Plant Cell Wall Deconstruction Research Articles

Selfish uptake versus extracellular arabinoxylan degradation in the primary degrader Ruminiclostridium cellulolyticum, a new string to its bow

BackgroundPrimary degraders of polysaccharides play a key role in anaerobic biotopes, where plant cell wall accumulates, providing extracellular enzymes to release fermentable carbohydrates to fuel themselves and other non-degrader species. Ruminiclostridium cellulolyticum is a model primary degrader growing amongst others on arabinoxylan. It produces large multi-enzymatic complexes called cellulosomes, which efficiently deconstruct arabinoxylan into fermentable monosaccharides.ResultsComplete extracellular arabinoxylan degradation was long thought to be required to fuel the bacterium during this plant cell wall deconstruction stage. We discovered and characterized a second system of “arabinoxylan” degradation in R. cellulolyticum, which challenged this paradigm. This “selfish” system is composed of an ABC transporter dedicated to the import of large and possibly acetylated arabinoxylodextrins, and a set of four glycoside hydrolases and two esterases. These enzymes show complementary action modes on arabinoxylo-dextrins. Two α-L-arabinofuranosidases target the diverse arabinosyl side chains, and two exo-xylanases target the xylo-oligosaccharides backbone either at the reducing or the non-reducing end. Together, with the help of two different esterases removing acetyl decorations, they achieve the depolymerization of arabinoxylo-dextrins in arabinose, xylose and xylobiose. The in vivo study showed that this new system is strongly beneficial for the fitness of the bacterium when grown on arabinoxylan, leading to the conclusion that a part of arabinoxylan degradation is achieved in the cytosol, even if monosaccharides are efficiently provided by the cellulosomes in the extracellular space. These results shed new light on the strategies used by anaerobic primary degrader bacteria to metabolize highly decorated arabinoxylan in competitive environments.ConclusionThe primary degrader model Ruminiclostridium cellulolyticum has developed a “selfish” strategy consisting of importing into the bacterium, large arabinoxylan–dextrin fractions released from a partial extracellular deconstruction of arabinoxylan, thus complementing its efficient extracellular arabinoxylan degradation system. Genetic studies suggest that this system is important to support fitness and survival in a competitive biotope. These results provide a better understanding of arabinoxylan catabolism in the primary degrader, with biotechnological application for synthetic microbial community engineering for the production of commodity chemicals from lignocellulosic biomass.

Low-abundance populations distinguish microbiome performance in plant cell wall deconstruction

BackgroundPlant cell walls are interwoven structures recalcitrant to degradation. Native and adapted microbiomes can be particularly effective at plant cell wall deconstruction. Although most understanding of biological cell wall deconstruction has been obtained from isolates, cultivated microbiomes that break down cell walls have emerged as new sources for biotechnologically relevant microbes and enzymes. These microbiomes provide a unique resource to identify key interacting functional microbial groups and to guide the design of specialized synthetic microbial communities.ResultsTo establish a system assessing comparative microbiome performance, parallel microbiomes were cultivated on sorghum (Sorghum bicolor L. Moench) from compost inocula. Biomass loss and biochemical assays indicated that these microbiomes diverged in their ability to deconstruct biomass. Network reconstructions from gene expression dynamics identified key groups and potential interactions within the adapted sorghum-degrading communities, including Actinotalea, Filomicrobium, and Gemmatimonadetes populations. Functional analysis demonstrated that the microbiomes proceeded through successive stages that are linked to enzymes that deconstruct plant cell wall polymers. The combination of network and functional analysis highlighted the importance of cellulose-degrading Actinobacteria in differentiating the performance of these microbiomes.ConclusionsThe two-tier cultivation of compost-derived microbiomes on sorghum led to the establishment of microbiomes for which community structure and performance could be assessed. The work reinforces the observation that subtle differences in community composition and the genomic content of strains may lead to significant differences in community performance.3Ck-Q-KzsX1uPWsLmUjZUvVideo

Enzyme Discovery in Anaerobic Fungi (Neocallimastigomycetes) Enables Lignocellulosic Biorefinery Innovation.

Lignocellulosic biorefineries require innovative solutions to realize their full potential, and the discovery of novel lignocellulose-active enzymes could improve biorefinery deconstruction processes. Enzymatic deconstruction of plant cell walls is challenging, as noncarbohydrate linkages in hemicellulosic sidechains and lignin protect labile carbohydrates from hydrolysis. Highly specialized microbes that degrade plant biomass are attractive sources of enzymes for improving lignocellulose deconstruction, and the anaerobic gut fungi (Neocallimastigomycetes) stand out as having great potential for harboring novel lignocellulose-active enzymes. We discuss the known aspects of Neocallimastigomycetes lignocellulose deconstruction, including their extensive carbohydrate-active enzyme content, proficiency at deconstructing complex lignocellulose, unique physiology, synergistic enzyme complexes, and sizeable uncharacterized gene content. Progress describing Neocallimastigomycetes and their enzymes has been rapid in recent years, and it will only continue to expand. In particular, direct manipulation of anaerobic fungal genomes, effective heterologous expression of anaerobic fungal enzymes, and the ability to directly relate chemical changes in lignocellulose to fungal gene regulation will accelerate the discovery and subsequent deployment of Neocallimastigomycetes lignocellulose-active enzymes.

Biodegradation of Gramineous Lignocellulose by Locusta migratoria manilensis (Orthoptera: Acridoidea)

Exploring an efficient and green pretreatment method is an important prerequisite for the development of biorefinery. It is well known that locusts can degrade gramineous lignocellulose efficiently. Locusts can be used as a potential resource for studying plant cell wall degradation, but there are few relative studies about locusts so far. Herein, some new discoveries were revealed about elucidating the process of biodegradation of gramineous lignocellulose in Locusta migratoria manilensis. The enzyme activity related to lignocellulose degradation and the content of cellulose, hemicellulose, and lignin in the different gut segments of locusts fed corn leaves were measured in this study. A series of characterization analyses were conducted on corn leaves and locust feces, which included field emission scanning electron microscopy (FE-SEM), Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction pattern (XRD), and thermogravimetric (TG) analysis. These results showed that the highest activities of carboxymethyl cellulase (CMCase), filter paper cellulase (FPA), and xylanase were obtained in the foregut of locusts, which strongly indicated that the foregut was the main lignocellulose degradation segment in locusts; furthermore, the majority of nutritional components were absorbed in the midgut of locusts. The activity of CMCase was significantly higher than that of xylanase, and manganese peroxidase (MnPase) activity was lowest, which might be due to the basic nutrition of locusts being cellulose and hemicellulose and not lignin based on the results of FE-SEM, FTIR, XRD, and TG analysis. Overall, these results provided a valuable insight into lignocellulosic degradation mechanisms for understanding gramineous plant cell wall deconstruction and recalcitrance in locusts, which could be useful in the development of new enzymatic pretreatment processes mimicking the locust digestive system for the biochemical conversion of lignocellulosic biomass to fuels and chemicals.

Mechanical processing effects on the secondary plant cell wall arrangement in Sorghum bicolor

Unlocking one third of plant biomass as a renewable feedstock for fuels and materials depends on the effective deconstruction of the secondary plant cell wall. Over 90% of the native secondary plant cell wall is composed of cellulose and hemicellulose polysaccharides and the lignin aromatic polymers. Deconstruction refers to the processes which digest polymers into desired subunits. Present deconstruction processes are centered on lignin-first extraction to overcome the recalcitrance of biomass: the accumulation of indigestible plant polymers during deconstruction.

Xylan Deconstruction by Thermophilic Thermoanaerobacterium bryantii Hemicellulases Is Stimulated by Two Oxidoreductases

Thermoanaerobacterium bryantii strain mel9T is a thermophilic bacterium isolated from a waste pile of a corn-canning factory. The genome of T. bryantii mel9T was sequenced and a hemicellulase gene cluster was identified. The cluster encodes seven putative enzymes, which are likely an endoxylanase, an α-glucuronidase, two oxidoreductases, two β-xylosidases, and one acetyl xylan esterase. These genes were designated tbxyn10A, tbagu67A, tbheoA, tbheoB, tbxyl52A, tbxyl39A, and tbaxe1A, respectively. Only TbXyn10A released reducing sugars from birchwood xylan, as shown by thin-layer chromatography analysis. The five components of the hemicellulase cluster (TbXyn10A, TbXyl39A, TbXyl52A, TbAgu67A, and TbAxe1A) functioned in synergy to hydrolyze birchwood xylan. Surprisingly, the two putative oxidoreductases increased the enzymatic activities of the gene products from the xylanolytic gene cluster in the presence of NADH and manganese ions. The two oxidoreductases were therefore named Hemicellulase-Enhancing Oxidoreductases (HEOs). All seven enzymes were thermophilic and acted in synergy to degrade xylans at 60 °C. Except for TbXyn10A, the other enzymes encoded by the gene cluster were conserved with high amino acid identities (85–100%) in three other Thermoanaerobacterium species. The conservation of the gene cluster is, therefore, suggestive of an important role of these enzymes in xylan degradation by these bacteria. The mechanism for enhancement of hemicellulose degradation by the HEOs is under investigation. It is anticipated, however, that the discovery of these new actors in hemicellulose deconstruction will have a significant impact on plant cell wall deconstruction in the biofuel industry.

Novel, recyclable Brønsted acidic deep eutectic solvent for mild fractionation of hemicelluloses

Acidic deep eutectic solvents (DESs) are promising media for lignin valorization and cellulose conversion due to their good ability in efficient deconstruction of plant cell wall. However, hemicellulose extraction from lignocellulose using acidic DESs remains a challenge. Herein, novel and green Brønsted acidic DESs (BDESs) were synthesized from natural organic acids and common polyols and successively adopted to deconstruct corncob for mild fractionation of hemicelluloses. Oxalic acid (OA)-based BDESs were preferred for corncob processing due to the high solubility of xylan. The results revealed that the suitable acidity of DESs and mild temperature effectively avoided the over-degradation of hemicelluloses. The chemical composition and structural features of the recovered hemicelluloses were investigated systematically. Moreover, after ethylene glycol (EG)-OA BDES was recycled and reused three times, the extraction still resulted in a satisfactory hemicellulose yield. The novel and eco-friendly processing offers a practical and sustainable route for hemicellulose extraction in acidic condition.

A circadian clock in Neurospora crassa functions during plant cell wall deconstruction

Circadian clocks are autonomous timers that are believed to confer organisms a selective advantage by enabling processes to occur at appropriate times of the day. In the model fungus Neurospora crassa, 20–40 % of its genes are reported to be under circadian regulation, as assayed in simple sugar media. Although it has been well-described that Neurospora efficiently deconstructs plant cell wall components, little is known regarding the status of the clock when Neurospora grows on cellulosic material, or whether such a clock has an impact on any of the genes involved in this process. Through luciferase-based reporters and fluorescent detection assays, we show that a clock is functioning when Neurospora grows on cellulose-containing wheat straw as the only carbon and nitrogen source. Additionally, we found that the major cellobiohydrolase encoding gene involved in plant cell wall deconstruction, cbh-1, is rhythmically regulated by the Neurospora clock, in a manner that depends on cellulose concentration and on the transcription factor CRE-1, known as a key player in carbon-catabolite repression in this fungus. Our findings are a step towards a more comprehensive understanding on how clock regulation modulates cellulose degradation, and thus Neurospora’s physiology.

Subcellular dissolution of xylan and lignin for enhancing enzymatic hydrolysis of microwave assisted deep eutectic solvent pretreated Pinus bungeana Zucc

The mechanism for enhancing enzymatic hydrolysis during microwave-assisted deep eutectic solvent (Mw-DES) pretreatment in deconstruction of plant cell wall was proposed by combining wet chemical analysis and microscopic measurements. Mw-DES pretreatment achieved significantly higher enzymatic conversion of 81.90% with lower lignin and comparable xylan removal (42.81% and 74.73%, respectively). While DES pretreated sample with higher lignin and xylan removal (66.59% and 74.93%, respectively) obtained limited sugar yield (45.67%). There were no significant differences with respect to chemical structures of lignin fraction between DES and Mw-DES pretreatment but primary discrepancies of topochemical and morphological changes were observed. Non- or low-substituted xylan was directly removed from secondary walls (SW) exposed more cellulose for enzyme attacking after Mw-DES pretreatment. Meanwhile, high-substituted xylan and lignin were synergistically dissolved from cell corner middle lamella (CCML). These topochemical changes of components resulted in cracked and porous cell wall structure, thus facilitating the accessibility of cellulose.

Multiple iron reduction by methoxylated phenolic lignin structures and the generation of reactive oxygen species by lignocellulose surfaces.

Chelator-mediated Fenton chemistry is capable of reducing non-stochiometric amounts of iron via hydroquinone oxidation. These types of reactions have previously been demonstrated to be promoted by some lignocellulose degrading fungi in generating hydroxyl radicals to permit lignified plant cell wall deconstruction. Here we demonstrate that lignocellulose surfaces, when exposed by chemical treatment or fragmentation, can promote a similar multi-oxidative mechanism in the presence of iron. Iron reduction by lignin surfaces permits the generation of hydroxyl radicals in the cell wall to help explain fungal non-enzymatic cell wall deconstruction, and it also provides an explanation for certain phenomenon such as the anthropogenic generation of formaldehyde by wood. The mechanism also provides a basis for the generation of electrons by lignin that are required by certain fungal redox enzymes active in plant cell wall degrading systems. Overall, the data demonstrate that iron found naturally in lignocellulose materials will promote the oxidation of phenolic lignin compounds in the naturally low pH environments occurring within lignified plant cell walls, and that this activity is promoted by cell wall fragmentation.

The endozoan, small-mammal reservoir hypothesis and the life cycle of Coccidioides species.

The prevailing hypothesis concerning the ecology of Coccidioides immitis and C. posadasii is that these human pathogenic fungi are soil fungi endemic to hot, dry, salty regions of the New World and that humans and the local, small-mammal fauna are only accidental hosts. Here we advance an alternative hypothesis that Coccidioides spp. live in small mammals as endozoans, which are kept inactive but alive in host granulomas and which transform into spore-producing hyphae when the mammal dies. The endozoan hypothesis incorporates results from comparative genomic analyses of Coccidioides spp. and related taxa that have shown a reduction in gene families associated with deconstruction of plant cell walls and an increase in those associated with digestion of animal protein, consistent with an evolutionary shift in substrate from plants to animals. If true, the endozoan hypothesis requires that models of the prevalence of human coccidioidomycosis account not only for direct effects of climate and soil parameters on the growth and reproduction of Coccidioides spp. but also consider indirect effects on these fungi that come from the plants that support the growth and reproduction of the small mammals that, in turn, support these endozoic fungi.

Mechanical fragmentation of wheat and rice straw at different scales: Energy requirement in relation to microstructure properties and enzymatic hydrolysis

Mechanical fragmentation is an important pretreatment step in lignocellulosic biorefinery. In this study, we explored the energy requirement (ER) for mechanical fragmentation of wheat and rice straw at different scales in relation to modifications of microstructure properties and enzymatic hydrolysis. The ER showed a reciprocal relationship with mean particle size (MPZ), but a negative linear relationship with cellulose crystallinity (CrI). An exponential relationship was observed between the ER with a specific surface area (SSA) and the oxygen-to-carbon surface ratio (O/C). The results showed that modification of the cellulose crystallinity required the most energy consumption, whereas reduction of particle size consumed the lowest energy. The order of importance of modifications of different microstructure parameters on the ER was: CrI > O/C > pore volume (PV) > SSA > MPZ. Sugar yield showed a clear, positive linear relationship with ER, indicating that grinding patterns with a high ER could significantly enhance the enzymatic hydrolysis yield owing to the plant cell wall deconstruction. The energy efficiency declined with an increasing ER because of the markedly lower increase in sugar yield compared with the corresponding increase in ER.

Hydrothermal pretreatment for deconstruction of plant cell wall: Part I. Effect on lignin‐carbohydrate complex

Hydrothermal pretreatment with characteristic of green chemistry is considered as promising technology in the biorefineries. Using material balance and multiscale characterization techniques, the effects of process severity factor and pH on chemical behaviors of lignin‐carbohydrate complex (LCC) were systematically studied. During pretreatment, spatial relocation of lignin with covalently linked xylan facilitated local cell wall collapse. A kinetic model was established to describe the behaviors of LCC components changing with severity factor. It was found that cleavage of LCC linkage was strongly pH‐dependent. Low pH dominated cleavage of coumarate/ferulate esters which cross‐linking lignin with xylan and repolymerization of aromatics either from furfural or lignin into polymers such as pseudo lignin, while high pH which allowed the existence of soluble LCC dominated the aldol condensations from xylose to aromatics and depolymerizaton of lignin to phenols. Detailed reaction pathways concerned with LCC were finally established to elucidate the underlying mechanism. © 2018 American Institute of Chemical Engineers AIChE J, 64: 1938–1953, 2018

Hydrothermal pretreatment for deconstruction of plant cell wall: Part II. Effect on cellulose structure and bioconversion

Influences of both ultrastructural modification of cellulose after hydrothermal pretreatment and products derived from lignin‐carbohydrate complex (LCC) on the subsequent enzymatic digestibility and fermentation were studied in this study. Under hydrothermal conditions, it was found that the rearrangement of hydrogen bonding pattern in cellulose via allomorph and conformational changes which was mainly severity‐dependent increased the numbers of water‐exposed glycosidic bond and the formation of “amorphous‐like” cellulose fibril facilitated enzymatic hydrolysis. Pseudo lignin, soluble xylo‐oligomers, phenols and degradation products from high severity impeded enzymatic digestion. LCC and phenols which were rich in pH‐controlled prehydrolyzate did not sufficiently inhibit yeast while furans and some aromatics which were rich in high‐severity prehydrolyzate might be potential inhibitors. Trade‐off phenomenon was solved by pH‐controlled operation and high yields in both glucose (83–93%) and xylose (75–80%) were simultaneously obtained. The final ethanol yield from cellulose to ethanol reached as high as 84–93%. © 2018 American Institute of Chemical Engineers AIChE J, 64: 1954–1964, 2018

Test of Efficacy of Cellulases for Biomass Degradation.

Testing of cellulases on real biomass samples is required to do a true assessment of their efficacy for biomass degradation. Cellulase enzymes belong to a number of different glycosyl hydrolase families, all with different activity, specificity and modes of action. The concerted and synergistic action of these different cellulases determines the efficacy for plant cell wall deconstruction and cellulose hydrolysis. However, the plant cell wall of lignocellulosic materials is a very complex matrix and the efficacy of a cellulase preparation to degrade lignocellulosic materials is influenced by many factors. In this chapter, two protocols for testing efficacy of cellulases on pretreated biomass samples are described. The first protocol describes a small-scale setup employing low solids concentration that easily enables the testing of a larger number of samples. The second protocol describes a method for testing the efficacy of cellulases at conditions more closely resembling industrial conditions, i.e., high solids concentrations. Both protocols can be used to test the cellulases under a variety of substrate types, substrate concentrations, enzyme loadings and process conditions. The protocols can also be used to evaluate different feedstocks.

Network reconstruction and systems analysis of plant cell wall deconstruction by Neurospora crassa

BackgroundPlant biomass degradation by fungal-derived enzymes is rapidly expanding in economic importance as a clean and efficient source for biofuels. The ability to rationally engineer filamentous fungi would facilitate biotechnological applications for degradation of plant cell wall polysaccharides. However, incomplete knowledge of biomolecular networks responsible for plant cell wall deconstruction impedes experimental efforts in this direction.ResultsTo expand this knowledge base, a detailed network of reactions important for deconstruction of plant cell wall polysaccharides into simple sugars was constructed for the filamentous fungus Neurospora crassa. To reconstruct this network, information was integrated from five heterogeneous data types: functional genomics, transcriptomics, proteomics, genetics, and biochemical characterizations. The combined information was encapsulated into a feature matrix and the evidence weighted to assign annotation confidence scores for each gene within the network. Comparative analyses of RNA-seq and ChIP-seq data shed light on the regulation of the plant cell wall degradation network, leading to a novel hypothesis for degradation of the hemicellulose mannan. The transcription factor CLR-2 was subsequently experimentally shown to play a key role in the mannan degradation pathway of N. crassa.ConclusionsHere we built a network that serves as a scaffold for integration of diverse experimental datasets. This approach led to the elucidation of regulatory design principles for plant cell wall deconstruction by filamentous fungi and a novel function for the transcription factor CLR-2. This expanding network will aid in efforts to rationally engineer industrially relevant hyper-production strains.

In vivo synergistic activity of a CAZyme cassette from Acidothermus cellulolyticus significantly improves the cellulolytic activity of the C. bescii exoproteome.

The use of microbial cells to convert plant biomass directly to fuels and chemicals is referred to as consolidated bioprocessing (CBP). Members of the bacterial genus, Caldicellulosiruptor (Gram-positive, anaerobic hyperthermophiles) are capable of deconstructing plant biomass without enzymatic or chemical pretreatment. This is accomplished by the production and secretion of free, multi-domain enzymes that outperform commercial enzyme cocktails on some substrates. Here, we show that the exoproteome of Caldicellulosiruptor bescii may be enhanced by the heterologous expression of enzymes from Acidothermus cellulolyticus that act synergistically to improve sugar release from complex substrates; as well as improve cell growth. In this work, co-expression of the A. cellulolyticus Acel_0615 β-glucanase (GH6 and GH12) and E1 endoglucanase (GH5) enzymes resulted in an increase in the activity of the exoproteome on Avicel; as well as an increase in growth of C. bescii on Avicel compared to the parental strain or the strain expressing the β-glucanase alone. Our ability to engineer the composition and effectiveness of the exoproteome of these bacteria provides insight into the natural mechanism of plant cell wall deconstruction, as well as future directions for improving CBP. Biotechnol. Bioeng. 2017;114: 2474-2480. © 2017 Wiley Periodicals, Inc.

Deletion of pH Regulator pac-3 Affects Cellulase and Xylanase Activity during Sugarcane Bagasse Degradation by Neurospora crassa.

Microorganisms play a vital role in bioethanol production whose usage as fuel energy is increasing worldwide. The filamentous fungus Neurospora crassa synthesize and secrete the major enzymes involved in plant cell wall deconstruction. The production of cellulases and hemicellulases is known to be affected by the environmental pH; however, the regulatory mechanisms of this process are still poorly understood. In this study, we investigated the role of the pH regulator PAC-3 in N. crassa during their growth on sugarcane bagasse at different pH conditions. Our data indicate that secretion of cellulolytic enzymes is reduced in the mutant Δpac-3 at alkaline pH, whereas xylanases are positively regulated by PAC-3 in acidic (pH 5.0), neutral (pH 7.0), and alkaline (pH 10.0) medium. Gene expression profiles, evaluated by real-time qPCR, revealed that genes encoding cellulases and hemicellulases are also subject to PAC-3 control. Moreover, deletion of pac-3 affects the expression of transcription factor-encoding genes. Together, the results suggest that the regulation of holocellulase genes by PAC-3 can occur as directly as in indirect manner. Our study helps improve the understanding of holocellulolytic performance in response to PAC-3 and should thereby contribute to the better use of N. crassa in the biotechnology industry.

Identification and characterization of the glucose dual-affinity transport system in Neurospora crassa: pleiotropic roles in nutrient transport, signaling, and carbon catabolite repression

BackgroundThe glucose dual-affinity transport system (low- and high-affinity) is a conserved strategy used by microorganisms to cope with natural fluctuations in nutrient availability in the environment. The glucose-sensing and uptake processes are believed to be tightly associated with cellulase expression regulation in cellulolytic fungi. However, both the identities and functions of the major molecular components of this evolutionarily conserved system in filamentous fungi remain elusive. Here, we systematically identified and characterized the components of the glucose dual-affinity transport system in the model fungus Neurospora crassa.ResultsUsing RNA sequencing coupled with functional transport analyses, we assigned GLT-1 (Km = 18.42 ± 3.38 mM) and HGT-1/-2 (Km = 16.13 ± 0.95 and 98.97 ± 22.02 µM) to the low- and high-affinity glucose transport systems, respectively. The high-affinity transporters hgt-1/-2 complemented a moderate growth defect under high glucose when glt-1 was deleted. Simultaneous deletion of hgt-1/-2 led to extensive derepression of genes for plant cell wall deconstruction in cells grown on cellulose. The suppression by HGT-1/-2 was connected to both carbon catabolite repression (CCR) and the cyclic adenosine monophosphate-protein kinase A pathway. Alteration of a residue conserved across taxa in hexose transporters resulted in a loss of glucose-transporting function, whereas CCR signal transduction was retained, indicating dual functions for HGT-1/-2 as “transceptors.”ConclusionsIn this study, GLT-1 and HGT-1/-2 were identified as the key components of the glucose dual-affinity transport system, which plays diverse roles in glucose transport and carbon metabolism. Given the wide conservation of the glucose dual-affinity transport system across fungal species, the identification of its components and their pleiotropic roles in this study shed important new light on the molecular basis of nutrient transport, signaling, and plant cell wall degradation in fungi.

Cloning, heterologous expression and biochemical characterization of a non-specific endoglucanase family 12 from Aspergillus terreus NIH2624

The cellulases from Glycoside Hydrolyses family 12 (GH12) play an important role in cellulose degradation and plant cell wall deconstruction being widely used in a number of bioindustrial processes. Aiming to contribute toward better comprehension of these class of the enzymes, here we describe a high-yield secretion of a endoglucanase GH12 from Aspegillus terreus (AtGH12), which was cloned and expressed in Aspergillus nidulans strain A773. The purified protein was used for complete biochemical and functional characterization. The optimal temperature and pH of the enzyme were 55°C and 5.0 respectively, which has high activity against β-glucan and xyloglucan and also is active toward glucomannan and CMC. The enzyme retained activity up to 60°C. AtGH12 is strongly inhibited by Cu2+, Fe2+, Cd2+, Mn2+, Ca2+, Zn2+ and EDTA, whereas K+, Tween, Cs+, DMSO, Triton X-100 and Mg2+ enhanced the enzyme activity. Furthermore, SAXS data reveal that the enzyme has a globular shape and CD analysis demonstrated a prevalence of a β-strand structure corroborating with typical β-sheets fold commonly found for other endoglucanases from GH12 family.