Multienzyme Cellulose Films as Sustainable and Self-Degradable Hydrogen Peroxide-Producing Material.

The use of hydrogen peroxide-releasing enzymes as a component to produce alternative and sustainable antimicrobial materials has aroused interest in the scientific community. However, the preparation of such materials requires an effective enzyme binding method that often involves the use of expensive and toxic chemicals. Here, we describe the development of an enzyme-based hydrogen peroxide-producing regenerated cellulose film (RCF) in which a cellobiohydrolase (TrCBHI) and a cellobiose dehydrogenase (MtCDHA) were efficiently adsorbed, 90.38 ± 2.2 and 82.40 ± 5.7%, respectively, without making use of cross-linkers. The enzyme adsorption kinetics and binding isotherm experiments showed high affinity of the proteins possessing cellulose-binding modules for RCF, suggesting that binding on regenerated cellulose via specific interactions can be an alternative method for enzyme immobilization. Resistance to compression and porosity at a micrometer scale were found to be tunable by changing cellulose concentration prior to film regeneration. The self-degradation process, triggered by stacking TrCBHI and MtCDHA (previously immobilized onto separate RCF), produced 0.15 nmol/min·cm2 of H2O2. Moreover, the production of H2O2 was sustained for at least 24 h reaching a concentration of ∼2 mM. The activity of MtCDHA immobilized on RCF was not affected by reuse for at least 3 days (1 cycle/day), suggesting that no significant enzyme leakage occurred in that timeframe. In the material herein designed, cellulose (regenerated from a 1-ethyl-3-methylimidazolium acetate/dimethyl sulfoxide (DMSO) solution) serves both as support and substrate for the immobilized enzymes. The sequential reaction led to the production of H2O2 at a micromolar-millimolar level revealing the potential use of the material as a self-degradable antimicrobial agent.

Redesigning the Aspergillus nidulans xylanase regulatory pathway to enhance cellulase production with xylose as the carbon and inducer source.

BACKGROUND: Biomass contains cellulose (C6-sugars), hemicellulose (C5-sugars) and lignin. Biomass ranks amongst the most abundant hydrocarbon resources on earth. However, biomass is recalcitrant to enzymatic digestion by cellulases. Physicochemical pretreatment methods make cellulose accessible but partially destroy hemicellulose, producing a C5-sugar-rich liquor. Typically, digestion of pretreated LCB is performed with commercial cellulase preparations, but C5-sugars could in principle be used for "on site" production of cellulases by genetically engineered microorganism, thereby reducing costs. RESULTS: Here we report a succession of genetic interventions in Aspergillus nidulans that redesign the natural regulatory circuitry of cellulase genes in such a way that recombinant strains use C5-sugar liquors (xylose) to grow a vegetative tissue and simultaneously accumulate large amounts of cellulases. Overexpression of XlnR showed that under xylose-induction conditions only xylanase C was produced. XlnR overexpression strains were constructed that use the xynCp promoter to drive the production of cellobiohydrolases, endoglucanases and ß-glucosidase. All five cellulases accumulated at high levels when grown on xylose. Production of cellulases in the presence of pretreated-biomass C5-sugar liquors was investigated, and cellulases accumulated to much higher enzyme titers than those obtained for traditional fungal cell factories with cellulase-inducing substrates. CONCLUSIONS: By replacing expensive substrates with a cheap by-product carbon source, the use of C5-sugar liquors directly derived from LCB pretreatment processes not only reduces enzyme production costs, but also lowers operational costs by eliminating the need for off-site enzyme production, purification, concentration, transport and dilution.

Xyloglucan breakdown by endo-xyloglucanase family 74 from Aspergillus fumigatus.

Xyloglucan is the most abundant hemicellulose in primary walls of spermatophytes except for grasses. Xyloglucan-degrading enzymes are important in lignocellulosic biomass hydrolysis because they remove xyloglucan, which is abundant in monocot-derived biomass. Fungal genomes encode numerous xyloglucanase genes, belonging to at least six glycoside hydrolase (GH) families. GH74 endo-xyloglucanases cleave xyloglucan backbones with unsubstituted glucose at the -1 subsite or prefer xylosyl-substituted residues in the -1 subsite. In this work, 137 GH74-related genes were detected by examining 293 Eurotiomycete genomes and Ascomycete fungi contained one or no GH74 xyloglucanase gene per genome. Another interesting feature is that the triad of tryptophan residues along the catalytic cleft was found to be widely conserved among Ascomycetes. The GH74 from Aspergillus fumigatus (AfXEG74) was chosen as an example to conduct comprehensive biochemical studies to determine the catalytic mechanism. AfXEG74 has no CBM and cleaves the xyloglucan backbone between the unsubstituted glucose and xylose-substituted glucose at specific positions, along the XX motif when linked to regions deprived of galactosyl branches. It resembles an endo-processive activity, which after initial random hydrolysis releases xyloglucan-oligosaccharides as major reaction products. This work provides insights on phylogenetic diversity and catalytic mechanism of GH74 xyloglucanases from Ascomycete fungi.

High-yield production of aryl alcohol oxidase under limited growth conditions in small-scale systems using a mutant Aspergillus nidulans strain.

Aryl alcohol oxidase (MtGloA) is an enzyme that belongs to the ligninolytic consortium and can play an important role in the bioenergy industry. This study investigated production of an MtGloA client enzyme by a mutant strain of Aspergillus nidulans unable to synthesize its own pyridoxine. Pyridoxine limitation can be used to control cell growth, diverting substrate to protein production. In agitated culture, enzyme production was similar when using media with 1 mg/L and without pyridoxine (26.64 ± 6.14 U/mg mycelia and 26.14 ± 8.39 U/mg mycelia using media with and without pyridoxine, respectively). However, the treatment lacking pyridoxine had to be supplemented with pyridoxine after 156 h of fermentation to sustain continued enzyme production. Use of extremely diluted pyridoxine levels allowed reduced fungal growth while maintaining steady enzyme production. Concentrations of 9 and 13.5 µg/L pyridoxine allowed MtGloA production with a growth rate of only 5% of that observed when using the standard 1 mg/L pyridoxine media.

A family of AA9 lytic polysaccharide monooxygenases in Aspergillus nidulans is differentially regulated by multiple substrates and at least one is active on cellulose and xyloglucan.

Fungal genomes contain multiple genes encoding AA9 lytic polysaccharide monooxygenases (LPMOs), a recently discovered class of enzymes known to be active on cellulose and expressed when grown on biomass. Because of extensive genetic and biochemical data already available, Aspergillus nidulans offers an excellent model system to study the need for multiple AA9 LPMOs and their activity during oxidative degradation of biomass. We provide the first report on regulation of the entire family of AA9 LPMOs in A. nidulans over a range of polysaccharides including xylan, xyloglucan, pectin, glucan, and cellulose. We have successfully cloned and expressed AN3046, an AA9 LPMO in A. nidulans that is active on cellulose. Additionally, we performed mass spectral analyses that show the enzyme is active on the hemicellulose xyloglucan. The AN3046 LPMO showed synergy with other hydrolases in degrading sorghum stover. Our data showing activity of the overexpressed LPMO on cellulose and xyloglucan provides further evidence for the breadth of substrates acted on by AA9 LPMOs.

Mechanistic strategies for catalysis adopted by evolutionary distinct family 43 arabinanases.

Arabinanases (ABNs, EC 3.2.1.99) are promising catalysts for environmentally friendly biomass conversion into energy and chemicals. These enzymes catalyze the hydrolysis of the α-1,5-linked L-arabinofuranoside backbone of plant cell wall arabinans releasing arabino-oligosaccharides and arabinose, the second most abundant pentose in nature. In this work, new findings about the molecular mechanisms governing activation, functional differentiation, and catalysis of GH43 ABNs are presented. Biophysical, mutational, and biochemical studies with the hyperthermostable two-domain endo-acting ABN from Thermotoga petrophila (TpABN) revealed how some GH43 ABNs are activated by calcium ions via hyperpolarization of the catalytically relevant histidine and the importance of the ancillary domain for catalysis and conformational stability. On the other hand, the two GH43 ABNs from rumen metagenome, ARN2 and ARN3, presented a calcium-independent mechanism in which sodium is the most likely substituent for calcium ions. The crystal structure of the two-domain endo-acting ARN2 showed that its ability to efficiently degrade branched substrates is due to a larger catalytic interface with higher accessibility than that observed in other ABNs with preference for linear arabinan. Moreover, crystallographic characterization of the single-domain exo-acting ARN3 indicated that its cleavage pattern producing arabinose is associated with the chemical recognition of the reducing end of the substrate imposed by steric impediments at the aglycone-binding site. By structure-guided rational design, ARN3 was converted into a classical endo enzyme, confirming the role of the extended Arg(203)-Ala(230) loop in determining its action mode. These results reveal novel molecular aspects concerning the functioning of GH43 ABNs and provide new strategies for arabinan degradation.

High-yield recombinant xylanase production by Aspergillus nidulans under pyridoxine limitation.

The present study investigated the limitation of pyridoxine on an Aspergillus nidulans culture that produces xylanase B (XynB) as a client enzyme and was unable to synthesize pyridoxine. This technique was used to limit cell growth and divert substrate to product formation for a surface grown culture that could be used in trickle bed reactors. It was observed that growth was limited when pyridoxine was absent, while enzyme production was unaffected. Enzyme production was 1,026 U after 480 h of continuous fermentation, which was similar to a culture that grew on medium with pyridoxine. Furthermore, the present study investigated the growth rate of A. nidulans with pyridoxine in the medium and determined the productivity of XynB production with and without pyridoxine. A maximum growth rate of 0.311/h was observed. The maximum XynB productivity of 21.14 U/g h was achieved when pyridoxine was not added to the medium.

Secretome Analysis of Thermothelomyces thermophilus LMBC 162 Cultivated with Tamarindus indica Seeds Reveals CAZymes for Degradation of Lignocellulosic Biomass.

The analysis of the secretome allows us to identify the proteins, especially carbohydrate-active enzymes (CAZymes), secreted by different microorganisms cultivated under different conditions. The CAZymes are divided into five classes containing different protein families. Thermothelomyces thermophilus is a thermophilic ascomycete, a source of many glycoside hydrolases and oxidative enzymes that aid in the breakdown of lignocellulosic materials. The secretome analysis of T. thermophilus LMBC 162 cultivated with submerged fermentation using tamarind seeds as a carbon source revealed 79 proteins distributed between the five diverse classes of CAZymes: 5.55% auxiliary activity (AAs); 2.58% carbohydrate esterases (CEs); 20.58% polysaccharide lyases (PLs); and 71.29% glycoside hydrolases (GHs). In the identified GH families, 54.97% are cellulolytic, 16.27% are hemicellulolytic, and 0.05 are classified as other. Furthermore, 48.74% of CAZymes have carbohydrate-binding modules (CBMs). Observing the relative abundance, it is possible to state that only thirteen proteins comprise 92.19% of the identified proteins secreted and are probably the main proteins responsible for the efficient degradation of the bulk of the biomass: cellulose, hemicellulose, and pectin.

Functional characterization and oligomerization of a recombinant xyloglucan-specific endo-ß-1,4-glucanase (GH12) from Aspergillus niveus.

Xyloglucan is a major structural polysaccharide of the primary (growing) cell wall of higher plants. It consists of a cellulosic backbone (beta-1,4-linked glucosyl residues) that is frequently substituted with side chains. This report describes Aspergillus nidulans strain A773 recombinant secretion of a dimeric xyloglucan-specific endo-ß-1,4-glucanohydrolase (XegA) cloned from Aspergillus niveus. The ORF of the A. niveus xegA gene is comprised of 714 nucleotides, and encodes a 238 amino acid protein with a calculated molecular weight of 23.5kDa and isoelectric point of 4.38. The optimal pH and temperature were 6.0 and 60°C, respectively. XegA generated a xyloglucan-oligosaccharides (XGOs) pattern similar to that observed for cellulases from family GH12, i.e., demonstrating that its mode of action includes hydrolysis of the glycosidic linkages between glucosyl residues that are not branched with xylose. In contrast to commercial lichenase, mixed linkage beta-glucan (lichenan) was not digested by XegA, indicating that the enzyme did not cleave glucan ß-1,3 or ß-1,6 bonds. The far-UV CD spectrum of the purified enzyme indicated a protein rich in ß-sheet structures as expected for GH12 xyloglucanases. Thermal unfolding studies displayed two transitions with mid-point temperatures of 51.3°C and 81.3°C respectively, and dynamic light scattering studies indicated that the first transition involves a change in oligomeric state from a dimeric to a monomeric form. Since the enzyme is a predominantly a monomer at 60°C, the enzymatic assays demonstrated that XegA is more active in its monomeric state.

Transcriptional profiling of Neurospora crassa Δmak-2 reveals that mitogen-activated protein kinase MAK-2 participates in the phosphate signaling pathway.

The filamentous fungus Neurospora crassa is an excellent model system for examining molecular responses to ambient signals in eukaryotic microorganisms. Inorganic phosphate (Pi) is an essential growth-limiting nutrient in nature and is crucial for the synthesis of nucleic acids and the flow of genetic information. The genetic and molecular mechanisms controlling the response to Pi starvation in N. crassa include at least four genes (nuc-2, preg, pogv, and nuc-1), which are involved in a hierarchical regulatory activation network. In a previous work, we identified a number of genes modulated by NUC-2 protein, including the mak-2 gene, which codes for a mitogen-activated protein kinase (MAPK), suggesting its participation in the phosphate signaling pathway. Thus, to identify other genes involved in metabolic responses to exogenous phosphate sensing and the functioning of the MAPK MAK-2, we performed microarray experiments using a mak-2 knockout strain (Δmak-2) grown under phosphate-shortage conditions by comparing its transcription profile to that of a control strain grown in low- and high-phosphate cultures. These experiments revealed 912 unique differentially expressed genes involved in a number of physiological processes related to phosphate transport, metabolism, and regulation as well as posttranslational modification of proteins, and MAPK signaling pathways. Quantitative Real-time PCR gene expression analysis of 18 selected genes, using independent RNA samples, validated our microarray results. A high Pearson correlation between microarray and quantitative Real-time PCR data was observed. The analysis of these differentially expressed genes in the Δmak-2 strain provide evidence that the mak-2 gene participates in the hierarchical phosphate-signaling pathway in N. crassa in addition to its involvement in other metabolic routes such as the isoprenylation pathway, thus revealing novel aspects of the N. crassa phosphorus-sensing network.

The genome of the anaerobic fungus Orpinomyces sp. strain C1A reveals the unique evolutionary history of a remarkable plant biomass degrader.

Anaerobic gut fungi represent a distinct early-branching fungal phylum (Neocallimastigomycota) and reside in the rumen, hindgut, and feces of ruminant and nonruminant herbivores. The genome of an anaerobic fungal isolate, Orpinomyces sp. strain C1A, was sequenced using a combination of Illumina and PacBio single-molecule real-time (SMRT) technologies. The large genome (100.95 Mb, 16,347 genes) displayed extremely low G+C content (17.0%), large noncoding intergenic regions (73.1%), proliferation of microsatellite repeats (4.9%), and multiple gene duplications. Comparative genomic analysis identified multiple genes and pathways that are absent in Dikarya genomes but present in early-branching fungal lineages and/or nonfungal Opisthokonta. These included genes for posttranslational fucosylation, the production of specific intramembrane proteases and extracellular protease inhibitors, the formation of a complete axoneme and intraflagellar trafficking machinery, and a near-complete focal adhesion machinery. Analysis of the lignocellulolytic machinery in the C1A genome revealed an extremely rich repertoire, with evidence of horizontal gene acquisition from multiple bacterial lineages. Experimental analysis indicated that strain C1A is a remarkable biomass degrader, capable of simultaneous saccharification and fermentation of the cellulosic and hemicellulosic fractions in multiple untreated grasses and crop residues examined, with the process significantly enhanced by mild pretreatments. This capability, acquired during its separate evolutionary trajectory in the rumen, along with its resilience and invasiveness compared to prokaryotic anaerobes, renders anaerobic fungi promising agents for consolidated bioprocessing schemes in biofuels production.

Molecular insights into substrate specificity and thermal stability of a bacterial GH5-CBM27 endo-1,4-ß-D-mannanase.

The breakdown of ß-1,4-mannoside linkages in a variety of mannan-containing polysaccharides is of great importance in industrial processes such as kraft pulp delignification, food processing and production of second-generation biofuels, which puts a premium on studies regarding the prospection and engineering of ß-mannanases. In this work, a two-domain ß-mannanase from Thermotoga petrophila that encompasses a GH5 catalytic domain with a C-terminal CBM27 accessory domain, was functionally and structurally characterized. Kinetic and thermal denaturation experiments showed that the CBM27 domain provided thermo-protection to the catalytic domain, while no contribution on enzymatic activity was observed. The structure of the catalytic domain determined by SIRAS revealed a canonical (α/ß)(8)-barrel scaffold surrounded by loops and short helices that form the catalytic interface. Several structurally related ligand molecules interacting with TpMan were solved at high-resolution and resulted in a wide-range representation of the subsites forming the active-site cleft with residues W134, E198, R200, E235, H283 and W284 directly involved in glucose binding.

Phanerochaete chrysosporium produces a diverse array of extracellular enzymes when grown on sorghum.

In an effort to understand how fungi degrade biomass, we grew Phanerochaete chrysosporium on sorghum stover and chronicled the growth of the fungus over the course of 14 days. The fungal mass grew steadily until the fifth day, reaching 0.06 mg of cells per milligram of dry mass, which fell by the seventh day and stayed at nearly the same level until day 14. After 1 day, hemicellulases, cellulases, and polygalacturonases were detected in the extracellular fluid at 1.06, 0.34, and 0.20 U/ml, respectively. Proteomic studies performed with the extracellular fluid using liquid chromatography­tandem mass spectrometry identified 57, 116, and 102 degradative enzymes targeting cellulose, hemicellulose, pectin, lignin, proteins, and lipids on days 1, 7, and 14, respectively. Significant concentrations of breakdown products of the sorghum polysaccharides were detected in the extracellular fluid indicating that the enzymes were breaking the polysaccharides, and after 14 days, almost 39% of the sorghum sugars had been used by the fungus. Our results suggest that P. chrysosporium produces a set of enzymes to degrade the components of lignocellulose from the beginning of its growth, but modifies the complement of enzymes it secretes over time to adapt to the particular substrate available.

Mode of operation and low-resolution structure of a multi-domain and hyperthermophilic endo-ß-1,3-glucanase from Thermotoga petrophila.

1,3-ß-Glucan depolymerizing enzymes have considerable biotechnological applications including biofuel production, feedstock-chemicals and pharmaceuticals. Here we describe a comprehensive functional characterization and low-resolution structure of a hyperthermophilic laminarinase from Thermotoga petrophila (TpLam). We determine TpLam enzymatic mode of operation, which specifically cleaves internal ß-1,3-glucosidic bonds. The enzyme most frequently attacks the bond between the 3rd and 4th residue from the non-reducing end, producing glucose, laminaribiose and laminaritriose as major products. Far-UV circular dichroism demonstrates that TpLam is formed mainly by beta structural elements, and the secondary structure is maintained after incubation at 90°C. The structure resolved by small angle X-ray scattering, reveals a multi-domain structural architecture of a V-shape envelope with a catalytic domain flanked by two carbohydrate-binding modules.

Comparison of transcriptional and translational changes caused by long-term menadione exposure in Aspergillus nidulans.

Under long-term oxidative stress caused by menadione sodium bisulfite, genome-wide transcriptional and proteome-wide translational changes were compared in Aspergillus nidulans vegetative cells. The comparison of proteomic and DNA microarray expression data demonstrated that global gene expression changes recorded with either flip-flop or dendrimer cDNA labeling techniques supported proteome changes moderately with 40% and 34% coincidence coefficients, respectively. Enzyme levels in the glycolytic pathway were alternating, which was a direct consequence of fluctuating gene expression patterns. Surprisingly, enzymes in the vitamin B2 and B6 biosynthetic pathways were repressed concomitantly with the repression of some protein folding chaperones and nuclear transport elements. Under long-term oxidative stress, the peroxide-detoxifying peroxiredoxins and cytochrome c peroxidase were replaced by thioredoxin reductase, a nitroreductase and a flavohemoprotein, and protein degradation became predominant to eliminate damaged proteins.

High-temperature enzymatic breakdown of cellulose.

Cellulose is an abundant and renewable biopolymer that can be used for biofuel generation; however, structural entrapment with other cell wall components hinders enzyme-substrate interactions, a key bottleneck for ethanol production. Biomass is routinely subjected to treatments that facilitate cellulase-cellulose contacts. Cellulases and glucosidases act by hydrolyzing glycosidic bonds of linear glucose ß-1,4-linked polymers, producing glucose. Here we describe eight high-temperature-operating cellulases (TCel enzymes) identified from a survey of thermobacterial and archaeal genomes. Three TCel enzymes preferentially hydrolyzed soluble cellulose, while two preferred insoluble cellulose such as cotton linters and filter paper. TCel enzymes had temperature optima ranging from 85°C to 102°C. TCel enzymes were stable, retaining 80% of initial activity after 120 h at 85°C. Two modes of cellulose breakdown, i.e., with endo- and exo-acting glucanases, were detected, and with two-enzyme combinations at 85°C, synergistic cellulase activity was observed for some enzyme combinations.

Phosphatidylinositol phospholipase C mediates carbon sensing and vegetative nuclear duplication rates in Aspergillus nidulans.

In this work, we disrupted one of three putative phosphatidylinositol phospholipase C genes of Aspergillus nidulans and studied its effect on carbon source sensing linked to vegetative mitotic nuclear division. We showed that glucose does not affect nuclear division rates during early vegetative conidial germination (6-7 h) in either the wild type or the plcA-deficient mutant. Only after 8 h of cultivation on glucose did the mutant strain present some decrease in nuclear duplication. However, decreased nuclear division rates were observed in the wild type when cultivated in media amended with polypectate, whereas our plcA-deficient mutant did not show slow nuclear duplication rates when grown on this carbon source, even though it requires induction and secretion of multiple pectinolytic enzymes to be metabolized. Thus, plcA appears to be directly linked to high-molecular-weight carbon source sensing.

Functional and biophysical characterization of a hyperthermostable GH51 α-L-arabinofuranosidase from Thermotoga petrophila.

A hyperthermostable glycoside hydrolase family 51 (GH51) α-L-arabinofuranosidase from Thermotoga petrophila RKU-1 (TpAraF) was cloned, overexpressed, purified and characterized. The recombinant enzyme had optimum activity at pH 6.0 and 70°C with linear α-1,5-linked arabinoheptaose as substrate. The substrate cleavage pattern monitored by capillary zone electrophoresis showed that TpAraF is a classical exo-acting enzyme producing arabinose as its end-product. Far-UV circular dichroism analysis displayed a typical spectrum of α/ß barrel proteins analogously observed for other GH51 α-L-arabinofuranosidases. Moreover, TpAraF was crystallized in two crystalline forms, which can be used to determine its crystallographic structure.

Exploring lignin depolymerization by a bi-enzyme system containing aryl alcohol oxidase and lignin peroxidase in aqueous biocompatible ionic liquids.

Enzymatic depolymerization of lignin to produce low molecular weight products requires mild reaction conditions and exhibits higher selectivity compared to thermochemical lignin depolymerization. However, it remains challenging to depolymerize lignin enzymatically, partially due to the low solubility of lignin in aqueous phase. This study aimed to develop a novel approach to combine aqueous lignin extraction with enzymatic lignin depolymerization in biocompatible ionic liquids. A bi-enzyme system containing aryl alcohol oxidase (AAO) and lignin peroxidase (LiP) was developed to depolymerize lignin. Temperature and pH profiles for LiP and AAO were determined. Biocompatibilities of LiP and AAO in different deep eutectic solvents and ionic liquids were investigated. Aqueous cholinium glycinate was found to be an efficient and suitable solvent to solubilize lignin and serve as a biocompatible medium for enzymes to work. LiP and AAO together reduced lignin molecular weight in both solid and liquid phase after enzymatic lignin depolymerization.

Thermal-induced conformational changes in the product release area drive the enzymatic activity of xylanases 10B: Crystal structure, conformational stability and functional characterization of the xylanase 10B from Thermotoga petrophila RKU-1.

Endo-xylanases play a key role in the depolymerization of xylan and recently, they have attracted much attention owing to their potential applications on biofuels and paper industries. In this work, we have investigated the molecular basis for the action mode of xylanases 10B at high temperatures using biochemical, biophysical and crystallographic methods. The crystal structure of xylanase 10B from hyperthermophilic bacterium Thermotoga petrophila RKU-1 (TpXyl10B) has been solved in the native state and in complex with xylobiose. The complex crystal structure showed a classical binding mode shared among other xylanases, which encompasses the -1 and -2 subsites. Interestingly, TpXyl10B displayed a temperature-dependent action mode producing xylobiose and xylotriose at 20°C, and exclusively xylobiose at 90°C as assessed by capillary zone electrophoresis. Moreover, circular dichroism spectroscopy suggested a coupling effect of temperature-induced structural changes with this particular enzymatic behavior. Molecular dynamics simulations supported the CD analysis suggesting that an open conformational state adopted by the catalytic loop (Trp297-Lys326) provokes significant modifications in the product release area (+1,+2 and +3 subsites), which drives the enzymatic activity to the specific release of xylobiose at high temperatures.