RESUMEN
The fungus Thermothelomyces thermophilus is a thermotolerant microorganism that has been explored as a reservoir for enzymes (hydrolytic enzymes and oxidoreductases). The functional analysis of a recombinant cellobiose dehydrogenase (MtCDHB) from T. thermophilus demonstrated a thermophilic behavior, an optimal pH in alkaline conditions for inter-domain electron transfer, and catalytic activity on cellooligosaccharides with different degree of polymerization. Its applicability was evaluated to the sustainable production of cellobionic acid (CBA), a potential pharmaceutical and cosmetic ingredient rarely commercialized. Dissolving pulp was used as a disaccharide source for MtCDHB. Initially, recombinant exoglucanases (MtCBHI and MtCBHII) from T. thermophilus hydrolyzed the dissolving pulp, resulting in 87% cellobiose yield, which was subsequently converted into CBA by MtCDHB, achieving a 66% CBA yield after 24 h. These findings highlight the potential of MtCDHB as a novel approach to obtaining CBA through the bioconversion of a plant-based source.
Asunto(s)
Deshidrogenasas de Carbohidratos , Proteínas Recombinantes , Deshidrogenasas de Carbohidratos/metabolismo , Proteínas Recombinantes/metabolismo , Concentración de Iones de Hidrógeno , Disacáridos/biosíntesis , Disacáridos/metabolismo , Temperatura , Celobiosa/metabolismo , Sordariales/enzimología , Hidrólisis , Eurotiales/enzimologíaRESUMEN
BACKGROUND: Trichoderma reesei is an organism extensively used in the bioethanol industry, owing to its capability to produce enzymes capable of breaking down holocellulose into simple sugars. The uptake of carbohydrates generated from cellulose breakdown is crucial to induce the signaling cascade that triggers cellulase production. However, the sugar transporters involved in this process in T. reesei remain poorly identified and characterized. RESULTS: To address this gap, this study used temporal membrane proteomics analysis to identify five known and nine putative sugar transporters that may be involved in cellulose degradation by T. reesei. Docking analysis pointed out potential ligands for the putative sugar transporter Tr44175. Further functional validation of this transporter was carried out in Saccharomyces cerevisiae. The results showed that Tr44175 transports a variety of sugar molecules, including cellobiose, cellotriose, cellotetraose, and sophorose. CONCLUSION: This study has unveiled a transporter Tr44175 capable of transporting cellobiose, cellotriose, cellotetraose, and sophorose. Our study represents the first inventory of T. reesei sugar transportome once exposed to cellulose, offering promising potential targets for strain engineering in the context of bioethanol production.
Asunto(s)
Celulasa , Glucanos , Hypocreales , Trichoderma , Celobiosa/metabolismo , Proteoma/metabolismo , Proteínas de la Membrana/metabolismo , Celulosa/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Saccharomyces cerevisiae/metabolismo , Celulasa/metabolismo , Azúcares/metabolismo , Oligosacáridos/metabolismo , Trichoderma/metabolismoRESUMEN
Concern over environmental impacts has spurred many efforts to replace fossil fuels with biofuels such as ethanol. However, for this to be possible, it is necessary to invest in other production technologies, such as second generation (2G) ethanol, in order to raise the levels of this product and meet the growing demand. Currently, this type of production is not yet economically feasible, due to the high costs of the enzyme cocktails used in saccharification stage of lignocellulosic biomass. In order to optimize these cocktails, the search for enzymes with superior activities has been the goal of several research groups. For this end, we have characterized the new ß-glycosidase AfBgl1.3 from A. fumigatus after expression and purification in Pichia pastoris X-33. Structural analysis by circular dichroism revealed that increasing temperature destructured the enzyme; the apparent Tm value was 48.5 °C. The percentages of α-helix (36.3%) and ß-sheet (12.4%) secondary structures at 25 °C were predicted. Biochemical characterization suggested that the optimal conditions for AfBgl1.3 were pH 6.0 and temperature of 40 °C. At 30 and 40 °C, the enzyme was stable and retained about 90% and 50% of its activity, respectively, after pre-incubation for 24 h. In addition, the enzyme was highly stable at pH between 5 and 8, retaining over 65% of its activity after pre-incubation for 48 h. AfBgl1.3 co-stimulation with 50-250 mM glucose enhanced its specific activity by 1.4-fold and revealed its high tolerance to glucose (IC50 = 2042 mM). The enzyme was active toward the substrates salicin (495.0 ± 49.0 U mg-1), pNPG (340.5 ± 18.6 U mg-1), cellobiose (89.3 ± 5.1 U mg-1), and lactose (45.1 ± 0.5 U mg-1), so it had broad specificity. The Vmax values were 656.0 ± 17.5, 706.5 ± 23.8, and 132.6 ± 7.1 U mg-1 toward p-nitrophenyl-ß-D-glucopyranoside (pNPG), D-(-)-salicin, and cellobiose, respectively. AfBgl1.3 displayed transglycosylation activity, forming cellotriose from cellobiose. The addition of AfBgl1.3 as a supplement at 0.9 FPU/g of cocktail Celluclast® 1.5L increased carboxymethyl cellulose (CMC) conversion to reducing sugars (g L-1) by about 26% after 12 h. Moreover, AfBgl1.3 acted synergistically with other Aspergillus fumigatus cellulases already characterized by our research group-CMC and sugarcane delignified bagasse were degraded, releasing more reducing sugars compared to the control. These results are important in the search for new cellulases and in the optimization of enzyme cocktails for saccharification.
Asunto(s)
Aspergillus fumigatus , Glicósido Hidrolasas , Aspergillus fumigatus/metabolismo , Glicósido Hidrolasas/metabolismo , Celobiosa , Glucosa/metabolismo , beta-Glucosidasa/metabolismo , Etanol/metabolismo , Concentración de Iones de Hidrógeno , HidrólisisRESUMEN
The recently discovered wild yeast Wickerhamomyces sp. UFFS-CE-3.1.2 was analyzed through a high-throughput experimental design to improve ethanol yields in synthetic media with glucose, xylose, and cellobiose as carbon sources and acetic acid, furfural, formic acid, and NaCl as fermentation inhibitors. After Plackett-Burman (PB) and central composite design (CCD), the optimized condition was used in a fermentation kinetic analysis to compare this yeast's performance with an industrial Saccharomyces cerevisiae strain (JDY-01) genetically engineered to achieve a higher xylose fermentation capacity and fermentation inhibitors tolerance by overexpressing the genes XYL1, XYL2, XKS1, and TAL1. Our results show that furfural and NaCl had no significant effect on sugar consumption by UFFS-CE-3.1.2. Surprisingly, acetic acid negatively affected glucose but not xylose and cellobiose consumption. In contrast, the pH positively affected all the analyzed responses, indicating a cell's preference for alkaline environments. In the CCD, sugar concentration negatively affected the yields of ethanol, xylitol, and cellular biomass. Therefore, fermentation kinetics were carried out with the average concentrations of sugars and fermentation inhibitors and the highest tested pH value (8.0). Although UFFS-CE-3.1.2 fermented glucose efficiently, xylose and cellobiose were mainly used for cellular growth. Interestingly, the genetically engineered strain JDY-01 consumed ~ 30% more xylose and produced ~ 20% more ethanol. Also, while UFFS-CE-3.1.2 only consumed 32% of the acetic acid of the medium, JDY-01 consumed > 60% of it, reducing its toxic effects. Thus, the overexpressed genes played an essential role in the inhibitors' tolerance, and the applied engineering strategy may help improve 2G ethanol production.
Asunto(s)
Celobiosa , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Etanol , Proyectos de Investigación , Furaldehído , Cinética , Cloruro de Sodio , Fermentación , Xilosa , GlucosaRESUMEN
ß-glucosidases (E.C. 3.2.1.21) are enzymes that hydrolyze ß-1,4-glycosidic bonds from non-reducing terminal residues in ß-D-glucosides, with the release of glucose. ß-glucosidases currently used for the saccharification of lignocellulosic biomass have low efficiency in hydrolyzing cellobiose and are inhibited by glucose, contrary to what would be desirable. In this work, we engineered Pichia pastoris strains to produce the ß-glucosidase Glu1B from the termite Coptotermes formosanus, and biochemically characterized the recombinant enzyme. After 36 h of methanol induction in shake flasks, the P. pastoris KM71BGlu strain produced and secreted 4.1 U/mL (approx. 26 mg/L) of N-glycosylated ß-glucosidase Glu1B. The recombinant product had an optimum pH of 5.0, optimum temperature of 50 °C, residual activity at 40 °C higher than 80 %, specific activity toward cellobiose of 431-597 U/mg protein, and a Ki for glucose of 166 mM. The protein structure was stabilized by Mn2+ and glycerol. The high specific activity of the recombinant ß-glucosidase Glu1B was correlated with the presence of specific residues in the glycone (Gln455) and aglycone (Thr193 and Hys252) binding sites, along with linker residues (Leu192, Ile251, and Phe333) between residues of these two sites. Moreover, the resistance to inhibition by glucose was correlated with the presence of specific gatekeeper residues in the active site (Met204, Gln360, Ala368, Ser369, Ser370, Leu450, and Arg451). Based on its biochemical properties and the possibility of its production in the P. pastoris expression system, the ß-glucosidase produced and described in this work could be suitable as a supplement in the enzymatic hydrolysis of cellulose for saccharification of lignocellulosic biomass.
Asunto(s)
Isópteros , beta-Glucosidasa , Animales , beta-Glucosidasa/química , Celobiosa/metabolismo , Isópteros/metabolismo , Pichia/metabolismo , Especificidad por Sustrato , Cinética , Glucosa/metabolismoRESUMEN
Lignocellulose hydrolysates are rich in fermentable sugars such as xylose, cellobiose and glucose, with high potential in the biotechnology industry to obtain bioproducts of higher economic value. Thus, it is important to search for and study new yeast strains that co-consume these sugars to achieve better yields and productivity in the processes. The yeast Clavispora lusitaniae CDBB-L-2031, a native strain isolated from mezcal must, was studied under various culture conditions to potentially produce ethanol and xylitol due to its ability to assimilate xylose, cellobiose and glucose. This yeast produced ethanol under microaerobic conditions with yields of 0.451 gethanol/gglucose and 0.344 gethanol/gcellobiose, when grown on 1% glucose or cellobiose, respectively. In mixtures (0.5% each) of glucose:xylose and glucose:xylose:cellobiose the yields were 0.367 gethanol/gGX and 0. 380 gethanol/gGXC, respectively. Likewise, in identical conditions, C. lusitaniae produced xylitol from xylose with a yield of 0.421 gxylitol/gxylose. In 5% glucose or xylose, this yeast had better ethanol and xylitol titers and yields, respectively. However, glucose negatively affected xylitol production in the mixture of both sugars (3% each), producing only ethanol. Xylose reductase (XR) and xylitol dehydrogenase (XDH) activities were evaluated in cultures growing on xylose or glucose, obtaining the highest values in cultures on xylose at 8 h (25.9 and 6.22 mU/mg, respectively). While in glucose cultures, XR and XDH activities were detected once this substrate was consumed (4.06 and 3.32 mU/mg, respectively). Finally, the XYL1 and XYL2 genes encoding xylose reductase and xylitol dehydrogenase, respectively, were up-regulated by xylose, whereas glucose down-regulated their expression.
Asunto(s)
Xilitol , Xilosa , Aldehído Reductasa/genética , Aldehído Reductasa/metabolismo , Celobiosa/metabolismo , D-Xilulosa Reductasa/genética , D-Xilulosa Reductasa/metabolismo , Etanol/metabolismo , Fermentación , Glucosa/metabolismo , Saccharomyces cerevisiae/genética , Saccharomycetales , Xilitol/metabolismo , Xilosa/metabolismoRESUMEN
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/metabolismoRESUMEN
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.
Asunto(s)
Celobiosa/química , Celulosa/química , Emulsionantes/química , Polisacáridos/química , Celulosa/ultraestructura , Cosméticos/química , Cristalización , Emulsiones , Tecnología de Alimentos/métodos , Humanos , Concentración de Iones de Hidrógeno , Cinética , Concentración Osmolar , Temperatura , Agua/químicaRESUMEN
Clostridium (Ruminiclostridium) thermocellum is recognized for its ability to ferment cellulosic biomass directly, but it cannot naturally grow on xylose. Recently, C. thermocellum (KJC335) was engineered to utilize xylose through expressing a heterologous xylose catabolizing pathway. Here, we compared KJC335's transcriptomic responses to xylose versus cellobiose as the primary carbon source and assessed how the bacteria adapted to utilize xylose. Our analyses revealed 417 differentially expressed genes (DEGs) with log2 fold change (FC) >|1| and 106 highly DEGs (log2 FC >|2|). Among the DEGs, two putative sugar transporters, cbpC and cbpD, were up-regulated, suggesting their contribution to xylose transport and assimilation. Moreover, the up-regulation of specific transketolase genes (tktAB) suggests the importance of this enzyme for xylose metabolism. Results also showed remarkable up-regulation of chemotaxis and motility associated genes responding to xylose feeding, as well as widely varying gene expression in those encoding cellulosomal enzymes. For the down-regulated genes, several were categorized in gene ontology terms oxidation-reduction processes, ATP binding and ATPase activity, and integral components of the membrane. This study informs potentially critical, enabling mechanisms to realize the conceptually attractive Next-Generation Consolidated BioProcessing approach where a single species is sufficient for the co-fermentation of cellulose and hemicellulose.
Asunto(s)
Celobiosa/metabolismo , Clostridium thermocellum/genética , Clostridium thermocellum/metabolismo , Transcriptoma/genética , Xilosa/metabolismo , Proteínas Bacterianas/metabolismo , Celulosa/metabolismo , Regulación Bacteriana de la Expresión Génica , Polisacáridos/metabolismoRESUMEN
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/genéticaRESUMEN
A new cellulase producer strain of Penicillium digitatum (RV 06) was previously obtained from rotten maize grains. This work aim was to optimize the production and characterize this microorganism produced cellulase. A CMCase maximum production (1.6 U/mL) was obtained in stationary liquid culture, with an initial pH of 5.0, at 25 °C, with 1% lactose as carbon source, and cultured for 5 days. The produced enzyme was purified by ammonium sulfate precipitation and exclusion chromatography. The purified enzyme optimal temperature and pH were 60 °C and 5.2, respectively. The experimental Tm of thermal inactivation was 63.68 °C, and full activity was recovered after incubation of 7 h at 50 °C. The purified 74 kDa CMCase presented KM for CMC of 11.2 mg/mL, Vmax of 0.13 µmol/min, kcat of 52 s-1, and kcat/KM of 4.7 (mg/mL)-1 s-1. The purified enzyme had a high specificity for CMC and p-nitrophenyl cellobioside and released glucose and cellobiose as final products of the CMC hydrolysis. The enzyme trypsin digestion produced peptides whose masses were obtained by MALDI-TOF/TOF mass spectrometry, which was also used to obtain two peptide sequences. These peptide sequences and the mass peak profile retrieved a CBHI within the annotated genome of P. digitatum PD1. Sequence alignments and phylogenetic analysis confirmed this enzyme as a CBHI of the glycoside hydrolase family 7. The P. digitatum PD1 protein in silico structural model revealed a coil and ß-conformation predominance, which was confirmed by circular dichroism of the P. digitatum RV 06 purified enzyme.
Asunto(s)
Celobiosa/metabolismo , Celulasa/biosíntesis , Celulosa 1,4-beta-Celobiosidasa/biosíntesis , Celulosa 1,4-beta-Celobiosidasa/aislamiento & purificación , Proteínas Fúngicas/biosíntesis , Penicillium/enzimología , Dicroismo Circular , Estabilidad de Enzimas , Genoma Fúngico , Glucosa/metabolismo , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Filogenia , Sensibilidad y Especificidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Especificidad por Sustrato , TemperaturaRESUMEN
First-generation (1G) fuel ethanol production in sugarcane-based biorefineries is an established economic enterprise in Brazil. Second-generation (2G) fuel ethanol from lignocellulosic materials, though extensively investigated, is currently facing severe difficulties to become economically viable. Some of the challenges inherent to these processes could be resolved by efficiently separating and partially hydrolysing the cellulosic fraction of the lignocellulosic materials into the disaccharide cellobiose. Here, we propose an alternative biorefinery, where the sucrose-rich stream from the 1G process is mixed with a cellobiose-rich stream in the fermentation step. The advantages of mixing are 3-fold: (i) decreased concentrations of metabolic inhibitors that are typically produced during pretreatment and hydrolysis of lignocellulosic materials; (ii) decreased cooling times after enzymatic hydrolysis prior to fermentation; and (iii) decreased availability of free glucose for contaminating microorganisms and undesired glucose repression effects. The iSUCCELL platform will be built upon the robust Saccharomyces cerevisiae strains currently present in 1G biorefineries, which offer competitive advantage in non-aseptic environments, and into which intracellular hydrolyses of sucrose and cellobiose will be engineered. It is expected that high yields of ethanol can be achieved in a process with cell recycling, lower contamination levels and decreased antibiotic use, when compared to current 2G technologies.
Asunto(s)
Biocombustibles , Fermentación , Microbiología Industrial/métodos , Saccharomyces cerevisiae/genética , Saccharum/microbiología , Brasil , Celobiosa/metabolismo , Etanol/metabolismo , Glucosa/metabolismo , Ingeniería Metabólica , Saccharomyces cerevisiae/metabolismo , Saccharum/metabolismo , Xilosa/metabolismoRESUMEN
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.
Asunto(s)
Lisina/química , Oxígeno/química , beta-Glucosidasa/química , Aspergillus niger/enzimología , Biomasa , Celobiosa/química , DEAE-Celulosa/química , Estabilidad de Enzimas , Enzimas Inmovilizadas/química , Fermentación , Glucólisis , Glicósidos , Glicosilación , Concentración de Iones de Hidrógeno , Hidrólisis , Sefarosa/química , Temperatura , Factores de TiempoRESUMEN
We isolated two Candida pseudointermedia strains from the Atlantic rain forest in Brazil, and analyzed cellobiose metabolization in their cells. After growth in cellobiose medium, both strains had high intracellular ß-glucosidase activity [~ 200 U (g cells)-1 for 200 mM cellobiose and ~ 100 U (g cells)-1 for 2 mM pNPßG] and negligible periplasmic cellobiase activity. During batch fermentation, the strain with the best performance consumed all the available cellobiose in the first 18 h of the assay, producing 2.7 g L-1 of ethanol. Kinetics of its cellobiase activity demonstrated a high-affinity hydrolytic system inside cells, with Km of 12.4 mM. Our data suggest that, unlike other fungal species that hydrolyze cellobiose extracellularly, both analyzed strains transport it to the cytoplasm, where it is then hydrolyzed by high-affinity intracellular ß-glucosidases. We believe this study increases the fund of knowledge regarding yeasts from Brazilian microbiomes.
Asunto(s)
Candida/enzimología , Celobiosa/metabolismo , Madera/metabolismo , Madera/microbiología , beta-Glucosidasa/metabolismo , Brasil , Candida/aislamiento & purificación , Candida/metabolismo , Metabolismo de los Hidratos de Carbono , Etanol/metabolismo , Fermentación , Hidrólisis , CinéticaRESUMEN
ß-glucosidases catalyze the hydrolysis ß-1,4, ß-1,3 and ß-1,6 glucosidic linkages from non-reducing end of short chain oligosaccharides, alkyl and aryl ß-D-glucosides and disaccharides. They catalyze the rate-limiting reaction in the conversion of cellobiose to glucose in the saccharification of cellulose for second-generation ethanol production, and due to this important role the search for glucose tolerant enzymes is of biochemical and biotechnological importance. In this study we characterize a family 3 glycosyl hydrolase (GH3) ß-glucosidase (Bgl) produced by Malbranchea pulchella (MpBgl3) grown on cellobiose as the sole carbon source. Kinetic characterization revealed that the MpBgl3 was highly tolerant to glucose, which is in contrast to many Bgls that are completely inhibited by glucose. A 3D model of MpBgl3 was generated by molecular modeling and used for the evaluation of structural differences with a Bgl3 that is inhibited by glucose. Taken together, our results provide new clues to understand the glucose tolerance in GH3 ß-glucosidases.
Asunto(s)
Celobiosa/metabolismo , Glucosa/metabolismo , Onygenales/metabolismo , beta-Glucosidasa/metabolismo , Carbono/metabolismo , Celulosa/metabolismo , Hidrólisis , Onygenales/enzimologíaRESUMEN
Agricultural practices generate lignocellulosic waste that can be bioconverted by fungi to generate value-added products such as biofuels. In this context, fungal enzymes are presented as an alternative for their use in the hydrolysis of cellulose to sugars that can be fermented to ethanol. The aim of this work was to characterize LBM 033 strain and to analyze its efficiency in the hydrolysis of cellulosic substrates, including barley straw. LBM 033 strain was identified as Trametes villosa by molecular techniques, through the use of the ITS and rbp2 markers and the construction of phylogenetic trees. The cell-free supernatant of T. villosa LBM 033 showed high titers of hydrolytic enzymatic activities, necessary for the hydrolysis of the holocellulosic substrates, hydrolyzing pure cellulose to cellobiose and glucose and also degraded the polysaccharides contained in barley straw to short soluble oligosaccharides. These results indicate that macro fungi from tropical soil environments, such as T. villosa LBM 033 can be a valuable resource for in-house, cost effective production of enzymes that can be applied in the hydrolysis stage, which could reduce the total cost of bioethanol production.
Asunto(s)
Hordeum/metabolismo , Trametes/enzimología , Biocatálisis , Biocombustibles , Biotecnología , Celobiosa/metabolismo , Celulosa/metabolismo , Glucosa/metabolismo , Hidrólisis , Filogenia , Trametes/genéticaRESUMEN
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.
Asunto(s)
Celobiosa/química , Nanopartículas de Magnetita/química , Ácidos Sulfónicos/química , Ácidos Carboxílicos/química , Celobiosa/análisis , Hidrólisis , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Twelve strains of a novel yeast species were isolated from rotting wood, mushrooms and fruit samples in Brazil and French Guiana. Analysis of the sequences of the internal transcribed spacer region and the D1/D2 domains of the large subunit rRNA gene showed that the novel species belongs to the Kurtzmaniella clade. The novel species differed from its closest relative, Candida natalensis, by 12 substitutions in the D1/D2 sequences. The novel species could be distinguished from C. natalensis by its inability to assimilate cellobiose and salicin, and growth at 50â% (w/w) glucose. The name Kurtzmaniella hittingeri f.a., sp. nov. is proposed for the novel species. The type strain of K. hittingeri sp. nov. is CBS 13469T (=UFMG CM-Y272T). The MycoBank number is 827183. We also propose the transfer of Candida fragi, Candida quercitrusa and Candida natalensis to the genus Kurtzmaniella as new combinations.
Asunto(s)
Candida/clasificación , Frutas/microbiología , Filogenia , Madera/microbiología , Alcoholes Bencílicos , Brasil , Candida/aislamiento & purificación , Celobiosa , ADN de Hongos/genética , ADN Espaciador Ribosómico/genética , Guyana Francesa , Glucósidos , Técnicas de Tipificación Micológica , Análisis de Secuencia de ADNRESUMEN
Lignocellulose feedstock constitutes the most abundant carbon source in the biosphere; however, its recalcitrance remains a challenge for microbial conversion into biofuel and bioproducts. Bacillus licheniformis is a microbial mesophilic bacterium capable of secreting a large number of glycoside hydrolase (GH) enzymes, including a glycoside hydrolase from GH family 9 (BlCel9). Here, we conducted biochemical and biophysical studies of recombinant BlCel9, and its low-resolution molecular shape was retrieved from small angle X-ray scattering (SAXS) data. BlCel9 is an endoglucanase exhibiting maximum catalytic efficiency at pH 7.0 and 60 °C. Furthermore, it retains 80% of catalytic activity within a broad range of pH values (5.5-8.5) and temperatures (up to 50 °C) for extended periods of time (over 48 h). It exhibits the highest hydrolytic activity against phosphoric acid swollen cellulose (PASC), followed by bacterial cellulose (BC), filter paper (FP), and to a lesser extent carboxymethylcellulose (CMC). The HPAEC-PAD analysis of the hydrolytic products demonstrated that the end product of the enzymatic hydrolysis is primarily cellobiose, and also small amounts of glucose, cellotriose, and cellotetraose are produced. SAXS data analysis revealed that the enzyme adopts a monomeric state in solution and has a molecular mass of 65.8 kDa as estimated from SAXS data. The BlCel9 has an elongated shape composed of an N-terminal family 3 carbohydrate-binding module (CBM3c) and a C-terminal GH9 catalytic domain joined together by 20 amino acid residue long linker peptides. The domains are closely juxtaposed in an extended conformation and form a relatively rigid structure in solution, indicating that the interactions between the CBM3c and GH9 catalytic domains might play a key role in cooperative cellulose biomass recognition and hydrolysis.
Asunto(s)
Bacillus licheniformis/enzimología , Bacillus licheniformis/metabolismo , Celulasa/metabolismo , Glicósido Hidrolasas/metabolismo , Lignina/metabolismo , Catálisis , Celobiosa/biosíntesis , Celulosa/análogos & derivados , Celulosa/biosíntesis , Glucosa/biosíntesis , Concentración de Iones de Hidrógeno , Dispersión del Ángulo Pequeño , Tetrosas/biosíntesis , Triosas/biosíntesis , Difracción de Rayos XRESUMEN
We investigated the yeast species associated with rotting wood samples obtained from Brazilian ecosystems, with a special focus on cellobiose-fermenting species. About 647 yeast strains were isolated from rotting wood samples collected from the areas of Atlantic rainforest, Cerrado, and Amazonian forest. Eighty-six known species and 47 novel species of yeasts were isolated. Candida boidinii, Cyberlindnera subsufficiens, Meyerozyma guilliermondii, Schwanniomyces polymorphus, Candida natalensis, and Debaryomyces hansenii were the most frequently isolated species. Among the cellobiose-fermenting yeasts, 14 known and three novel yeast species were identified. Scheffersomyces queiroziae, Sc. amazonensis, Yamadazyma sp.1, Hanseniaspora opuntiae, C. jaroonii, and Candida tammaniensis were the main ethanol-producing yeasts. These species also produced an intracellular ß-glucosidase responsible for cellobiose hydrolysis. In fermentation assays using a culture medium containing 50 g L-1 cellobiose, ethanol production was observed in all cases; Sc. queiroziae and Sc. amazonensis showed the highest yield, efficiency, and productivity. Candida jaroonii and Yamadazyma sp.1 strains also showed high efficiency in cellobiose fermentation, while C. tammaniensis and H. opuntiae strains produced low amounts of ethanol. This study shows the potential of rotting wood samples from Brazilian ecosystems as a source of yeasts, including new species as well as those with promising biotechnological properties.