Mapping secondary substrate-binding sites on the GH11 xylanase from Bacillus subtilis.

Xylanases are of significant interest for biomass conversion technologies. Here, we investigated the allosteric regulation of xylan hydrolysis by the Bacillus subtilis GH11 endoxylanase. Molecular dynamics simulations (MDS) in the presence of xylobiose identified binding to the active site and two potential secondary binding sites (SBS) around surface residues Asn54 and Asn151. Arabinoxylan titration experiments with single cysteine mutants N54C and N151C labeled with the thiol-reactive fluorophore acrylodan or the ESR spin-label MTSSL validated the MDS results. Ligand binding at the SBS around Asn54 confirms previous reports, and analysis of the second SBS around N151C discovered in the present study includes residues Val98/Ala192/Ser155/His156. Understanding the regulation of xylanases contributes to efforts for industrial decarbonization and to establishing a sustainable energy matrix.

Optimization of xylanase production by Pichia kudriavzevii and Candida tropicalis isolated from the wood product workshop.

Enzymatic compounds can be found abundantly and provide numerous advantages in microbial organisms. Xylanases are used in various pharmaceutical, food, livestock, poultry, and paper industries. This study aimed to investigate xylanase-producing yeasts, xylose concentration curve and their enzymatic activity under various factors including carbon and nitrogen sources, temperature, and pH. Enzyme activity was evaluated under different conditions before, during, and after purification. The yeast strains were obtained from the wood product workshop and were subsequently cultivated on YPD (yeast extract peptone dextrose) medium. Additionally, the growth curve of the yeast and its molecular identification were conducted. The optimization and design process of xylan isolated from corn wood involved the use of Taguchi software to test different parameters like carbon and nitrogen sources, temperature, and pH, with the goal of determining the most optimal conditions for enzyme production. In addition, the Taguchi method was utilized to conduct a multifactorial optimization of xylanase enzyme activity. The isolated species were partially purified using ammonium sulfate precipitation and dialysis bag techniques. The results indicated that 3 species (8S, 18S, and 16W) after molecular identification based on 18S rRNA gene sequencing were identified as Candida tropicalis SBN-IAUF-1, Candida tropicalis SBN-IAUF-3, and Pichia kudriavzevii SBN-IAUF-2, respectively. The optimal parameters for wheat carbon source and peptone nitrogen source were found at 50 °C and pH 9.0 through single-factor optimization. By using the Taguchi approach, the best combination for highest activity was rice-derived carbon source and peptone nitrogen source at 50 °C and pH 6.0. The best conditions for xylanase enzyme production in single-factor optimization of wheat bran were 2135.6 U/mL, peptone 4475.25 U/mL, temperature 50 °C 1868 U/mL, and pH 9.0 2002.4 U/mL. Among the tested yeast, Candida tropicalis strain SBN-IAUF-1 to the access number MZ816946.1 in NCBI was found to be the best xylanase product. The highest ratio of enzyme production at the end of the delayed phase and the beginning of the logarithmic phase was concluded by comparing the growth ratio of 8S, 16W, and 18S yeasts with the level of enzymatic activity. This is the first report on the production of xylan polymer with a relative purity of 80% in Iran. The extracellular xylanases purified from the yeast species of C. tropicalis were introduced as a desirable biocatalyst due to their high enzymatic activity for the degradation of xylan polymers.

Purification and characterization of the low molecular weight xylanase from Bacillus cereus L-1.

Xylanase is widely used in various industries such as food processing, paper, textiles, and leather tanning. In this study, Bacillus cereus L-1 strain was isolated and identified as capable of producing low molecular weight xylanase through 16 s rRNA sequencing. Maximum xylanase yield of 15.51 ± 2.08 U/mL was achieved under optimal fermentation conditions (5% inoculum, 20 g/L xylan, pH 6.0, for 24 h). After purification via ammonium sulfate precipitation and High-S ion exchange chromatography, electrophoretic purity xylanase was obtained with a 28-fold purification and specific activity of 244.97 U/mg. Xylanase had an optimal pH of 6.5 and temperature of 60 °C and displayed thermostability at 30 °C and 40 °C with 48.56% and 45.97% remaining activity after 180 min, respectively. The xylanase retained more than 82.97% of its activity after incubation for 24 h at pH 5.0 and was sensitive to metal ions, especially Mg2+ and Li+. Purified xylanase showed a molecular weight of 23 kDa on SDS-PAGE, and partial peptide sequencing revealed homology to the endo-1,4-beta-xylanase with a molecular weight of 23.3 kDa through LC/MS-MS (liquid chromatography-tandem mass spectrometry). This study suggests that the purified xylanase is easier to purify and enriches low molecular weight xylanases from bacteria source.

A genome-proteome-based approach for xylan degradation by Cohnella sp. AR92.

Several members of Cohnella genus have been reported as xylanolytic bacteria with significant capacity as carbohydrate-active enzyme producers (CAZymes), whose mechanisms involving xylan degradation are a key goal for suitable applications in bio-based industries. Using Cohnella sp. AR92 bacterium, we ensembled a genomic-proteomic approach to assess plant biomass conversion targeting its xylanolytic set of enzymes. Also, the genomic traits of the strain AR92 were compared to other Cohnella spp., showing a significant variability in terms of genome sizes and content of genes that code CAZymes. The AR92 strain genome harbours 209 CAZymes encoding sequences active on different polysaccharides, particularly directed towards xylans. Concurrent proteomic data recovered from cultures containing three kinds of lignocellulosic-derived substrates showed a broad set of xylan-degrading enzymes. The most abundant CAZymes expressed in the different conditions assayed were endo-ß-1,4-xylanases belonging to the GH11 and GH10 families, enzymes that were previously proved to be useful in the biotransformation of lignocellulosic biomass derived from sugarcane as well as onto xylan-enriched substrates. Therefore, considering the large reserve of CAZymes of Cohnella sp. AR92, a xylan processing model for AR92 strain is proposed.

Evaluation of endoglucanase and xylanase production by Aspergillus tamarii cultivated in agro-industrial lignocellulosic biomasses.

To better understand the production of enzymes of industrial interest from microorganisms with biotechnological potential using lignocellulosic biomass, we evaluated the production of endoglucanase and xylanase from Aspergillus tamarii. CAZymes domains were evaluated in the genome, and a screening of the enzymatic potential of A. tamarii in various agricultural biomasses was done. The enzymatic profile could be associated with the biomass complexity, with increased biomass recalcitrance yielding higher activity. A time-course profile defined 48 h of cultivation as the best period for cultivating A. tamarii in sugarcane bagasse reached 12.05 IU/mg for endoglucanase and 74.86 IU/mg for xylanase. Using 0.1% (w/v) tryptone as the only nitrogen source and 12 µmol/L CuSO4 addition had an overall positive effect on the enzymatic activity and protein production. A 22 factorial central composite design was used then to investigate the simultaneous influence of tryptone and CuSO4 on enzyme activity. Tryptone strongly affected enzymatic activity, decreasing endoglucanase activity but increasing xylanase activity. CuSO4 supplementation was advantageous for endoglucanases, increasing their activity, and it had a negative effect on xylanases. But overall, the experimental design increased the enzymatic activity of all biomasses used. For the clean cotton residue, the experimental design was able to reach the highest enzyme activity for endoglucanase and xylanase, with 1.195 IU/mL and 6.353 IU/mL, respectively. More experimental studies are required to investigate how the biomass induction effect impacts enzyme production.

Application of a recombinant GH10 endoxylanase from Thermoascus aurantiacus for xylooligosaccharide production from sugarcane bagasse and probiotic bacterial growth.

Xylooligosaccharides (XOs) are a promising class of prebiotics capable of selectively stimulating the growth of the beneficial intestinal microbiota against intestinal pathogens. They can be obtained from xylan present in residual lignocellulosic material from agriculture. Thus, in this study we produced XOs by extracting xylan from sugarcane bagasse and hydrolyzing it using the GH10 xylanase from Thermoascus aurantiacus expressed by Pichia pastoris. An alkaline method to extract xylan is described, which resulted in 83.40% of xylan recovery and low amounts of cellulose and lignin. The enzymatic hydrolysate exhibited a mixture of XOs containing mainly xylobiose, xylotriose and xylotetraose. These oligosaccharides stimulated the growth of Lactobacillus casei, L. rhamnosus, L. fermentum and L. bulgaricus strains, which were able to produce organic acids, especially acetic acid. These findings demonstrate the possibility to redirect crop by-products to produce XOs and their use as a supplement to stimulate the growth of probiotic strains.

Endo-xylanases from Cohnella sp. AR92 aimed at xylan and arabinoxylan conversion into value-added products.

The genus Cohnella belongs to a group of Gram-positive endospore-forming bacteria within the Paenibacillaceae family. Although most species were described as xylanolytic bacteria, the literature still lacks some key information regarding their repertoire of xylan-degrading enzymes. The whole genome sequence of an isolated xylan-degrading bacterium Cohnella sp. strain AR92 was found to contain five genes encoding putative endo-1,4-ß-xylanases, of which four were cloned, expressed, and characterized to better understand the contribution of the individual endo-xylanases to the overall xylanolytic properties of strain AR92. Three of the enzymes, CoXyn10A, CoXyn10C, and CoXyn11A, were shown to be effective at hydrolyzing xylans-derived from agro-industrial, producing oligosaccharides with substrate conversion values of 32.5%, 24.7%, and 10.6%, respectively, using sugarcane bagasse glucuronoarabinoxylan and of 29.9%, 19.1%, and 8.0%, respectively, using wheat bran-derived arabinoxylan. The main reaction products from GH10 enzymes were xylobiose and xylotriose, whereas CoXyn11A produced mostly xylooligosaccharides (XOS) with 2 to 5 units of xylose, often substituted, resulting in potentially prebiotic arabinoxylooligosaccharides (AXOS). The endo-xylanases assay displayed operational features (temperature optima from 49.9 to 50.4 °C and pH optima from 6.01 to 6.31) fitting simultaneous xylan utilization. Homology modeling confirmed the typical folds of the GH10 and GH11 enzymes, substrate docking studies allowed the prediction of subsites (- 2 to + 1 in GH10 and - 3 to + 1 in GH11) and identification of residues involved in ligand interactions, supporting the experimental data. Overall, the Cohnella sp. AR92 endo-xylanases presented significant potential for enzymatic conversion of agro-industrial by-products into high-value products.Key points• Cohnella sp. AR92 genome encoded five potential endo-xylanases.• Cohnella sp. AR92 enzymes produced xylooligosaccharides from xylan, with high yields.• GH10 enzymes from Cohnella sp. AR92 are responsible for the production of X2 and X3 oligosaccharides.• GH11 from Cohnella sp. AR92 contributes to the overall xylan degradation by producing substituted oligosaccharides.

Autodisplay of an endo-1,4-ß-xylanase from Clostridium cellulovorans in Escherichia coli for xylans degradation.

The goal of this work was the autodisplay of the endo ß-1,4-xylanase (XynA) from Clostridium cellulovorans in Escherichia coli using the AIDA system to carry out whole-cell biocatalysis and hydrolysate xylans. For this, pAIDA-xynA vector containing a synthetic xynA gene was fused to the signal peptide of the toxin subunit B Vibro cholere (ctxB) and the auto-transporter of the synthetic aida gene, which encodes for the connector peptide and ß-barrel of the auto-transporter (AT-AIDA). E. coli TOP10 cells were transformed and the biocatalyst was characterized using beechwood xylans as substrate. Optimal operational conditions were temperature of 55 °C and pH 6.5, and the Michaelis-Menten catalytic constants Vmax and Km were 149 U/gDCW and 6.01 mg/mL, respectively. Xylanase activity was inhibited by Cu2+, Zn2+ and Hg2+ as well as EDTA, detergents, and organic acids, and improved by Ca2+, Co2+ and Mn2+ ions. Ca2+ ion strongly enhanced the xylanolytic activity up to 2.4-fold when 5 mM CaCl2 were added. Also, Ca2+ improved enzyme stability at 60 and 70 °C. Results suggest that pAIDA-xynA vector has the ability to express functional xylanase to perform whole-cell biocatalysis in order to hydrolysate xylans from hemicellulose feedstock.

Characterization of a new bifunctional endo-1,4-ß-xylanase/esterase found in the rumen metagenome.

Metagenomic data mining of the Nellore cattle rumen microbiota identified a new bifunctional enzyme, endo-1,4-ß-xylanase/esterase, which was subsequently overexpressed in E. coli BL21 (DE3). This enzyme was stable at pH intervals of 5 to 6.5 and temperatures between 30 and 45 °C, and under the test conditions, it had a Vmax of 30.959 ± 2.334 µmol/min/mg, Km of 3.6 ± 0.6 mM and kcat of 2.323 ± 175 s-1. Additionally, the results showed that the enzyme is tolerant to NaCl and organic solvents and therefore is suitable for industrial environments. Xylanases are widely applicable, and the synergistic activity of endo-1,4-ß-xylanase/esterase in a single molecule will improve the degradation efficiency of heteroxylans via the creation of xylanase binding sites. Therefore, this new molecule has the potential for use in lignocellulosic biomass processing and as an animal feed food additive and could improve xylooligosaccharide production efficiency.

Purification and characterization of two thermostable xylanases from a halotolerant Bacillus sp. Asc6BA isolated from Salar de Ascotán, Atacama Desert.

Two extracellular xylanases, denominated X2 and X3, were purified and characterized from the halotolerant bacterium Bacillus sp. Asc6BA isolated from "Salar de Ascotán" in the Atacama Desert. Xylanases were purified by anion exchange, cation exchange and size exclusion liquid chromatography. Xylanase X2 and X3 were purified ~ 690-fold and ~ 629-fold, respectively, compared to the concentrated extracellular fraction with a final specific activity of 169 and 154 u mg-1, respectively. Optimal conditions of pH and temperature of xylanolytic activity were 6.0 and 60 °C for X2 and 7.0 and 60 °C for X3. Half-life of X2 xylanase was 30 min at 50 °C, while X3 xylanase was remarkably more thermostable, retaining more than 70% of its activity after 32 h of incubation at 50 °C. X2 exhibited Km, Vmax and kcat values of 7.17 mg mL-1, 1.28 mM min-1 mg-1 and 425.33 s-1, respectively. X3 exhibited Km, Vmax and kcat values of 6.00 mg mL-1, 19.25 mM min-1 mg-1 and 82,515 s-1, respectively. In addition to their thermal stabilities, these enzymes were shown to be resistant to freeze-drying. These stability properties, in addition to the ability of these enzymes to be active in a wide range of temperatures and pHs, make these xylanases good candidates for industrial applications.

Expression profiling of endo-xylanases during ripening of strawberry cultivars with contrasting softening rates. Influence of postharvest and hormonal treatments.

BACKGROUND: Softening is one of the main features that determine fruit quality during strawberry (Fragaria x ananassa, Duch.) ripening and storage. Being closely related to textural changes, the molecular and biochemical bases underlying strawberry cell-wall metabolism is a matter of interest. Here we investigated the abundance of transcripts encoding putative strawberry endo-xylanases in plant tissues, during fruit ripening and under postharvest and hormonal treatments. Total xylanase activity and expression of related genes in strawberry varieties with contrasting firmness were analyzed. RESULTS: FaXynA and FaXynC mRNA abundance was significantly higher than FaXynB in each plant tissue studied. Higher total xylanase activity was detected at the end of the ripening of the softer cultivar ('Toyonoka') in comparison with the firmer one ('Camarosa'), correlating with the abundance of FaXynA and FaXynC transcripts. Postharvest 1-methylcyclopropene treatment up-regulated FaXynA and FaXynC expressions. FaXynC mRNA abundance decreased with heat treatment but the opposite was observed for FaXynA. Calcium chloride treatment down-regulated FaXynA and FaXynC expression. Both genes responded differently to plant growth regulators' exposure. FaXynC expression was down-regulated by auxins and gibberellins treatment and up-regulated by abscisic acid. FaXynA was up-regulated by auxins, while no changes in mRNA levels were evident by abscisic acid and gibberellins treatment. Ethephon exposure did not change FaXynA and FaXynC expressions. CONCLUSION: New knowledge about the presence of xylanases in ripening strawberry fruit and their response to postharvest and hormonal treatments is provided. Our findings suggest a role for endo-xylanases in hemicelluloses depolymerization and possibly in strawberry fruit softening. © 2020 Society of Chemical Industry.

Mangrove soil as a source for novel xylanase and amylase as determined by cultivation-dependent and cultivation-independent methods.

Xylanase and α-amylase enzymes participate in the degradation of organic matter, acting in hemicellulose and starch mineralization, respectively, and are in high demand for industrial use. Mangroves represent a promising source for bioprospecting enzymes due to their unique characteristics, such as fluctuations in oxic/anoxic conditions and salinity. In this context, the present work aimed to bioprospect xylanases from mangrove soil using cultivation-dependent and cultivation-independent methods. Through screening from a metagenomic library, three potentially xylanolytic clones were obtained and sequenced, and reads were assembled into contigs and annotated. The contig MgrBr135 was affiliated with the Planctomycetaceae family and was one of 30 ORFs selected for subcloning that demonstrated only amylase activity. Through the cultivation method, 38 bacterial isolates with xylanolytic activity were isolated. Isolate 11 showed an enzymatic index of 10.9 using the plate assay method. Isolate 39 achieved an enzyme activity of 0.43 U/mL using the colorimetric method with 3,5-dinitrosalicylic acid. Isolate 39 produced xylanase on culture medium with salinity ranging from 1.25 to 5%. Partial 16S rRNA gene sequencing identified isolates in the Bacillus and Paenibacillus genera. The results of this study highlight the importance of mangroves as an enzyme source and show that bacterial groups can be used for starch and hemicellulose degradation.

Penicillium purpurogenum Produces a Set of Endoxylanases: Identification, Heterologous Expression, and Characterization of a Fourth Xylanase, XynD, a Novel Enzyme Belonging to Glycoside Hydrolase Family 10.

The fungus Penicillium purpurogenum grows on a variety of natural carbon sources and secretes a large number of enzymes which degrade the polysaccharides present in lignocellulose. In this work, the gene coding for a novel endoxylanase has been identified in the genome of the fungus. This gene (xynd) possesses four introns. The cDNA has been expressed in Pichia pastoris and characterized. The enzyme, XynD, belongs to family 10 of the glycoside hydrolases. Mature XynD has a calculated molecular weight of 40,997. It consists of 387 amino acid residues with an N-terminal catalytic module, a linker rich in ser and thr residues, and a C-terminal family 1 carbohydrate-binding module. XynD shows the highest identity (97%) to a putative endoxylanase from Penicillium subrubescens but its highest identity to a biochemically characterized xylanase (XYND from Penicillium funiculosum) is only 68%. The enzyme has a temperature optimum of 60 °C, and it is highly stable in its pH optimum range of 6.5-8.5. XynD is the fourth biochemically characterized endoxylanase from P. purpurogenum, confirming the rich potential of this fungus for lignocellulose biodegradation. XynD, due to its wide pH optimum and stability, may be a useful enzyme in biotechnological procedures related to this biodegradation process.

Engineered GH11 xylanases from Orpinomyces sp. PC-2 improve techno-functional properties of bread dough.

BACKGROUND: Endo-1,4-ß-xylanases have marked hydrolytic activity towards arabinoxylans. Xylanases (xynA) produced by the anaerobic fungus Orpinomyces sp. strain PC-2 have been shown to be superior in specific activity, which strongly suggests their applicability in the bakery industry for the processing of whole-wheat flour containing xylans. In the present study, two xylanases from this source, the small wild-type xylanase SWT and the small mutant xylanase SM2 (V108A, A199T), were expressed in Escherichia coli, purified, characterized, tested for their ability to hydrolyze whole-wheat flour and applied in dough processing. RESULTS: Both purified SM2 and SWT showed high specific activity against oat spelt xylan and wheat arabinoxylan, exhibiting maximum activity at pH 3-7 and 60 °C. SM2 was more thermostable than SWT, which suggests that the mutations enhanced its stability. Both SWT and SM2 were able to hydrolyze whole-wheat flour, and evaluation of their applicability in dough processing by the sponge method indicated that use of these enzymes increased dough volume by 60% and reduced texture hardness by more than 50%, while gumminess and chewiness were reduced by 40%. CONCLUSION: The recombinant xylanases showed potential for application in bakery processing and can improve techno-functional properties in sponges. © 2018 Society of Chemical Industry.

Regulation of Aspergillus nidulans CreA-Mediated Catabolite Repression by the F-Box Proteins Fbx23 and Fbx47.

The attachment of one or more ubiquitin molecules by SCF (Skp-Cullin-F-box) complexes to protein substrates targets them for subsequent degradation by the 26S proteasome, allowing the control of numerous cellular processes. Glucose-mediated signaling and subsequent carbon catabolite repression (CCR) are processes relying on the functional regulation of target proteins, ultimately controlling the utilization of this carbon source. In the filamentous fungus Aspergillus nidulans, CCR is mediated by the transcription factor CreA, which modulates the expression of genes encoding biotechnologically relevant enzymes. Although CreA-mediated repression of target genes has been extensively studied, less is known about the regulatory pathways governing CCR and this work aimed at further unravelling these events. The Fbx23 F-box protein was identified as being involved in CCR and the Δfbx23 mutant presented impaired xylanase production under repressing (glucose) and derepressing (xylan) conditions. Mass spectrometry showed that Fbx23 is part of an SCF ubiquitin ligase complex that is bridged via the GskA protein kinase to the CreA-SsnF-RcoA repressor complex, resulting in the degradation of the latter under derepressing conditions. Upon the addition of glucose, CreA dissociates from the ubiquitin ligase complex and is transported into the nucleus. Furthermore, casein kinase is important for CreA function during glucose signaling, although the exact role of phosphorylation in CCR remains to be determined. In summary, this study unraveled novel mechanistic details underlying CreA-mediated CCR and provided a solid basis for studying additional factors involved in carbon source utilization which could prove useful for biotechnological applications.IMPORTANCE The production of biofuels from plant biomass has gained interest in recent years as an environmentally friendly alternative to production from petroleum-based energy sources. Filamentous fungi, which naturally thrive on decaying plant matter, are of particular interest for this process due to their ability to secrete enzymes required for the deconstruction of lignocellulosic material. A major drawback in fungal hydrolytic enzyme production is the repression of the corresponding genes in the presence of glucose, a process known as carbon catabolite repression (CCR). This report provides previously unknown mechanistic insights into CCR through elucidating part of the protein-protein interaction regulatory system that governs the CreA transcriptional regulator in the reference organism Aspergillus nidulans in the presence of glucose and the biotechnologically relevant plant polysaccharide xylan.

Improvement of bread making quality by supplementation with a recombinant xylanase produced by Pichia pastoris.

Xylanases (EC 3.2.1.8) are hydrolytic enzymes, which randomly cleave the ß-1,4-linked xylose residues from xylan. The synthetic gene xynBS27 from Streptomyces sp. S27 was successfully cloned and expressed in Pichia pastoris. The full-length gene consists of 729 bp and encodes 243 amino acids including 51 residues of a putative signal peptide. This enzyme was purified in two steps and was shown to have a molecular weight of 20 kDa. The purified r-XynBS27 was active against beechwood xylan and oat spelt xylan as expected for GH 11 family. The optimum pH and temperature values for the enzyme were 6.0 and 75 °C, respectively. The Km and Vmax were 12.38 mg/mL and 13.68 µmol min/mg, respectively. The r-XynBS27 showed high xylose tolerance and was inhibited by some metal ions and by SDS. r-XynBS27 was employed as an additive in the bread making process. A decrease in firmness, stiffness and consistency, and improvements in specific volume and reducing sugar content were recorded.

Biochemical and biophysical characterization of novel GH10 xylanase prospected from a sugar cane bagasse compost-derived microbial consortia.

Environmental issues are promoting the development of innovative technologies for the production of renewable energy and "green products" from plant biomass residues. These technologies rely on the conversion of the plant cell wall (PCW) polysaccharides into simple sugars, which involve synergistic activities of different PCW degrading enzymes, including xylanases; these are widely applied in food and feed sectors, paper and textile industries, among others. We cloned, expressed and biochemically characterized a novel xylanase (Xyn10) from the GH10 identified in a metatranscriptome of compost-derived microbial consortia and determined its low-resolution SAXS molecular envelope in solution. Our results reveal that Xyn10 is a monomeric flexible globular enzyme, with high stability with a broad pH range from 4 to 10 and optimal activity conditions at pH 7 and 40 °C. Only 10% of activity loss was observed after the enzyme was incubated for 30 h at 40 °C with a pH ranging from 5 to 10. Moreover, Xyn10 maintained 100% of its initial activity after incubation for 120 h at 40 °C and 51% after incubation for 24 h at 50 °C (pH = 7.0). Xyn10 shows endocatalytic activity towards xylan and arabinoxylan, liberating xylose, xylobiose, 1,2-α-d-methylglucuronic acid decorated xylotriose, and 1,3-α-l-arabinofuranose decorated xylobiose and xylotriose oligosaccharides.

Impact of the removal of N-terminal non-structured amino acids on activity and stability of xylanases from Orpinomyces sp. PC-2.

Xylanases catalyze the random hydrolysis of xylan backbone from plant biomass and thus, they have application in the production of biofuels, Kraft pulps biobleaching and feed industry. Here, xylanases derived from Orpinomyces sp. PC-2 were engineered guided by molecular dynamics methods to obtain more thermostable enzymes. Based on these models, 27 amino acid residues from the N-terminal were predicted to reduce protein stability and the impact of this removal was validated to two enzyme constructs: small xylanase Wild-Type (SWT) obtained from Wild-Type xylanase (WT) and small xylanase Mutant (SM2) generated from M2 mutant xylanase (V135A, A226T). The tail removal promoted increase in specific activity of purified SWT and SM2, which achieved 5,801.7 and 5,106.8Umg-1 of protein, respectively, while the WT activity was 444.1Umg-1 of protein. WT, SWT and SM2 showed half-life values at 50°C of 0.8, 2.3 and 29.5h, respectively. Overall, in view of the results, we propose that the presence of non-structured amino acid in the N-terminal leads to destabilization of the xylanases and may promote less access of the substrate to the active site. Therefore, its removal may promote increased stability and enzymatic activity, interesting properties that make them suitable for biotechnological applications.

One-step combined focused epPCR and saturation mutagenesis for thermostability evolution of a new cold-active xylanase.

Enzymes active at low temperature are of great interest for industrial bioprocesses due to their high efficiency at a low energy cost. One of the particularities of naturally evolved cold-active enzymes is their increased enzymatic activity at low temperature, however the low thermostability presented in this type of enzymes is still a major drawback for their application in biocatalysis. Directed evolution of cold-adapted enzymes to a more thermostable version, appears as an attractive strategy to fulfill the stability and activity requirements for the industry. This paper describes the recombinant expression and characterization of a new and highly active cold-adapted xylanase from the GH-family 10 (Xyl-L), and the use of a novel one step combined directed evolution technique that comprises saturation mutagenesis and focused epPCR as a feasible semi-rational strategy to improve the thermostability. The Xyl-L enzyme was cloned from a marine-Antarctic bacterium, Psychrobacter sp. strain 2-17, recombinantly expressed in E. coli strain BL21(DE3) and characterized enzymatically. Molecular dynamic simulations using a homology model of the catalytic domain of Xyl-L were performed to detect flexible regions and residues, which are considered to be the possible structural elements that define the thermolability of this enzyme. Mutagenic libraries were designed in order to stabilize the protein introducing mutations in some of the flexible regions and residues identified. Twelve positive mutant clones were found to improve the T5015 value of the enzyme, in some cases without affecting the activity at 25°C. The best mutant showed a 4.3°C increase in its T5015. The efficiency of the directed evolution approach can also be expected to work in the protein engineering of stereoselectivity.

The role of local residue environmental changes in thermostable mutants of the GH11 xylanase from Bacillus subtilis.

A thermostable variant of the mesophilic xylanase A from Bacillus subtilis (BsXynA-G3_4x) contains the four mutations Gln7His, Gly13Arg, Ser22Pro, and Ser179Cys. The crystal structure of the BsXynA-G3_4x has been solved, and the local environments around each of these positions investigated by molecular dynamics (MD) simulations at 328K and 348K. The structural and MD simulation results were correlated with thermodynamic data of the wild-type enzyme, the 4 single mutants and the BsXynA-G3_4x. This analysis suggests that the overall stabilizing effect is entropic, and is consistent with solvation of charged residues and reduction of main-chain flexibility. Furthermore, increased protein-protein hydrogen bonding and hydrophobic interactions also contribute to stabilize the BsXynA-G3_4x. The study revealed that a combination of several factors is responsible for increased thermostability of the BsXynA-G3_4x; (i) introduction of backbone rigidity in regions of high flexibility, (ii) solvation effects and (iii) hydrophobic contacts.