Olive Yield and Physicochemical Properties of Olives and Oil in Response to Nutrient Application under Rainfed Conditions.

The effects of mineral fertilizers on the physicochemical properties of olives and oil under rainfed conditions is scarce. In this three-year study, the results of a nitrogen (N), phosphorus (P), potassium (K) and boron (B) fertilization trial carried out in a young rainfed olive grove and arranged as a nutrient omission trial are reported. The control consisted of the application of N, P, K and B (NPKB) and four other treatments corresponded to the removal of one of them (N0, P0, K0 and B0). Olive yield and several variables associated with the physicochemical properties of olives and oil were evaluated. The NPKB treatment increased olive yield compared to the treatment that did not receive N (N0). Although dependent on the climate conditions of the crop season, the NPKB treatment increased fruit weight and the pulp/pit ratio and its fruits tended to accumulate more oil than K0. However, the phenolics concentrations on fruits and oil tended to be lower. All olive oil samples were classified in the "extra virgin" category and all showed a decrease in its stability between 3 and 15 months of storage, regardless of treatment, especially in N0, P0 and B0 treatments. The results of the sensorial analysis indicate that all the oils fell into the medium fruitiness and greenly-fruity category. Only in P0 and B0 were defects detected, namely muddy sediment. Thus, this study seems to indicate the importance of N application, but also a balanced nutrient application and that further studies are needed, given the difficulty in finding clear trends in the response of measured variables to fertilizer treatments.

A dual cohesin-dockerin complex binding mode in Bacteroides cellulosolvens contributes to the size and complexity of its cellulosome.

The Cellulosome is an intricate macromolecular protein complex that centralizes the cellulolytic efforts of many anaerobic microorganisms through the promotion of enzyme synergy and protein stability. The assembly of numerous carbohydrate processing enzymes into a macromolecular multiprotein structure results from the interaction of enzyme-borne dockerin modules with repeated cohesin modules present in noncatalytic scaffold proteins, termed scaffoldins. Cohesin-dockerin (Coh-Doc) modules are typically classified into different types, depending on structural conformation and cellulosome role. Thus, type I Coh-Doc complexes are usually responsible for enzyme integration into the cellulosome, while type II Coh-Doc complexes tether the cellulosome to the bacterial wall. In contrast to other known cellulosomes, cohesin types from Bacteroides cellulosolvens, a cellulosome-producing bacterium capable of utilizing cellulose and cellobiose as carbon sources, are reversed for all scaffoldins, i.e., the type II cohesins are located on the enzyme-integrating primary scaffoldin, whereas the type I cohesins are located on the anchoring scaffoldins. It has been previously shown that type I B. cellulosolvens interactions possess a dual-binding mode that adds flexibility to scaffoldin assembly. Herein, we report the structural mechanism of enzyme recruitment into B. cellulosolvens cellulosome and the identification of the molecular determinants of its type II cohesin-dockerin interactions. The results indicate that, unlike other type II complexes, these possess a dual-binding mode of interaction, akin to type I complexes. Therefore, the plasticity of dual-binding mode interactions seems to play a pivotal role in the assembly of B. cellulosolvens cellulosome, which is consistent with its unmatched complexity and size.

Fenestrated endovascular aortic aneurysm repair promotes positive infrarenal neck remodeling and greater sac shrinkage compared with endovascular aortic aneurysm repair.

OBJECTIVE: Endovascular aortic aneurysm repair (EVAR) has become the standard of care for abdominal aortic aneurysms (AAAs) in the modern era. Although numerous devices exist for standard infrarenal AAA repair, fenestrated EVAR (fEVAR) offers a minimally invasive alternative to traditional open repair for patients with a short infrarenal neck. Over time, aortic neck dilation can occur, leading to loss of the proximal seal, endoleaks, and AAA sac growth. In the present study, we analyzed aortic remodeling after EVAR vs fEVAR and further evaluated whether fEVAR confers a benefit in terms of sac shrinkage. METHODS: A retrospective review of prospectively collected data from 120 patients who had undergone EVAR was performed. Of these 120 patients, 30 had been treated with fEVAR (Zenith fenestrated; Cook Medical Inc, Bloomington, IN) and 90 patients were treated with EVAR devices (30 each with Endurant [Medtronic, Dublin, Ireland], Excluder [W.L. Gore & Associates, Flagstaff, AZ], and Zenith [Cook Medical Inc]). The demographic data were recorded. Also, anatomic measurements were performed for each patient preoperatively, at 30 days postoperatively, and at the longest follow-up point using three-dimensional reconstruction software. RESULTS: No significant differences were found in demographic data between the four groups. fEVAR had been used more often in aortas with large necks and irregular morphology (P = .004). At the longest follow-up, the suprarenal aorta encompassing 5, 10, and 15 mm above the lowest renal artery had dilated the most for the fEVAR group vs all EVAR groups. However, the infrarenal segment had tended to increase the least, or to even have regressed, for fEVAR compared with all three EVAR groups and was associated with the overall greatest proportion of sac shrinkage for the fEVAR group compared with the Medtronic, Gore, and Cook devices (-13.90% vs -5.75% vs -2.31% vs -4.68%, respectively; P = .025). CONCLUSIONS: Compared with EVAR, the patients treated with fEVAR had experienced greater suprarenal dilation over time, consistent with an overall greater burden of disease in the proximal native aorta. However, the infrarenal segment had dilated significantly less over time in the fEVAR group compared with all three EVAR groups, suggesting that fEVAR might stabilize the infrarenal neck, promoting positive sac remodeling, which was evidenced by the greatest degree of decrease in the largest AAA diameter in the fEVAR group.

Generation of a Library of Carbohydrate-Active Enzymes for Plant Biomass Deconstruction.

In nature, the deconstruction of plant carbohydrates is carried out by carbohydrate-active enzymes (CAZymes). A high-throughput (HTP) strategy was used to isolate and clone 1476 genes obtained from a diverse library of recombinant CAZymes covering a variety of sequence-based families, enzyme classes, and source organisms. All genes were successfully isolated by either PCR (61%) or gene synthesis (GS) (39%) and were subsequently cloned into Escherichia coli expression vectors. Most proteins (79%) were obtained at a good yield during recombinant expression. A significantly lower number (p < 0.01) of proteins from eukaryotic (57.7%) and archaeal (53.3%) origin were soluble compared to bacteria (79.7%). Genes obtained by GS gave a significantly lower number (p = 0.04) of soluble proteins while the green fluorescent protein tag improved protein solubility (p = 0.05). Finally, a relationship between the amino acid composition and protein solubility was observed. Thus, a lower percentage of non-polar and higher percentage of negatively charged amino acids in a protein may be a good predictor for higher protein solubility in E. coli. The HTP approach presented here is a powerful tool for producing recombinant CAZymes that can be used for future studies of plant cell wall degradation. Successful production and expression of soluble recombinant proteins at a high rate opens new possibilities for the high-throughput production of targets from limitless sources.

Structure-function analyses generate novel specificities to assemble the components of multienzyme bacterial cellulosome complexes.

The cellulosome is a remarkably intricate multienzyme nanomachine produced by anaerobic bacteria to degrade plant cell wall polysaccharides. Cellulosome assembly is mediated through binding of enzyme-borne dockerin modules to cohesin modules of the primary scaffoldin subunit. The anaerobic bacterium Acetivibrio cellulolyticus produces a highly intricate cellulosome comprising an adaptor scaffoldin, ScaB, whose cohesins interact with the dockerin of the primary scaffoldin (ScaA) that integrates the cellulosomal enzymes. The ScaB dockerin selectively binds to cohesin modules in ScaC that anchors the cellulosome onto the cell surface. Correct cellulosome assembly requires distinct specificities displayed by structurally related type-I cohesin-dockerin pairs that mediate ScaC-ScaB and ScaA-enzyme assemblies. To explore the mechanism by which these two critical protein interactions display their required specificities, we determined the crystal structure of the dockerin of a cellulosomal enzyme in complex with a ScaA cohesin. The data revealed that the enzyme-borne dockerin binds to the ScaA cohesin in two orientations, indicating two identical cohesin-binding sites. Combined mutagenesis experiments served to identify amino acid residues that modulate type-I cohesin-dockerin specificity in A. cellulolyticus Rational design was used to test the hypothesis that the ligand-binding surfaces of ScaA- and ScaB-associated dockerins mediate cohesin recognition, independent of the structural scaffold. Novel specificities could thus be engineered into one, but not both, of the ligand-binding sites of ScaB, whereas attempts at manipulating the specificity of the enzyme-associated dockerin were unsuccessful. These data indicate that dockerin specificity requires critical interplay between the ligand-binding surface and the structural scaffold of these modules.

Complexity of the Ruminococcus flavefaciens cellulosome reflects an expansion in glycan recognition.

The breakdown of plant cell wall (PCW) glycans is an important biological and industrial process. Noncatalytic carbohydrate binding modules (CBMs) fulfill a critical targeting function in PCW depolymerization. Defining the portfolio of CBMs, the CBMome, of a PCW degrading system is central to understanding the mechanisms by which microbes depolymerize their target substrates. Ruminococcus flavefaciens, a major PCW degrading bacterium, assembles its catalytic apparatus into a large multienzyme complex, the cellulosome. Significantly, bioinformatic analyses of the R. flavefaciens cellulosome failed to identify a CBM predicted to bind to crystalline cellulose, a key feature of the CBMome of other PCW degrading systems. Here, high throughput screening of 177 protein modules of unknown function was used to determine the complete CBMome of R. flavefaciens The data identified six previously unidentified CBM families that targeted ß-glucans, ß-mannans, and the pectic polysaccharide homogalacturonan. The crystal structures of four CBMs, in conjunction with site-directed mutagenesis, provide insight into the mechanism of ligand recognition. In the CBMs that recognize ß-glucans and ß-mannans, differences in the conformation of conserved aromatic residues had a significant impact on the topology of the ligand binding cleft and thus ligand specificity. A cluster of basic residues in CBM77 confers calcium-independent recognition of homogalacturonan, indicating that the carboxylates of galacturonic acid are key specificity determinants. This report shows that the extended repertoire of proteins in the cellulosome of R. flavefaciens contributes to an extended CBMome that supports efficient PCW degradation in the absence of CBMs that specifically target crystalline cellulose.

Stability and Ligand Promiscuity of Type A Carbohydrate-binding Modules Are Illustrated by the Structure of Spirochaeta thermophila StCBM64C.

Deconstruction of cellulose, the most abundant plant cell wall polysaccharide, requires the cooperative activity of a large repertoire of microbial enzymes. Modular cellulases contain non-catalytic type A carbohydrate-binding modules (CBMs) that specifically bind to the crystalline regions of cellulose, thus promoting enzyme efficacy through proximity and targeting effects. Although type A CBMs play a critical role in cellulose recycling, their mechanism of action remains poorly understood. Here we produced a library of recombinant CBMs representative of the known diversity of type A modules. The binding properties of 40 CBMs, in fusion with an N-terminal GFP domain, revealed that type A CBMs possess the ability to recognize different crystalline forms of cellulose and chitin over a wide range of temperatures, pH levels, and ionic strengths. A Spirochaeta thermophila CBM64, in particular, displayed plasticity in its capacity to bind both crystalline and soluble carbohydrates under a wide range of extreme conditions. The structure of S. thermophila StCBM64C revealed an untwisted, flat, carbohydrate-binding interface comprising the side chains of four tryptophan residues in a co-planar linear arrangement. Significantly, two highly conserved asparagine side chains, each one located between two tryptophan residues, are critical to insoluble and soluble glucan recognition but not to bind xyloglucan. Thus, CBM64 compact structure and its extended and versatile ligand interacting platform illustrate how type A CBMs target their appended plant cell wall-degrading enzymes to a diversity of recalcitrant carbohydrates under a wide range of environmental conditions.

Single Binding Mode Integration of Hemicellulose-degrading Enzymes via Adaptor Scaffoldins in Ruminococcus flavefaciens Cellulosome.

The assembly of one of Nature's most elaborate multienzyme complexes, the cellulosome, results from the binding of enzyme-borne dockerins to reiterated cohesin domains located in a non-catalytic primary scaffoldin. Generally, dockerins present two similar cohesin-binding interfaces that support a dual binding mode. The dynamic integration of enzymes in cellulosomes, afforded by the dual binding mode, is believed to incorporate additional flexibility in highly populated multienzyme complexes. Ruminococcus flavefaciens, the primary degrader of plant structural carbohydrates in the rumen of mammals, uses a portfolio of more than 220 different dockerins to assemble the most intricate cellulosome known to date. A sequence-based analysis organized R. flavefaciens dockerins into six groups. Strikingly, a subset of R. flavefaciens cellulosomal enzymes, comprising dockerins of groups 3 and 6, were shown to be indirectly incorporated into primary scaffoldins via an adaptor scaffoldin termed ScaC. Here, we report the crystal structure of a group 3 R. flavefaciens dockerin, Doc3, in complex with ScaC cohesin. Doc3 is unusual as it presents a large cohesin-interacting surface that lacks the structural symmetry required to support a dual binding mode. In addition, dockerins of groups 3 and 6, which bind exclusively to ScaC cohesin, display a conserved mechanism of protein recognition that is similar to Doc3. Groups 3 and 6 dockerins are predominantly appended to hemicellulose-degrading enzymes. Thus, single binding mode dockerins interacting with adaptor scaffoldins exemplify an evolutionary pathway developed by R. flavefaciens to recruit hemicellulases to the sophisticated cellulosomes acting in the gastrointestinal tract of mammals.

Gene design, fusion technology and TEV cleavage conditions influence the purification of oxidized disulphide-rich venom peptides in Escherichia coli.

BACKGROUND: Animal venoms are large, complex libraries of bioactive, disulphide-rich peptides. These peptides, and their novel biological activities, are of increasing pharmacological and therapeutic importance. However, recombinant expression of venom peptides in Escherichia coli remains difficult due to the significant number of cysteine residues requiring effective post-translational processing. There is also an urgent need to develop high-throughput recombinant protocols applicable to the production of reticulated peptides to enable efficient screening of their drug potential. Here, a comprehensive study was developed to investigate how synthetic gene design, choice of fusion tag, compartment of expression, tag removal conditions and protease recognition site affect levels of solubility of oxidized venom peptides produced in E. coli. RESULTS: The data revealed that expression of venom peptides imposes significant pressure on cysteine codon selection. DsbC was the best fusion tag for venom peptide expression, in particular when the fusion was directed to the bacterial periplasm. While the redox activity of DsbC was not essential to maximize expression of recombinant fusion proteins, redox activity did lead to higher levels of correctly folded target peptides. With the exception of proline, the canonical TEV protease recognition site tolerated all other residues at its C-terminus, confirming that no non-native residues, which might affect activity, need to be incorporated at the N-terminus of recombinant peptides for tag removal. CONCLUSIONS: This study reveals that E. coli is a convenient heterologous host for the expression of soluble and functional venom peptides. Using the optimal construct design, a large and diverse range of animal venom peptides were produced in the µM scale. These results open up new possibilities for the high-throughput production of recombinant disulphide-rich peptides in E. coli.

Family 46 Carbohydrate-binding Modules Contribute to the Enzymatic Hydrolysis of Xyloglucan and ß-1,3-1,4-Glucans through Distinct Mechanisms.

Structural carbohydrates comprise an extraordinary source of energy that remains poorly utilized by the biofuel sector as enzymes have restricted access to their substrates within the intricacy of plant cell walls. Carbohydrate active enzymes (CAZYmes) that target recalcitrant polysaccharides are modular enzymes containing noncatalytic carbohydrate-binding modules (CBMs) that direct enzymes to their cognate substrate, thus potentiating catalysis. In general, CBMs are functionally and structurally autonomous from their associated catalytic domains from which they are separated through flexible linker sequences. Here, we show that a C-terminal CBM46 derived from BhCel5B, a Bacillus halodurans endoglucanase, does not interact with ß-glucans independently but, uniquely, acts cooperatively with the catalytic domain of the enzyme in substrate recognition. The structure of BhCBM46 revealed a ß-sandwich fold that abuts onto the region of the substrate binding cleft upstream of the active site. BhCBM46 as a discrete entity is unable to bind to ß-glucans. Removal of BhCBM46 from BhCel5B, however, abrogates binding to ß-1,3-1,4-glucans while substantially decreasing the affinity for decorated ß-1,4-glucan homopolymers such as xyloglucan. The CBM46 was shown to contribute to xyloglucan hydrolysis only in the context of intact plant cell walls, but it potentiates enzymatic activity against purified ß-1,3-1,4-glucans in solution or within the cell wall. This report reveals the mechanism by which a CBM can promote enzyme activity through direct interaction with the substrate or by targeting regions of the plant cell wall where the target glucan is abundant.

Cell-surface Attachment of Bacterial Multienzyme Complexes Involves Highly Dynamic Protein-Protein Anchors.

Protein-protein interactions play a pivotal role in the assembly of the cellulosome, one of nature's most intricate nanomachines dedicated to the depolymerization of complex carbohydrates. The integration of cellulosomal components usually occurs through the binding of type I dockerin modules located at the C terminus of the enzymes to cohesin modules located in the primary scaffoldin subunit. Cellulosomes are typically recruited to the cell surface via type II cohesin-dockerin interactions established between primary and cell-surface anchoring scaffoldin subunits. In contrast with type II interactions, type I dockerins usually display a dual binding mode that may allow increased conformational flexibility during cellulosome assembly. Acetivibrio cellulolyticus produces a highly complex cellulosome comprising an unusual adaptor scaffoldin, ScaB, which mediates the interaction between the primary scaffoldin, ScaA, through type II cohesin-dockerin interactions and the anchoring scaffoldin, ScaC, via type I cohesin-dockerin interactions. Here, we report the crystal structure of the type I ScaB dockerin in complex with a type I ScaC cohesin in two distinct orientations. The data show that the ScaB dockerin displays structural symmetry, reflected by the presence of two essentially identical binding surfaces. The complex interface is more extensive than those observed in other type I complexes, which results in an ultra-high affinity interaction (Ka ∼10(12) M). A subset of ScaB dockerin residues was also identified as modulating the specificity of type I cohesin-dockerin interactions in A. cellulolyticus. This report reveals that recruitment of cellulosomes onto the cell surface may involve dockerins presenting a dual binding mode to incorporate additional flexibility into the quaternary structure of highly populated multienzyme complexes.

Combined Crystal Structure of a Type I Cohesin: MUTATION AND AFFINITY BINDING STUDIES REVEAL STRUCTURAL DETERMINANTS OF COHESIN-DOCKERIN SPECIFICITIES.

Cohesin-dockerin interactions orchestrate the assembly of one of nature's most elaborate multienzyme complexes, the cellulosome. Cellulosomes are produced exclusively by anaerobic microbes and mediate highly efficient hydrolysis of plant structural polysaccharides, such as cellulose and hemicellulose. In the canonical model of cellulosome assembly, type I dockerin modules of the enzymes bind to reiterated type I cohesin modules of a primary scaffoldin. Each type I dockerin contains two highly conserved cohesin-binding sites, which confer quaternary flexibility to the multienzyme complex. The scaffoldin also bears a type II dockerin that anchors the entire complex to the cell surface by binding type II cohesins of anchoring scaffoldins. In Bacteroides cellulosolvens, however, the organization of the cohesin-dockerin types is reversed, whereby type II cohesin-dockerin pairs integrate the enzymes into the primary scaffoldin, and type I modules mediate cellulosome attachment to an anchoring scaffoldin. Here, we report the crystal structure of a type I cohesin from B. cellulosolvens anchoring scaffoldin ScaB to 1.84-Å resolution. The structure resembles other type I cohesins, and the putative dockerin-binding site, centered at ß-strands 3, 5, and 6, is likely to be conserved in other B. cellulosolvens type I cohesins. Combined computational modeling, mutagenesis, and affinity-based binding studies revealed similar hydrogen-bonding networks between putative Ser/Asp recognition residues in the dockerin at positions 11/12 and 45/46, suggesting that a dual-binding mode is not exclusive to the integration of enzymes into primary cellulosomes but can also characterize polycellulosome assembly and cell-surface attachment. This general approach may provide valuable structural information of the cohesin-dockerin interface, in lieu of a definitive crystal structure.

Unveiling the structure of polytetraruthenated nickel porphyrin by Raman spectroelectrochemistry.

The structure of polytetraruthenated nickel porphyrin was unveiled for the first time by electrochemistry, Raman spectroelectrochemistry, and a hydroxyl radical trapping assay. The electrocatalytic active material, precipitated on the electrode surface after successive cycling of [NiTPyP{Ru(bipy)2Cl}4](4+) species in strong aqueous alkaline solution (pH 13), was found to be a peroxo-bridged coordination polymer. The electropolymerization process involves hydroxyl radicals (as confirmed by the characteristic set of DMPO/(•)OH adduct EPR peaks) as reaction intermediates, electrocatalytically generated in the 0.80-1.10 V range, that induce the formation of Ni-O-O-Ni coordination polymers, as evidenced by Raman spectroelectrochemistry and molecular modeling studies. The film growth is halted above 1.10 V due to the formation of oxygen gas bubbles.

Understanding how noncatalytic carbohydrate binding modules can display specificity for xyloglucan.

Plant biomass is central to the carbon cycle and to environmentally sustainable industries exemplified by the biofuel sector. Plant cell wall degrading enzymes generally contain noncatalytic carbohydrate binding modules (CBMs) that fulfil a targeting function, which enhances catalysis. CBMs that bind ß-glucan chains often display broad specificity recognizing ß1,4-glucans (cellulose), ß1,3-ß1,4-mixed linked glucans and xyloglucan, a ß1,4-glucan decorated with α1,6-xylose residues, by targeting structures common to the three polysaccharides. Thus, CBMs that recognize xyloglucan target the ß1,4-glucan backbone and only accommodate the xylose decorations. Here we show that two closely related CBMs, CBM65A and CBM65B, derived from EcCel5A, a Eubacterium cellulosolvens endoglucanase, bind to a range of ß-glucans but, uniquely, display significant preference for xyloglucan. The structures of the two CBMs reveal a ß-sandwich fold. The ligand binding site comprises the ß-sheet that forms the concave surface of the proteins. Binding to the backbone chains of ß-glucans is mediated primarily by five aromatic residues that also make hydrophobic interactions with the xylose side chains of xyloglucan, conferring the distinctive specificity of the CBMs for the decorated polysaccharide. Significantly, and in contrast to other CBMs that recognize ß-glucans, CBM65A utilizes different polar residues to bind cellulose and mixed linked glucans. Thus, Gln(106) is central to cellulose recognition, but is not required for binding to mixed linked glucans. This report reveals the mechanism by which ß-glucan-specific CBMs can distinguish between linear and mixed linked glucans, and show how these CBMs can exploit an extensive hydrophobic platform to target the side chains of decorated ß-glucans.

Outcome and quality of life after endovascular abdominal aortic aneurysm repair in octogenarians.

OBJECTIVE: This study determined outcome and quality of life (QOL) in octogenarians, compared with patients aged <80 years, 1 year after endovascular aortic aneurysm repair (EVAR). METHODS: From March 2009 until April 2011, 1263 patients in the Endurant Stent Graft Natural Selection Global Postmarket Registry (ENGAGE) registry with an abdominal aortic aneurysm were treated with EVAR using the Endurant endograft (Medtronic Cardiovascular, Santa Rosa, Calif). The patients were grouped according to those aged ≥80 years (290 [22.9%]) and those aged <80 years (973 [77.1%]) at the time of the procedure. QOL was assessed using composite EuroQoL 5-Dimensions Questionnaire (EQ-5D) index scores. Baseline, perioperative, and follow-up data were analyzed at 1 year. RESULTS: Octogenarians had poorer anatomic characteristics. The technical success rate was almost 99% for both cohorts, with no deaths. The duration of the implant procedure was significantly longer in the elderly patients (P = .002), with significant differences in overall (P < .001) and postprocedure (P < .001) hospital stays in favor of the younger group. At 1 year, there was a significant difference in all-cause mortality (P = .002) and in the number of major adverse events (P = .003), including secondary rupture (P = .01), to the detriment of octogenarians. There were no significant differences in conversion to open surgery or in overall secondary endovascular procedures. The octogenarians scored lower in their overall health care perception (P < .001) but with no significant difference in the EQ-5D index. Compared with the group aged <80 years, they had still not completely recovered their QOL after 1 year (P = .01). CONCLUSIONS: Octogenarians are more difficult to treat by EVAR than younger patients due to poorer anatomic suitability and a higher incidence of complications. Recovery of QOL in octogenarians takes longer (>12 months) than expected.

Novel Clostridium thermocellum type I cohesin-dockerin complexes reveal a single binding mode.

Protein-protein interactions play a pivotal role in a large number of biological processes exemplified by the assembly of the cellulosome. Integration of cellulosomal components occurs through the binding of type I cohesin modules located in a non-catalytic molecular scaffold to type I dockerin modules located at the C terminus of cellulosomal enzymes. The majority of type I dockerins display internal symmetry reflected by the presence of two essentially identical cohesin-binding surfaces. Here we report the crystal structures of two novel Clostridium thermocellum type I cohesin-dockerin complexes (CohOlpC-Doc124A and CohOlpA-Doc918). The data revealed that the two dockerins, Doc918 and Doc124A, are unusual because they lack the structural symmetry required to support a dual binding mode. Thus, in both cases, cohesin recognition is dominated by residues located at positions 11, 12, and 19 of one of the dockerin binding surfaces. The alternative binding mode is not possible (Doc918) or highly limited (Doc124A) because residues that assume the critical interacting positions, when dockerins are reoriented by 180°, make steric clashes with the cohesin. In common with a third dockerin (Doc258) that also presents a single binding mode, Doc124A directs the appended cellulase, Cel124A, to the surface of C. thermocellum and not to cellulosomes because it binds preferentially to type I cohesins located at the cell envelope. Although there are a few exceptions, such as Doc918 described here, these data suggest that there is considerable selective pressure for the evolution of a dual binding mode in type I dockerins that direct enzymes into cellulosomes.

Overproduction, purification, crystallization and preliminary X-ray characterization of the C-terminal family 65 carbohydrate-binding module (CBM65B) of endoglucanase Cel5A from Eubacterium cellulosolvens.

The rumen anaerobic cellulolytic bacterium Eubacterium cellulosolvens produces a large range of cellulases and hemicellulases responsible for the efficient hydrolysis of plant cell wall polysaccharides. One of these enzymes, endoglucanase Cel5A, comprises a tandemly repeated carbohydrate-binding module (CBM65) fused to a glycoside hydrolase family 5 (Cel5A) catalytic domain, joined by flexible linker sequences. The second carbohydrate-binding module located at the C-terminus side of the endoglucanase (CBM65B) has been co-crystallized with either cellohexaose or xyloglucan heptasaccharide. The crystals belong to the hexagonal space group P6(5) and tetragonal space group P4(3)2(1)2, containing a single molecule in the asymmetric unit. The structures of CBM65B have been solved by molecular replacement.

Overexpression, crystallization and preliminary X-ray crystallographic analysis of glucuronoxylan xylanohydrolase (Xyn30A) from Clostridium thermocellum.

The modular carbohydrate-active enzyme belonging to glycoside hydrolase family 30 (GH30) from Clostridium thermocellum (CtXynGH30) is a cellulosomal protein which plays an important role in plant cell-wall degradation. The full-length CtXynGH30 contains an N-terminal catalytic module (Xyn30A) followed by a family 6 carbohydrate-binding module (CBM6) and a dockerin at the C-terminus. The recombinant protein has a molecular mass of 45 kDa. Preliminary structural characterization was carried out on Xyn30A crystallized in different conditions. All tested crystals belonged to space group P1 with one molecule in the asymmetric unit. Molecular replacement has been used to solve the Xyn30A structure.

Numerical Simulations of the Low-Velocity Impact Response of Semicylindrical Woven Composite Shells.

This paper presents an efficient and reliable approach to study the low-velocity impact response of woven composite shells using 3D finite element models that account for the physical intralaminar and interlaminar progressive damage. The authors' previous work on the experimental assessment of the effect of thickness on the impact response of semicylindrical composite laminated shells served as the basis for this paper. Therefore, the finite element models were put to the test in comparison to the experimental findings. A good agreement was obtained between the numerical predictions and experimental data for the load and energy histories as well as for the maximum impact load, maximum displacement, and contact time. The use of the mass-scaling technique was successfully implemented, reducing considerably the computing cost of the solutions. The maximum load, maximum displacement, and contact time are negligibly affected by the choice of finite element mesh discretization. However, it has an impact on the initiation and progression of interlaminar damage. Therefore, to accurately compute delamination, its correct definition is of upmost importance. The validation of these finite element models opens the possibility for further numerical studies on of woven composite shells and enables shortening the time and expenses associated with the experimental testing.

Electron Beam Irradiation on the Production of a Si- and Zr-Based Hybrid Material: A Study by FTIR and WDXRF.

Sol-gel production of hybrid materials has, to some extent, revolutionised materials' engineering and the way science and technology perceive the creation of new materials. Despite that, the method presents some limitations that are circumvented by radiation processing. Electron beam irradiation was used to promote synthesis of hybrid structures while using silanol-terminated PDMS, TEOS and TPOZ as precursors. Evaluation of the method's performance was executed by gel fraction determination, WDXRF and FTIR-ATR. Results showed that, although there is some pre-irradiation reactivity between precursors, radiolysis induces scission on multiple sites of precursor's structures, which induces hybrid network formation to a greater extent. Characterisation allowed determining electron beam irradiation to be effective in the creation of Si-O-Zr bonds, resulting in the production of a Class II hybrid material.