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Glycoside hydrolase family 7 (GH7) cellulases are key enzymes responsible for carbon cycling on earth through their role in cellulose degradation and constitute highly important industrial enzymes as well. Although these enzymes are found in a wide variety of evolutionarily distant organisms across eukaryotes, they exhibit remarkably conserved features within two groups: exo‐acting cellobiohydrolases and endoglucanases. However, recently reports have emerged of a separate clade of GH7 endoglucanases from protist symbionts of termites that are 60–80 amino acids shorter. In this work, we describe the first crystal structure of a short GH7 endoglucanase, RsSymEG1, from a symbiont of the lower termite Reticulitermes speratus. A more open flat surface and shorter loops around the non‐reducing end of the cellulose‐binding cleft indicate enhanced access to cellulose chains on the surface of cellulose microfibrils. Additionally, when comparing activities on polysaccharides to a typical fungal GH7 endoglucanase (Trichoderma longibrachiatum Cel7B), RsSymEG1 showed significantly faster initial hydrolytic activity. We also examine the prevalence and diversity of GH7 enzymes that the symbionts provide to the termite host, compare overall structures and substrate binding between cellobiohydrolase and long and short endoglucanase, and highlight the presence of similar short GH7s in other organisms. [ABSTRACT FROM AUTHOR]

Understanding rye and wheat digestion is vital for evaluating impacts on nutrient availability and glycaemic responses. This study investigated the disintegration of processed high-fibre rye foods and refined wheat products during simulated intestinal digestion, aiming to link product characteristics with nutrient liberation. The overarching aim was to elucidate how these rye products contribute to the observed benefits in human intervention studies, particularly regarding satiety, weight loss, and metabolism. Analysis included four wholegrain rye products and three refined wheat products, spanning yeast-fermented breads and un-leavened cereal products. Microstructure examination revealed larger, partially intact digesta particles in wholegrain rye products after 120 min of digestion, alongside more aggregated and less degraded starch granules compared to refined wheat bread. Fermented rye bread exhibited greater degradation of subaleurone cell wall fragments than un-fermented rye bread. Viscosity assessments indicated lower viscosity for wheat products than for rye products, with yeast-fermented soft rye bread and rye crispbread showing notably lower viscosity than unfermented rye products. Post-digestion carbohydrate analysis uncovered higher glucose and maltose release during digestion for wheat products. PCA analysis confirmed negative correlations between glucose and maltose release and rye products, characterized by larger post-digestion bolus particles and higher dietary fibre. The elevated cell wall content in rye products acted as a protective barrier for starch granules, mitigating swelling and amylose release, explaining the observed viscosity differences between wheat and rye products and potentially influencing starch digestibility. Consequently, rye products undergo slower and less complete digestion than wheat, aligning with findings from human intervention studies. [Display omitted] • A comparison of rye and wheat food products before and after in vitro digestion. • Liquid part after in vitro digestion have the highest viscosity in non-fermented rye. • Imaging during digestion show faster starch granule degradation in wheat products. • Glucose and maltose are easier released during digestion in wheat products. • PCA analysis validates observed correlations between analysed product parameters. [ABSTRACT FROM AUTHOR]

Improving the quality of Bachelor's and Master's theses while at the same time increasing the number of theses without expanding the existing resources proportionately is a huge challenge faced by higher educational institutions. The aim of this study is to investigate the effect of multiple change processes on Bachelors and Masters level thesis work in a selected higher educational institution. The following research questions were studied: (1) How has the thesis quality changed? (2) How has the number of completed theses changed? and, (3) How has the ratio of completed theses per supervisor changed? The change processes were introduced into the thesis process in the Department of Computer and Systems Sciences (DSV), Stockholm University during 2008–2014. The results show that the quality and the number of completed theses have significantly increased. The multiple change processes including a purpose built ICT system named SciPro, which was introduced and improved incrementally during 2010–2014 are discussed and evaluated in relation to these results. [ABSTRACT FROM AUTHOR]

The glycoside hydrolase family 3 (GH3) β‐glucosidases are a structurally diverse family of enzymes. Cel3A from Neurospora crassa (NcCel3A) belongs to a subfamily of key enzymes that are crucial for industrial biomass degradation. β‐Glucosidases hydrolyse the β‐1,4 bond at the nonreducing end of cellodextrins. The hydrolysis of cellobiose is of special importance as its accumulation inhibits other cellulases acting on crystalline cellulose. Here, the crystal structure of the biologically relevant dimeric form of NcCel3A is reported. The structure has been refined to 2.25 Å resolution, with an Rcryst and Rfree of 0.18 and 0.22, respectively. NcCel3A is an extensively N‐glycosylated glycoprotein that shares 46% sequence identity with Hypocrea jecorina Cel3A, the structure of which has recently been published, and 61% sequence identity with the thermophilic β‐glucosidase from Rasamsonia emersonii. NcCel3A is a three‐domain protein with a number of extended loops that deepen the active‐site cleft of the enzyme. These structures characterize this subfamily of GH3 β‐glucosidases and account for the high cellobiose specificity of this subfamily. The glycoside hydrolase family 3 β‐glucosidase Cel3A from Neurospora crassa (NcCel3A) is highly specific for cellobiose. The crystal structure of NcCel3A has been solved to 2.25 Å resolution. The structure is a dimer and exhibits a high degree of N‐glycosylation. [ABSTRACT FROM AUTHOR]

For decades, the enzymes of the fungus Hypocrea jecorina have served as a model system for the breakdown of cellulose. Three-dimensional structures for almost all H. jecorina cellulose- degrading enzymes are available, except for HjLPMO9A, belonging to the AA9 family of lytic polysaccharide monooxygenases (LPMOs). These enzymes enhance the hydrolytic activity of cellulases and are essential for cost-efficient conversion of lignocellulosic biomass. Here, using structural and spectroscopic analyses, we found that native HjLPMO9A contains a catalytic domain and a family-1 carbohydrate-binding module (CBM1) connected via a linker sequence. A C terminally truncated variant of HjLPMO9A containing 21 residues of the predicted linker was expressed at levels sufficient for analysis. Here, using structural, spectroscopic, and biochemical analyses, we found that this truncated variant exhibited reduced binding to and activity on cellulose compared with the full-length enzyme. Importantly, a 0.95-Å resolution X-ray structure of truncated HjLPMO9Arevealed that the linker forms an integral part of the catalytic domain structure, covering a hydrophobic patch on the catalytic AA9 module. We noted that the oxidized catalytic center contains a Cu(II) coordinated by two His ligands, one of which has a His-brace in which the His-1 terminal amine group also coordinates to a copper. The final equatorial position of the Cu(II) is occupied by a water-derived ligand. The spectroscopic characteristics of the truncated variant were not measurably different from those of full-length HjLPMO9A, indicating that the presence of the CBM1 module increases the affinity of HjLPMO9A for cellulose binding, but does not affect the active site. [ABSTRACT FROM AUTHOR]

Common problems identified by students during their interaction with supervisors are too little instructions as well as infrequent and insufficient supervisor feedback. The SciPro system has been developed to tackle these problems. This paper describes, analyze and discuss the interaction between students and supervisors using the SciPro system. Through two use cases involving two supervisors and 38 bachelor and master students, the authors show that the structure implemented in SciPro supports and allows for different pedagogical approaches and supervision styles. Ten different types of interactions in the thesis process are identified. In order to explain why some students do not complete within the timeframe, the authors identify six categories of problems: 1) language skills, 2) inability to apply research methods, 3) ability for self-study, 4) lack of creativity, 5) limited knowledge about how to write academic work, and 6) lack of motivation. The collaborative stage of group-supervision may be one possible way to deal with the different type of support for the process of managing student theses. [ABSTRACT FROM AUTHOR]

The filamentous fungus Hypocrea jecorina produces a number of cellulases and hemicellulases that act in a concerted fashion on biomass and degrade it into monomeric or oligomeric sugars. β-Glucosidases are involved in the last step of the degradation of cellulosic biomass and hydrolyse the β-glycosidic linkage between two adjacent molecules in dimers and oligomers of glucose. In this study, it is shown that substituting the β-glucosidase from H. jecorina (HjCel3A) with the β-glucosidase Cel3A from the thermophilic fungus Rasamsonia emersonii (ReCel3A) in enzyme mixtures results in increased efficiency in the saccharification of lignocellulosic materials. Biochemical characterization of ReCel3A, heterologously produced in H. jecorina, reveals a preference for disaccharide substrates over longer gluco-oligosaccharides. Crystallographic studies of ReCel3A revealed a highly N-glycosylated three-domain dimeric protein, as has been observed previously for glycoside hydrolase family 3 β-glucosidases. The increased thermal stability and saccharification yield and the superior biochemical characteristics of ReCel3A compared with HjCel3A and mixtures containing HjCel3A make ReCel3A an excellent candidate for addition to enzyme mixtures designed to operate at higher temperatures. [ABSTRACT FROM AUTHOR]

There is a need for development of on-site wastewater treatment technologies suitable to "dry-process" industries, such as the wooden floor sector. Due to the nature of their activities, these industries generate low volumes of highly polluted and recalcitrant wastewaters due to washing and cleaning surfaces and machinery. Advanced oxidation processes such as Fenton and photo-Fenton are potentially feasible options for the treatment of wastewaters with not easily biodegradable pollutants. The wastewater from a wooden floor industry with initial COD value of 4956 mg/L and TOC value of 2730 mg/L was treated with Fenton (Fe/H2O2) and photo-Fenton (Fe/H2O2/UV) applying a 2-level full-factorial experimental design. The highest removals of COD and TOC (79% and 62% respectively) were achieved when photo-Fenton was applied. In conclusion, Fenton and photo- Fenton are promising treatment options for these highly recalcitrant wastewaters, photo-Fenton being a more promising option according to the results. [ABSTRACT FROM AUTHOR]

There is a need for the development of on-site wastewater treatment technologies suitable for "dry-process industries," such as the wood-floor sector. Due to the nature of their activities, these industries generate lower volumes of highly polluted wastewaters after cleaning activities. Advanced oxidation processes such as Fenton and photo-Fenton, are potentially feasible options for treatment of these wastewaters. One of the disadvantages of the Fenton process is the formation of large amounts of ferrous iron sludge, a constraint that might be overcome with the use of nanoscale zero-valent iron (nZVI) powder. Wastewater from a wood-floor industry with initial COD of 4956 mg/L and TOC of 2730 mg/L was treated with dark-Fenton (nZVI/H2O2) and photo-Fenton (nZVI/H2O2/UV) applying a 2-level full-factorial experimental design. The highest removal of COD and TOC (80% and 60%, resp.) was achieved using photo-Fenton. The supply of the reactants in more than one dose during the reaction time had significant and positive effects on the treatment efficiency. According to the results, Fenton and mostly photo-Fenton are promising treatment options for these highly recalcitrant wastewaters. Future investigations should focus on optimizing treatment processes and assessing toxic effects that residual pollutants and the nZVI might have. The feasibility of combining advanced oxidation processes with biological treatment is also recommended. [ABSTRACT FROM AUTHOR]

Abstract: The glycoside hydrolase (GH) family 61 is a long-recognized, but still recondite, class of proteins, with little known about the activity, mechanism or function of its more than 70 members. The best-studied GH family 61 member, Cel61A of the filamentous fungus Hypocrea jecorina, is known to be an endoglucanase, but it is not clear if this represents the main activity or function of this family in vivo. We present here the first structure for this family, that of Cel61B from H. jecorina. The best-quality crystals were formed in the presence of nickel, and the crystal structure was solved to 1.6 Å resolution using a single-wavelength anomalous dispersion method with nickel as the source of anomalous scatter. Cel61B lacks a carbohydrate-binding module and is a single-domain protein that folds into a twisted β-sandwich. A structure-aided sequence alignment of all GH family 61 proteins identified a highly conserved group of residues on the surface of Cel61B. Within this patch of mostly polar amino acids was a site occupied by the intramolecular nickel hexacoordinately bound in the solved structure. In the Cel61B structure, there is no easily identifiable carbohydrate-binding cleft or pocket or catalytic center of the types normally seen in GHs. A structural comparison search showed that the known structure most similar to Cel61B is that of CBP21 from the Gram-negative soil bacterium Serratia marcescens, a member of the carbohydrate-binding module family 33 proteins. A polar surface patch highly conserved in that structural family has been identified in CBP21 and shown to be involved in chitin binding and in the protein''s enhancement of chitinase activities. The analysis of the Cel61B structure is discussed in light of our continuing research to better understand the activities and function of GH family 61. [Copyright &y& Elsevier]

More than 30 proteins and peptides have been found to form amyloid fibrils in human diseases. Fibrils formed by transthyretin (TTR) are associated with ATTR amyloidosis, affecting many vital organs, including the heart and peripheral nervous system. Congo red staining is the gold standard method for detection of amyloid deposits in tissue. However, Congo red staining and amyloid typing methods such as immunofluorescence labelling are limited to relatively large deposits. Detection of small ATTR deposits present at an early stage of the disease could enable timely treatment and prevent severe tissue damage. In this study, we developed an enhanced ATTR amyloid detection method that uses functionalised protein nanofibrils. Using this method, we achieved sensitive detection of monomeric TTR in a microplate immunoassay and immunofluorescence labelling of ex vivo tissue from two patients containing ATTR aggregates. The system's utility was confirmed on sections from a patient with AA amyloidosis and liver sections from inflamed mouse. These results suggest that the detection system constitutes important new technology for highly sensitive detection of microscopic amounts of ATTR amyloid deposited in tissue. [ABSTRACT FROM AUTHOR]

Lactose, glucose and galactose contents in milk and fermented milk products and their lactose-free alternatives, were determined. Storage of products up to the best-before dates had no effect on carbohydrate composition. Total galactose content in yoghurt, i.e., free galactose plus the galactose moiety of lactose, was 2.3–2.4 g 100 g −1 (94–95% of that in milk), whereas that in Swedish soured milk and kefir was 1.9–2.0 g 100 g −1 (75–79% of that in milk). Lactose levels in lactose-free milk and fermented milk products were below or close to detection limits for all products. Only small differences in total galactose content existed for the lactose-free alternatives of milk, yoghurt and filmjölk, all with average total galactose contents of 1.3–1.4 g 100 g −1 (55–60% of that in milk). In contrast, lactose-free kefir had the same galactose content as milk, 2.4 g 100 g −1 . [ABSTRACT FROM AUTHOR]

Glycoside hydrolase family 7 (GH7) cellobiohydrolases (CBHs) play a key role in biomass recycling in nature. They are typically the most abundant enzymes expressed by potent cellulolytic fungi, and are also responsible for the majority of hydrolytic potential in enzyme cocktails for industrial processing of plant biomass. The thermostability of the enzyme is an important parameter for industrial utilization. In this study, Cel7 enzymes from different fungi were expressed in a fungal host and assayed for thermostability, including Hypocrea jecorina Cel7A as a reference. The most stable of the homologues, Humicola grisea var. thermoidea Cel7A, exhibits a 10°C higher melting temperature ( Tm of 72.5°C) and showed a 4-5 times higher initial hydrolysis rate than H. jecorina Cel7A on phosphoric acid-swollen cellulose and showed the best performance of the tested enzymes on pretreated corn stover at elevated temperature (65°C, 24 h). The enzyme shares 57% sequence identity with H. jecorina Cel7A and consists of a GH7 catalytic module connected by a linker to a C-terminal CBM1 carbohydrate-binding module. The crystal structure of the H. grisea var. thermoidea Cel7A catalytic module (1.8 Å resolution; Rwork and Rfree of 0.16 and 0.21, respectively) is similar to those of other GH7 CBHs. The deviations of several loops along the cellulose-binding path between the two molecules in the asymmetric unit indicate higher flexibility than in the less thermostable H. jecorina Cel7A. [ABSTRACT FROM AUTHOR]

Background: Many teachers consider it challenging to teach children with autism spectrum disorder (ASD) in an inclusive classroom due to their unique needs and challenges. The integration of information communication technology (ICT) in the education system allows children with ASD to improve their learning. However, these ICT tools should meet their needs to lead a productive life. Objective: This study aimed to examine the possibilities of re-creating and adapting digital content to improve the learning of numeracy among children with ASD in inclusive school settings. Methods: We conducted 7 focus group discussions (FGDs) with 56 teachers from 7 schools and 14 parents from April to November 2019. Each of the FGDs took around 1 hour. Two clustered sets of questions were used: (1) general knowledge about teaching children with ASD and (2) analysis of selected online educational video content of early math (specifically, counting numbers). The researchers used video to understand current methodologies used in teaching children with ASD, possibilities of adaptation of the content in the current teaching environment, future challenges when the content is adapted, and possible solutions to overcome those challenges. All data, including audio recordings, field notes, and participants’ comments, were transcribed, recorded, and analyzed following the steps recommended in qualitative data analysis. Results: The researchers identified ten themes from the analysis of the data: (1) awareness of the existence of ASD among children in schools and the community, (2) acceptance of children with ASD in an inclusive classroom and the community, (3) methods and models used when teaching children with ASD, (4)realia used to improve the learning of children with ASD, (5) the design of educational digital content, (6) the accessibility of online educational content, (7) quality of the content of the educational multimedia, (8) the opportunity of using the translated and re-created content inside and outside the classroom, (9) the relevance of the digital content in the Rwandan educational system, and (10) enhancement of the accessibility and quality of the digital content. We found that participants assumed that the content translation, gamification, and re-creation would help teach children with ASD. Moreover, they recommended contextualizing the content, increasing access to digital devices, and further research in the education of different subjects. Conclusions: Although many studies have identified the possibilities of using ICT to support children with ASD, few studies have documented the possibilities of integrating the existing technologies tested in the international community. This study is charting new territory to investigate online content to suit the context of schools. This study recommends further exploration of possible methodologies, such as applied behavior analysis or verbal behavior therapy, and the development of contextualized technologies that respond to the educational needs of children with ASD. [ABSTRACT FROM AUTHOR]

Xylan is the most common hemicellulose in plant cell walls, though the structure of xylan polymers differs between plant species. Here, to gain a better understanding of fungal xylan degradation systems, which can enhance enzymatic saccharification of plant cell walls in industrial processes, we conducted a comparative study of two glycoside hydrolase family 3 (GH3) β-xylosidases (Bxls), one from the basidiomycete Phanerochaete chrysosporium (PcBxl3), and the other from the ascomycete Trichoderma reesei (TrXyl3A). A comparison of the crystal structures of the two enzymes, both with saccharide bound at the catalytic center, provided insight into the basis of substrate binding at each subsite. PcBxl3 has a substratebinding pocket at subsite -1, while TrXyl3A has an extra loop that contains additional binding subsites. Furthermore, kinetic experiments revealed that PcBxl3 degraded xylooligosaccharides faster than TrXyl3A, while the KM values of TrXyl3A were lower than those of PcBxl3. The relationship between substrate specificity and degree of polymerization of substrates suggested that PcBxl3 preferentially degrades xylobiose (X2), while TrXyl3A degrades longer xylooligosaccharides. Moreover, docking simulation supported the existence of extended positive subsites of TrXyl3A in the extra loop located at the N-terminus of the protein. Finally, phylogenetic analysis suggests that wood-decaying basidiomycetes use Bxls such as PcBxl3 that act efficiently on xylan structures from woody plants, whereas molds use instead Bxls that efficiently degrade xylan from grass. Our results provide added insights into fungal efficient xylan degradation systems. [ABSTRACT FROM AUTHOR]

In an effort to characterise the whole transcriptome of the fungus Hypocrea jecorina, cDNA clones of this fungus were identified that encode for previously unknown proteins that are likely to function in biomass degradation. One of these newly identified proteins, found to be co-regulated with the major H. jecorina cellulases, is a protein that was denoted Cellulose induced protein 1 (Cip1). This protein consists of a glycoside hydrolase family 1 carbohydrate binding module connected via a linker region to a domain with yet unknown function. After cloning and expression of Cip1 in H. jecorina, the protein was purified and biochemically characterised with the aim of determining a potential enzymatic activity for the novel protein. No hydrolytic activity against any of the tested plant cell wall components was found. The proteolytic core domain of Cip1 was then crystallised, and the three-dimensional structure of this was determined to 1.5 Å resolution utilising sulphur single-wavelength anomalous dispersion phasing (sulphor-SAD). A calcium ion binding site was identified in a sequence conserved region of Cip1 and is also seen in other proteins with the same general fold as Cip1, such as many carbohydrate binding modules. The presence of this ion was found to have a structural role. The Cip1 structure was analysed and a structural homology search was performed to identify structurally related proteins. The two published structures with highest overall structural similarity to Cip1 found were two poly-lyases: CsGL, a glucuronan lyase from H. jecorina and vAL-1, an alginate lyase from the Chlorella virus. This indicates that Cip1 may be a lyase. However, initial trials did not detect significant lyase activity for Cip1. Cip1 is the first structure to be solved of the 23 currently known Cip1 sequential homologs (with a sequence identity cut-off of 25%), including both bacterial and fungal members. [ABSTRACT FROM AUTHOR]

The occurrence of binaural interaction in humans has been demonstrated using auditory brainstem response (ABR). A distinctly binaural potential, β, is derived by subtracting the ABR recording evoked by binaural clicks from the monaural aggregate, i.e., the sum of the two corresponding ABR recordings evoked by monaural clicks. However, few clinical data are available, possibly because the β-wave is considered an elusive response due to a low signal-to-noise-ratio. In the present study, β-wave latency, amplitude and area were evaluated for 10 subjects with normal hearing using automatic analysis and averaging based on a large number of stimulations. The efficacy of the β-wave measures was assessed using different stimulus rates, as binaural interaction is known to decrease with increasing stimulus rate. It was found that the β-wave given by automatic analysis demonstrated known characteristics of binaural interaction in human ABR, i.e. the absence of binaural interaction during wave III, significant binaural interaction during wave V and a significant decrease in binaural interaction when the stimulus rate was increased. These findings suggest that a β-wave in the binaural difference waveform can be detected and quantified using automatic analysis, thus it is suitable for clinical studies, at least for patients with normal hearing thresholds. [ABSTRACT FROM AUTHOR]

ß-Glucosidases enhance enzymatic biomass conversion by relieving cellobiose inhibition of endoglucanases and cellobiohydrolases. However, the susceptibility of these enzymes to inhibition and transglycosylation at high glucose or cellobiose concentrations severely limits their activity and, consequently, the overall efficiency of enzyme mixtures. We determined the impact of these two processes on the hydrolytic activity of the industrially relevant family 3 ß-glucosidases from Hypocrea jecorina, HjCel3A and HjCel3B, and investigated the underlying molecular mechanisms through kinetic studies, binding free energy calculations, and molecular dynamics (MD) simulations. HjCel3B had a 7-fold higher specificity for cellobiose than HjCel3A but greater tendency for glucose inhibition. Energy decomposition analysis indicated that cellobiose has relatively weak electrostatic interactions with binding site residues, allowing it to be easily displaced by glucose and free to inhibit other hydrolytic enzymes. HjCel3A is, thus, preferable as an industrial ß-glucosidase despite its lower activity caused by transglycosylation. This competing pathway to hydrolysis arises from binding of glucose or cellobiose at the product site after formation of the glycosyl-enzyme intermediate. MD simulations revealed that binding is facilitated by hydrophobic interactions with Trp-37, Phe-260, and Tyr-443. Targeting these aromatic residues for mutation to reduce substrate affinity at the product site would therefore potentially mitigate transglycosidic activity. Engineering improved variants of HjCel3A and other structurally similar ß-glucosidases would have a significant economic effect on enzymatic biomass conversion in terms of yield and production cost as the process can be consequently conducted at higher substrate loadings. [ABSTRACT FROM AUTHOR]

Background: The ascomycete fungus Trichoderma reesei is the predominant source of enzymes for industrial conversion of lignocellulose. Its glycoside hydrolase family 7 cellobiohydrolase (GH7 CBH) TreCel7A constitutes nearly half of the enzyme cocktail by weight and is the major workhorse in the cellulose hydrolysis process. The orthologs from Trichoderma atroviride (TatCel7A) and Trichoderma harzianum (ThaCel7A) show high sequence identity with TreCel7A, ~ 80%, and represent naturally evolved combinations of cellulose-binding tunnel-enclosing loop motifs, which have been suggested to influence intrinsic cellobiohydrolase properties, such as endo-initiation, processivity, and off-rate. Results: The TatCel7A, ThaCel7A, and TreCel7A enzymes were characterized for comparison of function. The catalytic domain of TatCel7A was crystallized, and two structures were determined: without ligand and with thio-cellotriose in the active site. Initial hydrolysis of bacterial cellulose was faster with TatCel7A than either ThaCel7A or TreCel7A. In synergistic saccharification of pretreated corn stover, both TatCel7A and ThaCel7A were more efficient than TreCel7A, although TatCel7A was more sensitive to thermal inactivation. Structural analyses and molecular dynamics (MD) simulations were performed to elucidate important structure/function correlations. Moreover, reverse conservation analysis (RCA) of sequence diversity revealed divergent regions of interest located outside the cellulose-binding tunnel of Trichoderma spp. GH7 CBHs. Conclusions: We hypothesize that the combination of loop motifs is the main determinant for the observed differences in Cel7A activity on cellulosic substrates. Fine-tuning of the loop flexibility appears to be an important evolutionary target in Trichoderma spp., a conclusion supported by the RCA data. Our results indicate that, for industrial use, it would be beneficial to combine loop motifs from TatCel7A with the thermostability features of TreCel7A. Furthermore, one region implicated in thermal unfolding is suggested as a primary target for protein engineering. [ABSTRACT FROM AUTHOR]

Secreted mixtures of Hypocrea jecorina cellulases are able to efficiently degrade cellulosic biomass to fermentable sugars at large, commercially relevant scales. H. jecorina Cel7A, cellobiohydrolase I, from glycoside hydrolase family 7, is the workhorse enzyme of the process. However, the thermal stability of Cel7A limits its use to processes where temperatures are no higher than 50 °C. Enhanced thermal stability is desirable to enable the use of higher processing temperatures and to improve the economic feasibility of industrial biomass conversion. Here, we enhanced the thermal stability of Cel7A through directed evolution. Sites with increased thermal stability properties were combined, and a Cel7A variant (FCA398) was obtained, which exhibited a 10.4 °C increase in Tm and a 44-fold greater half-life compared with the wild-type enzyme. This Cel7A variant contains 18 mutated sites and is active under application conditions up to at least 75 °C. The X-ray crystal structure of the catalytic domain was determined at 2.1 Å resolution and showed that the effects of the mutations are local and do not introduce major backbone conformational changes. Molecular dynamics simulations revealed that the catalytic domain of wild-type Cel7A and the FCA398 variant exhibit similar behavior at 300 K, whereas at elevated temperature (475 and 525 K), the FCA398 variant fluctuates less and maintains more native contacts over time. Combining the structural and dynamic investigations, rationales were developed for the stabilizing effect at many of the mutated sites. [ABSTRACT FROM AUTHOR]

The filamentous fungus Hypocrea jecorina (anamorph of Trichoderma reesei) is the predominant source of enzymes for industrial saccharification of lignocellulose biomass. The major enzyme, cellobiohydrolase Cel7A, constitutes nearly half of the total protein in the secretome. The performance of such enzymes is susceptible to inhibition by compounds liberated by physico-chemical pre-treatment if the biomass is kept unwashed. Xylan and xylo-oligosaccharides ( XOS) have been proposed to play a key role in inhibition of cellobiohydrolases of glycoside hydrolase family 7. To elucidate the mechanism behind this inhibition at a molecular level, we used X-ray crystallography to determine structures of H. jecorina Cel7A in complex with XOS. Structures with xylotriose, xylotetraose and xylopentaose revealed a predominant binding mode at the entrance of the substrate-binding tunnel of the enzyme, in which each xylose residue is shifted ~ 2.4 Å towards the catalytic center compared with binding of cello-oligosaccharides. Furthermore, partial occupancy of two consecutive xylose residues at subsites -2 and -1 suggests an alternative binding mode for XOS in the vicinity of the catalytic center. Interestingly, the -1 xylosyl unit exhibits an open aldehyde conformation in one of the structures and a ring-closed pyranoside in another complex. Complementary inhibition studies with p-nitrophenyl lactoside as substrate indicate mixed inhibition rather than pure competitive inhibition. Database The atomic coordinates and structure factors are available in the Protein Data Bank under accession number ( H. jecorina Cel7A E212Q variant, complex with xylotriose), ( H. jecorina Cel7A E217Q variant, complex with xylotriose), ( H. jecorina Cel7A E212Q variant, complex with xylopentaose), ( H. jecorina Cel7A E217Q variant, complex with xylopentaose), (wild-type H. jecorina Cel7A, complex with xylopentaose) and ( H. jecorina Cel7A E217Q variant, complex with xylotetraose). [ABSTRACT FROM AUTHOR]

The article discusses a research on fungal cellulases. Topics include structures, microfibrils, and substrates of cellulose, glycoside hydrolase catalytic mechanisms, and early developments in fungal cellulases. Other topics include carbohydrate-binding modules and linkers, structural studies and catalytic function of glycoside hydrolases, and kinetic modeling and visualization.

Cellulase mixtures from Hypocrea jecorina are commonly used for the saccharification of cellulose in biotechnical applications. The most abundant β-glucosidase in the mesophilic fungus Hypocrea jecorina is HjCel3A, which hydrolyzes the β-linkage between two adjacent molecules in dimers and short oligomers of glucose. It has been shown that enhanced levels of HjCel3AinH. jecorina cellulase mixtures benefit the conversion of cellulose to glucose. Biochemical characterization of HjCel3A shows that the enzyme efficiently hydrolyzes (1,4)- as well as (1,2)-, (1,3)-, and (1,6)β-D-linked disaccharides. For crystallization studies, HjCel3A was produced in both H. jecorina (HjCel3A) and Pichia pastoris (Pp-HjCel3A).Whereas the thermostabilities of HjCel3A and Pp-HjCel3A are the same, Pp-HjCel3A has a higher degree of N-linked glycosylation. Here, we present x-ray structures of HjCel3A with and without glucose bound in the active site. The structures have a three-domain architecture as observed previously for other glycoside hydrolase family 3 β-glucosidases. Both production hosts resulted in HjCel3A structures that have N-linked glycosylations at Asn208 and Asn310. In H. jecorina-produced HjCel3A, a single N-acetylglucosamine is present at both sites, whereas in Pp-HjCel3A, the P. pastoris-produced HjCel3A enzyme, the glycan chains consist of 8 or 4 saccharides. The glycosylations are involved in intermolecular contacts in the structures derived fromeither host. Due to the different sizes of the glycosylations, the interactions result in different crystal forms for the two protein forms. [ABSTRACT FROM AUTHOR]

Cellobiohydrolases (CBHs) are typically major components of natural enzyme cocktails for biomass degradation. Their active sites are enclosed in a tunnel, enabling processive hydrolysis of cellulose chains. Glycoside hydrolase Family 6 (GH6) CBHs act from nonreducing ends by an inverting mechanism and are present in many cellulolytic fungi and bacteria. The bacterial Thermobifida fusca Cel6B (TfuCel6B) exhibits a longer and more enclosed active site tunnel than its fungal counterparts. Here, we determine the structures of two TfuCel6B mutants co-crystallized with cellobiose, D274A (catalytic acid), and the double mutant D226A/S232A, which targets the putative catalytic base and a conserved serine that binds the nucleophilic water. The ligand binding and the structure of the active site are retained when compared with the wild type structure, supporting the hypothesis that these residues are directly involved in catalysis. One structure exhibits crystallographic waters that enable construction of a model of the α-anomer product after hydrolysis. Interestingly, the product sites of TfuCel6B are completely enclosed by an "exit loop" not present in fungal GH6 CBHs and by an extended "bottom loop". From the structures, we hypothesize that either of the loops enclosing the product subsites in the TfuCel6B active site tunnel must open substantially for product release. With simulation, we demonstrate that both loops can readily open to allow product release with equal probability in solution or when the enzyme is engaged on cellulose. Overall, this study reveals new structural details of GH6 CBHs likely important for functional differences among enzymes from this important family. [ABSTRACT FROM AUTHOR]

Abstract: Cellulases, glycoside hydrolases that catalyze the degradation of cellulose, are classified as either endoglucanases or cellobiohydrolases (CBHs) based on their architecture and mode of action on the cellulose. CBHs bind the cellulose chain in a more or less closed tunnel and cleave off cellobiose units processively from one end of the cellulosic polymer, while endoglucanases have their active sites in a more or less open cleft and show a higher tendency to cut bonds internally in the polymer. The CBH Cel6A (also called CBH2) from the ascomycete Hypocrea jecorina has a much shorter substrate-binding tunnel and seems less processive than the CBH Cel7A (CBH1), from the same fungus. Here, we present the X-ray crystal structure of the catalytic domain of the CBH Cel6B, also called E3, from the soil bacterium Thermobifida fusca, both in its apo form and co-crystallized with cellobiose. The enzyme structure reveals that the Cel6B enzyme has a much longer substrate-binding site than its fungal GH6 counterparts. The tunnel is comparable in length to that of GH7 CBHs. In the ligand structure with cellobiose, the tunnel exit is completely closed by a 13-residue loop not present in fungal GH6 enzymes. The loop needs to be displaced to allow cellobiose product release for a processive action by the enzyme. When ligand is absent, seven of these residues are not visible in the electron density and the tunnel exit is open. [Copyright &y& Elsevier]

Methylumbelliferyl-β-cellobioside ( MUF- G2) is a convenient fluorogenic substrate for certain β-glycoside hydrolases ( GH). However, hydrolysis of the aglycone is poor with GH family 6 enzymes ( GH6), despite strong binding. Prediction of the orientation of the aglycone of MUF- G2 in the +1 subsite of Hypocrea jecorina Cel6 A by automated docking suggested umbelliferyl modifications at C4 and C6 for improved recognition. Four modified umbelliferyl-β-cellobiosides [6-chloro-4-methyl- (Cl MUF); 6-chloro-4-trifluoromethyl- (ClF3 MUF); 4-phenyl- (Ph UF); 6-chloro-4-phenyl- (ClPh UF)] were synthesized and tested with GH6, GH7, GH9, GH5 and GH45 cellulases. Indeed the rate of aglycone release by H. jecorina Cel6 A was 10-150 times higher than with MUF- G2, although it was still three orders of magnitude lower than with H. jecorina Cel7 B. The 4-phenyl substitution drastically reduced the fluorescence intensity of the free aglycone, while Cl MUF- G2 could be used for determination of kcat and KM for H. jecorina Cel6 A and Thermobifida fusca Cel6 A. Crystal structures of H. jecorina Cel6 A D221 A mutant soaked with the MUF-, Cl MUF- and ClPh UF-β-cellobioside substrates show that the modifications turned the umbelliferyl group 'upside down', with the glycosidic bond better positioned for protonation than with MUF- G2. Database Structural data have been submitted to the Protein Data Bank under accession numbers pdb 4AU0, 4AX7, 4AX6 Structured digital abstract • http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7260296 • Cel6A and Cel6A bind by x-ray crystallography (View Interaction: 1, 2) [ABSTRACT FROM AUTHOR]

Purpose: The objective of this study was to compare different methods of adjusted indirect comparisons that can be used to investigate the relative bioavailability of different generic products. To achieve this goal, generic artemether/lumefantrine 20/120 mg tablets that have been prequalified by the World Health Organization (WHO) were selected as model products for study. Methods: Data from three bioequivalence studies conducted independently that compared three generics with the same reference product were used to indirectly determine the relative bioavailability between the generics themselves. Results: The different methods of indirect comparison examined in this study provide consistent results. Methods based on the assumption of a large sample size give slightly narrower 90 % confidence intervals. Therefore, the use of methods based on the t test is recommended. Given the precision of the area under the time-concentration curve (AUC) data, it is possible to conclude that the extent of exposure of artemether and lumefantrine is bioequivalent between the different generics studied. However, given the precision of the drug peak concentration (C) data, it is not possible to demonstrate equivalence within the conventional acceptance range for all comparisons; it is possible to conclude bioequivalence within the widened acceptance range 75-133 %. Conclusions: From a clinical viewpoint, not only are these prequalified generics bioequivalent and interchangeable with the reference product (Coartem, Novartis), but also the existing indirect evidence makes it possible to conclude that these WHO prequalified products are bioequivalent between themselves with respect to the AUC. The lack of the necessary precision to demonstrate bioequivalence between generics with respect to the C within the conventional acceptance range does not preclude considering them as interchangeable, if necessary, since C is considered to be of less clinical relevance for the relevant therapy. [ABSTRACT FROM AUTHOR]

Abstract: Using standard laboratory equipment, thermochemically pretreated oat straw was enzymatically saccharified and fermented to ethanol, and after removal of ethanol the remaining material was subjected to biogas digestion. A detailed mass balance calculation shows that, for steam explosion pretreatment, this combined ethanol fermentation and biogas digestion converts 85–87% of the higher heating value (HHV) of holocellulose (cellulose and hemicellulose) in the oat straw into biofuel energy. The energy (HHV) yield of the produced ethanol and methane was 9.5–9.8MJ/(kg dry oat straw), which is 28–34% higher than direct biogas digestion that yielded 7.3–7.4MJ/(kg dry oat straw). The rate of biogas formation from the fermentation residues was also higher than from the corresponding pretreated but unfermented oat straw, indicating that the biogas digestion could be terminated after only 24days. This suggests that the ethanol process acts as an additional pretreatment for the biogas process. [Copyright &y& Elsevier]

Glycosynthases are precise molecular instruments for making specifically linked oligosaccharides. X-ray crystallography screening of ligands bound to the 1,3(4)-β-o-glucanase nucleophile mutant E115S of Phanerochaete chtysosporium Laminarinase 16A (Lam16A) showed that laminariheptaose (L7) bound in an arch with the reducing and non reducing ends occupying either side of the catalytic cleft of the enzyme. The X-ray structure of Lam16A E115S in complex with α-laminariheptaosyl fluoride (αL7F) revealed how αL7F could make a nucleophilic attack upon itself. Indeed, when Lam16A E115S was allowed to react with αL7F the major product was a cyclic β-1,3-heptaglucan, as shown by mass spectrometry. NMR confirmed uniquely β-1,3-linkages and no reducing end. Molecular dynamics simulations indicate that the cyclic laminariheptaose molecule is not completely planar and that torsion angles at the glycosidic linkages fluctuate between two energy minima. This is the first report of a glycosynthase that joins the reducing and nonreducing ends of a single oligosaccharide and the first reported synthesis of cyclic β-glucan. [ABSTRACT FROM AUTHOR]

Microsomal prostaglandin E synthase type 1 (mPGES-1) converts prostaglandin endoperoxides, generated from arachidonic acid by cyclooxygenases, into prostaglandin E[sub2]. This enzyme belongs to the membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG) family of integral membrane proteins, and because of its link to inflammatory conditions and preferential coupling to cyclooxygenase 2, it has received considerable attention as a drug target. Based on the high resolution crystal structure of human leukotriene C4 synthase, a model of mPGES-1 has been constructed in which the tripeptide co-substrate glutathione is bound in a horseshoe-shaped conformation with its thiol group positioned in close proximity to Arg-126. Mutation of Arg-126 into an Ala or Gln strongly reduces the enzyme's prostaglandin E synthase activity (85-95%), whereas mutation of a neighboring Arg-122 does not have any significant effect. Interestingly, R126A and R126Q mPGES-1 exhibit a novel, glutathione-dependent, reductase activity, which allows conversion of prostaglandin H[sub2] into prostaglandin F[sub2]α. Our data show that Arg-126 is a catalytic residue in mPGES-1 and suggest that MAPEG enzymes share significant structural components of their active sites. [ABSTRACT FROM AUTHOR]

The three-dimensional structure of a complete Hypocrea jecorina glucoamylase has been determined at 1.8 Å resolution. The presented structure model includes the catalytic and starch binding domains and traces the course of the 37-residue linker segment. While the structures of other fungal and yeast glucoamylase catalytic and starch binding domains have been determined separately, this is the first intact structure that allows visualization of the juxtaposition of the starch binding domain relative to the catalytic domain. The detailed interactions we see between the catalytic and starch binding domains are confirmed in a second independent structure determination of the enzyme in a second crystal form. This second structure model exhibits an identical conformation compared to .the first structure model, which suggests that the H jecorina glucoamylase structure we report is independent of crystal lattice contact restraints and represents the three-dimensional structure found in solution. The proposed starch binding regions for the starch binding domain are aligned with the catalytic domain in the three-dimensional structure in a manner that supports the hypothesis that the starch binding domain serves to target the glucoamylase at sites where the starch granular matrix is disrupted and where the enzyme might most effectively function. [ABSTRACT FROM AUTHOR]

Tec family protein tyrosine kinases (TFKs) play a central role in hematopoietic cellular signaling. Initial activation takes place through specific tyrosine phosphorylation situated in the activation loop. Further activation occurs within the SH3 domain via a transphosphorylation mechanism, which for Bruton''s tyrosine kinase (Btk) affects tyrosine 223. We found that TFKs phosphorylate preferentially their own SH3 domains, but differentially phosphorylate other member family SH3 domains, whereas non-related SH3 domains are not phosphorylated. We demonstrate that SH3 domains are good and reliable substrates. We observe that transphosphorylation is selective not only for SH3 domains, but also for dual SH3SH2 domains. However, the dual domain is phosphorylated more effectively. The major phosphorylation sites were identified as conserved tyrosines, for Itk Y180 and for Bmx Y215, both sites being homologous to the Y223 site in Btk. There is, however, one exception because the Tec-SH3 domain is phosphorylated at a non-homologous site, nevertheless a conserved tyrosine, Y206. Consistent with these findings, the 3D structures for SH3 domains point out that these phosphorylated tyrosines are located on the ligand-binding surface. Because a number of Tec family kinases are coexpressed in cells, it is possible that they could regulate the activity of each other through transphosphorylation. [Copyright &y& Elsevier]