The dynamic water vapor sorption of untreated, acetylated (W
), and formaldehyde-treated (W
) Scots pine (
L.) sapwood was studied in a dynamic vapor sorption apparatus to assess the effects of cell ...wall bulking and cross-linking. Both modifications resulted in a considerable reduction of reduced equilibrium moisture content (EMC
), the corresponding equilibrium times, and hysteresis in the hydroscopic range of wood. Acetylation reduced the adsorption and desorption of water at each given relative humidity (RH) step from 0% to 95% RH, whereas formalization affected the sorption behavior of wood solely above 20% RH. From 20% to 95% RH, the EMC ratio of W
to its control steadily decreased, whereas the EMC ratio of W
was still constant in this RH range. Below 20% RH, the sorption behavior of W
was governed by hydroxyl blocking, whereas that of W
was hardly influenced compared with the control. Above 20% RH, the sorption behavior of W
was solely determined by cell wall bulking, whereas that of W
was governed by the increased matrix stiffness due to the cross-linking of cell wall polymers.
The water vapour sorption data of untreated (W
), acetylated (W
) and formaldehyde-treated (W
) Scots pine (
L.) sapwood were analysed in terms of their sorption kinetics and were transformed into ...excess surface work (ESW) isotherms. The sorption kinetics were studied by fitting the non-linear parallel exponential kinetics (PEK) model to the experimental data in which the sorption kinetics curve is composed of two processes (fast and slow components). W
and W
showed evident differences in their sorption kinetics and their thermodynamic sorption behaviour. In contrast to acetylation, formalisation influenced both the extent of the slow sorption process and the shape of its pseudoisotherm. For W
and W
, it appears that some water associated with the slow process is adsorbed at sites for fast sorption newly generated upon swelling (previously postulated as extra water) and subsequently desorbed by the fast process. For W
, the formation of extra water hardly occurs. ESW was reduced through acetylation with a constant factor over the whole hydroscopic range, whereas the ESW of W
was reduced only after reaching the monolayer capacity compared to its control. The sorption behaviour of W
was solely determined by cell wall bulking, whereas that of W
was governed by the increased matrix stiffness due to cross-linking of the cell wall polymers.
Three-layer polymeric diphenyl-methane-diisocyanate (pMDI)-bonded particleboards (PBs) were produced with different proportions of simulated recovered wood (rW) in the core layers (cLs) to assess the ...effect of rW on the formaldehyde (FA) release of PB. A pre-test was conducted on furniture and particle mixtures of rW to determine the range of expectable FA emission of rW. The FA content of the raw particle mixtures could be predicted from the contents of the individual raw material and did not change compared to the PB. FA content correlated strongly with PB-FA emission. It was possible to predict the maximum PB-FA contents, which should not be exceeded according to F*** and CARB 2 by linear regression models. At moderate and high total FA emission levels, the FA emission of the particle mixtures was approximately 60% higher than the emission of PB blocks. At low total FA level, the flask method and the gas analysis method exhibited different results with regard to the emissions from particles and their respective PBs.
Several standardized methods exist to determine formaldehyde (HCHO) release from wood-based panels (WBPs). These methods were developed decades ago to be used in manufacturers laboratories to provide ...a mean of production control. They are robust and take several hours to yield results. Modern WBP panel production, however, is a continuous process. Therefore the established methods are too time-consuming for process control and process optimization with respect to HCHO release. Moreover, there is a strong trend of lowering the regulatory HCHO emission limits. Thus, there is a need for a comparatively fast and precise method which is suitable for the use on-site in a WBP manufacturers laboratory. In this work, an optimization of the solid phase micro extraction gas chromatography high field asymmetric waveform ion mobility spectrometry (SPME-GC-FAIMS) method is presented with respect to GC-FAIMS settings and the calibration procedure. It is also shown that, in addition to WBP block samples, also particles can be used for the measurement. The industrial applicability of SPME-GC-FAIMS system was demonstrated by testing the HCHO release of freshly produced WBPs on-site in the manufacturers laboratory.
The inhibitory action of lignin on cellulase cocktails is a major challenge to the biological saccharification of plant cell wall polysaccharides. Although the mechanism remains unclear, hydrophobic ...interactions between enzymes and lignin are hypothesized to drive adsorption. Here we evaluate the role of hydrophobic interactions in enzyme-lignin binding. The hydrophobicity of the enzyme surface was quantified using an estimation of the clustering of nonpolar atoms, identifying potential interaction sites. The adsorption of enzymes to lignin surfaces, measured using the quartz crystal microbalance, correlates to the hydrophobic cluster scores. Further, these results suggest a minimum hydrophobic cluster size for a protein to preferentially adsorb to lignin. The impact of electrostatic contribution was ruled out by comparing the isoelectric point (pI) values to the adsorption of proteins to lignin surfaces. These results demonstrate the ability to predict enzyme-lignin adsorption and could potentially be used to design improved cellulase cocktails, thus lowering the overall cost of biofuel production.
Background: Lignin is a plant cell wall polymer that inhibits enzymatic saccharification of polysaccharides for the production of biofuel.
Results: The adsorption of enzymes to lignin surfaces correlates to solvent-exposed hydrophobic clusters.
Conclusion: Hydrophobicity, not surface charge, identifies proteins that preferentially adsorb to lignin.
Significance: The method could be used to design improved cellulase cocktails to lower the cost of biofuel production.
In nature, many microbes secrete mixtures of glycoside hydrolases, oxidoreductases, and accessory enzymes to deconstruct polysaccharides and lignin in plants. These enzymes are often decorated with ...N- and O-glycosylation, the roles of which have been broadly attributed to protection from proteolysis, as the extracellular milieu is an aggressive environment. Glycosylation has been shown to sometimes affect activity, but these effects are not fully understood. Here, we examine N- and O-glycosylation on a model, multimodular glycoside hydrolase family 7 cellobiohydrolase (Cel7A), which exhibits an O-glycosylated carbohydrate-binding module (CBM) and an O-glycosylated linker connected to an N- and O-glycosylated catalytic domain (CD)—a domain architecture common to many biomass-degrading enzymes. We report consensus maps for Cel7A glycosylation that include glycan sites and motifs. Additionally, we examine the roles of glycans on activity, substrate binding, and thermal and proteolytic stability. N-glycan knockouts on the CD demonstrate that N-glycosylation has little impact on cellulose conversion or binding, but does have major stability impacts. O-glycans on the CBM have little impact on binding, proteolysis, or activity in the whole-enzyme context. However, linker O-glycans greatly impact cellulose conversion via their contribution to proteolysis resistance. Molecular simulations predict an additional role for linker O-glycans, namely that they are responsible for maintaining separation between ordered domains when Cel7A is engaged on cellulose, as models predict α-helix formation and decreased cellulose interaction for the nonglycosylated linker. Overall, this study reveals key roles for N- and O-glycosylation that are likely broadly applicable to other plant cell-wall–degrading enzymes.
Glycoside Hydrolase Family 7 cellobiohydrolases (GH7 CBHs) catalyze cellulose depolymerization in cellulolytic eukaryotes, making them key discovery and engineering targets. However, there remains a ...lack of robust structure-activity relationships for these industrially important cellulases. Here, we compare CBHs from Trichoderma reesei (TrCel7A) and Penicillium funiculosum (PfCel7A), which exhibit a multi-modular architecture consisting of catalytic domain (CD), carbohydrate-binding module, and linker. We show that PfCel7A exhibits 60% greater performance on biomass than TrCel7A. To understand the contribution of each domain to this improvement, we measure enzymatic activity for a library of CBH chimeras with swapped subdomains, demonstrating that the enhancement is mainly caused by PfCel7A CD. We solve the crystal structure of PfCel7A CD and use this information to create a second library of TrCel7A CD mutants, identifying a TrCel7A double mutant with near-equivalent activity to wild-type PfCel7A. Overall, these results reveal CBH regions that enable targeted activity improvements.
The crystalline nature of cellulose microfibrils is one of the key factors influencing biomass recalcitrance which is a key technical and economic barrier to overcome to make cellulosic biofuels a ...commercial reality. To date, all known fungal enzymes tested have great difficulty degrading highly crystalline cellulosic substrates. We have demonstrated that the CelA cellulase from Caldicellulosiruptor bescii degrades highly crystalline cellulose as well as low crystallinity substrates making it the only known cellulase to function well on highly crystalline cellulose. Unlike the secretomes of cellulolytic fungi, which typically comprise multiple, single catalytic domain enzymes for biomass degradation, some bacterial systems employ an alternative strategy that utilizes multi-catalytic domain cellulases. Additionally, CelA is extremely thermostable and highly active at elevated temperatures, unlike commercial fungal cellulases. Furthermore we have determined that the factors negatively affecting digestion of lignocellulosic materials by C. bescii enzyme cocktails containing CelA appear to be significantly different from the performance barriers affecting fungal cellulases. Here, we explore the activity and degradation mechanism of CelA on a variety of pretreated substrates to better understand how the different bulk components of biomass, such as xylan and lignin, impact its performance.
Summary
T‐regulatory cells (Tregs) have a fundamental role in the establishment and maintenance of peripheral tolerance. There is now compelling evidence that deficits in the numbers and/or function ...of different types of Tregs can lead to autoimmunity, allergy, and graft rejection, whereas an over‐abundance of Tregs can inhibit anti‐tumor and anti‐pathogen immunity. Experimental models in mice have demonstrated that manipulating the numbers and/or function of Tregs can decrease pathology in a wide range of contexts, including transplantation, autoimmunity, and cancer, and it is widely assumed that similar approaches will be possible in humans. Research into how Tregs can be manipulated therapeutically in humans is most advanced for two main types of CD4+ Tregs: forkhead box protein 3 (FOXP3)+ Tregs and interleukin‐10‐producing type 1 Tregs (Tr1 cells). The aim of this review is to highlight current information on the characteristics of human FOXP3+ Tregs and Tr1 cells that make them an attractive therapeutic target. We discuss the progress and limitations that must be overcome to develop methods to enhance Tregs in vivo, expand or induce them in vitro for adoptive transfer, and/or inhibit their function in vivo. Although many technical and theoretical challenges remain, the next decade will see the first clinical trials testing whether Treg‐based therapies are effective in humans.
Biomass deconstruction remains integral for enabling second‐generation biofuel production at scale. However, several steps necessary to achieve significant solubilization of biomass, notably harsh ...pretreatment conditions, impose economic barriers to commercialization. By employing hyperthermostable cellulase machinery, biomass deconstruction can be made more efficient, leading to milder pretreatment conditions and ultimately lower production costs. The hyperthermophilic bacterium Caldicellulosiruptor bescii produces extremely active hyperthermostable cellulases, including the hyperactive multifunctional cellulase CbCel9A/Cel48A. Recombinant CbCel9A/Cel48A components have been previously produced in Escherichia coli and integrated into synthetic hyperthermophilic designer cellulosome complexes. Since then, glycosylation has been shown to be vital for the high activity and stability of CbCel9A/Cel48A. Here, we studied the impact of glycosylation on a hyperthermostable designer cellulosome system in which two of the cellulosomal components, the scaffoldin and the GH9 domain of CbCel9A/Cel48A, were glycosylated as a consequence of employing Ca. bescii as an expression host. Inclusion of the glycosylated components yielded an active cellulosome system that exhibited long‐term stability at 75 °C. The resulting glycosylated designer cellulosomes showed significantly greater synergistic activity compared to the enzymatic components alone, as well as higher thermostability than the analogous nonglycosylated designer cellulosomes. These results indicate that glycosylation can be used as an essential engineering tool to improve the properties of designer cellulosomes. Additionally, Ca. bescii was shown to be an attractive candidate for production of glycosylated designer cellulosome components, which may further promote the viability of this bacterium both as a cellulase expression host and as a potential consolidated bioprocessing platform organism.
Enzymatic breakdown of cellulose is a major challenge in the production of second‐generation biofuels. Hyperthermostable enzymes from Caldicellulosiruptor bescii, a very efficient cellulolytic bacterium, were assembled into highly active cellulose deconstruction complexes: designer cellulosomes. We transformed Ca. bescii to produce designer cellulosomes composed of glycosylated scaffoldins and chimaeric Ca. bescii enzymes. This study demonstrated that glycosylation on these designer cellulosomes improved their thermostability and activity.
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