Comparison of glycoside hydrolase family 3 ß-xylosidases from basidiomycetes and ascomycetes reveals evolutionarily distinct xylan degradation systems.

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) ß-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 substrate-binding 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.

Kinetic and molecular dynamics study of inhibition and transglycosylation in Hypocrea jecorina family 3 ß-glucosidases.

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.

Oxygen Activation by Cu LPMOs in Recalcitrant Carbohydrate Polysaccharide Conversion to Monomer Sugars.

Natural carbohydrate polymers such as starch, cellulose, and chitin provide renewable alternatives to fossil fuels as a source for fuels and materials. As such, there is considerable interest in their conversion for industrial purposes, which is evidenced by the established and emerging markets for products derived from these natural polymers. In many cases, this is achieved via industrial processes that use enzymes to break down carbohydrates to monomer sugars. One of the major challenges facing large-scale industrial applications utilizing natural carbohydrate polymers is rooted in the fact that naturally occurring forms of starch, cellulose, and chitin can have tightly packed organizations of polymer chains with low hydration levels, giving rise to crystalline structures that are highly recalcitrant to enzymatic degradation. The topic of this review is oxidative cleavage of carbohydrate polymers by lytic polysaccharide mono-oxygenases (LPMOs). LPMOs are copper-dependent enzymes (EC 1.14.99.53-56) that, with glycoside hydrolases, participate in the degradation of recalcitrant carbohydrate polymers. Their activity and structural underpinnings provide insights into biological mechanisms of polysaccharide degradation.

High-resolution structure of a lytic polysaccharide monooxygenase from Hypocrea jecorina reveals a predicted linker as an integral part of the catalytic domain.

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 HjLPMO9A revealed 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.

Improving the thermal stability of cellobiohydrolase Cel7A from Hypocrea jecorina by directed evolution.

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.

Biochemical characterization and crystal structures of a fungal family 3 ß-glucosidase, Cel3A from Hypocrea jecorina.

Cellulase mixtures from Hypocrea jecorina are commonly used for the saccharification of cellulose in biotechnical applications. The most abundant ß-glucosidase in the mesophilic fungus Hypocrea jecorina is HjCel3A, which hydrolyzes the ß-linkage between two adjacent molecules in dimers and short oligomers of glucose. It has been shown that enhanced levels of HjCel3A in H. 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)-ß-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 ß-glucosidases. Both production hosts resulted in HjCel3A structures that have N-linked glycosylations at Asn(208) and Asn(310). 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 from either host. Due to the different sizes of the glycosylations, the interactions result in different crystal forms for the two protein forms.

Photo-Fenton and Fenton Oxidation of Recalcitrant Wastewater from the Wooden Floor Industry.

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.

Structural, biochemical, and computational characterization of the glycoside hydrolase family 7 cellobiohydrolase of the tree-killing fungus Heterobasidion irregulare.

Root rot fungi of the Heterobasidion annosum complex are the most damaging pathogens in temperate forests, and the recently sequenced Heterobasidion irregulare genome revealed over 280 carbohydrate-active enzymes. Here, H. irregulare was grown on biomass, and the most abundant protein in the culture filtrate was identified as the only family 7 glycoside hydrolase in the genome, which consists of a single catalytic domain, lacking a linker and carbohydrate-binding module. The enzyme, HirCel7A, was characterized biochemically to determine the optimal conditions for activity. HirCel7A was crystallized and the structure, refined at 1.7 Å resolution, confirms that HirCel7A is a cellobiohydrolase rather than an endoglucanase, with a cellulose-binding tunnel that is more closed than Phanerochaete chrysosporium Cel7D and more open than Hypocrea jecorina Cel7A, suggesting intermediate enzyme properties. Molecular simulations were conducted to ascertain differences in enzyme-ligand interactions, ligand solvation, and loop flexibility between the family 7 glycoside hydrolase cellobiohydrolases from H. irregulare, H. jecorina, and P. chrysosporium. The structural comparisons and simulations suggest significant differences in enzyme-ligand interactions at the tunnel entrance in the -7 to -4 binding sites and suggest that a tyrosine residue at the tunnel entrance of HirCel7A may serve as an additional ligand-binding site. Additionally, the loops over the active site in H. jecorina Cel7A are more closed than loops in the other two enzymes, which has implications for the degree of processivity, endo-initiation, and substrate dissociation. Overall, this study highlights molecular level features important to understanding this biologically and industrially important family of glycoside hydrolases.

Loop motions important to product expulsion in the Thermobifida fusca glycoside hydrolase family 6 cellobiohydrolase from structural and computational studies.

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.

Expression, crystal structure and cellulase activity of the thermostable cellobiohydrolase Cel7A from the fungus Humicola grisea var. thermoidea.

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 (T(m) 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 Šresolution; R(work) and R(free) 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.

The crystal structure of RsSymEG1 reveals a unique form of smaller GH7 endoglucanases alongside GH7 cellobiohydrolases in protist symbionts of termites.

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.

Statistical approaches to indirectly compare bioequivalence between generics: a comparison of methodologies employing artemether/lumefantrine 20/120 mg tablets as prequalified by WHO.

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(max)) 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(max) within the conventional acceptance range does not preclude considering them as interchangeable, if necessary, since C(max) is considered to be of less clinical relevance for the relevant therapy.

Adapting the Use of Digital Content to Improve the Learning of Numeracy Among Children With Autism Spectrum Disorder in Rwanda: Thematic Content Analysis Study.

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.

Can resting-state functional MRI serve as a complement to task-based mapping of sensorimotor function? A test-retest reliability study in healthy volunteers.

PURPOSE: To investigate if resting-state functional MRI (fMRI) reliably can serve as a complement to task-based fMRI for presurgical mapping of the sensorimotor cortex. MATERIALS AND METHODS: Functional data were obtained in 10 healthy volunteers using a 3 Tesla MRI system. Each subject performed five bilateral finger tapping experiments interleaved with five resting-state experiments. Following preprocessing, data from eight volunteers were further analyzed with the general linear model (finger tapping data) and independent component analysis (rest data). Test-retest reliability estimates (hit rate and false alarm rate) for resting-state fMRI activation of the sensorimotor network were compared with the reliability estimates for task-evoked activation of the sensorimotor cortex. The reliability estimates constituted a receiver operating characteristics curve from which the area under the curve (AUC) was calculated. Statistical testing was performed to compare the two groups with respect to reliability. RESULTS: The AUC was generally higher for the task experiments, although median AUC was not significantly different on a group level. Also, the two groups showed comparable levels of within-group variance. CONCLUSION: Test-retest reliability was comparable between resting-state measurements and task-based fMRI, suggesting that presurgical mapping of functional networks can be a supplement to task-based fMRI in cases where patient status excludes task-based fMRI.

Enhanced detection of ATTR amyloid using a nanofibril-based assay.

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.

Synthesis of cyclic beta-glucan using laminarinase 16A glycosynthase mutant from the basidiomycete Phanerochaete chrysosporium.

Glycosynthases are precise molecular instruments for making specifically linked oligosaccharides. X-ray crystallography screening of ligands bound to the 1,3(4)-beta-D-glucanase nucleophile mutant E115S of Phanerochaete chrysosporium Laminarinase 16A (Lam16A) showed that laminariheptaose (L7) bound in an arch with the reducing and nonreducing ends occupying either side of the catalytic cleft of the enzyme. The X-ray structure of Lam16A E115S in complex with alpha-laminariheptaosyl fluoride (alphaL7F) revealed how alphaL7F could make a nucleophilic attack upon itself. Indeed, when Lam16A E115S was allowed to react with alphaL7F the major product was a cyclic beta-1,3-heptaglucan, as shown by mass spectrometry. NMR confirmed uniquely beta-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 beta-glucan.

Three-dimensional structure of an intact glycoside hydrolase family 15 glucoamylase from Hypocrea jecorina.

The three-dimensional structure of a complete Hypocrea jecorina glucoamylase has been determined at 1.8 A 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.

Production of Ready-To-Use Functionalized Sup35 Nanofibrils Secreted by Komagataella pastoris.

Amyloid nanofibrils are excellent scaffolds for designable materials that can be endowed with biotechnologically relevant functions. However, most of all excellent ideas and concepts that have been reported in the literature might never see real-world implementation in biotechnological applications. One bottleneck is the large-scale production of these materials. In this paper, we present an attempt to create a generic and scalable platform for producing ready-to-use functionalized nanofibrils directly from a eukaryotic organism. As a model material, we assembled Sup35(1-61) amyloid nanofibrils from Saccharomyces cerevisiae decorated with the Z-domain dimer, which has a high affinity toward antibody molecules. To this end, Komagataella pastoris was engineered by inserting gene copies of Sup35(1-61) and the protein chimera Sup35(1-61)-ZZ into the genome. This strain has the capability to constantly secrete amyloidogenic proteins into the extracellular medium, where the mature functionalized fibrils form, with a production yield of 35 mg/L culture. Another striking feature of this strategy is that the separation of the fibril material from the cells requires only centrifugation and resuspension in saline water. The fast production rates, minimal hands-on time, and high stability of the assembled material are some highlights that make the direct assembly of functionalized fibrils in the extracellular medium an alternative to production methods that are not suitable for large-scale production of designed amyloids.

Structural studies of a glycoside hydrolase family 3 ß-glucosidase from the model fungus Neurospora crassa.

The glycoside hydrolase family 3 (GH3) ß-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. ß-Glucosidases hydrolyse the ß-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 Šresolution, 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 ß-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 ß-glucosidases and account for the high cellobiose specificity of this subfamily.