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Dienelactone hydrolase (DLH) is one of numerous hydrolytic enzymes with an α/ß-hydrolase fold, which catalyze the hydrolysis of dienelactone to maleylacetate. The DLHs share remarkably similar tertiary structures and a conserved arrangement of catalytic residues. This study presents the crystal structure and comprehensive functional characterization of a novel thermostable DLH from the bacterium Hydrogenobacter thermophilus (HtDLH). The crystal structure of the HtDLH, solved at a resolution of about 1.67â¯Å, exhibits a canonical α/ß-hydrolase fold formed by eight ß-sheet strands in the core, with one buried α-helix and six others exposed to the solvent. The structure also confirmed the conserved catalytic triad of DHLs formed by Cys121, Asp170, and His202 residues. The HtDLH forms stable homodimers in solution. Functional studies showed that HtDLH has the expected esterase activity over esters with short carbon chains, such as p-nitrophenyl acetate, reaching optimal activity at pH 7.5 and 70⯰C. Furthermore, HtDLH maintains more than 50â¯% of its activity even after incubation at 90⯰C for 16â¯h. Interestingly, HtDLH exhibits catalytic activity towards polyethylene terephthalate (PET) monomers, including bis-1,2-hydroxyethyl terephthalate (BHET) and 1-(2-hydroxyethyl) 4-methyl terephthalate, as well as other aliphatic and aromatic esters. These findings associated with the lack of activity on amorphous PET indicate that HtDLH has characteristic of a BHET-degrading enzyme. This work expands our understanding of enzyme families involved in PET degradation, providing novel insights for plastic biorecycling through protein engineering, which could lead to eco-friendly solutions to reduce the accumulation of plastic in landfills and natural environments.
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Plastics derived from fossil fuels are used ubiquitously owing to their exceptional physicochemical characteristics. However, the extensive and short-term use of plastics has caused environmental challenges. The biotechnological plastic conversion can help address the challenges related to plastic pollution, offering sustainable alternatives that can operate using bioeconomic concepts and promote socioeconomic benefits. In this context, using soil from a plastic-contaminated landfill, two consortia were established (ConsPlastic-A and -B) displaying versatility in developing and consuming polyethylene or polyethylene terephthalate as the carbon source of nutrition. The ConsPlastic-A and -B metagenomic sequencing, taxonomic profiling, and the reconstruction of 79 draft bacterial genomes significantly expanded the knowledge of plastic-degrading microorganisms and enzymes, disclosing novel taxonomic groups associated with polymer degradation. The microbial consortium was utilized to obtain a novel Pseudomonas putida strain (BR4), presenting a striking metabolic arsenal for aromatic compound degradation and assimilation, confirmed by genomic analyses. The BR4 displays the inherent capacity to degrade polyethylene terephthalate (PET) and produce polyhydroxybutyrate (PHB) containing hydroxyvalerate (HV) units that contribute to enhanced copolymer properties, such as increased flexibility and resistance to breakage, compared with pure PHB. Therefore, BR4 is a promising strain for developing a bioconsolidated plastic depolymerization and upcycling process. Collectively, our study provides insights that may extend beyond the artificial ecosystems established during our experiments and supports future strategies for effectively decomposing and valorizing plastic waste. Furthermore, the functional genomic analysis described herein serves as a valuable guide for elucidating the genetic potential of microbial communities and microorganisms in plastic deconstruction and upcycling.
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Biodegradação Ambiental , Microbiota , Plásticos , Plásticos/metabolismo , Microbiologia do Solo , Polietilenotereftalatos/metabolismo , Poluentes do Solo/metabolismo , Polímeros/metabolismo , Bactérias/metabolismo , Bactérias/genética , Plásticos Biodegradáveis/metabolismo , Consórcios Microbianos , Pseudomonas putida/metabolismo , Pseudomonas putida/genéticaRESUMO
OBJECTIVES: Apiosidases are enzymes that cleave the glycosidic bond between the monosaccharides linked to apiose, a branched chain furanose found in the cell walls of vascular plants and aquatic monocots. There is biotechnological interest in this enzyme group because apiose is the flavor-active compound of grapes, fruit juice, and wine, and the monosaccharide is found to be a plant secondary metabolite with pharmaceutical properties. However, functional and structural studies of this enzyme family are scarce. Recently, a glycoside hydrolase family member GH140 was isolated from Bacteroides thetaiotaomicron and identified as an endo-apiosidase. RESULTS: The structural characterization and functional identification of a second GH140 family enzyme, termed MmApi, discovered through mangrove soil metagenomic approach, are described. Among the various substrates tested, MmApi exhibited activity on an apiose-containing oligosaccharide derived from the pectic polysaccharide rhamnogalacturonan-II. While the crystallographic model of MmApi was similar to the endo-apiosidase from Bacteroides thetaiotaomicron, differences in the shape of the binding sites indicated that MmApi could cleave apioses within oligosaccharides of different compositions. CONCLUSION: This enzyme represents a novel tool for researchers interested in studying the physiology and structure of plant cell walls and developing biocatalytic strategies for drug and flavor production.
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Microbiota , Polissacarídeos , Oligossacarídeos/química , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/química , MonossacarídeosRESUMO
The isolation and selection of yeast strains to improve the quality of the cachaça-Brazilian Spirit-have been studied in our research group. Our strategy considers Saccharomyces cerevisiae as the predominant species involved in sugarcane juice fermentation and the presence of different stressors (osmolarity, temperature, ethanol content, and competition with other microorganisms). It also considers producing balanced concentrations of volatile compounds (higher alcohols and acetate and/or ethyl esters), flocculation capacity, and ethanol production. Since the genetic bases behind these traits of interest are not fully established, the whole genome sequencing of 11 different Saccharomyces cerevisiae strains isolated and selected from different places was analyzed to identify the presence of a specific genetic variation common to cachaça yeast strains. We have identified 20,128 single-nucleotide variants shared by all genomes. Of these shared variants, 37 were new variants (being six missenses), and 4,451 were identified as missenses. We performed a detailed functional annotation (using enrichment analysis, protein-protein interaction network analysis, and database and in-depth literature searches) of these new and missense variants. Many genes carrying these variations were involved in the phenotypes of flocculation, tolerance to fermentative stresses, and production of volatile compounds and ethanol. These results demonstrate the existence of a genetic profile shared by the 11 strains under study that could be associated with the applied selective strategy. Thus, this study points out genes and variants that may be used as molecular markers for selecting strains well suited to the fermentation process, including genetic improvement by genome editing, ultimately producing high-quality beverages and adding value.IMPORTANCEThis work demonstrates the existence of new genetic markers related to different phenotypes used to select yeast strains and mutations in genes directly involved in producing flavoring compounds and ethanol, and others related to flocculation and stress resistance.
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Perfil Genético , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Fermentação , Etanol , Fenótipo , GenômicaRESUMO
Cellulases from anaerobic fungi are enzymes less-studied biochemically and structurally than cellulases from bacteria and aerobic fungi. Currently, only thirteen GH5 cellulases from anaerobic fungi were biochemically characterized and two crystal structures were reported. In this context, here, we report the functional and biophysical characterization of a novel multi-modular cellulosomal GH5 endoglucanase from the anaerobic gut fungus Piromyces finnis (named here PfGH5). Multiple sequences alignments indicate that PfGH5 is composed of a GH5 catalytic domain and a CBM1 carbohydrate-binding module connected through a CBM10 dockerin module. Our results showed that PfGH5 is an endoglucanase from anaerobic fungus with a large spectrum of activity. PfGH5 exhibited preference for hydrolysis of oat ß-glucan, followed by galactomannan, carboxymethyl cellulose, mannan, lichenan and barley ß-glucan, therefore displaying multi-functionality. For oat ß-glucan, PfGH5 reaches its optimum enzymatic activity at 40 °C and pH 5.5, with Km of 7.1 µM. Ion exchange chromatography analyzes revealed the production of oligosaccharides with a wide degree of polymerization indicated that PfGH5 has endoglucanase activity. The ability to bind and cleave different types of carbohydrates evidence the potential of PfGH5 for use in biotechnology and provide a useful basis for future investigation and application of new anaerobic fungi enzymes.
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Celulase , Celulases , Celulase/química , Anaerobiose , FungosRESUMO
Simultaneous intracellular depolymerization of xylo-oligosaccharides (XOS) and acetate fermentation by engineered Saccharomyces cerevisiae offers significant potential for more cost-effective second-generation (2G) ethanol production. In the present work, the previously engineered S. cerevisiae strain, SR8A6S3, expressing enzymes for xylose assimilation along with an optimized route for acetate reduction, was used as the host for expressing two ß-xylosidases, GH43-2 and GH43-7, and a xylodextrin transporter, CDT-2, from Neurospora crassa, yielding the engineered SR8A6S3-CDT-2-GH34-2/7 strain. Both ß-xylosidases and the transporter were introduced by replacing two endogenous genes, GRE3 and SOR1, that encode aldose reductase and sorbitol (xylitol) dehydrogenase, respectively, and catalyse steps in xylitol production. The engineered strain, SR8A6S3-CDT-2-GH34-2/7 (sor1Δ gre3Δ), produced ethanol through simultaneous XOS, xylose, and acetate co-utilization. The mutant strain produced 60% more ethanol and 12% less xylitol than the control strain when a hemicellulosic hydrolysate was used as a mono- and oligosaccharide source. Similarly, the ethanol yield was 84% higher for the engineered strain using hydrolysed xylan, compared with the parental strain. Xylan, a common polysaccharide in lignocellulosic residues, enables recombinant strains to outcompete contaminants in fermentation tanks, as XOS transport and breakdown occur intracellularly. Furthermore, acetic acid is a ubiquitous toxic component in lignocellulosic hydrolysates, deriving from hemicellulose and lignin breakdown. Therefore, the consumption of XOS, xylose, and acetate expands the capabilities of S. cerevisiae for utilization of all of the carbohydrate in lignocellulose, potentially increasing the efficiency of 2G biofuel production.
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Saccharomyces cerevisiae , Xilosidases , Saccharomyces cerevisiae/metabolismo , Xilanos/metabolismo , Xilose/metabolismo , Etanol/metabolismo , Engenharia Metabólica , Xilitol/metabolismo , Oligossacarídeos/metabolismo , Fermentação , D-Xilulose Redutase/genética , D-Xilulose Redutase/metabolismo , Xilosidases/metabolismo , Acetatos/metabolismoRESUMO
The biorefinery concept, in which biomass is utilized for the production of fuels and chemicals, emerges as an eco-friendly, cost-effective, and renewable alternative to petrochemical-based production. The hydroxycinnamic acid fraction of lignocellulosic biomass represents an untapped source of aromatic molecules that can be converted to numerous high-value products with industrial applications, including in the flavor and fragrance sector and pharmaceuticals. This review describes several biochemical pathways useful in the development of a biorefinery concept based on the biocatalytic conversion of the hydroxycinnamic acids ferulic, caffeic, and p-coumaric acid into high-value molecules. KEY POINTS: ⢠The phenylpropanoids bioconversion pathways in the context of biorefineries ⢠Description of pathways from hydroxycinnamic acids to high-value compounds ⢠Metabolic engineering and synthetic biology advance hydroxycinnamic acid-based biorefineries.
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Vias Biossintéticas , Ácidos Cumáricos , Ácidos Cumáricos/metabolismo , Biomassa , Biocatálise , Engenharia MetabólicaRESUMO
BACKGROUND: Lignin is an attractive alternative for producing biobased chemicals. It is the second major component of the plant cell wall and is an abundant natural source of aromatic compounds. Lignin degradation using microbial oxidative enzymes that depolymerize lignin and catabolize aromatic compounds into central metabolic intermediates is a promising strategy for lignin valorization. However, the intrinsic heterogeneity and recalcitrance of lignin severely hinder its biocatalytic conversion. In this context, examining microbial degradation systems can provide a fundamental understanding of the pathways and enzymes that are useful for lignin conversion into biotechnologically relevant compounds. RESULTS: Lignin-degrading catabolism of a novel Rhodosporidium fluviale strain LM-2 was characterized using multi-omic strategies. This strain was previously isolated from a ligninolytic microbial consortium and presents a set of enzymes related to lignin depolymerization and aromatic compound catabolism. Furthermore, two catabolic routes for producing 4-vinyl guaiacol and vanillin were identified in R. fluviale LM-2. CONCLUSIONS: The multi-omic analysis of R. fluviale LM-2, the first for this species, elucidated a repertoire of genes, transcripts, and secreted proteins involved in lignin degradation. This study expands the understanding of ligninolytic metabolism in a non-conventional yeast, which has the potential for future genetic manipulation. Moreover, this work unveiled critical pathways and enzymes that can be exported to other systems, including model organisms, for lignin valorization.
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Deciphering how enzymes interact, modify, and recognize carbohydrates has long been a topic of interest in academic, pharmaceutical, and industrial research. Carbohydrate-binding modules (CBMs) are noncatalytic globular protein domains attached to carbohydrate-active enzymes that strengthen enzyme affinity to substrates and increase enzymatic efficiency via targeting and proximity effects. CBMs are considered auspicious for various biotechnological purposes in textile, food, and feed industries, representing valuable tools in basic science research and biomedicine. Here, we present the first crystallographic structure of a CBM8 family member (CBM8), DdCBM8, from the slime mold Dictyostelium discoideum, which was identified attached to an endo-ß-1,4-glucanase (glycoside hydrolase family 9). We show that the planar carbohydrate-binding site of DdCBM8, composed of aromatic residues, is similar to type A CBMs that are specific for crystalline (multichain) polysaccharides. Accordingly, pull-down assays indicated that DdCBM8 was able to bind insoluble forms of cellulose. However, affinity gel electrophoresis demonstrated that DdCBM8 also bound to soluble (single chain) polysaccharides, especially glucomannan, similar to type B CBMs, although it had no apparent affinity for oligosaccharides. Therefore, the structural characteristics and broad specificity of DdCBM8 represent exceptions to the canonical CBM classification. In addition, mutational analysis identified specific amino acid residues involved in ligand recognition, which are conserved throughout the CBM8 family. This advancement in the structural and functional characterization of CBMs contributes to our understanding of carbohydrate-active enzymes and protein-carbohydrate interactions, pushing forward protein engineering strategies and enhancing the potential biotechnological applications of glycoside hydrolase accessory modules.
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Dictyostelium , Carboidratos/química , Cristalografia por Raios X , Dictyostelium/metabolismo , Glucanos/metabolismo , Glicosídeo Hidrolases , Ligantes , Polissacarídeos/metabolismoRESUMO
The biocatalytic production of fuels and chemicals from plant biomass represents an attractive alternative to fossil fuel-based refineries. In this context, the mining and characterization of novel biocatalysts can promote disruptive innovation opportunities in the field of lignocellulose conversion and valorization. In the present work, we conducted the biochemical and structural characterization of two novel hydroxycinnamic acid catabolic enzymes, isolated from a lignin-degrading microbial consortium, a feruloyl-CoA synthetase, and a feruloyl-CoA hydratase-lyase, named LM-FCS2 and LM-FCHL2, respectively. Besides establishing the homology model structures for novel FCS and FCHL members with unique characteristics, the enzymes presented interesting biochemical features: LM-FCS2 showed stability in alkaline pHs and was able to convert a wide array of p-hydroxycinnamic acids to their respective CoA-thioesters, including sinapic acid; LM-FCHL2 efficiently converted feruloyl-CoA and p-coumaroyl-CoA into vanillin and 4-hydroxybenzaldehyde, respectively, and could produce vanillin directly from ferulic acid. The coupled reaction of LM-FCS2 and LM-FCHL2 produced vanillin, not only from commercial ferulic acid but also from a crude lignocellulosic hydrolysate. Collectively, this work illuminates the structure and function of two critical enzymes involved in converting ferulic acid into high-value molecules, thus providing valuable concepts applied to the development of plant biomass biorefineries. KEY POINTS: ⢠Comprehensive characterization of feruloyl-CoA synthetase from metagenomic origin. ⢠Novel low-resolution structures of hydroxycinnamate catabolic enzymes. ⢠Production of vanillin via enzymatic reaction using lignocellulosic hydrolysates.
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Lignina , Metagenoma , Escherichia coli/genética , Hiperlipidemia Familiar Combinada , Lignina/metabolismo , SoloRESUMO
Although lignocellulose is the most abundant and renewable natural resource for biofuel production, its use remains under exploration because of its highly recalcitrant structure. Its deconstruction into sugar monomers is mainly driven by carbohydrate-active enzymes (CAZymes). To develop highly efficient and fast strategies to discover biomass-degrading enzymes for biorefinery applications, an enrichment process combined with integrative omics approaches was used to identify new CAZymes. The lignocellulolytic-enriched mangrove microbial community (LignoManG) established on sugarcane bagasse (SB) was enriched with lignocellulolytic bacteria and fungi such as Proteobacteria, Bacteroidetes, Basidiomycota, and Ascomycota. These microbial communities were able to degrade up to 55 % of the total SB, indicating the production of lignocellulolytic enzymes. Metagenomic analysis revealed that the LignoManG harbors 18.042 CAZyme sequences such as of cellulases, hemicellulases, carbohydrate esterases, and lytic polysaccharide monooxygenase. Similarly, our metaproteomic analysis depicted several enzymes from distinct families of different CAZy families. Based on the LignoManG data, a xylanase (coldXynZ) was selected, amplified, cloned, expressed, and biochemically characterized. The enzyme displayed psicrofilic properties, with the highest activity at 15⯰C, retaining 77 % of its activity when incubated at 0⯰C. Moreover, molecular modeling in silico indicated that coldXynZ is composed of a TIM barrel, which is a typical folding found in the GH10 family, and displayed similar structural features related to cold-adapted enzymes. Collectively, the data generated in this study represent a valuable resource for lignocellulolytic enzymes with potential biotechnological applications.
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Ascomicetos , Celulases , Saccharum , Bacteroidetes , Basidiomycota , Biomassa , Metabolismo dos Carboidratos , Esterases , Sedimentos Geológicos , Glicosídeo Hidrolases , Metagenoma , Proteobactérias , Áreas AlagadasRESUMO
BACKGROUND: Wood-decay basidiomycetes are effective for the degradation of highly lignified and recalcitrant plant substrates. The degradation of lignocellulosic materials by brown-rot strains is carried out by carbohydrate-active enzymes and non-enzymatic Fenton mechanism. Differences in the lignocellulose catabolism among closely related brown rots are not completely understood. Here, a multi-omics approach provided a global understanding of the strategies employed by L. sulphureus ATCC 52600 for lignocellulose degradation. RESULTS: The genome of Laetiporus sulphureus ATCC 52600 was sequenced and phylogenomic analysis supported monophyletic clades for the Order Polyporales and classification of this species within the family Laetiporaceae. Additionally, the plasticity of its metabolism was revealed in growth analysis on mono- and disaccharides, and polysaccharides such as cellulose, hemicelluloses, and polygalacturonic acid. The response of this fungus to the presence of lignocellulosic substrates was analyzed by transcriptomics and proteomics and evidenced the occurrence of an integrated oxidative-hydrolytic metabolism. The transcriptomic profile in response to a short cultivation period on sugarcane bagasse revealed 125 upregulated transcripts, which included CAZymes (redox enzymes and hemicellulases) as well as non-CAZy redox enzymes and genes related to the synthesis of low-molecular-weight compounds. The exoproteome produced in response to extended cultivation time on Avicel, and steam-exploded sugarcane bagasse, sugarcane straw, and Eucalyptus revealed 112 proteins. Contrasting with the mainly oxidative profile observed in the transcriptome, the secretomes showed a diverse hydrolytic repertoire including constitutive cellulases and hemicellulases, in addition to 19 upregulated CAZymes. The secretome induced for 7 days on sugarcane bagasse, representative of the late response, was applied in the saccharification of hydrothermally pretreated grass (sugarcane straw) and softwood (pine) by supplementing a commercial cocktail. CONCLUSION: This study shows the singularity of L. sulphureus ATCC 52600 compared to other Polyporales brown rots, regarding the presence of cellobiohydrolase and peroxidase class II. The multi-omics analysis reinforces the oxidative-hydrolytic metabolism involved in lignocellulose deconstruction, providing insights into the overall mechanisms as well as specific proteins of each step.
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Glycoside hydrolases (GHs) are involved in the degradation of a wide diversity of carbohydrates and present several biotechnological applications. Many GH families are composed of enzymes with a single well-defined specificity. In contrast, enzymes from the GH16 family can act on a range of different polysaccharides, including ß-glucans and galactans. SCLam, a GH16 member derived from a soil metagenome, an endo-ß-1,3(4)-glucanase (EC 3.2.1.6), can cleave both ß-1,3 and ß-1,4 glycosidic bonds in glucans, such as laminarin, barley ß-glucan, and cello-oligosaccharides. A similar cleavage pattern was previously reported for other GH16 family members. However, the molecular mechanisms for this dual cleavage activity on (1,3)- and (1,4)-ß-D-glycosidic bonds by laminarinases have not been elucidated. In this sense, we determined the X-ray structure of a presumably inactive form of SCLam cocrystallized with different oligosaccharides. The solved structures revealed general bound products that are formed owing to residual activities of hydrolysis and transglycosylation. Biochemical and biophysical analyses and molecular dynamics simulations help to rationalize differences in activity toward different substrates. Our results depicted a bulky aromatic residue near the catalytic site critical to select the preferable configuration of glycosidic bonds in the binding cleft. Altogether, these data contribute to understanding the structural basis of recognition and hydrolysis of ß-1,3 and ß-1,4 glycosidic linkages of the laminarinase enzyme class, which is valuable for future studies on the GH16 family members and applications related to biomass conversion into feedstocks and bioproducts.
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Proteínas de Bactérias/metabolismo , Celulases/metabolismo , Glucanos/metabolismo , Proteínas de Bactérias/química , Sequência de Carboidratos , Domínio Catalítico , Celulases/química , Cristalografia por Raios X/métodos , Glucanos/classificação , Glicosídeos/química , Glicosídeos/metabolismo , Hidrólise , Simulação de Dinâmica Molecular , Microbiologia do Solo , Especificidade por SubstratoRESUMO
The physicochemical pretreatment is an important step to reduce biomass recalcitrance and facilitate further processing of plant lignocellulose into bioproducts. This process results in soluble and insoluble biomass fractions, and both may contain by-products that inhibit enzymatic biocatalysts and microbial fermentation. These fermentation inhibitory compounds (ICs) are produced during the degradation of lignin and sugars, resulting in phenolic and furanic compounds, and carboxylic acids. Therefore, detoxification steps may be required to improve lignocellulose conversion by microoganisms. Several physical and chemical methods, such as neutralization, use of activated charcoal and organic solvents, have been developed and recommended for removal of ICs. However, biological processes, especially enzyme-based, have been shown to efficiently remove ICs with the advantage of minimizing environmental issues since they are biogenic catalysts and used in low quantities. This review focuses on describing several enzymatic approaches to promote detoxification of lignocellulosic hydrolysates and improve the performance of microbial fermentation for the generation of bioproducts. Novel strategies using classical carbohydrate active enzymes (CAZymes), such as laccases (AA1) and peroxidases (AA2), as well as more advanced strategies using prooxidant, antioxidant and detoxification enzymes (dubbed as PADs), i.e. superoxide dismutases, are discussed as perspectives in the field.
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Biomassa , Lignina/metabolismo , Ácidos Carboxílicos/metabolismo , Fermentação , Lacase/metabolismo , Peroxidases/metabolismo , Superóxido Dismutase/metabolismoRESUMO
Arabinanases from glycoside hydrolase family GH93 are enzymes with exo-activity that hydrolyze the α-1,5 bonds between arabinose residues present on arabinan. Currently, several initiatives aiming to use byproducts rich in arabinan such as pectin and sugar beet pulp as raw material to produce various compounds of interest are being developed. However, it is necessary to use robust enzymes that have an optimal performance under pH and temperature conditions used in the industrial processes. In this work, the first GH93 from the thermophilic fungus Thermothielavioides terrestris (Abn93T) was heterologously expressed in Aspergillus nidulans, purified and biochemically characterized. The enzyme is a thermophilic glycoprotein (optimum activity at 70 °C) with prolonged stability in acid pHs (4.0 to 6.5). The presence of glycosylation affected slightly the hydrolytic capacity of the enzyme, which was further increased by 34% in the presence of 1 mM CoCl2. Small-angle X-ray scattering results show that Abn93T is a globular-like-shaped protein with a slight bulge at one end. The hydrolytic mechanism of the enzyme was elucidated using capillary zone electrophoresis and molecular docking calculations. Abn93T has an ability to produce (in synergism with arabinofuranosidases) arabinose and arabinobiose from sugar beet arabinan, which can be explored as fermentable sugars and prebiotics. KEY POINTS: ⢠Thermophilic exo-arabinanase from family GH93 ⢠Molecular basis of arabinan depolymerization.
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Arabinose , Glicosídeo Hidrolases , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Simulação de Acoplamento Molecular , Sordariales , Especificidade por SubstratoRESUMO
Bioproducts production using monomeric sugars derived from lignocellulosic biomass presents several challenges, such as to require a physicochemical pretreatment to improve its conversion yields. Hydrothermal lignocellulose pretreatment has several advantages and results in solid and liquid streams. The former is called hemicellulosic hydrolysate (HH), which contains inhibitory phenolic compounds and sugar degradation products that hinder microbial fermentation products from pentose sugars. Here, we developed and applied a novel enzyme process to detoxify HH. Initially, the design of experiments with different redox activities enzymes was carried out. The enzyme mixture containing the peroxidase (from Armoracia rusticana) together with superoxide dismutase (from Coptotermes gestroi) are the most effective to detoxify HH derived from sugarcane bagasse. Butanol fermentation by the bacteria Clostridium saccharoperbutylacetonicum and ethanol production by the yeast Scheffersomyces stipitis increased by 24.0× and 2.4×, respectively, relative to the untreated hemicellulosic hydrolysates. Detoxified HH was analyzed by chromatographic and spectrometric methods elucidating the mechanisms of phenolic compound modifications by enzymatic treatment. The enzyme mixture degraded and reduced the hydroxyphenyl- and feruloyl-derived units and polymerized the lignin fragments. This strategy uses biocatalysts under environmentally friendly conditions and could be applied in the fuel, food, and chemical industries.
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Clostridium/metabolismo , Peroxidase/química , Polissacarídeos/química , Saccharum/química , Superóxido Dismutase/química , Leveduras/metabolismo , Biocatálise , Butanóis/metabolismo , Celulose/química , Celulose/metabolismo , Fermentação , Microbiologia Industrial , Peroxidase/metabolismo , Polissacarídeos/metabolismo , Saccharum/microbiologia , Superóxido Dismutase/metabolismoRESUMO
In the context of increasing demand for renewable alternatives of fuels and chemicals, the valorization of lignin emerges as a value-adding strategy in biorefineries and an alternative to petroleum-derived molecules. One of the compounds derived from lignin is ferulic acid (FA), which can be converted into valuable molecules such as vanillin. In microorganisms, FA biotransformation into vanillin can occur via a two-step reaction catalyzed by the sequential activity of a feruloyl-CoA synthetase (FCS) and an feruloyl-CoA hydratase-lyase (FCHL), which could be exploited industrially. In this study, a prokaryotic FCHL derived from a lignin-degrading microbial consortium (named LM-FCHL) was cloned, successfully expressed in soluble form and purified. The crystal structure was solved and refined at 2.1 Å resolution. The LM-FCHL is a hexamer composed of a dimer of trimers, which showed to be quite stable under extreme pH conditions. Finally, small angle X-ray scattering corroborates the hexameric state in solution and indicates flexibility in the protein structure. The present study contributes to the field of lignin valorization to valuable molecules by establishing the biophysical and structural characterization for a novel FCHL member of unique characteristics.
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Benzaldeídos/metabolismo , Ácidos Cumáricos/metabolismo , Hidroliases/química , Lignina/metabolismo , Acil Coenzima A/metabolismo , Hidroliases/metabolismo , Concentração de Íons de Hidrogênio , Consórcios Microbianos , Modelos Moleculares , Multimerização ProteicaRESUMO
Xylanases decrease the xylan content in pretreated biomass releasing it from hemicellulose, thus improving the accessibility of cellulose for cellulases. In this work, an endo-ß-1,4-xylanase from Aspergillus fumigatus var. niveus (AFUMN-GH10) was successfully expressed. The structural analysis and biochemical characterization showed this AFUMN-GH10 does not contain a carbohydrate-binding module. The enzyme retained its activity in a pH range from 4.5 to 7.0, with an optimal temperature at 60⯰C. AFUMN-GH10 showed the highest activity in beechwood xylan. The mode of action of AFUMN-GH10 was investigated by hydrolysis of APTS-labeled xylohexaose, which resulted in xylotriose and xylobiose as the main products. AFUMN-GH10 released 27% of residual xylan from hydrothermally-pretreated corn stover and 14% of residual xylan from hydrothermally-pretreated sugarcane bagasse. The results showed that environmentally friendly pretreatment followed by enzymatic hydrolysis with AFUMN-GH10 in low concentration is a suitable method to remove part of residual and recalcitrant hemicellulose from biomass.
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Ferulic acid (FA), a low-molecular weight aromatic compound derived from lignin, represents a high-value molecule, used for applications in the cosmetic and pharmaceutical industries. FA can be further enzymatically converted in other commercially interesting molecules, such as vanillin and bioplastics. In several organisms, these transformations often start with a common step of FA activation via CoA-thioesterification, catalyzed by feruloyl-CoA synthetases (Fcs). In this context, these enzymes are of biotechnological interest for conversion of lignin-derived FA into high value chemicals. In this study, we describe the first structural characterization of a prokaryotic Fcs, named FCS1, isolated from a lignin-degrading microbial consortium. The FCS1 optimum pH and temperature were 9 and 37°C, respectively, with Km of 0.12 mM and Vmax of 36.82 U/mg. The circular dichroism spectra indicated a notable secondary structure stability at alkaline pH values and high temperatures. This secondary structure stability corroborates the activity data, which remains high until pH 9. The Small Angle X-Ray Scattering analyses resulted on the tertiary/quaternary structure and the low-resolution envelope in solution of FCS1, which was modeled as a homodimer using the hyperthermophilic nucleoside diphosphate-forming acetyl-CoA synthetase from Candidatus Korachaeum cryptofilum. This study contributes to the field of research by establishing the first biophysical and structural characterization for Fcs, and our data may be used for comparison against novel enzymes of this class that to be studied in the future.
Assuntos
Archaea , Proteínas Arqueais , Coenzima A Ligases , Lignina/química , Metagenoma , Microbiologia do Solo , Archaea/enzimologia , Archaea/genética , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Benzaldeídos/química , Benzaldeídos/metabolismo , Coenzima A Ligases/química , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Ácidos Cumáricos/química , Ácidos Cumáricos/metabolismo , Concentração de Íons de Hidrogênio , Lignina/metabolismo , Domínios Proteicos , SoloRESUMO
The evolution of the symbiotic association with microbes allowed termites to decompose ingested lignocellulose from plant-derived substrates, including herbivore dung and soil humus. Representatives of the Syntermitinae (Termitidae) range in their feeding habits from wood and litter-feeding to humus-feeding species. However, only limited information is available about their feeding ecology and associated microbial communities. Here we conducted a study of the microbial communities associated to the termite Procornitermes araujoi using Illumina sequencing of the 16S and ITS rRNA genes. This species has been previously included in different feeding guilds. However, most aspects of its feeding ecology are unknown, especially those associated to its symbiotic microbiota. Our results showed that the microbial communities of termite guts and nest substrates of P. araujoi differed significantly for bacteria and fungi. Firmicutes dominated the bacterial gut community of both workers and soldiers, whereas Actinobacteria was found in higher prevalence in the nest walls. Sordariomycetes was the most abundant fungal class in both gut and nest samples and distinguish P. araujoi from the grass/litter feeding Cornitermes cumulans. Our results also showed that diversity of gut bacteria were higher in P. araujoi and Silvestritermes euamignathus than in the grass/litter feeders (C. cumulans and Syntermes dirus), that could indicate an adaptation of the microbial community of polyphagous termites to the higher complexity of their diets.