RESUMEN

ß-glucosidases play a pivotal role in second-generation biofuel (2G-biofuel) production. For this application, thermostable enzymes are essential due to the denaturing conditions on the bioreactors. Random amino acid substitutions have originated new thermostable ß-glucosidases, but without a clear understanding of their molecular mechanisms. Here, we probe by different molecular dynamics simulation approaches with distinct force fields and submitting the results to various computational analyses, the molecular bases of the thermostabilization of the Paenibacillus polymyxa GH1 ß-glucosidase by two-point mutations E96K (TR1) and M416I (TR2). Equilibrium molecular dynamic simulations (eMD) at different temperatures, principal component analysis (PCA), virtual docking, metadynamics (MetaDy), accelerated molecular dynamics (aMD), Poisson-Boltzmann surface analysis, grid inhomogeneous solvation theory and colony method estimation of conformational entropy allow to converge to the idea that the stabilization carried by both substitutions depend on different contributions of three classic mechanisms: (i) electrostatic surface stabilization; (ii) efficient isolation of the hydrophobic core from the solvent, with energetic advantages at the solvation cap; (iii) higher distribution of the protein dynamics at the mobile active site loops than at the protein core, with functional and entropic advantages. Mechanisms i and ii predominate for TR1, while in TR2, mechanism iii is dominant. Loop A integrity and loops A, C, D, and E dynamics play critical roles in such mechanisms. Comparison of the dynamic and topological changes observed between the thermostable mutants and the wildtype protein with amino acid co-evolutive networks and thermostabilizing hotspots from the literature allow inferring that the mechanisms here recovered can be related to the thermostability obtained by different substitutions along the whole family GH1. We hope the results and insights discussed here can be helpful for future rational approaches to the engineering of optimized ß-glucosidases for 2G-biofuel production for industry, biotechnology, and science.

Asunto(s)

Biocombustibles , beta-Glucosidasa , beta-Glucosidasa/genética , beta-Glucosidasa/química , beta-Glucosidasa/metabolismo , Sustitución de Aminoácidos , Simulación de Dinámica Molecular , Dominio Catalítico

RESUMEN

The enzymatic conversion of lignocellulosic biomass to fermentable sugars is determined by the enzymatic activity of cellulases; consequently, improving enzymatic activity has attracted great interest in the scientific community. Cocktails of commercial cellulase often have low ß-glucosidase content, leading to the accumulation of cellobiose. This accumulation inhibits the activity of the cellulolytic complex and can be used to determine the enzymatic efficiency of commercial cellulase cocktails. Here, a novel codon optimized ß-glucosidase gene (B-glusy) from Trichoderma reesei QM6a was cloned and expressed in three strains of Escherichia coli (E. coli). The synthetic sequence containing an open reading frame (ORF) of 1491 bp was used to encode a polypeptide of 497 amino acid residues. The ß-glucosidase recombinant protein that was expressed (57 kDa of molecular weight) was purified by Ni agarose affinity chromatography and visualized by SDS-PAGE. The recombinant protein was better expressed in E. coli BL21 (DE3), and its enzymatic activity was higher at neutral pH and 30 °C (22.4 U/mg). Subsequently, the ß-glucosidase was immobilized using magnetite nano-support, after which it maintained >65% of its enzymatic activity from pH 6 to 10, and was more stable than the free enzyme above 40 °C. The maximum immobilization yield had enzyme activity of 97.2%. In conclusion, ß-glucosidase is efficiently expressed in the microbial strain E. coli BL21 (DE3) grown in a simplified culture medium.

Asunto(s)

Enzimas Inmovilizadas , Escherichia coli , Proteínas Fúngicas , Expresión Génica , Hypocreales/genética , Nanopartículas de Magnetita/química , beta-Glucosidasa , Estabilidad de Enzimas , Enzimas Inmovilizadas/biosíntesis , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/genética , Enzimas Inmovilizadas/aislamiento & purificación , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/aislamiento & purificación , Hypocreales/enzimología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , beta-Glucosidasa/biosíntesis , beta-Glucosidasa/química , beta-Glucosidasa/genética , beta-Glucosidasa/aislamiento & purificación

RESUMEN

ß-glucosidases (EC 3.2.1.21) have been described as essential to second-generation biofuel production. They act in the last step of the lignocellulosic saccharification, cleaving the ß - 1,4 glycosidic bonds in cellobiose to produce two molecules of glucose. However, ß-glucosidases have been described as strongly inhibited by glucose, causing an increment of cellobiose concentration. Also, cellobiose is an inhibitor of other enzymes used in this process, such as exoglucanases and endoglucanases. Hence, the engineering of thermostable and glucose-tolerant ß-glucosidases has been targeted by many studies. In this study, we performed high sampling accelerated molecular dynamics for a wild glucose-tolerant GH1 ß-glucosidase (Bgl1A), a wild non-tolerant (Bgl1B), and a set of glucose-tolerant Bgl1B's mutants: V302F, N301Q/V302F, F172I, V227M, G246S, T299S, and H228T. Our results suggest that point mutations promissory to induce glucose tolerance trend to enhance the mobility of the flexible loops around the active site. Mutations affected B and C loops regions, and an αß-hairpin motif between them. Conformational clusters and free energy landscape profiles suggest that the mobility acquired by mutants allows a higher closure of the substrate channel. This closure is compatible with a higher impedance for glucose entrance and stimulus of its withdrawal. Based on mutants' structural analyses, we inferred that both the direct stereochemical effect on the glucose path and the changes in the mobility affect glucose tolerance. We hope these results be useful for the rational design of glucose-tolerant and industrially promising enzymes.Communicated by Ramaswamy H. Sarma.

Asunto(s)

Celobiosa , Mutación Puntual , Biocombustibles , Glucosa , Especificidad por Sustrato , beta-Glucosidasa/genética , beta-Glucosidasa/metabolismo

RESUMEN

In this study, a GH3 family ß-glucosidase (Bgl7226) from metagenomic sequences of the Syntermes wheeleri gut, a Brazilian Cerrado termite, was expressed, purified and characterized. The enzyme showed two optimum pHs (pH 7 and pH 10), and a maximum optimum temperature of about 40 °C using 4-Nitrophenyl ß-D-glucopyranoside (pNPG) as substrate. Bgl7226 showed higher enzymatic activity at basic pH, but higher affinity (Km) at neutral pH. However, at neutral pH the Bgl7226 enzyme showed higher catalytic efficiency (kcat/Km) for pNPG substrate. Predictive analysis about the enzyme structure-function relationship by sequence alignment suggested the presence of multi-domains and conserved catalytic sites. Circular dichroism results showed that the secondary structure composition of the enzyme is pH-dependent. Small conformational changes occurred close to the optimum temperature of 40 o C, and seem important for the highest activity of Bgl7226 observed at pH 7 and 10. In addition, the small transition in the unfolding curves close to 40 o C is typical of intermediates associated with proteins structured in several domains. Bgl7226 has significant ß-glucosidase activity which could be attractive for biotechnological applications, such as plant roots detoxification; specifically, our group is interested in cassava roots (Manihot esculenta) detoxification.

Asunto(s)

Microbioma Gastrointestinal , Isópteros/microbiología , Metagenoma , beta-Glucosidasa , Animales , Estabilidad de Enzimas , Especificidad por Sustrato , beta-Glucosidasa/química , beta-Glucosidasa/genética

RESUMEN

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 .

Asunto(s)

Biocombustibles/microbiología , Bases de Datos de Compuestos Químicos , beta-Glucosidasa , Secuencia de Aminoácidos , Bacterias/genética , Bacterias/metabolismo , Celobiosa/química , Genes Bacterianos , Glucosa/efectos adversos , Glucosa/química , Lignina/metabolismo , Modelos Moleculares , Simulación del Acoplamiento Molecular , Mutación , Paenibacillus polymyxa/genética , Paenibacillus polymyxa/metabolismo , Conformación Proteica , Streptomyces/genética , Streptomyces/metabolismo , beta-Glucosidasa/síntesis química , beta-Glucosidasa/química , beta-Glucosidasa/genética

RESUMEN

BACKGROUND: Gaucher disease (GD) is a lysosomal disorder caused by biallelic pathogenic mutations in the GBA1 gene that encodes beta-glucosidase (GCase), and more rarely, by a deficiency in the GCase activator, saposin C. Clinically, GD manifests with heterogeneous multiorgan involvement mainly affecting hematological, hepatic and neurological axes. This disorder is divided into three types, based on the absence (type I) or presence and severity (types II and III) of involvement of the central nervous system. At the cellular level, deficiency of GBA1 disturbs lysosomal storage with buildup of glucocerebroside. The consequences of disturbed lysosomal metabolism on biochemical pathways that require lysosomal processing are unknown. Abnormal systemic markers of cobalamin (Cbl, B12) metabolism have been reported in patients with GD, suggesting impairments in lysosomal handling of Cbl or in its downstream utilization events. METHODS: Cultured skin fibroblasts from control humans (n = 3), from patients with GD types I (n = 1), II (n = 1) and III (n = 1) and an asymptomatic carrier of GD were examined for their GCase enzymatic activity and lysosomal compartment intactness. Control human and GD fibroblasts were cultured in growth medium with and without 500 nM hydroxocobalamin supplementation. Cellular cobalamin status was examined via determination of metabolomic markers in cell lysate (intracellular) and conditioned culture medium (extracellular). The presence of transcobalamin (TC) in whole cell lysates was examined by Western blot. RESULTS: Cultured skin fibroblasts from GD patients exhibited reduced GCase activity compared to healthy individuals and an asymptomatic carrier of GD, demonstrating a preserved disease phenotype in this cell type. The concentrations of total homocysteine (tHcy), methylmalonic acid (MMA), cysteine (Cys) and methionine (Met) in GD cells were comparable to control levels, except in one patient with GD III. The response of these metabolomic markers to supplementation with hydroxocobalamin (HOCbl) yielded variable results. The content of transcobalamin in whole cell lysates was comparable in control human and GD patients. CONCLUSIONS: Our results indicate that cobalamin transport and cellular processing pathways are overall protected from lysosomal storage damage in GD fibroblasts. Extending these studies to hepatocytes, macrophages and plasma will shed light on cell- and compartment-specific vitamin B12 metabolism in Gaucher disease.

Asunto(s)

Enfermedad de Gaucher/genética , Glucosilceramidasa/genética , Vitamina B 12/metabolismo , beta-Glucosidasa/genética , Técnicas de Cultivo de Célula , Femenino , Fibroblastos/metabolismo , Enfermedad de Gaucher/metabolismo , Enfermedad de Gaucher/patología , Homocisteína/metabolismo , Humanos , Lisosomas/metabolismo , Lisosomas/patología , Masculino , Ácido Metilmalónico/metabolismo , Mutación , Fenotipo , Saposinas/genética , Transcobalaminas/metabolismo

RESUMEN

The use of plant biomass for biofuel production will require efficient utilization of the sugars in lignocellulose, primarily cellobiose, because it is the major soluble by-product of cellulose and acts as a strong inhibitor, especially for cellobiohydrolase, which plays a key role in cellulose hydrolysis. Commonly used ethanologenic yeast Saccharomyces cerevisiae is unable to utilize cellobiose; accordingly, genetic engineering efforts have been made to transfer ß-glucosidase genes enabling cellobiose utilization. Nonetheless, laboratory yeast strains have been employed for most of this research, and such strains may be difficult to use in industrial processes because of their generally weaker resistance to stressors and worse fermenting abilities. The purpose of this study was to engineer industrial yeast strains to ferment cellobiose after stable integration of tabgl1 gene that encodes a ß-glucosidase from Thermoascus aurantiacus (TaBgl1). The recombinant S. cerevisiae strains obtained in this study secrete TaBgl1, which can hydrolyze cellobiose and produce ethanol. This study clearly indicates that the extent of glycosylation of secreted TaBgl1 depends from the yeast strains used and is greatly influenced by carbon sources (cellobiose or glucose). The recombinant yeast strains showed high osmotolerance and resistance to various concentrations of ethanol and furfural and to high temperatures. Therefore, these yeast strains are suitable for ethanol production processes with saccharified lignocellulose.

Asunto(s)

Fermentación , Saccharomyces cerevisiae/genética , Thermoascus/enzimología , beta-Glucosidasa/biosíntesis , Biocombustibles , Biomasa , Ingeniería Genética , Microbiología Industrial , Lignina/metabolismo , Thermoascus/genética , beta-Glucosidasa/genética

RESUMEN

ß-glucosidases play a critical role among the enzymes in enzymatic cocktails designed for plant biomass deconstruction. By catalysing the breakdown of ß-1, 4-glycosidic linkages, ß-glucosidases produce free fermentable glucose and alleviate the inhibition of other cellulases by cellobiose during saccharification. Despite this benefit, most characterised fungal ß-glucosidases show weak activity at high glucose concentrations, limiting enzymatic hydrolysis of plant biomass in industrial settings. In this study, structural analyses combined with site-directed mutagenesis efficiently improved the functional properties of a GH1 ß-glucosidase highly expressed by Trichoderma harzianum (ThBgl) under biomass degradation conditions. The tailored enzyme displayed high glucose tolerance levels, confirming that glucose tolerance can be achieved by the substitution of two amino acids that act as gatekeepers, changing active-site accessibility and preventing product inhibition. Furthermore, the enhanced efficiency of the engineered enzyme in terms of the amount of glucose released and ethanol yield was confirmed by saccharification and simultaneous saccharification and fermentation experiments using a wide range of plant biomass feedstocks. Our results not only experimentally confirm the structural basis of glucose tolerance in GH1 ß-glucosidases but also demonstrate a strategy to improve technologies for bioethanol production based on enzymatic hydrolysis.

Asunto(s)

Lignina/metabolismo , Trichoderma/enzimología , beta-Glucosidasa/química , Dominio Catalítico , Escherichia coli , Etanol/metabolismo , Fermentación , Glucosa/metabolismo , Hidrólisis , Mutagénesis Sitio-Dirigida , Trichoderma/genética , beta-Glucosidasa/genética

RESUMEN

With the use of genetic engineering, modified and sometimes more efficient enzymes can be created for different purposes, including industrial applications. However, building modified enzymes depends on several in vitro experiments, which may result in the process being expensive and time-consuming. Therefore, computational approaches could reduce costs and accelerate the discovery of new technological products. In this study, we present a method, called structural signature variation (SSV), to propose mutations for improving enzymes' activity. SSV uses the structural signature variation between target enzymes and template enzymes (obtained from the literature) to determine if randomly suggested mutations may provide some benefit for an enzyme, such as improvement of catalytic activity, half-life, and thermostability, or resistance to inhibition. To evaluate SSV, we carried out a case study that suggested mutations in ß-glucosidases: Essential enzymes used in biofuel production that suffer inhibition by their product. We collected 27 mutations described in the literature, and manually classified them as beneficial or not. SSV was able to classify the mutations with values of 0.89 and 0.92 for precision and specificity, respectively. Then, we used SSV to propose mutations for Bgl1B, a low-performance ß-glucosidase. We detected 15 mutations that could be beneficial. Three of these mutations (H228C, H228T, and H228V) have been related in the literature to the mechanism of glucose tolerance and stimulation in GH1 ß-glucosidase. Hence, SSV was capable of detecting promising mutations, already validated by in vitro experiments, that improved the inhibition resistance of a ß-glucosidase and, consequently, its catalytic activity. SSV might be useful for the engineering of enzymes used in biofuel production or other industrial applications.

Asunto(s)

Biología Computacional/métodos , Mutación/genética , beta-Glucosidasa/química , beta-Glucosidasa/genética , Candida/enzimología , Lipasa/genética , Modelos Moleculares

RESUMEN

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.

Asunto(s)

Proteínas Bacterianas/química , Dominio Catalítico/genética , beta-Glucosidasa/química , Animales , Proteínas Bacterianas/genética , Celobiosa/química , Pruebas de Enzimas , Glicósidos/química , Hidrólisis , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Nitrofenoles/química , Estructura Terciaria de Proteína/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Spodoptera , beta-Glucosidasa/genética

RESUMEN

The Amazon region holds most of the biological richness of Brazil. Despite their ecological and biotechnological importance, studies related to microorganisms from this region are limited. Metagenomics leads to exciting discoveries, mainly regarding non-cultivable microorganisms. Herein, we report the discovery of a novel ß-glucosidase (glycoside hydrolase family 1) gene from a metagenome from Lake Poraquê in the Amazon region. The gene encodes a protein of 52.9 kDa, named AmBgl-LP, which was recombinantly expressed in Escherichia coli and biochemically and structurally characterized. Although AmBgl-LP hydrolyzed the synthetic substrate p-nitrophenyl-ß-d-glucopyranoside (pNPßG) and the natural substrate cellobiose, it showed higher specificity for pNPßG (kcat/Km = 6 s-1·mM-1) than cellobiose (kcat/Km = 0.6 s-1·mM-1). AmBgl-LP showed maximum activity at 40 °C and pH 6.0 when pNPßG was used as the substrate. Glucose is a competitive inhibitor of AmBgl-LP, presenting a Ki of 14 mM. X-ray crystallography and Small Angle X-ray Scattering were used to determine the AmBgl-LP three-dimensional structure and its oligomeric state. Interestingly, despite sharing similar active site architecture with other structurally characterized GH1 family members which are monomeric, AmBgl-LP forms stable dimers in solution. The identification of new GH1 members by metagenomics might extend our understanding of the molecular mechanisms and diversity of these enzymes, besides enabling us to survey their industrial applications.

Asunto(s)

Lagos/microbiología , Metagenoma , Microbiología del Agua , beta-Glucosidasa/química , Brasil , beta-Glucosidasa/genética , beta-Glucosidasa/metabolismo

RESUMEN

Proteins that fold as (ß/α)8 barrels are thought to have evolved from half-barrels that underwent duplication and fusion events. The evidence is particularly clear for small barrels, which have almost identical halves. Additionally, computational calculations of the thermodynamic stability of these structures in the presence of denaturants have revealed that (ß/α)8 barrels contain two subunits or domains corresponding to half-barrels. Hence, within (ß/α)8 barrels, half-barrels are self-contained units. Here, we tested this hypothesis using ß-glucosidase from the bacterium Thermotoga maritima (bglTm), which has a (ß/α)8 barrel structure. Mutations were introduced to disrupt the noncovalent contacts between its halves and reveal the presence of two domains within bglTm, thus resulting in the creation of mutants T1 (containing W12A and I217A mutations) and T2 (containing W12A, H195A, I217A and F404A mutations). Mutants T1 and T2 were properly folded, as indicated by their fluorescence spectra and enzyme kinetic parameters. T1 and wild-type bglTm were equally stable, as shown by the results of thermal inactivation, differential scanning fluorimetry and guanidine hydrochloride denaturation experiments. However, T2 showed a first-order inactivation at 80°C, a single melting temperature of 82°C and only one transition concentration (c50) in 2.4 M guanidine hydrochloride. Additionally, T1 and T2 exhibited a cooperative denaturation process that followed a two-state model (m-values equal to 1.4 and 1.6 kcal/mol/M, respectively), similar to that of wild-type bglTm (1.2 kcal/mol/M). Hence, T1 and T2 each denatured as a single unit, although they contained different degrees of disruption between their halves. In conclusion, bglTm halves are equivalent in terms of their thermal and chemical stability; thus, their separate contributions to (ß/α)8 barrel unfolding cannot be disentangled.

Asunto(s)

beta-Glucosidasa/química , beta-Glucosidasa/metabolismo , Activación Enzimática , Cinética , Modelos Moleculares , Mutación , Conformación Proteica en Lámina beta , Dominios Proteicos , Temperatura , Thermotoga maritima/enzimología , beta-Glucosidasa/genética

RESUMEN

The activity of the GH1 ß-glucosidase from Humicola insolens (Bglhi) against p-nitrophenyl-ß-D-glucopyranoside (pNP-Glc) and cellobiose is enhanced 2-fold by glucose and/or xylose. Kinetic and transglycosylation data showed that hydrolysis is preferred in the absence of monosaccharides. Stimulation involves allosteric interactions, increased transglycosylation and competition of the substrate and monosaccharides for the -1 glycone and the +1/+2 aglycone binding sites. Protein directed evolution has been used to generate 6 mutants of Bglhi with altered stimulation patterns. All mutants contain one of three substitutions (N235S, D237V or H307Y) clustered around the +1/+2 aglycone binding sites. Two mutants with the H307Y substitution preferentially followed the transglycosylation route in the absence of xylose or glucose. The strong stimulation of their pNP-glucosidase and cellobiase activities was accompanied by increased transglycosylation and higher monosaccharide tolerance. The D237V mutation favoured hydrolysis over transglycosylation and the pNP-glucosidase activity, but not the cellobiase activity, was stimulated by xylose. The substitution N235S abolished the preference for hydrolysis or transglycosylation; the cellobiase, but not the pNP-glucosidase activity of the mutants was strongly inhibited by xylose. Both the D237V and N235S mutations lowered tolerance to the monosaccharides. These results provide evidence that the fine modulation of the activity of Bglhi and mutants by glucose and/or xylose is regulated by the relative affinities of the glycone and aglycone binding sites for the substrate and the free monosaccharides.

Asunto(s)

Glucosa/metabolismo , Mycoplasma/enzimología , Ingeniería de Proteínas , Xilosa/metabolismo , beta-Glucosidasa/metabolismo , Celobiosa/metabolismo , Glicosilación , Cinética , Mutagénesis Sitio-Dirigida , Especificidad por Sustrato , beta-Glucosidasa/genética

RESUMEN

Lignin is a major obstacle for cost-effective conversion of cellulose into fermentable sugars. Non-productive adsorption onto insoluble lignin fragments and interactions with soluble phenols are important inhibition mechanisms of cellulases, including ß-glucosidases. Here, we examined the inhibitory effect of tannic acid (TAN), a model polyphenolic compound, on ß-glucosidases from the bacterium Thermotoga petrophila (TpBGL1 and TpBGL3) and archaeon Pyrococcus furiosus (PfBGL1). The results revealed that the inhibition effects on ß-glucosidases were TAN concentration-dependent. TpBGL1 and TpBGL3 were more tolerant to the presence of TAN when compared with PfBGL1, while TpBGL1 was less inhibited when compared with TpBGL3. In an attempt to better understand the inhibitory effect, the interaction between TAN and ß-glucosidases were analyzed by isothermal titration calorimetry (ITC). Furthermore, the exposed hydrophobic surface areas in ß-glucosidases were analyzed using a fluorescent probe and compared with the results of inhibition and ITC. The binding constants determined by ITC for the interactions between TAN and ß-glucosidases presented the same order of magnitude. However, the number of binding sites and exposed hydrophobic surface areas varied for the ß-glucosidases studied. The binding between TAN and ß-glucosidases were driven by enthalpic effects and with an unfavorable negative change in entropy upon binding. Furthermore, the data suggest that there is a high correlation between exposed hydrophobic surface areas and the number of binding sites on the inhibition of microbial ß-glucosidases by TAN. These studies can be useful for biotechnological applications.

Asunto(s)

Inhibidores Enzimáticos/farmacología , Bacilos Gramnegativos Anaerobios Rectos, Curvos y Espirales/enzimología , Pyrococcus furiosus/enzimología , Taninos/farmacología , beta-Glucosidasa/metabolismo , Proteínas Arqueales/antagonistas & inhibidores , Proteínas Arqueales/química , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Calorimetría , Relación Dosis-Respuesta a Droga , Escherichia coli , Bacilos Gramnegativos Anaerobios Rectos, Curvos y Espirales/química , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Unión Proteica , Pyrococcus furiosus/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tensoactivos/farmacología , beta-Glucosidasa/antagonistas & inhibidores , beta-Glucosidasa/química , beta-Glucosidasa/genética

RESUMEN

Bioethanol is one of the main biofuels produced from the fermentation of saccharified agricultural waste; however, this technology needs to be optimized for profitability. Because the commonly used ethanologenic yeast strains are unable to assimilate cellobiose, several efforts have been made to express cellulose hydrolytic enzymes in these yeasts to produce ethanol from lignocellulose. The C. flavigenabglA gene encoding ß-glucosidase catalytic subunit was optimized for preferential codon usage in S. cerevisiae. The optimized gene, cloned into the episomal vector pRGP-1, was expressed, which led to the secretion of an active ß-glucosidase in transformants of the S. cerevisiae diploid strain 2-24D. The volumetric and specific extracellular enzymatic activities using pNPG as substrate were 155 IU L-1 and 222 IU g-1, respectively, as detected in the supernatant of the cultures of the S. cerevisiae RP2-BGL transformant strain growing in cellobiose (20 g L-1) as the sole carbon source for 48 h. Ethanol production was 5 g L-1 after 96 h of culture, which represented a yield of 0.41 g g-1 of substrate consumed (12 g L-1), equivalent to 76% of the theoretical yield. The S. cerevisiae RP2-BGL strain expressed the ß-glucosidase extracellularly and produced ethanol from cellobiose, which makes this microorganism suitable for application in ethanol production processes with saccharified lignocellulose.

Asunto(s)

Biocombustibles , Celobiosa/metabolismo , Cellulomonas/enzimología , Etanol/metabolismo , Saccharomyces cerevisiae/genética , beta-Glucosidasa/genética , Celulosa/metabolismo , Codón , Lignina/metabolismo , Saccharomyces cerevisiae/metabolismo , beta-Glucosidasa/metabolismo

RESUMEN

New ß-glucosidases with product (glucose) or ethanol tolerances are greatly desired to make industrial processes more marketable and efficient. Therefore, this report describes the in silico/vitro characterization of Bg10, a metagenomically derived homodimeric ß-glucosidase that exhibited a Vmax of 10.81 ± 0.43 µM min-1, Kcat of 175.1± 6.91 min-1, and Km of 0.49 ± 0.12 mM at a neutral pH and 37°C when pNP-ß-D-glucopyranoside was used as the substrate, and the enzyme retained greater than 80% activity within the respective pH and temperature ranges of 6.5 to 8.0 and 35 to 40°C. The enzyme was stimulated by its product, glucose; consequently, the Bg10 activity against 50 and 100 mM of glucose were increased by 36.8% and 22%, respectively, while half of the activity was retained at 350 mM. Moreover, the Bg10 was able to hydrolyse 55% (milk sample) and 100% (purified sugar) of the lactose at low (6°C) and optimum (37°C) temperatures, respectively, suggesting the possibility of further optimization of the reaction for lactose-free dairy production. In addition, the enzyme was able to fully hydrolyse 40 mM of cellobiose at one hour and was tolerant to ethanol up to concentrations of 500 mM (86% of activity), while a 1 M concentration still resulted in 41% residual activity, which could be interesting for biofuel production.

Asunto(s)

Etanol/química , Glucosa/metabolismo , Lactosa/química , Leche/metabolismo , beta-Glucosidasa/metabolismo , Animales , Celobiosa/metabolismo , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Metagenómica , Filogenia , Estabilidad Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Especificidad por Sustrato , Temperatura , beta-Glucosidasa/clasificación , beta-Glucosidasa/genética

RESUMEN

Environments where lignocellulosic biomass is naturally decomposed are sources for discovery of new hydrolytic enzymes that can reduce the high cost of enzymatic cocktails for second-generation ethanol production. Metagenomic analysis was applied to discover genes coding carbohydrate-depleting enzymes from a microbial laboratory subculture using a mix of sugarcane bagasse and cow manure in the thermophilic composting phase. From a fosmid library, 182 clones had the ability to hydrolyse carbohydrate. Sequencing of 30 fosmids resulted in 12 contigs encoding 34 putative carbohydrate-active enzymes belonging to 17 glycosyl hydrolase (GH) families. One third of the putative proteins belong to the GH3 family, which includes ß-glucosidase enzymes known to be important in the cellulose-deconstruction process but present with low activity in commercial enzyme preparations. Phylogenetic analysis of the amino acid sequences of seven selected proteins, including three ß-glucosidases, showed low relatedness with protein sequences deposited in databases. These findings highlight microbial consortia obtained from a mixture of decomposing biomass residues, such as sugar cane bagasse and cow manure, as a rich resource of novel enzymes potentially useful in biotechnology for saccharification of lignocellulosic substrate.

Asunto(s)

Celulasas/metabolismo , Celulosa/metabolismo , Lignina/metabolismo , Estiércol/microbiología , Consorcios Microbianos/genética , Saccharum/microbiología , Animales , Bacterias/enzimología , Bacterias/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biomasa , Bovinos , Celulasas/genética , Activación Enzimática , Etanol/metabolismo , Metagenómica , Filogenia , Saccharum/metabolismo , Análisis de Secuencia de ADN , beta-Glucosidasa/genética , beta-Glucosidasa/metabolismo

RESUMEN

Shewanella sp. G5, a psychrotolerant marine bacterium, has a cold-shock protein (CspA) and three ß-glucosidases, two of which were classified in the glycosyl hydrolase families 1 and 3 and are encoded by bgl-A and bgl genes, respectively. Shewanella sp. G5 was cultured on Luria-Bertani (LB) and Mineral Medium Brunner (MMB) media with glucose and cellobiose at various temperatures and pH 6 and 8. Relative quantification of the expression levels of all three genes was studied by real-time PCR with the comparative Ct method (2(-ΔΔCt)) using the gyrB housekeeping gene as a normalizer. Results showed that the genes had remarkably different genetic expression levels under the conditions evaluated, with increased expression of all genes obtained on MMB with cellobiose at 30 °C. Specific growth rate and specific ß-glucosidase activity were also determined for all the culture conditions. Shewanella sp. G5 was able to grow on both media at 4 °C, showing the maximum specific growth rate on LB with cellobiose at 37 °C. The specific ß-glucosidase activity obtained on MMB with cellobiose at 30 °C was 25 to 50 % higher than for all other conditions. At pH 8, relative activity was 34, 60, and 63 % higher at 30 °C than at 10 °C, with three peaks at 10, 25, and 37 °C on both media. Enzyme activity increased by 61 and 47 % in the presence of Ca(2+) and by 24 and 31 % in the presence of Mg(2+) on LB and MMB at 30 °C, respectively, but it was totally inhibited by Hg(2+), Cu(2+), and EDTA. Moreover, this activity was slightly decreased by SDS, Zn(2+), and DTT, all at 5 mM. Ethanol (14 % v/v) and glucose (100 mM) also reduced the activity by 63 and 60 %, respectively.

Asunto(s)

Proteínas Bacterianas/genética , Celobiosa/metabolismo , Regulación Bacteriana de la Expresión Génica , Microbiología Industrial , Shewanella/genética , beta-Glucosidasa/genética , Adaptación Fisiológica/genética , Organismos Acuáticos , Proteínas Bacterianas/metabolismo , Cationes Bivalentes , Celobiosa/farmacología , Girasa de ADN/genética , Girasa de ADN/metabolismo , Fermentación , Genes Esenciales , Glucosa/metabolismo , Glucosa/farmacología , Concentración de Iones de Hidrógeno , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Magnesio/metabolismo , Salinidad , Shewanella/efectos de los fármacos , Shewanella/enzimología , Shewanella/crecimiento & desarrollo , Temperatura , beta-Glucosidasa/metabolismo

RESUMEN

Psychrophilic enzymes evolved from a plethora of structural scaffolds via multiple molecular pathways. Elucidating their adaptive strategies is instrumental to understand how life can thrive in cold ecosystems and to tailor enzymes for biotechnological applications at low temperatures. In this work, we used X-ray crystallography, in solution studies and molecular dynamics simulations to reveal the structural basis for cold adaptation of the GH1 ß-glucosidase from Exiguobacterium antarcticum B7. We discovered that the selective pressure of low temperatures favored mutations that redesigned the protein surface, reduced the number of salt bridges, exposed more hydrophobic regions to the solvent and gave rise to a tetrameric arrangement not found in mesophilic and thermophilic homologues. As a result, some solvent-exposed regions became more flexible in the cold-adapted tetramer, likely contributing to enhance enzymatic activity at cold environments. The tetramer stabilizes the native conformation of the enzyme, leading to a 10-fold higher activity compared to the disassembled monomers. According to phylogenetic analysis, diverse adaptive strategies to cold environments emerged in the GH1 family, being tetramerization an alternative, not a rule. These findings reveal a novel strategy for enzyme cold adaptation and provide a framework for the semi-rational engineering of ß-glucosidases aiming at cold industrial processes.

Asunto(s)

Adaptación Fisiológica/genética , Proteínas Bacterianas/química , Firmicutes/enzimología , Filogenia , beta-Glucosidasa/química , Organismos Acuáticos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Clonación Molecular , Frío , Cristalografía por Rayos X , Estabilidad de Enzimas , Escherichia coli/genética , Escherichia coli/metabolismo , Firmicutes/clasificación , Firmicutes/genética , Expresión Génica , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Simulación de Dinámica Molecular , Dominios Proteicos , Multimerización de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Termodinámica , beta-Glucosidasa/genética , beta-Glucosidasa/metabolismo

RESUMEN

Network structural analysis, known as residue interaction networks or graphs (RIN or RIG, respectively) or protein structural networks or graphs (PSN or PSG, respectively), comprises a useful tool for detecting important residues for protein function, stability, folding and allostery. In RIN, the tertiary structure is represented by a network in which residues (nodes) are connected by interactions (edges). Such structural networks have consistently presented a few central residues that are important for shortening the pathways linking any two residues in a protein structure. To experimentally demonstrate that central residues effectively participate in protein properties, mutations were directed to seven central residues of the ß-glucosidase Sfßgly (ß-D-glucoside glucohydrolase; EC 3.2.1.21). These mutations reduced the thermal stability of the enzyme, as evaluated by changes in transition temperature (Tm ) and the denaturation rate at 45 °C. Moreover, mutations directed to the vicinity of a central residue also caused significant decreases in the Tm of Sfßgly and clearly increased the unfolding rate constant at 45 °C. However, mutations at noncentral residues or at surrounding residues did not affect the thermal stability of Sfßgly. Therefore, the data reported in the present study suggest that the perturbation of the central residues reduced the stability of the native structure of Sfßgly. These results are in agreement with previous findings showing that networks are robust, whereas attacks on central nodes cause network failure. Finally, the present study demonstrates that central residues underlie the functional properties of proteins.

Asunto(s)

Proteínas/química , Sustitución de Aminoácidos , Animales , Dicroismo Circular , Estabilidad de Enzimas , Calor , Proteínas de Insectos/química , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Conformación Proteica , Desnaturalización Proteica , Pliegue de Proteína , Mapas de Interacción de Proteínas , Proteínas/genética , Proteínas/metabolismo , Espectrometría de Fluorescencia , Spodoptera/enzimología , Spodoptera/genética , beta-Glucosidasa/química , beta-Glucosidasa/genética , beta-Glucosidasa/metabolismo