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[Abstract] Archaea are a domain of prokaryotic organisms that often inhabit extremophilic environments. Their enzymes are therefore of enormous biotechnological interest, due to their stability and activity in harsh conditions. Although bioprospection by the metagenomics of extreme environments has yielded many novel xylanases (endo-xylanases and β-xylosidades), most show a highest sequence similarity to bacteria or unculturable microorganisms and not to archaea. This graphical review focuses on the very promising but underexploited (hyper)thermophilic archaeal xylanases. There are only a few examples of xylanases isolated from culturable archaea. So far, 5 different (hyper)thermophilic archaeal strains with xylanase activity have been described, of which 4 present endo-xylanase activity and one shows both endo-xylanase and β-xylosidase activity. All of them show enzymatic activity under a high optimum temperature, neutral optimum pH (with one exception). These facts, together with the important applications of xylanases, reveal archaeal extremophilic xylanases to be a hidden treasure of biotechnology. Bioprospection of archaeal endo-xylanases constitutes a relatively unexploited field, full of potential. This research was funded by XUNTA DE GALICIA “Consolidación GRC” co-financed by FEDER, Grant No ED431C 2020/08; and MINISTERIO DE CIENCIA, INNOVACIÓN Y UNIVERSIDADES (MICINN), Grant Number RTI2018-099249-B-I00 Xunta de Galicia; ED431C 2020/08

Xylo-oligosaccharides (XOS) are considered as functional oligosaccharides and have great prebiotic potential. XOS are the degraded products of xylan prepared via chemical, physical or enzymatic degradation. They are mainly composed of xylose units linked by β-1, 4 bonds. XOS not only exhibit some specific physicochemical properties such as excellent water solubility and high temperature resistance, but also have a variety of functional biological activities including anti-inflammation, antioxidative, antitumor, antimicrobial properties and so on. Numerous studies have revealed in the recent decades that XOS can be applied to many food and feed products and exert their nutritional benefits. XOS have also been demonstrated to reduce the occurrence of human health-related diseases, improve the growth and resistance to diseases of animals. These effects open a new perspective on XOS potential applications for human consumption and animal production. Herein, this review aims to provide a general overview of preparation methods for XOS, and will also discuss the current application of XOS to human and animal health field. ispartof: FRONTIERS IN NUTRITION vol:8 ispartof: location:Switzerland status: published

Red alga dulse possesses a unique xylan, which is composed of a linear &beta, (1&rarr, 3)/&beta, 4)-xylosyl linkage. We previously prepared characteristic xylooligosaccharide (DX3, (&beta, 3)-xylosyl-xylobiose)) from dulse. In this study, we evaluated the prebiotic effect of DX3 on enteric bacterium. Although DX3 was utilized by Bacteroides sp. and Bifidobacterium adolescentis, Bacteroides Ksp. grew slowly as compared with &beta, 4)-xylotriose (X3) but B. adolescentis grew similar to X3. Therefore, we aimed to find the key DX3 hydrolysis enzymes in B. adolescentis. From bioinformatics analysis, two enzymes from the glycoside hydrolase family 43 (BAD0423: subfamily 12 and BAD0428: subfamily 11) were selected and expressed in Escherichia coli. BAD0423 hydrolyzed &beta, 3)-xylosyl linkage in DX3 with the specific activity of 2988 mU/mg producing xylose (X1) and xylobiose (X2), and showed low activity on X2 and X3. BAD0428 showed high activity on X2 and X3 producing X1, and the activity of BAD0428 on DX3 was 1298 mU/mg producing X1. Cooperative hydrolysis of DX3 was found in the combination of BAD0423 and BAD0428 producing X1 as the main product. From enzymatic character, hydrolysis of X3 was completed by one enzyme BAD0428, whereas hydrolysis of DX3 needed more than two enzymes.

Concerns over the environment are a central driver for designing cell-free enzymatic cascade reactions that synthesize non–petrol-based commodity compounds. An often-suggested strategy that would demonstrate the economic competitiveness of this technology is recycling of valuable enzymes through their immobilization. For this purpose, amyloid nanofibrils are an ideal scaffold to realize chemistry-free covalent enzyme immobilization on a material that offers a large surface area. However, in most instances, only single enzyme–functionalized amyloid fibrils have so far been studied. To embark on the next stage, here we displayed xylanase A, β-xylosidase, and an aldose sugar dehydrogenase on Sup35(1–61) nanofibrils to convert beechwood xylan to xylonolactone. We characterized this enzymatic cascade by measuring the time-dependent accumulation of xylose, xylooligomers, and xylonolactone. Furthermore, we studied the effects of relative enzyme concentrations, pH, temperature, and agitation on product formation. Our investigations revealed that a modular cascade with a mixture of xylanase and β-xylosidase, followed by product removal and separate oxidation of xylose with the aldose sugar dehydrogenase, is more productive than an enzyme mix containing all of these enzymes together. Moreover, we found that the nanofibril-coupled enzymes do not lose activity compared with their native state. These findings provide proof of concept of the feasibility of functionalized Sup35(1–61) fibrils as a molecular scaffold for biocatalytic cascades consisting of reusable enzymes that can be used in biotechnology.

Agradecimentos: We thank the LNBio Mass Spectrometry staff for their assistance with LC–MS/MS and Professor Hernandes Faustino de Carvalho Laboratory for his assistance with the immunohistochemistry methodology. We would like to thank Paul Whitford for the simulation and analysis program codes. The simulations were performed at CENAPAD-SP (Centro Nacional de Processamento de Alto Desempenho em São Paulo), under the UNICAMP/FINEP-MCT project. Research was supported by LNBR-Brazilian Biorenewables National Laboratory (CNPEM/MCTIC) using the Characterization of Macromolecules (MAC) open-access facility. The authors thank Espaço da Escrita-Pró-Reitoria de Pesquisa, UNICAMP for the language services provided Abstract: ß-Xylosidases are glycoside hydrolases (GHs) that cleave xylooligosaccharides and/or xylobiose into shorter oligosaccharides and xylose. Aspergillus nidulans is an established genetic model and good source of carbohydrate-active enzymes (CAZymes). Most fungal enzymes are N-glycosylated, which influences their secretion, stability, activity, signalization, and protease protection. A greater understanding of the N-glycosylation process would contribute to better address the current bottlenecks in obtaining high secretion yields of fungal proteins for industrial applications. In this study, BxlB - a highly secreted GH3 ß-xylosidase from A. nidulans, presenting high activity and several N-glycosylation sites - was selected for N-glycosylation engineering. Several glycomutants were designed to investigate the influence of N-glycans on BxlB secretion and function. The non-glycosylated mutant (BxlBnon-glyc) showed similar levels of enzyme secretion and activity compared to the wild-type (BxlBwt), while a partially glycosylated mutant (BxlBN1;5;7) exhibited increased activity. Additionally, there was no enzyme secretion in the mutant in which the N-glycosylation context was changed by the introduction of four new N-glycosylation sites (BxlBCC), despite the high transcript levels. BxlBwt, BxlBnon-glyc, and BxlBN1;5;7 formed similar secondary structures, though the mutants had lower melting temperatures compared to the wild type. Six additional glycomutants were designed based on BxlBN1;5;7, to better understand its increased activity. Among them, the two glycomutants which maintained only two N-glycosylation sites each (BxlBN1;5 and BxlBN5;7) showed improved catalytic efficiency, whereas the other four mutants’ catalytic efficiencies were reduced. The N-glycosylation site N5 is important for improved BxlB catalytic efficiency, but needs to be complemented by N1 and/or N7. Molecular dynamics simulations of BxlBnon-glyc and BxlBN1;5 reveals that the mobility pattern of structural elements in the vicinity of the catalytic pocket changes upon N1 and N5 N-glycosylation sites, enhancing substrate binding properties which may underlie the observed differences in catalytic efficiency between BxlBnon-glyc and BxlBN1;5. This study demonstrates the influence of N-glycosylation on A. nidulans BxlB production and function, reinforcing that protein glycoengineering is a promising tool for enhancing thermal stability, secretion, and enzymatic activity. Our report may also support biotechnological applications for N-glycosylation modification of other CAZymes FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQ Fechado

An intracellular β-xylosidase (AbXyl), fromthe thermoalkaline Anoxybacillus sp. 3M,was purified and characterized. The homodimeric enzyme (140 kDa) was optimally active at 65 °C and pH 5.5, exhibited half life of 10 h at 60 °C, 78 and 88% residual activity after 24 h, at pH 4.5 and 8.0, respectively. Fe2+, Cu2+, Al3+, Ag+ and Hg2+inhibited the enzyme; the activity was moderately stimulated by SDS and not influenced by β-mercaptoethanol. In the presence of p-nitrophenyl-β-D-xylopyranoside, AbXyl exhibited Km of 0.19 mM, Kcat of 453.29 s−1, KcatKm−1 of 2322 s−1mMandwas moderately influenced by xylose (Ki 21.25mM). The enzyme hydrolyzed xylo-oligomers into xylose and catalyzed transxylosilation reactions also in presence of alcohols as acceptors, producing xylo-oligosaccharides and alkyl-xylosides. Finally AbXyl was applied towards a statistically optimized process of brewery's spent grain bioconversion, highlighting the important role of this biocatalyst in reaching high yields of fermentable sugars. info:eu-repo/semantics/publishedVersion

A highly xylanolytic bacterium, Sphingobacterium sp. HP455, was isolated from the hindgut of soildwelling Holotrichia parallela larvae. The endo-xylanase (Xyn455) gene of the glycoside hydrolase (GH) family 10 and p-xylosidase (Xy1455) gene of the GH43 family were cloned and expressed in vitro from this highly xylanolytic bacterium. Both the Xyn455 and Xy1455 enzymes acted on a broad range of hemicelluloses. Xyn455 cleaved xylan to liberate xylooligosaccharides (XOS), and the XOS were subsequently cleaved into xylose through the action of Xy1455. This synergistic action significantly increased the xylan hydrolysis to 62.8%, which is higher than the sum of hydrolysis achieved by the enzymes individually (26.65%). Furthermore, Xy1455 is a bifunctional enzyme with both beta-D-xylosidase and alpha-L-arabinofuranosidase activities. Xy1455 also exhibits high xylose tolerance and a broad pH stability. The pH-dependent half-lives of Xy1455 range from 8.77 h to 43.52 h after pre-incubation for 1 h at 4 degrees C in buffers ranging from pH 3.0 to 9.0. These results suggest that both recombinant Xyn455 and Xy1455 and the bacterium are potential candidates to be used in commercial biomass conversion.

Knowledge of Antarctic habitat biodiversity, both marine and terrestrial, has increased considerably in recent years, causing considerable development in the studies of life science related to Antarctica. In the Austral summer 1986−1987, a new thermophilic bacterium, Parageobacillus thermantarcticus strain M1 was isolated from geothermal soil of the crater of Mount Melbourne (74°22′ S, 164°40′ E) during the Italian Antarctic Expedition. In addition to the biotechnological potential due to the production of exopolysaccharides and thermostable enzymes, successful studies have demonstrated its use in the green chemistry for the transformation and valorization of residual biomass and its employment as a suitable microbial model for astrobiology studies. The recent acquisition of its genome sequence opens up new opportunities for the use of this versatile bacterium in still unexplored biotechnology sectors.

In order to use corn fiber as a source for bioethanol production the enzymatic hydrolysis of the complex glucuronoarabinoxylans present has to be improved. Several oligosaccharides present in the supernatant of mild acid pretreated and enzymatically saccharified corn fiber that resist the current available enzymes were (semi)purified for structural analysis by NMR or ESI-MSn. The structural features of 21 recalcitrant oligosaccharides are presented. A common feature of almost all these oligosaccharides is that they contain (part of) an α- l -galactopyranosyl-(1→2)-β- d -xylopyranosyl-(1→2)-5-O-trans-feruloyl- l -arabinofuranose side chain attached to the O-3 position of the β-1–4 linked xylose backbone. Several of the identified oligosaccharides contained an ethyl group at the reducing end hypothesized to be formed during SSF. The ethyl glycosides found are far more complex than previously described structures. A new feature present in more than half of the oligosaccharides is an acetyl group attached to the O-2 position of the same xylose to which the oligomeric side chain was attached to the O-3 position. Finding enzymes attacking these large side chains and the dense substituted xylan backbone will boost the hydrolysis of corn fiber glucuronoxylan.

International audience; Various enzymatic cocktails were produced from two Trichoderma reesei strains, a cellulase hyperproducer strain and a strain with β-glucosidase activity overexpression. By using various carbon sources (lactose, glucose, xylose, hemi-cellulosic hydrolysate) for strains growth, contrasted enzymatic activities were obtained. The enzymatic cocktails presented various levels of efficiency for the hydrolysis of cellulose Avicel into glucose, in presence of xylans, or not. These latter were also hydrolyzed with different extents according to cocktails. The most efficient cocktails (TR1 and TR3) on Avicel were richer in filter paper activity (FPU) and presented a low ratio FPU/β-glucosidase activity. Cocktails TR2 and TR5 which were produced on the higher amount of hemicellulosic hydrolysate, possess both high xylanase and β-xylosidase activities, and were the most efficient for xylans hydrolysis. When hydrolysis of Avicel was conducted in presence of xylans, a decrease of glucose release occurred for all cocktails compared to hydrolysis of Avicel alone. Mixing TR1 and TR5 cocktails with two different ratios of proteins (1/1 and 1/4) resulted in a gain of efficiency for glucose release during hydrolysis of Avicel in presence of xylans compared to TR5 alone. Our results demonstrate the importance of combining hemicellulase and cellulase activities to improve the yields of glucose release from Avicel in presence of xylans. In this context, strategies involving enzymes production with carbon sources comprising mixed C5 and C6 sugars or combining different cocktails produced on C5 or on C6 sugars are of interest for processes developed in the context of lignocellulosic biorefinery.

TM0077 from Thermotoga maritima is a member of the carbohydrate esterase family 7 and is active on a variety of acetylated compounds, including cephalosporin C. TM0077 esterase activity is confined to short-chain acyl esters (C2-C3), and is optimal around 100°C and pH 7.5. The positional specificity of TM0077 was investigated using 4-nitrophenyl-ß-D-xylopyranoside monoacetates as substrates in a ß-xylosidase-coupled assay. TM0077 hydrolyzes acetate at positions 2, 3, and 4 with equal efficiency. No activity was detected on xylan or acetylated xylan, which implies that TM0077 is an acetyl esterase and not an acetyl xylan esterase as currently annotated. Selenomethionine-substituted and native structures of TM0077 were determined at 2.1 and 2.5 Å resolution, respectively, revealing a classic a/ß-hydrolase fold. TM0077 assembles into a doughnut-shaped hexamer with small tunnels on either side leading to an inner cavity, which contains the six catalytic centers. Structures of TM0077 with covalently bound phenylmethylsulfonyl fluoride and paraoxon were determined to 2.4 and 2.1 Å, respectively, and confirmed that both inhibitors bind covalently to the catalytic serine (Ser188). Upon binding of inhibitor, the catalytic serine adopts an altered conformation, as observed in other esterase and lipases, and supports a previously proposed catalytic mechanism in which Ser hydroxyl rotation prevents reversal of the reaction and allows access of a water molecule for completion of the reaction

Aspergillus niger is an important organism for the production of industrial enzymes such as hemicellulases and pectinases. The xylan-backbone monomer, d -xylose, is an inducing substance for the coordinate expression of a large number of polysaccharide-degrading enzymes. In this study, the responses of 22 genes to low (1 mM) and high (50 mM) d -xylose concentrations were investigated. These 22 genes encode enzymes that function as xylan backbone-degrading enzymes, accessory enzymes, cellulose-degrading enzymes, or enzymes involved in the pentose catabolic pathway in A. niger . Notably, genes encoding enzymes that have a similar function (e.g., xylan backbone degradation) respond in a similar manner to different concentrations of d -xylose. Although low d -xylose concentrations provoke the greatest change in transcript levels, in particular, for hemicellulase-encoding genes, transcript formation in the presence of high concentrations of d -xylose was also observed. Interestingly, a high d -xylose concentration is favorable for certain groups of genes. Furthermore, the repressing influence of CreA on the transcription and transcript levels of a subset of these genes was observed regardless of whether a low or high concentration of d -xylose was used. Interestingly, the decrease in transcript levels of certain genes on high d -xylose concentrations is not reflected by the transcript level of their activator, XlnR. Regardless of the d -xylose concentration applied and whether CreA was functional, xlnR was constitutively expressed at a low level.

International audience; The ability of GH 39 beta-D-Xylosidase (XylBH39) from Bacillus halodurans to catalyze transxylosylation reactions, particularly for the production of alkyl xylosides, has been investigated using p-nitrophenyl (pNP) beta-D-xylopyranoside, xylobiose and xylotriose. Furthermore wheat bran, being rich in xylo-oligosaccharides after enzymatic treatment with an endoxylanase. was used as a source of xylosyl donors. The autocondensation of pNP beta-D-Xylopyranoside was characterized by poor regioselectivity and the production of a variety of short (di- and trimers) oligosaccharides. Compared to xylobiose, xylotriose was a better donor molecule for XylBH39-mediated transxylosylation of primary alcohols presenting chain length from 1- to 5-carbon atoms. Using a wheat bran hydrolysate, generated using an endoxylanase, it was possible to perform XylBH39-mediated transxylosylation of propan-1-ol to produce propyl beta-D-xylopyranoside. This enzymatic process led to synthesis of 0.9 g of propyl beta-D-Xylopyranoside from 20 g of destarched wheat bran.

International audience; To investigate the concept of a xylosidase-based process for the continuous production of xylose from arabinoxylan-containing feedstocks, a beta-xylosidase from Bacillus halodurans C-125 was immobilized and deployed in packed bed reactor (PBR). Among the several immobilization methods tested, glutaraldehyde-mediated immobilization on chitosan was the best both in terms of immobilization and activity yields (91% and 72.9%, respectively). In batch experiments the immobilized enzyme hydrolyzed wheat bran hydrolysates quite efficiently, consuming nearly all xylobiose and xylotriose after 6 h. Its reusability showed only a 50% decrease of its activity after 92 h. Using the chitosan-immobilized beta-xylosidase in a PBR, xylose productivity was 7.2 g xylose l(-1) h(-1) and the conversion factor was 0.55 (derived from initial xylose in the substrate). The operational stability of the PBR was good, because only 25% of productivity was lost after the treatment of three batches of substrate over a 72-h period.

The Aspergillus nidulans xlnR gene encodes a Zn(2)Cys(6) transcription activator necessary for the synthesis of the main xylanolytic enzymes, i.e. endo-xylanases X(22), X(24) and X(34), and beta-xilosidase XlnD. Expression of xlnR is not sufficient for induction of genes encoding the xylanolytic complex, the presence of xylose is absolutely required. It has been established previously that the wide-domain carbon catabolite repressor CreA indirectly represses xlnA (encodes X(22)) and xlnB (encodes X(24)) genes as well as exerting direct repression on xlnA. This work provides evidence that CreA-mediated indirect repression occurs through repression of xlnR: (i) the xlnR gene promoter is repressed by glucose and this repression is abolished in creA(d)30 mutant strains and (ii) deregulated expression of xlnR completely relieves glucose repression of xlnA and xlnB. Thus, CreA and XlnR form a transcriptional cascade regulating A. nidulans xylanolytic genes.

Aims: It is evaluated the effectiveness of the combined action of two highly thermostable enzymes for the hydrolysis of xylans at high temperature in order to produce D-xylose. Methods and Results: Xylans from different sources were hydrolyzed at high degree at 70°C by co-action of a xylanase from the thermophilic bacterium Anoxybacillus flavithermus BC and the novel β-xylosidase/α-arabinosidase from the hyperthermophilic crenarchaeon Sulfolobus solfataricus Oα. Beechwood xylan was the best substrate among the xylans tested giving, by incubation only with xylanase, 32·8 % hydrolysis after 4 h. The addition of the β-xylosidase/α-arabinosidase significantly improved the rate of hydrolysis, yielding 63·6% conversion after 4 h incubation, and the main sugar identified was xylose. Conclusions: This study demonstrates that a significant degree of xylan degradation was reached at high temperature by co-action of the two enzymes. Xylose was obtained as a final product in considerable yield. Significance and Impact of the Study: Although the xylan represents the second most abundant polysaccharide in nature, it still doesn't have significant utilization for the difficulties encountered in its hydrolysis. Its successful hydrolysis to xylose in only one stage process could make of it a cheap sugar source and could have an enormous economic potential for the conversion of plant biomass into fuels and chemicals.

By deletion across the promoter region of the xynF1 gene encoding the major Aspergillus oryzae xylanase, a 53-bp DNA fragment containing the XlnR binding sequence GGCTAAA as well as two similar sequences was shown to confer xylan inducibility on the gene. Complementary and genomic DNAs encoding the Aspergillus niger xlnR homologous gene, abbreviated AoxlnR, were cloned from A. oryzae and sequenced. AoXlnR comprised 971 amino acids with a zinc binuclear cluster domain at the N-terminal region and revealed 77.5% identity to the A. niger XlnR. Recombinant AoXlnR protein encompassing the zinc cluster region of the N-terminal part bound to both the consensus binding sequence and its cognate sequence, GGCTGA, with an approximately 10 times lower affinity. GGCTA/GA is more appropriate as the XlnR consensus binding sequence. Both sequences functioned independently in vivo in XlnR-mediating induction of the xynF1 gene. This was further confirmed by using an AoxlnR disruptant. Neither the xynF1 nor the xylA gene was expressed in the disruptant, suggesting that the xylan-inducible genes in A. oryzae may also be controlled in the same manner as described for A. niger.

The use of lignocellulosic biomass for second generation biofuels requires optimization of enzymatic breakdown of plant cell walls. In this work, cellulolytic bacteria were isolated from a native and two cultivated forest soil samples. Amplification of glycosyl hydrolases was attempted by using a low stringency-degenerate primer PCR strategy, using total soil DNA and bulk DNA pooled from positive colonies as template. A set of primers was designed based on Acidothermus cellulolyticus genome, by search of conserved domains of glycosyl hydrolases (GH) families of interest. Using this approach, a fragment containing an open reading frame (ORF) with 98% identity to a putative GH43 beta-xylosidase coding gene from Enterobacter cloacae was amplified and cloned. The full protein was expressed in Escherichia coli as N-terminal or C-terminal His-tagged fusions and purified under native conditions. Only N-terminal fusion protein, His-Xyl43, presented beta-xylosidase activity. On pNPX, optimal activity was achieved at pH 6 and 40°C and Km and Kcat values were 2.92mM and 1.32seg-1, respectively. Activity was also demonstrated on xylobiose (X2), with Km 17.8mM and Kcat 380s-1. These results demonstrated that Xyl43 is a functional beta-xylosidase and it is the first evidence of this activity for Enterobacter sp. Fil: Campos, Eleonora. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina Fil: Negro Alvarez, María José. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas; España Fil: Sabarís Di Lorenzo, Gonzalo Julián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina Fil: González, Sergio Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina Fil: Rorig, Marcela. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación de Recursos Naturales. Instituto de Suelos; Argentina Fil: Talia, Paola. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina Fil: Grasso, Daniel Hector. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación de Recursos Naturales. Instituto de Suelos; Argentina Fil: Sáez, Felicia. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas; España Fil: Manzanares Secades, Paloma. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas; España Fil: Ballesteros Perdices, Mercedes. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas; España Fil: Cataldi, Ángel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina

The GH39 beta-xylosidase from Bacillus halodurans (BhXy139) was previously reported to catalyze the synthesis of alkyl xylosides from donors such as pNP beta-D-xylopyranoside or xylobiose and from aliphatic alcohols acting as acceptors with chain length inferior to five carbons. In the present study, the role played by aromatic residues present in the aglycone binding subsite of BhXy139 in the hydrolysis and transglycosylation reactions of the enzyme was investigated. In this way, site-directed mutagenesis was carried out in order to highlight the role of three targeted hydrophobic residues F116, F167 and Y284. These residues were replaced by alanine to decrease the steric hindrance or were mutated into serine to evaluate the impact of the presence of a polar residue into the aglycone binding subsite of BhXy139. Taking into account kinetic parameters and yields of transglycosylation, the function of each mutated residue in the catalytic mechanism was studied. Results concerning transglycosylation reactions in the presence of pentan-1-ol and octan-1-ol indicated that yields of transglycosylation were impacted both by the position and the nature of the mutated residues. These results were consistent with molecular docking performed with both acceptors which notably confirms that among the three targeted residues, F116 represents the most interesting one for mutagenesis to increase the transglycosylation reactions in presence of long chain alcohols. (C) 2013 Elsevier B.V. All rights reserved.

The xylanase biosynthesis is induced by its substrate—xylan. The high xylan content in some wastes such as wheat residues (wheat bran and wheat straw) makes them accessible and cheap sources of inducers to be mainly applied in great volumes of fermentation, such as those of industrial bioreactors. Thus, in this work, the main proposal was incorporated in the nutrient medium wheat straw particles decomposed to soluble compounds (liquor) through treatment of lignocellulosic materials in autohydrolysis process, as a strategy to increase and undervalue xylanase production by Aspergillus ochraceus. The wheat straw autohydrolysis liquor produced in several conditions was used as a sole carbon source or with wheat bran. The best conditions for xylanase and β-xylosidase production were observed when A. ochraceus was cultivated with 1% wheat bran added of 10% wheat straw liquor (produced after 15 min of hydrothermal treatment) as carbon source. This substrate was more favorable when compared with xylan, wheat bran, and wheat straw autohydrolysis liquor used separately. The application of this substrate mixture in a stirred tank bioreactor indicated the possibility of scaling up the process to commercial production., This work was supported by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP/Brazil), Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq/Brazil), National System for Research on Biodiversity (SISBIOTA-Brazil, CNPq 563260/2010-6/FAPESP no. 2010/52322-3), and Fundacao para a Ciencia e a Tecnologia (FCT/Portugal).

TM0077 from Thermotoga maritima is a member of the carbohydrate esterase family 7 and is active on a variety of acetylated compounds, including cephalosporin C. TM0077 esterase activity is confined to short-chain acyl esters (C2-C3), and is optimal around 100°C and pH 7.5. The positional specificity of TM0077 was investigated using 4-nitrophenyl-ß-D-xylopyranoside monoacetates as substrates in a ß-xylosidase-coupled assay. TM0077 hydrolyzes acetate at positions 2, 3, and 4 with equal efficiency. No activity was detected on xylan or acetylated xylan, which implies that TM0077 is an acetyl esterase and not an acetyl xylan esterase as currently annotated. Selenomethionine-substituted and native structures of TM0077 were determined at 2.1 and 2.5 Å resolution, respectively, revealing a classic a/ß-hydrolase fold. TM0077 assembles into a doughnut-shaped hexamer with small tunnels on either side leading to an inner cavity, which contains the six catalytic centers. Structures of TM0077 with covalently bound phenylmethylsulfonyl fluoride and paraoxon were determined to 2.4 and 2.1 Å, respectively, and confirmed that both inhibitors bind covalently to the catalytic serine (Ser188). Upon binding of inhibitor, the catalytic serine adopts an altered conformation, as observed in other esterase and lipases, and supports a previously proposed catalytic mechanism in which Ser hydroxyl rotation prevents reversal of the reaction and allows access of a water molecule for completion of the reaction

La bagasse de canne à sucre est l’un des co-produits d’industries agricoles les plus générés de la planète. Elle constitue, du fait de sa composition, une source renouvelable de matière lignocellulosique pouvant être d’un grand intérêt pour la production de molécules à haute valeur ajoutée. Une démarche valorisation de la bagasse qui ne lui ferait pas perdre sa valeur énergétique et s’intégrerait parfaitement à l’activité principale – la sucrerie – consisterait à se baser sur le concept de la bioraffinerie. On installerait alors un atelier de fabrication de xylose qui recevrait la bagasse à la sortie des moulins ou des diffusions de canne à sucre et la restituerait après passage dans des presses à la sucrerie. Un petit atelier de fermentation fongique sur milieu solide constitué de bagasse permettrait de préparer les enzymes nécessaires au traitement. La bagasse ayant servi à ce travail provient de l’Ile de la Réunion. Elle a été fournie par le CERF (Centre d’Etude, Recherche et Formation sur la canne à sucre). Sa teneur élevée (26%) en xylane pratiquement pur (92% xylose) en fait une bonne source d’extraction de ce pentose. De plus, la bagasse de canne à sucre semble être intéressante pour une utilisation comme source de carbone pour l’induction de production d’activités enzymatiques par des microorganismes. Pour extraire le xylose, un traitement hydrothermique optimisé (170°C – 2h) a été appliqué. Cela a permis la solubilisation de la fraction hemicellulosique de la bagasse qui a par la suite été soumise à l’hydrolyse enzymatique.Deux enzymes ont été utilisées pour catalyser le fractionnement des hémicelluloses de la bagasse : une enzyme purifiée de Thermobacillus xylanilyticus et un surnageant de culture d’Humicola insolens à activité xylanolytique prédominante. Une comparaison entre ces deux enzymes a été réalisée et a montré l’intérêt d’utiliser le surnageant de culture plutôt qu’une enzyme purifiée. Un surnageant de culture de Trichoderma Reesei riche en β-xylosidase a été utilisé pour poursuivre la saccharification jusqu’à obtenir du xylose. Les concentrations optimales d’enzymes et de substrat ont été déterminées. Les conditions d’obtention d’un bon rendement, de l’ordre de 60% de xylose en présence d’un taux réduit d’impuretés sont définies., Sugarcane bagasse is one of the co-products of agro-industries the most widely present on earth. Because of its composition and its availability as a renewable resource, bagasse constitutes a good candidate for valorization in view of the production of added value molecules. A reasonable approach of bagasse valorization should be integrated to the principal activity using sugarcane, namely, sugar factory with application of the concept of biorefinery. Such an approach would consist in installing a workshop of extraction of xylose which will be fed with bagasse coming out of the crushers or diffusion of sugarcane and will give it back to the factory for traditional use in vapor and electricity generators after dewatering in presses. A small workshop can be installed for enzyme production in a solid fermentor using bagasse as a carbon source for the chosen fungi. The sample of bagasse used in this work was supplied by CERF, a center of research on sugar cane at La Réunion. The high content of xylan (26%) particularly rich in xylose (92%) of the experimented bagasse makes it a good source of extraction of this pentose. Moreover, we have shown the validity of bagasse as a carbon source for induction of different enzyme activities by different microorganisms among which xylanase and ß-glucosidase are the most important. To extract xylose, an optimized hydro-thermal treatment (170°C – 2h) was applied. It allowed solubilization of the hemicellulotic fraction of bagasse. This step was followed by an enzymatic treatment. Two enzymes were used to catalyse hydrolysis of xylans in the soluble fraction. These enzymes were a purified endo-xylanase from Thermobacillus xylanilyticus and a supernatant from Humicola insolens fungal broth rich in endo-xylanase. Comparison between the 2 enzymes incited us to use the non purified supernatant. Another supernatant of Trichoderma Reesei was used because it was rich in β-xylosidase. It allowed hydrolyzing the oligo-xylan fractions liberated by the endo-xylanase into xylose. Optimization of enzyme concentrations was achieved. Conditions for obtaining a yield of about 60% of xylose were defined.

The thermostable β-xylosidase from Sulfolobus solfataricus , expressed in Escherichia coli , was immobilized by entrapment into alginate with full recovery of activity and tested for xylose production from xylan hydrolysates. Since the recombinant activity was also cell bound, alginate beads entrapping E. coli whole cells were also prepared. The immobilized preparations exhibited higher thermostability at 90 °C compared to their free counterparts. The half-lives of the immobilized enzyme and cells were 21 and 23 h, respectively, while half of the inactivation was reached after 10 and 11.5 h for free enzyme and whole cells. Interestingly, thermophilicity increased from 85 up to 100 °C and the optimal pH shifted to higher values for immobilized preparations. Results obtained from xylo-oligosaccharides hydrolysis in subsequent batch experiments of recycling, indicated that the immobilized enzyme had good operational stability, retaining 84% of its initial activity after four cycles. Here we report on the immobilization of the β-xylosidase into alginate and its characterization.

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Aspergillus awamori showed high extracellular endoxylanase (100 U/ml) and beta-xylosidase activities (3.5 U/ml) when grown on milled sugar cane bagasse as the principal carbon source without treatment. Partial characterization of xylanases showed that the apparent values of Km were 3.12 ± 0.05 mg/ml for endoxylanase (in birchwood xylan) and 0.45 ± 0.05 mM for beta-xylosidase (in p -nitrophenyl beta-D-xylanopiranoside). Corresponding values of Vmax were 6.63 ± 0.02 and 0.078 ± 0.02 mumol/min. Gradual repression of endoxylanase activity was observed when increasing concentrations of glucose and xylose (1, 2, 4, 6 and 8 g of carbohydrate / 4 g of sugar cane bagasse) were added to production media. In contrast, beta-xylosidase activity was stimulated using low levels of carbohydrates (1 g xylose or glucose/ 4 g of sugar cane bagasse). O cultivo de Aspergillus awamori em bagaço de cana, finamente dividido e sem tratamento prévio, favoreceu a produção de níveis elevados de endo-xilanase (100 U/ml) e beta-xilosidase (3.5 U/ml) extracelulares. A caracterização parcial das enzimas xilanolíticas permitiu estabelecer os parâmetros cinéticos aparentes, sendo Km igual a 3.12 ( 0.05 mg/ml para endox-xilanase (xilana birchwood) e 0.45 ( 0.05 mM para beta-xylosidase (p-nitrofenil beta-D-xilanopiranosideo). Os correspondentes valores de Vm foram 6.63 ( 0.02 e 0.078 ( 0.02 mimol/min. Observou-se uma repressão gradual da atividade endo-xilanásica quando concentrações crescentes de glicose e xilose (1, 2, 4, 6 e 8 g de carboidrato / 4 g de bagaço de cana) foram adicionadas aos meios de produção. Contrariamente, a atividade beta-xilosidásica foi estimulada quando níveis baixos de açúcares simples foram utilizados (1 g de xilose ou glicose/ 4 g de bagaço de cana).

The recalcitrance of lignocellulose to bioprocessing represents the core problem and remains the limiting factor in creating an economy based on lignocellulosic ethanol production. Lignin is responsible for unproductive interactions with enzymes, and understanding how lignin impairs the susceptibility of biomass to enzymatic hydrolysis represents a significant aim in optimising the biological deconstruction of lignocellulose. The objective of this study was to develop methodologies based on surface plasmon resonance (SPR), which provide novel insights into the interactions between xylanase (Tx-xyn11) and phenolic compounds or lignin oligomers. In a first approach, Tx-xyn11 was fixed onto sensor surfaces, and phenolic molecules were applied in the liquid phase. The results demonstrated weak affinity and over-stoichiometric binding, as several phenolic molecules bound to each xylanase molecule. This approach, requiring the use of soluble molecules in the liquid phase, is not applicable to insoluble lignin oligomers, such as the dehydrogenation polymer (DHP). An alternative approach was developed in which a lignin oligomer was fixed onto a sensor surface. Due to their hydrophobic properties, the preparation of stable lignin layers on the sensor surfaces represented a considerable challenge. Among the various chemical and physico-chemical approaches assayed, two approaches (physisorption via the Langmuir-Blodgett technique onto self-assembled monolayer (SAM)-modified gold and covalent coupling to a carboxylated dextran matrix) led to stable lignin layers, which allowed the study of its interactions with Tx-xyn11 in the liquid phase. Our results indicated the presence of weak and non-specific interactions between Tx-xyn11 and DHP.