Photosynthesis of hybrid silver-based nanoparticles mediated lectins and evaluation of their hemagglutinating properties.

Lectins are carbohydrate-binding proteins belonging to the Leguminosae family. In this family stand out proteins extracted from species belonging to Diocleinae subtribe, which includes, for example, the seed lectin from Dioclea violacea (DVL) and the jack bean lectin Concanavalin A (ConA). Here, we report the photosynthesis of silver/silver chloride nanoparticles (NPs) assisted by ConA and DVL. The syntheses were simple processes using a green-chemistry approach. Under electron microscopy, NPs heterogeneous in size, nearly spherical and covered by a thin lectin corona, were observed. Both NPs assisted by lectins were capable to cause strong rabbit erythrocytes agglutination with the same titers of hemagglutinating activities. These results indicate that both lectins maintained their biological activities even after association with the NPs and therefore are able to interact with biological membrane carbohydrates. However, for rabbit erythrocytes treated with proteolytic enzymes were observed different titers of hemagglutinating activities, suggesting differences in the spatial arrangement of the lectins on the surface of the NPs. This study provides evidences that these hybrid lectin-coated silver/silver chloride NPs can be used for selective recognition and interaction with membrane carbohydrates and others biotechnological applications.

The periplasmic expression and purification of AA15 lytic polysaccharide monooxygenases from insect species in Escherichia coli.

Lytic polysaccharide monooxygenases (LPMOs) are metalloenzymes that cleave structural polysaccharides through an oxidative mechanism. The enzymatic activity of LPMOs relies on the presence of a Cu2+ histidine-brace motif in their flat catalytic surface. Upon reduction by an external electron donor and in the presence of its co-substrates, O2 or H2O2, LPMOs can generate reactive oxygen species to oxidize the substrates. Fungal and bacterial LPMOs are involved in the catabolism of polysaccharides, such as chitin, cellulose, and hemicelluloses, and virulence mechanisms. Based on the reports on the discovery of LPMOs from the family AA15 in termites, firebrats, and flies, the functional role of the LPMO in the biosphere could expand, as these enzymes may be correlated with chitin remodeling and molting in insects. However, there is limited knowledge of AA15 LPMOs due to difficulties in recombinant expression of soluble proteins and purification protocols. In this study, we describe a protocol for the cloning, expression, and purification of insect AA15 LPMOs from Arthropoda, mainly from termites, followed by the expression and purification of an AA15 LPMO from the silkworm Bombyx mori, which contains a relatively high number of disulfide bonds. We also report the recombinant expression and purification of a protein with homology to AA15 family from the western European honeybee Apis mellifera, an LPMO-like enzyme lacking the canonical histidine brace. Therefore, this work can support future studies concerning the role of LPMOs in the biology of insects and inspire molecular entomologists and insect biochemists in conducting activities in this field.

Recombinant expression, purification and characterization of an active bacterial feruloyl-CoA synthase with potential for application in vanillin production.

The ferulic acid (FA) represents a high-value molecule with applications in the cosmetic and pharmaceutical industries. This aromatic molecule is derived from lignin and can be enzymatically converted in other commercially interesting molecules, such as vanillin and bioplastics. This process starts with a common step of FA activation via CoA-thioesterification, catalyzed by feruloyl-CoA synthetases. Therefore, here, we report the successfully expression, purification as well as the initial structural and biochemical characterization of a stable, correctly folded, and catalytically active bacterial feruloyl-CoA synthase (here named FCS3) isolated from a lignin-degrading microbial consortium. The purification of recombinant FCS3 to near homogeneity was achieved using affinity chromatography. The FCS3 structure is composed of a mixture of α and ß secondary structures and most likely forms stable homodimers in solution. The FCS3 presented a notable structural stability at alkaline pH values and it was able to convert FA and coenzyme A (CoA) into feruloyl-CoA complex at room temperature. This study should provide a useful basis for future biotechnological applications of FCS3, especially in the field of conversion of lignin-derived FA into high value compounds.

Multi-omic Directed Discovery of Cellulosomes, Polysaccharide Utilization Loci, and Lignocellulases from an Enriched Rumen Anaerobic Consortium.

Lignocellulose is one of the most abundant renewable carbon sources, representing an alternative to petroleum for the production of fuel and chemicals. Nonetheless, the lignocellulose saccharification process, to release sugars for downstream applications, is one of the most crucial factors economically challenging to its use. The synergism required among the various carbohydrate-active enzymes (CAZymes) for efficient lignocellulose breakdown is often not satisfactorily achieved with an enzyme mixture from a single strain. To overcome this challenge, enrichment strategies can be applied to develop microbial communities with an efficient CAZyme arsenal, incorporating complementary and synergistic properties, to improve lignocellulose deconstruction. We report a comprehensive and deep analysis of an enriched rumen anaerobic consortium (ERAC) established on sugarcane bagasse (SB). The lignocellulolytic abilities of the ERAC were confirmed by analyzing the depolymerization of bagasse by scanning electron microscopy, enzymatic assays, and mass spectrometry. Taxonomic analysis based on 16S rRNA sequencing elucidated the community enrichment process, which was marked by a higher abundance of Firmicutes and Synergistetes species. Shotgun metagenomic sequencing of the ERAC disclosed 41 metagenome-assembled genomes (MAGs) harboring cellulosomes and polysaccharide utilization loci (PULs), along with a high diversity of CAZymes. The amino acid sequences of the majority of the predicted CAZymes (60% of the total) shared less than 90% identity with the sequences found in public databases. Additionally, a clostridial MAG identified in this study produced proteins during consortium development with scaffoldin domains and CAZymes appended to dockerin modules, thus representing a novel cellulosome-producing microorganism.IMPORTANCE The lignocellulolytic ERAC displays a unique set of plant polysaccharide-degrading enzymes (with multimodular characteristics), cellulosomal complexes, and PULs. The MAGs described here represent an expansion of the genetic content of rumen bacterial genomes dedicated to plant polysaccharide degradation, therefore providing a valuable resource for the development of biocatalytic toolbox strategies to be applied to lignocellulose-based biorefineries.

The Hydroxyquinol Degradation Pathway in Rhodococcus jostii RHA1 and Agrobacterium Species Is an Alternative Pathway for Degradation of Protocatechuic Acid and Lignin Fragments.

Deletion of the pcaHG genes, encoding protocatechuate 3,4-dioxygenase in Rhodococcus jostii RHA1, gives a gene deletion strain still able to grow on protocatechuic acid as the sole carbon source, indicating a second degradation pathway for protocatechuic acid. Metabolite analysis of wild-type R. jostii RHA1 grown on medium containing vanillin or protocatechuic acid indicated the formation of hydroxyquinol (benzene-1,2,4-triol) as a downstream product. Gene cluster ro01857-ro01860 in Rhodococcus jostii RHA1 contains genes encoding hydroxyquinol 1,2-dioxygenase and maleylacetate reductase for degradation of hydroxyquinol but also putative mono-oxygenase (ro01860) and putative decarboxylase (ro01859) genes, and a similar gene cluster is found in the genome of lignin-degrading Agrobacterium species. Recombinant R. jostii mono-oxygenase and decarboxylase enzymes in combination were found to convert protocatechuic acid to hydroxyquinol. Hence, an alternative pathway for degradation of protocatechuic acid via oxidative decarboxylation to hydroxyquinol is proposed.IMPORTANCE There is a well-established paradigm for degradation of protocatechuic acid via the ß-ketoadipate pathway in a range of soil bacteria. In this study, we have found the existence of a second pathway for degradation of protocatechuic acid in Rhodococcus jostii RHA1, via hydroxyquinol (benzene-1,2,4-triol), which establishes a metabolic link between protocatechuic acid and hydroxyquinol. The presence of this pathway in a lignin-degrading Agrobacterium sp. strain suggests the involvement of the hydroxyquinol pathway in the metabolism of degraded lignin fragments.

Functional genomic analysis of bacterial lignin degraders: diversity in mechanisms of lignin oxidation and metabolism.

Although several bacterial lignin-oxidising enzymes have been discovered in recent years, it is not yet clear whether different lignin-degrading bacteria use similar mechanisms for lignin oxidation and degradation of lignin fragments. Genome sequences of 13 bacterial lignin-oxidising bacteria, including new genome sequences for Microbacterium phyllosphaerae and Agrobacterium sp., were analysed for the presence of lignin-oxidising enzymes and aromatic degradation gene clusters that could be used to metabolise the products of lignin degradation. Ten bacterial genomes contain DyP-type peroxidases, and ten bacterial strains contain putative multi-copper oxidases (MCOs), both known to have activity for lignin oxidation. Only one strain lacks both MCOs and DyP-type peroxidase genes. Eleven bacterial genomes contain aromatic degradation gene clusters, of which ten contain the central ß-ketoadipate pathway, with variable numbers and types of degradation clusters for other aromatic substrates. Hence, there appear to be diverse metabolic strategies used for lignin oxidation in bacteria, while the ß-ketoadipate pathway appears to be the most common route for aromatic metabolism in lignin-degrading bacteria.

Draft genome sequence of Wickerhamomyces anomalus LBCM1105, isolated from cachaça fermentation.

Wickerhamomyces anomalus LBCM1105 is a yeast isolated from cachaça distillery fermentation vats, notable for exceptional glycerol consumption ability. We report its draft genome with 20.5x in-depth coverage and around 90% extension and completeness. It harbors the sequences of proteins involved in glycerol transport and metabolism.

Suppression of a single BAHD gene in Setaria viridis causes large, stable decreases in cell wall feruloylation and increases biomass digestibility.

Feruloylation of arabinoxylan (AX) in grass cell walls is a key determinant of recalcitrance to enzyme attack, making it a target for improvement of grass crops, and of interest in grass evolution. Definitive evidence on the genes responsible is lacking so we studied a candidate gene that we identified within the BAHD acyl-CoA transferase family. We used RNA interference (RNAi) silencing of orthologs in the model grasses Setaria viridis (SvBAHD01) and Brachypodium distachyon (BdBAHD01) and determined effects on AX feruloylation. Silencing of SvBAHD01 in Setaria resulted in a c. 60% decrease in AX feruloylation in stems consistently across four generations. Silencing of BdBAHD01 in Brachypodium stems decreased feruloylation much less, possibly due to higher expression of functionally redundant genes. Setaria SvBAHD01 RNAi plants showed: no decrease in total lignin, approximately doubled arabinose acylated by p-coumarate, changes in two-dimensional NMR spectra of unfractionated cell walls consistent with biochemical estimates, no effect on total biomass production and an increase in biomass saccharification efficiency of 40-60%. We provide the first strong evidence for a key role of the BAHD01 gene in AX feruloylation and demonstrate that it is a promising target for improvement of grass crops for biofuel, biorefining and animal nutrition applications.

Molecular basis of substrate recognition and specificity revealed in family 12 glycoside hydrolases.

Fungal GH12 enzymes are classified as xyloglucanases when they specifically target xyloglucans, or promiscuous endoglucanases when they exhibit catalytic activity against xyloglucan and ß-glucan chains. Several structural and functional studies involving GH12 enzymes tried to explain the main patterns of xyloglucan activity, but what really determines xyloglucanase specificity remains elusive. Here, three fungal GH12 enzymes from Aspergillus clavatus (AclaXegA), A. zonatus (AspzoGH12), and A. terreus (AtEglD) were studied to unveil the molecular basis for substrate specificity. Using functional assays, site-directed mutagenesis, and molecular dynamics simulations, we demonstrated that three main regions are responsible for substrate selectivity: (i) the YSG group in loop 1; (ii) the SST group in loop 2; and (iii) loop A3-B3 and neighboring residues. Functional assays and sequence alignment showed that while AclaXegA is specific to xyloglucan, AtEglD cleaves ß-glucan, and xyloglucan. However, AspzoGH12 was also shown to be promiscuous contrarily to a sequence alignment-based prediction. We find that residues Y111 and R93 in AtEglD harbor the substrate in an adequate orientation for hydrolysis in the catalytic cleft entrance and that residues Y19 in AclaXegA and Y30 in AspzoGH12 partially compensate the absence of the YSG segment, typically found in promiscuous enzymes. The results point out the multiple structural factors underlying the substrate specificity of GH12 enzymes. Biotechnol. Bioeng. 2016;113: 2577-2586. © 2016 Wiley Periodicals, Inc.

Mechanistic strategies for catalysis adopted by evolutionary distinct family 43 arabinanases.

Arabinanases (ABNs, EC 3.2.1.99) are promising catalysts for environmentally friendly biomass conversion into energy and chemicals. These enzymes catalyze the hydrolysis of the α-1,5-linked L-arabinofuranoside backbone of plant cell wall arabinans releasing arabino-oligosaccharides and arabinose, the second most abundant pentose in nature. In this work, new findings about the molecular mechanisms governing activation, functional differentiation, and catalysis of GH43 ABNs are presented. Biophysical, mutational, and biochemical studies with the hyperthermostable two-domain endo-acting ABN from Thermotoga petrophila (TpABN) revealed how some GH43 ABNs are activated by calcium ions via hyperpolarization of the catalytically relevant histidine and the importance of the ancillary domain for catalysis and conformational stability. On the other hand, the two GH43 ABNs from rumen metagenome, ARN2 and ARN3, presented a calcium-independent mechanism in which sodium is the most likely substituent for calcium ions. The crystal structure of the two-domain endo-acting ARN2 showed that its ability to efficiently degrade branched substrates is due to a larger catalytic interface with higher accessibility than that observed in other ABNs with preference for linear arabinan. Moreover, crystallographic characterization of the single-domain exo-acting ARN3 indicated that its cleavage pattern producing arabinose is associated with the chemical recognition of the reducing end of the substrate imposed by steric impediments at the aglycone-binding site. By structure-guided rational design, ARN3 was converted into a classical endo enzyme, confirming the role of the extended Arg(203)-Ala(230) loop in determining its action mode. These results reveal novel molecular aspects concerning the functioning of GH43 ABNs and provide new strategies for arabinan degradation.

Oligomeric state and structural stability of two hyperthermophilic ß-glucosidases from Thermotoga petrophila.

The ß-glucosidases are enzymes essential for several industrial applications, especially in the field of plant structural polysaccharides conversion into bioenergy and bioproducts. In a recent study, we have provided a biochemical characterization of two hyperthermostable ß-glucosidases from Thermotoga petrophila belonging to the families GH1 (TpBGL1) and GH3 (TpBGL3). Here, as part of a continuing investigation, the oligomeric state, the net charge, and the structural stability, at acidic pH, of the TpBGL1 and TpBGL3 were characterized and compared. Enzymatic activity is directly related to the balance between protonation and conformational changes. Interestingly, our results indicated that there were no significant changes in the secondary, tertiary and quaternary structures of the ß-glucosidases at temperatures below 80 °C. Furthermore, the results indicated that both the enzymes are stable homodimers in solution. Therefore, the observed changes in the enzymatic activities are due to variations in pH that modify protonation of the enzymes residues and the net charge, directly affecting the interactions with ligands. Finally, the results showed that the two ß-glucosidases displayed different pH dependence of thermostability at temperatures above 80 °C. TpBGL1 showed higher stability at pH 6 than at pH 4, while TpBGL3 showed similar stability at both pH values. This study provides a useful comparison of the structural stability, at acidic pH, of two different hyperthermostable ß-glucosidases and how it correlates with the activity of the enzymes. The information described here can be useful for biotechnological applications in the biofuel and food industries.

Recombinant Trichoderma harzianum endoglucanase I (Cel7B) is a highly acidic and promiscuous carbohydrate-active enzyme.

Trichoderma filamentous fungi have been investigated due to their ability to secrete cellulases which find various biotechnological applications such as biomass hydrolysis and cellulosic ethanol production. Previous studies demonstrated that Trichoderma harzianum IOC-3844 has a high degree of cellulolytic activity and potential for biomass hydrolysis. However, enzymatic, biochemical, and structural studies of cellulases from T. harzianum are scarce. This work reports biochemical characterization of the recombinant endoglucanase I from T. harzianum, ThCel7B, and its catalytic core domain. The constructs display optimum activity at 55 °C and a surprisingly acidic pH optimum of 3.0. The full-length enzyme is able to hydrolyze a variety of substrates, with high specific activity: 75 U/mg for ß-glucan, 46 U/mg toward xyloglucan, 39 U/mg for lichenan, 26 U/mg for carboxymethyl cellulose, 18 U/mg for 4-nitrophenyl ß-D-cellobioside, 16 U/mg for rye arabinoxylan, and 12 U/mg toward xylan. The enzyme also hydrolyzed filter paper, phosphoric acid swollen cellulose, Sigmacell 20, Avicel PH-101, and cellulose, albeit with lower efficiency. The ThCel7B catalytic domain displays similar substrate diversity. Fluorescence-based thermal shift assays showed that thermal stability is highest at pH 5.0. We determined kinetic parameters and analyzed a pattern of oligosaccharide substrates hydrolysis, revealing cellobiose as a final product of C6 degradation. Finally, we visualized effects of ThCel7B on oat spelt using scanning electron microscopy, demonstrating the morphological changes of the substrate during the hydrolysis. The acidic behavior of ThCel7B and its considerable thermostability hold a promise of its industrial applications and other biotechnological uses under extremely acidic conditions.

Aspergillus niger ß-glucosidase has a cellulase-like tadpole molecular shape: insights into glycoside hydrolase family 3 (GH3) ß-glucosidase structure and function.

Aspergillus niger is known to secrete large amounts of ß-glucosidases, which have a variety of biotechnological and industrial applications. Here, we purified an A. niger ß-glucosidase (AnBgl1) and conducted its biochemical and biophysical analyses. Purified enzyme with an apparent molecular mass of 116 kDa forms monomers in solution as judged by native gel electrophoresis and has a pI value of 4.55, as found for most of the fungi of ß-glucosidases. Surprisingly, the small angle x-ray experiments reveal that AnBgl1 has a tadpole-like structure, with the N-terminal catalytic domain and C-terminal fibronectin III-like domain (FnIII) connected by the long linker peptide (∼100 amino acid residues) in an extended conformation. This molecular organization resembles the one adopted by other cellulases (such as cellobiohydrolases, for example) that frequently contain a catalytic domain linked to the cellulose-binding module that mediates their binding to insoluble and polymeric cellulose. The reasons why AnBgl1, which acts on the small soluble substrates, has a tadpole molecular shape are not entirely clear. However, our enzyme pulldown assays with different polymeric substrates suggest that AnBgl1 has little or no capacity to bind to and to adsorb cellulose, xylan, and starch, but it has high affinity to lignin. Molecular dynamics simulations suggested that clusters of residues located in the C-terminal FnIII domain interact strongly with lignin fragments. The simulations showed that numerous arginine residues scattered throughout the FnIII surface play an important role in the interaction with lignin by means of cation-π stacking with the lignin aromatic rings. These results indicate that the C-terminal FnIII domain could be operational for immobilization of the enzyme on the cell wall and for the prevention of unproductive binding of cellulase to the biomass lignin.

Assembling a xylanase-lichenase chimera through all-atom molecular dynamics simulations.

Multifunctional enzyme engineering can improve enzyme cocktails for emerging biofuel technology. Molecular dynamics through structure-based models (SB) is an effective tool for assessing the tridimensional arrangement of chimeric enzymes as well as for inferring the functional practicability before experimental validation. This study describes the computational design of a bifunctional xylanase-lichenase chimera (XylLich) using the xynA and bglS genes from Bacillus subtilis. In silico analysis of the average solvent accessible surface area (SAS) and the root mean square fluctuation (RMSF) predicted a fully functional chimera, with minor fluctuations and variations along the polypeptide chains. Afterwards, the chimeric enzyme was built by fusing the xynA and bglS genes. XylLich was evaluated through small-angle X-ray scattering (SAXS) experiments, resulting in scattering curves with a very accurate fit to the theoretical protein model. The chimera preserved the biochemical characteristics of the parental enzymes, with the exception of a slight variation in the temperature of operation and the catalytic efficiency (kcat/Km). The absence of substantial shifts in the catalytic mode of operation was also verified. Furthermore, the production of chimeric enzymes could be more profitable than producing a single enzyme separately, based on comparing the recombinant protein production yield and the hydrolytic activity achieved for XylLich with that of the parental enzymes.

Flavonoid supplementation affects the expression of genes involved in cell wall formation and lignification metabolism and increases sugar content and saccharification in the fast-growing eucalyptus hybrid E. urophylla x E. grandis.

BACKGROUND: Eucalyptus species are the most widely planted hardwood species in the world and are renowned for their rapid growth and adaptability. In Brazil, one of the most widely grown Eucalyptus cultivars is the fast-growing Eucalyptus urophylla x Eucalyptus grandis hybrid. In a previous study, we described a chemical characterization of these hybrids when subjected to flavonoid supplementation on 2 distinct timetables, and our results revealed marked differences between the wood composition of the treated and untreated trees. RESULTS: In this work, we report the transcriptional responses occurring in these trees that may be related to the observed chemical differences. Gene expression was analysed through mRNA-sequencing, and notably, compared to control trees, the treated trees display differential down-regulation of cell wall formation pathways such as phenylpropanoid metabolism as well as differential expression of genes involved in sucrose, starch and minor CHO metabolism and genes that play a role in several stress and environmental responses. We also performed enzymatic hydrolysis of wood samples from the different treatments, and the results indicated higher sugar contents and glucose yields in the flavonoid-treated plants. CONCLUSIONS: Our results further illustrate the potential use of flavonoids as a nutritional complement for modifying Eucalyptus wood, since, supplementation with flavonoids alters its chemical composition, gene expression and increases saccharification probably as part of a stress response.

Understanding the function of conserved variations in the catalytic loops of fungal glycoside hydrolase family 12.

Enzymes that cleave the xyloglucan backbone at unbranched glucose residues have been identified in GH families 5, 7, 12, 16, 44, and 74. Fungi produce enzymes that populate 20 of 22 families that are considered critical for plant biomass deconstruction. We searched for GH12-encoding genes in 27 Eurotiomycetes genomes. After analyzing 50 GH12-related sequences, the conserved variations of the amino acid sequences were examined. Compared to the endoglucanases, the endo-xyloglucanase-associated YSG deletion at the negative subsites of the catalytic cleft with a SST insertion at the reducing end of the substrate-binding crevice is highly conserved. In addition, a highly conserved alanine residue was identified in all xyloglucan-specific enzymes, and this residue is substituted by arginine in more promiscuous glucanases. To understand the basis for the xyloglucan specificity displayed by certain GH12 enzymes, two fungal GH12 endoglucanases were chosen for mutagenesis and functional studies: an endo-xyloglucanase from Aspergillus clavatus (AclaXegA) and an endoglucanase from A. terreus (AtEglD). Comprehensive molecular docking studies and biochemical analyses were performed, revealing that mutations at the entrance of the catalytic cleft in AtEglD result in a wider binding cleft and the alteration of the substrate-cleavage pattern, implying that a trio of residues coordinates the interactions and binding to linear glycans. The loop insertion at the crevice-reducing end of AclaXegA is critical for catalytic efficiency to hydrolyze xyloglucan. The understanding of the structural elements governing endo-xyloglucanase activity on linear and branched glucans will facilitate future enzyme modifications with potential applications in industrial biotechnology.

Development of hemicellulolytic enzyme mixtures for plant biomass deconstruction on target biotechnological applications.

An essential step in the conversion of lignocellulosic biomass to ethanol and other biorefinery products is conversion of cell wall polysaccharides into fermentable sugars by enzymatic hydrolysis. The objective of the present study was to understand the mode of action of hemicellulolytic enzyme mixtures for pretreated sugarcane bagasse (PSB) deconstruction and wheat arabinoxylan (WA) hydrolysis on target biotechnological applications. In this study, five hemicellulolytic enzymes-two endo-1,4-xylanases (GH10 and GH11), two α-L-arabinofuranosidases (GH51 and GH54), and one ß-xylosidase (GH43)-were submitted to combinatorial assays using the experimental design strategy, in order to analyze synergistic and antagonistic effects of enzyme interactions on biomass degradation. The xylooligosaccharides (XOSs) released from hydrolysis were analyzed by capillary electrophoresis and quantified by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Based on this analysis, it was possible to define which enzymatic combinations favor xylose (X1) or XOS production and thus enable the development of target biotechnological applications. Our results demonstrate that if the objective is X1 production from WA, the best enzymatic combination is GH11 + GH54 + GH43, and for xylobiose (X2) production from WA, it is best to combine GH11 + GH51. However, if the goal is to produce XOS, the five enzymes used in WA hydrolysis are important, but for PSB hydrolysis, only GH11 is sufficient. If the final objective is bioethanol production, GH11 is responsible for hydrolyzing 64.3 % of hemicellulose from PSB. This work provides a basis for further studies on enzymatic mechanisms for XOS production, and the development of more efficient and less expensive enzymatic mixtures, targeting commercially viable lignocellulosic ethanol production and other biorefinery products.

Identification of novel interaction between ADAM17 (a disintegrin and metalloprotease 17) and thioredoxin-1.

ADAM17, which is also known as TNFα-converting enzyme, is the major sheddase for the EGF receptor ligands and is considered to be one of the main proteases responsible for the ectodomain shedding of surface proteins. How a membrane-anchored proteinase with an extracellular catalytic domain can be activated by inside-out regulation is not completely understood. We characterized thioredoxin-1 (Trx-1) as a partner of the ADAM17 cytoplasmic domain that could be involved in the regulation of ADAM17 activity. We induced the overexpression of the ADAM17 cytoplasmic domain in HEK293 cells, and ligands able to bind this domain were identified by MS after protein immunoprecipitation. Trx-1 was also validated as a ligand of the ADAM17 cytoplasmic domain and full-length ADAM17 recombinant proteins by immunoblotting, immunolocalization, and solid phase binding assay. In addition, using nuclear magnetic resonance, it was shown in vitro that the titration of the ADAM17 cytoplasmic domain promotes changes in the conformation of Trx-1. The MS analysis of the cross-linked complexes showed cross-linking between the two proteins by lysine residues. To further evaluate the functional role of Trx-1, we used a heparin-binding EGF shedding cell model and observed that the overexpression of Trx-1 in HEK293 cells could decrease the activity of ADAM17, activated by either phorbol 12-myristate 13-acetate or EGF. This study identifies Trx-1 as a novel interaction partner of the ADAM17 cytoplasmic domain and suggests that Trx-1 is a potential candidate that could be involved in ADAM17 activity regulation.

Biomass-to-bio-products application of feruloyl esterase from Aspergillus clavatus.

The structural polysaccharides contained in plant cell walls have been pointed to as a promising renewable alternative to petroleum and natural gas. Ferulic acid is a ubiquitous component of plant polysaccharides, which is found in either monomeric or dimeric forms and is covalently linked to arabinosyl residues. Ferulic acid has several commercial applications in food and pharmaceutical industries. The study herein introduces a novel feruloyl esterase from Aspergillus clavatus (AcFAE). Along with a comprehensive functional and biophysical characterization, the low-resolution structure of this enzyme was also determined by small-angle X-ray scattering. In addition, we described the production of phenolic compounds with antioxidant capacity from wheat arabinoxylan and sugarcane bagasse using AcFAE. The ability to specifically cleave ester linkages in hemicellulose is useful in several biotechnological applications, including improved accessibility to lignocellulosic enzymes for biofuel production.

Dissecting structure-function-stability relationships of a thermostable GH5-CBM3 cellulase from Bacillus subtilis 168.

Cellulases participate in a number of biological events, such as plant cell wall remodelling, nematode parasitism and microbial carbon uptake. Their ability to depolymerize crystalline cellulose is of great biotechnological interest for environmentally compatible production of fuels from lignocellulosic biomass. However, industrial use of cellulases is somewhat limited by both their low catalytic efficiency and stability. In the present study, we conducted a detailed functional and structural characterization of the thermostable BsCel5A (Bacillus subtilis cellulase 5A), which consists of a GH5 (glycoside hydrolase 5) catalytic domain fused to a CBM3 (family 3 carbohydrate-binding module). NMR structural analysis revealed that the Bacillus CBM3 represents a new subfamily, which lacks the classical calcium-binding motif, and variations in NMR frequencies in the presence of cellopentaose showed the importance of polar residues in the carbohydrate interaction. Together with the catalytic domain, the CBM3 forms a large planar surface for cellulose recognition, which conducts the substrate in a proper conformation to the active site and increases enzymatic efficiency. Notably, the manganese ion was demonstrated to have a hyper-stabilizing effect on BsCel5A, and by using deletion constructs and X-ray crystallography we determined that this effect maps to a negatively charged motif located at the opposite face of the catalytic site.