336, mutation S337F also caused a decrease in noncovalent interactions involved in transition state stabilization. Therefore, we suggest that mutations A263F, A264F, V336F and S337F may directly perturb the position of the hub F251, which could propagate these perturbations into the Sfßgly active site through short connection pathways along the protein network.
Assuntos
Proteínas de Bactérias/química , Domínio Catalítico/genética , beta-Glucosidase/química , Animais , Proteínas de Bactérias/genética , Celobiose/química , Ensaios Enzimáticos , Glicosídeos/química , Hidrólise , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Nitrofenóis/química , Estrutura Terciária de Proteína/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Spodoptera , beta-Glucosidase/genéticaRESUMO
In this work, a ß-glucosidase of Aspergillus awamori with a molecular weight of 180â¯kDa was produced in solid-state cultures using a mixture of pineapple crown leaves and wheat bran. Maximum production of the enzyme (820⯱â¯30â¯U/g substrate) was obtained after 8â¯days of culture at 28⯰C and initial moisture of 80%. The crude enzyme was efficiently immobilized on glutaraldehyde cross-linked commercial gelatin. Immobilization changed the kinetics of the enzyme, whose behavior could no longer be described by a saturation function of the Michaelis-Menten type. Comparative evaluation of the free and immobilized enzyme showed that the immobilized enzyme was more thermostable and less inhibited by glucose than the free form. In consequence of these properties, the immobilized enzyme was able to hydrolyze cellobiose more extensively. In association with Trichoderma reesei cellulase, the free and immobilized ß-glucosidase increased the liberation of glucose from cellulose 3- and 5-fold, respectively. Immobilization of the A. awamori ß-glucosidase on glutaraldehyde cross-linked commercial gelatin is an efficient and cheap method allowing the reuse of the enzyme by at least 10 times.
Assuntos
Aspergillus/enzimologia , Enzimas Imobilizadas/química , Gelatina/química , beta-Glucosidase/química , Celobiose/química , Celulose/química , Glucose/química , Hidrólise , Cinética , TemperaturaRESUMO
Enzymatic conversion of lignocellulosic biomass into biofuels and chemicals constitutes a potential route for sustainable development. Cellobiohydrolases are key enzymes used in industrial cocktails for depolymerization of crystalline cellulose, and their mechanism of action has been intensely studied in the past several years. Provided with a tunnel-like substrate-binding cavity, cellobiohydrolases possess the ability to processively hydrolyze glycosidic bonds of crystalline cellulose, yielding one molecule of cellobiose per catalytic cycle. As such, cellobiose expulsion from the product binding site is a necessary step in order to allow for the processive hydrolysis mechanism. However, the high-affinity binding of cellobiose to the enzyme impairs the process and causes activity inhibition due to reaction products. Here, we use molecular dynamics simulations to study the binding of cellobiose to the Trichoderma reesei Cel7A (TrCel7A) cellobiohydrolase and the effects of mutations that reduce cellobiose binding, without affecting the structural and dynamical integrities of the enzyme. We observe that the product binding site exhibits an intrinsic flexibility that can sterically hinder cellobiose release. Several point mutations in the product binding site reduce cellobiose-enzyme interactions, but not all modifications are able to maintain the structural integrity of the enzyme. In particular, mutation of charged residues in the TrCel7A product binding site causes perturbations that affect the structure of the loops that form the substrate-binding tunnel of the enzyme and, hence, may affect TrCel7A function in other steps of the hydrolysis mechanism. Our results suggest there is a trade-off between product inhibition and catalytic efficiency, and they provide directions for cellulases engineering.
Assuntos
Celulose 1,4-beta-Celobiosidase/química , Celulose 1,4-beta-Celobiosidase/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Simulação de Dinâmica Molecular , Mutação , Sítios de Ligação , Celobiose/química , Probabilidade , Conformação Proteica , TrichodermaRESUMO
ß-Glucosidases represent an important group of enzymes due to their pivotal role in various biotechnological processes. One of the most prominent is biomass degradation for the production of fuel ethanol from cellulosic agricultural residues and wastes, where the use of immobilized biocatalysts may prove advantageous. Within such scope, the present work aimed to evaluate the feasibility of entrapping ß-glucosidase in either sol-gel or in Lentikats supports for application in cellobiose hydrolysis, and to perform the characterization of the resulting bioconversion systems. The activity and stability of the immobilized biocatalyst over given ranges of temperature and pH values were assessed, as well as kinetic data, and compared to the free form, and the operational stability was evaluated. Immobilization increased the thermal stability of the enzyme, with a 10 °C shift to an optimal temperature in the case of sol-gel support. Mass transfer hindrances as a result of immobilization were not significant, for sol-gel support. Lentikats-entrapped glucosidase was used in 19 consecutive batch runs for cellobiose hydrolysis, without noticeable decrease in product yield. Moreover, encouraging results were obtained for continuous operation. In the overall, the feasibility of using immobilized biocatalysts for cellobiose hydrolysis was established.