Добірка наукової літератури з теми "GH10 Xylanases"

ABSTRACTThe ascomyceteTrichoderma reeseiis a paradigm for the regulation and production of plant cell wall-degrading enzymes, including xylanases. Four xylanases, including XYN1 and XYN2 of glycosyl hydrolase family 11 (GH11), the GH10 XYN3, and the GH30 XYN4, were already described. By genome mining, we identified a fifth xylanase, XYN5, belonging to GH11. Transcriptional analysis reveals that the expression of all xylanases butxyn3is induced byd-xylose, dependent on the cellulase and xylanase regulator XYR1 and negatively regulated by the carbon catabolite repressor CRE1. Impairment ofd-xylose catabolism at thed-xylose reductase and xylitol dehydrogenase step strongly enhanced induction byd-xylose. Knockout of thel-xylulose reductase-encoding genelxr3, which connects thed-xylose andl-arabinose catabolic pathways, had no effect on xylanase induction. Besides the induction byd-xylose, theT. reeseixylanases were also induced byl-arabinose, and this induction was also enhanced in knockout mutants inl-arabinose reductase (xyl1),l-arabitol dehydrogenase (lad1), andl-xylulose reductase (lxr3). Induction byl-arabinose was also XYR1 dependent. Analysis of intracellular polyols revealed accumulation of xylitol in all strains only during incubation withd-xylose and accumulation ofl-arabitol only during incubation withl-arabinose. Induction byl-arabinose could be further stimulated by addition ofd-xylose. We conclude that the expression of theT. reeseixylanases can be induced by bothd-xylose andl-arabinose, but independently of each other and by using different inducing metabolites.

Endo-1,4-beta-xylanases (xylanases) are classified into 9 glycoside hydrolase families, GH5, 8, 10, 11, 30, 43, 51, 98, and 141 based on the CAZy database. The probe sequences representing the enzymes were constructed from published sequences of actual experimental studies with xylan decomposition activity. From online databases, we found one sequence belonging to the GH5 family, 6 sequences belonging to the GH8 family and 5 sequences belonging to the GH30 family exhibiting xylanase activity. Thus specific probes for xylanase GH8 and GH30 families were designed with the length of 351 and 425 amino acids respectively. The reference values for the probe of the GH8 family were defined as the sequences with maximum score greater than 168, the lowest coverage was 84%, the lowest similarity was 36%; for the probe GH30, the maximum score was greater than 316, the coverage was greater than 98%, the similarity was greater than 41%. Using the built probes, including the probe of the two GH10 and GH11 families, we found 41 xylanase-encoding sequences from the metagenomic DNA data of bacteria in Vietnamese goats’rumen. Of the 41 exploited sequences, 19 were identical to the BGI company's annotation result based on KEGG database, whereas there were 16 sequences that are not annotated by the BGI company. Total 28 of 41 exploited sequences were complete open reading frames, of which the predicted ternary structure was highly similar to the published structures of xylanase.

ABSTRACTXylanases are crucial for lignocellulosic biomass deconstruction and generally contain noncatalytic carbohydrate-binding modules (CBMs) accessing recalcitrant polymers. Understanding how multimodular enzymes assemble can benefit protein engineering by aiming at accommodating various environmental conditions. Two multimodular xylanases, XynA and XynB, which belong to glycoside hydrolase families 11 (GH11) and GH10, respectively, have been identified fromCaldicellulosiruptorsp. strain F32. In this study, both xylanases and their truncated mutants were overexpressed inEscherichia coli, purified, and characterized. GH11 XynATM1 lacking CBM exhibited a considerable improvement in specific activity (215.8 U nmol−1versus 94.7 U nmol−1) and thermal stability (half-life of 48 h versus 5.5 h at 75°C) compared with those of XynA. However, GH10 XynB showed higher enzyme activity and thermostability than its truncated mutant without CBM. Site-directed mutagenesis of N-terminal amino acids resulted in a mutant, XynATM1-M, with 50% residual activity improvement at 75°C for 48 h, revealing that the disordered region influenced protein thermostability negatively. The thermal stability of both xylanases and their truncated mutants were consistent with their melting temperature (Tm), which was determined by using differential scanning calorimetry. Through homology modeling and cross-linking analysis, we demonstrated that for XynB, the resistance against thermoinactivation generally was enhanced through improving both domain properties and interdomain interactions, whereas for XynA, no interdomain interactions were observed. Optimized intramolecular interactions can accelerate thermostability, which provided microbes a powerful evolutionary strategy to assemble catalysts that are adapted to various ecological conditions.

This study describes the catalytic properties of a GH30_7 xylanase produced by the fungus Talaromyces leycettanus. The enzyme is an ando-β-1,4-xylanase, showing similar specific activity towards glucuronoxylan, arabinoxylan, and rhodymenan (linear β-1,3-β-1,4-xylan). The heteroxylans are hydrolyzed to a mixture of linear as well as branched β-1,4-xylooligosaccharides that are shorter than the products generated by GH10 and GH11 xylanases. In the rhodymenan hydrolyzate, the linear β-1,4-xylooligosaccharides are accompanied with a series of mixed linkage homologues. Initial hydrolysis of glucuronoxylan resembles the action of other GH30_7 and GH30_8 glucuronoxylanases, resulting in a series of aldouronic acids of a general formula MeGlcA2Xyln. Due to the significant non-specific endoxylanase activity of the enzyme, these acidic products are further attacked in the unbranched regions, finally yielding MeGlcA2Xyl2-3. The accommodation of a substituted xylosyl residue in the −2 subsite also applies in arabinoxylan depolymerization. Moreover, the xylose residue may be arabinosylated at both positions 2 and 3, without negatively affecting the main chain cleavage. The catalytic properties of the enzyme, particularly the great tolerance of the side-chain substituents, make the enzyme attractive for biotechnological applications. The enzyme is also another example of extraordinarily great catalytic diversity among eukaryotic GH30_7 xylanases.

ABSTRACT Pseudomonas cellulosa is a highly efficient xylan-degrading bacterium. Genes encoding five xylanases, and several accessory enzymes, which remove the various side chains that decorate the xylan backbone, have been isolated from the pseudomonad and characterized. The xylanase genes consist of xyn10A, xyn10B, xyn10C, xyn10D, and xyn11A, which encode Xyn10A, Xyn10B, Xyn10C, Xyn10D, and Xyn11A, respectively. In this study a sixth xylanase gene, xyn11B, was isolated which encodes a 357-residue modular enzyme, designated Xyn11B, comprising a glycoside hydrolase family 11 catalytic domain appended to a C-terminal X-14 module, a homologue of which binds to xylan. Localization studies showed that the two xylanases with glycoside hydrolase family (GH) 11 catalytic modules, Xyn11A and Xyn11B, are secreted into the culture medium, whereas Xyn10C is membrane bound. xyn10C, xyn10D, xyn11A, and xyn11B were all abundantly expressed when the bacterium was cultured on xylan or β-glucan but not on medium containing mannan, whereas glucose repressed transcription of these genes. Although all of the xylanase genes were induced by the same polysaccharides, temporal regulation of xyn11A and xyn11B was apparent on xylan-containing media. Transcription of xyn11A occurred earlier than transcription of xyn11B, which is consistent with the predicted mode of action of the encoded enzymes. Xyn11A, but not Xyn11B, exhibits xylan esterase activity, and the removal of acetate side chains is required for xylanases to hydrolyze the xylan backbone. A transposon mutant of P. cellulosa in which xyn11A and xyn11B were inactive displayed greatly reduced extracellular but normal cell-associated xylanase activity, and its growth rate on medium containing xylan was indistinguishable from wild-type P. cellulosa. Based on the data presented here, we propose a model for xylan degradation by P. cellulosa in which the GH11 enzymes convert decorated xylans into substituted xylooligosaccharides, which are then hydrolyzed to their constituent sugars by the combined action of cell-associated GH10 xylanases and side chain-cleaving enzymes.

Xylanase inhibitors inhibit the activities of microbial xylanases and seriously compromise the efficacy of microbial xylanases added to modify cereals. Cereal endogenous xylanases are unaffected by these xylanase inhibitors, but little information is available regarding their effects in improving cereal quality, a neglected potential application. As a strategy for circumventing the negative effects of xylanase inhibitors, the objective of this study was to use genetic engineering to obtain sufficient amounts of active endo-1,4-β-D-xylanase from wheat to analyze the characteristics of its structure. The endo-1,4-β-D-xylanase from wheat was heterologously expressed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, MALDI-TOF/TOF (MS) analyses, and enzyme activity determination confirmed 2 active endo-1,4-β-D-xylanases (EXY3 and EXY4) were successfully obtained. The molecular weights (MW) and isoelectric point (pI) of EXY3 were 36.108 kDa and 5.491, while those of the EXY4 protein were 41.933 kDa and 5.726. They both contained the same catalytic domain of GH10 xylanases from G266 to V276 and have the same catalytic site, Glu273. They shared the same putative N-glycosylation sites (N62-T63-S64 and N280–V281–S282) and 3 putative O-glycosylation sites (Ser8, Ser9, and Thr21), but EXY4 had an additional O-glycosylation site (Thr358). EXY3 was smaller than EXY4 by 51 amino acids because of a nonsense mutation and premature termination. They both had the 8-fold beta/alpha-barrel (TIM-barrel) fold. The specific activities of EXY3 and EXY4 were 152.0891 and 67.2928 U/mg, respectively. This work demonstrates a promising way to obtain wheat xylanases by genetic engineering; the properties of the enzymes indicate their potential application in cereal-based industries.

ABSTRACTXanthomonas citripv. citri strain 306 (Xcc306), a causative agent of citrus canker, produces endoxylanases that catalyze the depolymerization of cell wall-associated xylans. In the sequenced genomes of all plant-pathogenic xanthomonads, genes encoding xylanolytic enzymes are clustered in three adjacent operons. InXcc306, these consecutive operons contain genes encoding the glycoside hydrolase family 10 (GH10) endoxylanases Xyn10A and Xyn10C, theagu67gene, encoding a GH67 α-glucuronidase (Agu67), thexyn43Egene, encoding a putative GH43 α-l-arabinofuranosidase, and thexyn43Fgene, encoding a putative β-xylosidase. Recombinant Xyn10A and Xyn10C convert polymeric 4-O-methylglucuronoxylan (MeGXn) to oligoxylosides methylglucuronoxylotriose (MeGX3), xylotriose (X3), and xylobiose (X2).Xcc306 completely utilizes MeGXnpredigested with Xyn10A or Xyn10C but shows little utilization of MeGXn.Xcc306 with a deletion in the gene encoding α-glucuronidase (Xcc306 Δagu67) will not utilize MeGX3for growth, demonstrating the role of Agu67 in the complete utilization of GH10-digested MeGXn. Preferential growth on oligoxylosides compared to growth on polymeric MeGXnindicates that GH10 xylanases, either secreted byXcc306in plantaor produced by the plant host, generate oligoxylosides that are processed by Xyn10 xylanases and Agu67 residing in the periplasm. Coordinate induction by oligoxylosides ofxyn10,agu67,cirA, thetonBreceptor, and other genes within these three operons indicates that they constitute a regulon that is responsive to the oligoxylosides generated by the action ofXcc306 GH10 xylanases on MeGXn. The combined expression of genes in this regulon may allow scavenging of oligoxylosides derived from cell wall deconstruction, thereby contributing to the tissue colonization and/or survival ofXcc306 and, ultimately, to plant disease.

Glycoside hydrolase family 10 (GH10) xylanases are responsible for enzymatic cleavage of the internal glycosidic linkages of the xylan backbone, to generate xylooligosaccharides (XOS) and xyloses. The topologies of active-site cleft determine the substrate preferences and product profiles of xylanases. In this study, positional bindings and substrate interactions of TmxB, one of the most thermostable xylanases characterized from Thermotoga maritima to date, was investigated by docking simulations. XOS with backbone lengths of two to five (X2–X5) were docked into the active-site cleft of TmxB by AutoDock The modeled complex structures provided a series of snapshots of the interactions between XOS and TmxB. Changes in binding energy with the length of the XOS backbone indicated the existence of four effective subsites in TmxB. The interaction patterns at subsites −2 to +1 in TmxB were conserved among GH10 xylanases whereas those at distal aglycone subsite +2, consisting of the hydrogen bond network, was unique for TmxB. This work helps in obtaining an in-depth understanding of the substrate-binding property of TmxB and provides a basis for rational design of mutants with desired product profiles.

Abstract The cost-efficient degradation of xylan to fermentable sugars is of particular interest in second generation bioethanol production, feed, food, and pulp and paper industries. Multiple potentially secreted enzymes involved in polysaccharide deconstruction are encoded in the genome of Paenibacillus sp. A59, a xylanolytic soil bacterium, such as three endoxylanases, seven GH43 β-xylosidases, and two GH30 glucuronoxylanases. In secretome analysis of xylan cultures, ten glycoside hydrolases were identified, including the three predicted endoxylanases, confirming their active role. The two uni-modular xylanases, a 32-KDa GH10 and a 20-KDa GH11, were recombinantly expressed and their activity on xylan was confirmed (106 and 85 IU/mg, respectively), with differences in their activity pattern. Both endoxylanases released mainly xylobiose (X2) and xylotriose (X3) from xylan and pre-treated biomasses (wheat straw, barley straw, and sweet corn cob), although only rGH10XynA released xylose (X1). rGH10XynA presented optimal conditions at pH 6, with thermal stability at 45–50 °C, while rGH11XynB showed activity in a wider range of pH, from 5 to 9, and was thermostable only at 45 °C. Moreover, GH11XynB presented sigmoidal kinetics on xylan, indicating possible cooperative binding, which was further supported by the structural model. This study provides a detailed analysis of the complete set of carbohydrate-active enzymes encoded in Paenibacillus sp. A59 genome and those effectively implicated in hemicellulose hydrolysis, contributing to understanding the mechanisms necessary for the bioconversion of this polysaccharide. Moreover, the two main free secreted xylanases, rGH10XynA and rGH11XynB, were fully characterized, supporting their potential application in industrial bioprocesses on lignocellulosic biomass.

Dans le contexte de la bioéconomie, la découverte et la caractérisation des enzymes capables de dégrader la paroi végétale est particulièrement intéressante pour l’utilisation de la biomasse lignocellulosique dans l’industrie. A cet égard, la métagénomique fonctionnelle est un outil puissantpour découvrir de nouvelles enzymes à partir d’écosystèmes microbiens variés, comme l’illustrent les travaux sur le tube digestif du termite Pseudacanthotermes militaris. Cette étude a fourni une mine d’informations et identifié un hypothétique locus d’utilisation du xylane (XUS), codant pour cinq glycosides hydrolases (GH) et une carbohydrate esterase (CE) de Bacteroidales.Le XUS du métagénome de Pseudacanthotermes militaris contient une xylanase de la famille GH10 qui possède une organisation modulaire complexe dans laquelle la séquence du domaine GH10 est interrompue par une insertion de deux carbohydrate binding modules (CBM). Des travaux préliminaires ont montré que cette enzyme modulaire, désignée Pm25, est active sur xylane. Par conséquent, un des objectifs de cette étude a été la caractérisation détaillée des propriétés biochimiques et catalytiques de Pm25. Le rôle des CBM a également été examiné en quantifiant les interactions protéines-sucres et permettant ainsi une meilleure compréhension du rôle spécifique de ces modules, les résultats obtenus permettent de cerner l’impact de la modularité de Pm25 sur ses propriétés fonctionnelles.Dans une deuxième partie de l’étude, nous avons entrepris d’étudier la fonction de Pm25 dans le contexte du cluster XUS. Pour ce faire, nous avons étudié les enzymes adjacentes à Pm25 sur le locus,une autre GH10, une GH11, une GH115 et une GH43. La comparaison des paramètres cinétiques et une étude détaillée des produits d’hydrolyse ont été analysés par spectrométrie de masse et ont révélé que la GH10 et la GH11 étaient les enzymes clefs de la dépolymérisation en étant 20 fois plus efficaces que Pm25. En parallèle, nous avons développé un protocole pour l’utilisation de la micro-thermophorèse (MST) pour quantifier les interactions CBM-sucres, une approche intéressante qui nécessite peut d’échantillon et de ligand contrairement à d’autres méthodes biophysiques. Dans l’ensemble, cette étude a révélé le rôle important de Pm25 et ses homologues dans les locus d’utilisation des xylanes chez les Bacteroidetes et a permis d’identifié le sens de cette architecture particulière
In the context of bioeconomy, the discovery and study of plant-cell wall degrading enzymes is particularly relevant for the use of lignocellulosic biomass for industrial purposes. In this respect, functional metagenomics has proven to be a powerful tool to discover new enzymes from a variety of microbial ecosystems, as exemplified by work performed on the gut of the termite Pseudacanthotermes militaris. This study provided a wealth of information and identified an interesting hypothetical xylan utilization system, encoding five glycoside hydrolases (GH) and one carbohydrate esterase (CE) annotated from bacteroidales. The Pseudacanthotermes militaris-derived putative XUS cluster contains a GH10 xylanase that displays a quite complex modular arrangement wherein the GH10 catalytic module contains two insertional carbohydrate binding modules (CBM). During the preliminary work, this modular enzyme, designated Pm25, was shown to be active on xylan, thus in the present research we set out to more thoroughly characterize its biochemical and catalytic properties.The role of the CBM was also investigated, quantifying protein-carbohydrate interactions and thus providing better insight into the specific role of the modules. Taken together, the results obtained provide insight into how Pm25 modularity translates into functional properties. In second part of our study, we set out investigate the function of Pm25 in the context of the XUS cluster. To achieve this we studied a xylan utilization system, which is constituted by another GH10, GH11, GH115 and GH43. The comparison of kinetic parameters and a detailed end product analysis by mass spectrometry showed that GH10 and GH11 outweigh over 20 fold Pm25 catalytic efficiency. In parallel, we developed the use of MicroScale Thermophoresis (MST) to quantify CBM-carbohydrates interactions, an interesting approach requiring smaller concentration of proteinsand ligands compared to other biophysical methods. Overall this study highlighted the important role of Pm25 homologs in the xylan utilization system in Bacteroidetes, and pinpointed the meaning of its unusual architecture

La création de nouvelles enzymes pour l’hydrolyse de la biomasse est une stratégie clé pour ledéveloppement du bioraffinage. Dans ce contexte, les xylanases de la famille GH11 sont déjàdéployées dans de nombreux procédés industriels et donc bien positionnées pour jouer un rôleimportant dans ces procédés. La cible de cette étude, la xylanase GH11 (Tx-Xyl) de la bactérieThermobacillus xylanilyticus, est une enzyme thermostable et donc une bonne candidate pour destravaux d’ingénierie visant l’amélioration de son activité sur des substrats ligno-cellulosiques.Dans cette étude, deux stratégies d’ingénierie des enzymes ont été employées afin d’obtenir denouvelles informations portants sur les relations structure-fonction au sein de Tx-Xyl. La premièrestratégie a consisté en l’utilisation d’une approche de mutagenèse aléatoire, couplée à l’emploi deméthodes de recombinaison in vitro. Ces travaux avaient pour objectif d’améliorer la capacitéhydrolytique de Tx-Xyl sur la paille de blé. La deuxième stratégie mise en oeuvre s’est appuyée surune approche semi-rationnelle visant la création d’une enzyme chimérique, qui bénéficierait d’uneamélioration des interactions enzyme-substrat au niveau du sous-site -3.Le premier résultat majeur de cette thèse concerne le développement d’une méthode de criblagequi permet l’analyse à haut débit de banques de mutants pour la détection de variants quiprésentent une activité hydrolytique accrue directement sur paille de blé. A l’aide de ce crible, nousavons pu analyser plusieurs banques de mutants, représentant un total de six générations demutants, et identifier une série de combinaisons de mutations différentes. D’un côté, un variant,comportant deux mutations silencieuses, permet une meilleure expression de Tx-Xyl, alors qued’autres enzymes mutées présentent des modifications intrinsèques de leurs aptitudes catalytiques.Comparés à l’enzyme parentale Tx-Xyl, certains mutants solubilisent davantage les arabinoxylanes dela paille et, lorsqu’ils sont déployés avec un cocktail de cellulases, participent à une réactionsynergique qui permet un accroissement du rendement des pentoses et du glucose libérés.A l’aide d’une approche semi-rationnelle, une séquence de 17 acides aminés en provenance d’unexylanase GH11 fongique a été ajoutée à l’extrémité N-terminale de Tx-Xyl, afin de créer de nouveauxbrins β. L’enzyme chimérique a pu être exprimée avec succès et caractérisée. Néanmoins, l’analysede ses propriétés catalytiques a révélé que celle-ci ne présente pas davantage d’interactions avec sonsubstrat dans le sous-site -3, mais les résultats obtenus fournissent de nombreux renseignements surles relations structure-fonction au sein de l’enzyme. De plus, ces travaux nous permettent depostuler que Tx-Xyl posséderait un site de fixation secondaire pour les xylanes, un élement jusqu’iciinsoupçonné dans cette enzyme. Par ailleurs, l’analyse de nos résultats nous permet de proposer uneexplication rationnelle pour l’échec de notre stratégie initiale
Engineering new and powerful enzymes for biomass hydrolysis is one area that will facilitate thefuture development of biorefining. In this respect, xylanases from family GH11 are already importantindustrial biocatalysts that can contribute to 2nd generation biorefining. The target of this study, theGH11 xylanase (Tx-Xyl) from Thermobacillus xylanilyticus is thermostable, and is thus an interestingtarget for enzyme engineering, aiming at increasing its specific activity on lignocellulosic biomass,such as wheat straw. Nevertheless, the action of xylanases on complex biomass is not yet wellunderstood, and thus the use of a rational engineering approach is not really feasible.In this doctoral study, to gain new insight into structure-function relationships, two enzymeengineering strategies have been deployed. The first concerns the development of a randommutagenesis and in vitro DNA shuffling approach, which was used in order to improve the hydrolyticpotency of Tx-Xyl on wheat straw, while the second strategy consisted in the creation of a chimericenzyme, with the aim of probing and improving -3 subsite binding, and ultimately improvinghydrolytic activity.The first key results that has been obtained is the development of a novel high-throughputscreening method, which was devised in order to reliably pinpoint mutants that can better hydrolyzewheat straw. Using this screening method, several generations of mutant libraries have beenanalyzed and a series of improved enzyme variants have been identified. One mutant, bearing silentmutations, actually leads to higher gene expression, while others have intrinsically altered catalyticproperties. Testing of mutants has shown that some of the enzyme variants can improve thesolubilization of wheat straw arabinoxylans and can work in synergy with cellulose cocktails torelease both pentose sugars and glucose.Using a semi-rational approach, 17 amino acids have been added to the N-terminal of Tx-Xyl, withthe aim of adding two extra β-strands coming from a GH11 fungal xylanase. A chimeric enzyme hasbeen successfully expressed and purified and its catalytic properties have been investigated.Although this approach has failed to create increased -3 subsite binding, the data presented revealsimportant information on structure-function relationships and suggest that Tx-Xyl may possess ahitherto unknown secondary substrate binding site. Moreover, a rational explanation for the failureof the original strategy is proposed

A dinâmica estrutural fundamentando a função das xilanases GH11 ainda não está clara. Novo conhecimento sobre a dinâmica catalítica dessas enzimas é crucial para a engenharia de novas enzimas melhoradas beneficiando, assim, diversas indústrias biotecnológicas e de química verde. Com base nesse fato, esse trabalho teve por objetivo obter novas informações acerca da dinâmica catalítica de uma xilanase GH11, através do uso de um conjunto de diversas técnicas avançadas de biofísica molecular em nível bulk e em nível de molécula única (inglês single molecule ou sm). Para isso, foram projetadas xilanases GH11 de Bacillus subtilis ssp. subtilis 168 (XynA) com mutações únicas de cisteína para a marcação dos resíduos D119 e R122 no domínio polegar, do resíduo N54 no domínio dedos, e do resíduo N151 na alfa-hélice, seguidas pela sua construção e produção por métodos de biologia molecular. Esses mutantes foram marcados em seus respectivos grupos tióis com a sonda fluorescente sensível à polaridade Acrylodan, com a sonda de spin MTSSL, e com a sonda fluorescente fotoestável AttoOxa11. A xilanase tipo selvagem for marcada em seu N-terminal com a sonda fotoestável Alexa Fluor® 488 5-SDP Ester. Foram utilizados ensaios de espectrofotometria de fluorescência em nível bulk e de espectroscopia de ressonância paramagnética eletrônica para investigar como a dinâmica do domínio polegar da xilanase GH11, temperatura, e ligação ao substrato se correlacionam um com o outro. Os resultados atestaram que um estado do domínio polegar controlado por temperatura, aberto, dinâmico e flexível tem mais chances de se ligar efetivamente ao substrato de uma maneira produtiva, o que está em completo acordo com estudos anteriores de simulação de dinâmica molecular, cristalografia, desnaturação térmica, e análise funcional por desenho racional de mutantes de domínio polegar de xilanases GH11. Com base nas evidências adquiridas e em estudos anteriores, nós propomos um conjunto de hipóteses e modelos para a dinâmica catalítica da xilanase, focando no papel do domínio polegar nesse processo. No intuito de determinar a constante de afinidade da xilanase por seu substrato e os tempos de relaxamento e constantes de velocidade dos movimentos do domínio polegar, foram feitas medidas de espectroscopia de correlação de fluorescência simples e combinada com transferência eletrônica fotoinduzida, usando as xilanases marcadas com as sondas fluorescentes fotoestáveis, na presença e na ausência de substrato. Os resultados mostraram tempos de difusão muito maiores para as xilanases na presença de substrato, como efeito da afinidade da enzima pelo mesmo. Entretanto, não foi verificada nenhuma curva de decaimento como efeito de supressão dinâmica da sonda por PET. Esses mesmos conjugados foram aplicados com sucesso em microscopia por imagem de tempo de vida de fluorescência, no intuito de analisar sistematicamente a afinidade da xilanase por partículas insolúveis e filmes de substrato, e por fragmentos insolúveis de frações de processos de deslignificação e desestruturação de bagaço de cana-de-açúcar, assim como para a análise da composição, estrutura e topologia desses materiais. Foi possível verificar a presença de xilano na maioria das frações desse bagaço tratado, mas em quantidades variáveis
The structural dynamics underlying the function of GH11 xylanases is still unclear. New insights into the catalytic dynamics of these enzymes are crucial for engineering novel improved enzymes benefiting biotechnological and green chemistry industries. The objective of this work was to obtain new information concerning the catalytic dynamics of a GH11 xylanase, by using a combination of advanced molecular biophysics techniques, both at the bulk level and at the single molecule level (sm). Mutant GH11 xylanases from Bacillus subtilis ssp. subtilis 168 (XynA) were designed with single point cysteine mutations for labeling the residues D119 and R122 on the thumb domain, N54 on the fingers domain, and N151 on the alpha helix, followed by their construction and production by molecular biology methods. These mutants were labeled at their respective thiol groups by the polarity sensitive fluorescent probe Acrylodan, by the electron spin probe MTSSL, and by the photostable fluorescent probe AttoOxa11. The wild-type xylanase was labeled at its N-terminus by the photostable fluorescent probe Alexa Fluor® 488 5-SDP Ester. Bulk fluorescence spectrophotometry and electron paramagnetic resonance assays were used to investigate how the thumb domain dynamics of the GH11 xylanase, temperature and substrate binding were correlated. These results demonstrated that a temperature controlled, open, dynamical and flexible thumb domain state is more likely to effectively bind the substrate in a productive way, which is in complete agreement with previous studies from molecular dynamics simulations, crystallography, thermal denaturation, and function analysis by the rational design of thumb mutants for GH11 xylanases. Based on this evidence and previous studies, we proposed a hypothesis for the xylanase catalytic dynamics, focusing on the role of the thumb domain. In order to determine the xylanase affinity constant for its substrate and the relaxation times and rate constants of the thumb domain movements, fluorescence correlation spectroscopy measurements were performed. Both simple and combined measurements with photoinduced electron transfer were performed, using the xylanases labeled with photostable fluorescent probes, in the presence and absence of substrate. The results have shown longer diffusion times for the xylanases in the presence of substrate, as an effect of the enzyme affinity for it. However, it was not verified any decay curve as an effect of the dynamic suppression of the probe via PET. The same conjugates were successfully applied to fluorescence-lifetime imaging microscopy, aiming to systematically analyze the affinity for xylanase of substrates in the form of insoluble particles and films, and for water insoluble fractions from sugarcane bagasse delignification processes. In addition, the composition, structure and topology of these materials was examined. It was possible to verify the presence of xylan in most fractions of this treated bagasse, although in variable quantities

Xylanases (EC 3.2.1.8) are glycosyl hydrolases that catalyze the hydrolysis of internal β-1,4 glycosidic bonds of xylan backbones, and have potential economical and environment friendly applications in the paper pulp, food, animal feed, detergent industries, bio-ethanol and bio-energy production systems. A xylanase from Bacillus sp. NG-27 (BSX), which is an extracellular endoxylanase, belonging to glycosyl hydrolase family 10 (GH10), shows optimum activity at a temperature of 70 °C and at a pH 8.5. It has a (β/α)8-triosephosphate isomerase (TIM) barrel fold, which has been studied concerning its function, structural properties, design and evolution. BSX, apart from thermo-alkalophilic features, shows resistance to SDS denaturation and protease K degradation. Hence, BSX serves as an important model system for fundamental understanding of the structure-stability-evolution relations of the ubiquitous TIM barrel fold. While the factors responsible for the thermal stability of GH10 xylanases have been analyzed, the improvement of thermostability of already thermostable enzymes is an important challenge. In general, there are large differences in optimal temperature (Tm) between hyperthermostable proteins with respect to their mesophilic homologs, indicating considerable scope available for introducing novel protein engineering approaches to improve protein stability. Thermostability and thermotolerance are of particular importance for industrial enzymes, because higher operating temperatures allow higher reactivity, higher bioavailability, higher process yield, lower viscosity, and reducing the risk of contamination. Thus, finding enzymes that can function at high temperatures has immense industrial importance and constitutes an active area of research. Earlier studies on enzymatic activity and thermostability of a recombinant BSX (RBSX) with different extreme N-terminus mutants by biochemical/biophysical methods showed that a single amino acid substitution (Val1→Leu) markedly enhanced the thermostability of recombinant xylanase from 70 °C to 75 °C without compromising its catalytic activity and showed higher cooperativity in the thermal unfolding transition. Conversely, substitution of Val1→Ala (V1A) at the same position decreased the stability of the protein from 70 °C to 68 °C. Furthermore, it was observed that substitution of Phe4 by Ala decreased the stability by ~4 °C whereas substitution of Trp6→Ala and Tyr343→Ala decreased the stability by ~10 °C with respect to RBSX. On the other hand, substitution of Phe4 by another aromatic residue Trp (F4W) did not change the stability and activity of RBSX. However, structural details were not available at that time, precluding any structure-based rationalization of stability changes resulting from a single amino acid substitution. The thesis reports the crystal structures of a recombinant xylanase from Bacillus sp. NG-27 (RBSX) and its various N-terminal and C-terminal mutants namely V1A, V1L, F4A, F4W, W6A, and Y343A. The crystal structure of RBSX (PDB ID: 4QCE) was solved at a resolution of 2.35 Å whereas those of V1A mutant (PDB ID: 4QCF) and V1L mutant (PDB ID: 4QDM) were solved at a resolution of 2.26 Å and 1.99 Å respectively. On the other hand, the crystal structure of F4A was solved at a resolution of 2.23 Å whereas F4W, W6A, and Y343A mutants were solved at a resolution of 2.22 Å, 1.67 Å, and 2.30 Å respectively. The availability of experimentally determined RBSX structure and its various mutant structures has enabled a critical examination including from a network perspective, of factors influencing thermal stability. The crystal structures in combination with computational analysis have provided valuable insights into the structural features that govern protein thermostability. The thesis candidate established a link between N-terminal to C-terminal contacts and RBSX thermostability. The study reveals that augmenting N-terminal to C-terminal noncovalent interactions is associated with enhancement of the stability of the enzyme. Perhaps, for the first time, the study provides a network perspective of N-terminal to C-terminal interactions and shows that the stabilizing interactions are not restricted to terminal regions but propagate to different parts of the protein structure. Furthermore, analysis of structures of different aromatic mutants of RBSX and structural bioinformatics studies were combined to understand the role of long-range aromatic cluster in the form of 'aromatic-clique' in the thermal stabilization of proteins. The results highlight an additional source of stability in thermophilic proteins, which could arise due to the prevalence of aromatic-cliques. In addition, the work exemplifies the experimental evidence specifically through long-range aromatic clique, in reiterating the role of interactions between N- and C-termini in protein stabilization. The thesis candidate demonstrated the experimental evidence depicting the role of partially solvent exposed tryptophan residues in shielding a surface pocket, which influenced the solvation of backbone atoms and stability of the RBSX enzyme. The candidate carried out a comprehensive database analysis of available crystal structures to look into the possible role of partially exposed tryptophan in hyperthermophilic proteins. The study provides strong evidence that partially exposed tryptophan side-chain is recruited in hyperthermophilic proteins for occluding potential surface pockets, to provide backbone solvent shielding and local stabilization. The overall structure of this thesis is further explained through a chapter wise description below: Chapter 1 | An introduction and outline of the thesis This chapter starts with a general introduction about the diversity of microorganisms and their ability to thrive in extreme environments such as high temperature. The research on these enterprising organisms offers not just the insights into the resilience of life on earth or possibilities of life elsewhere in the universe but also can provide exciting opportunities for a variety of industrial, environmental, biomedical, and pharmaceutical applications. While the adaptation of the cell inventory is important, it is a challenge for proteins to overcome high temperature in order to remain folded in the correct three-dimensional structure while maintaining adequate flexibility for their desired function. Hence, elucidation of the molecular basis of protein stability at extreme temperature continues to attract researcher over a board range of disciplines. The various structural features responsible for protein stability are outlined and the basic structural and molecular strategies for the adaptation to high temperatures revealed by structure analysis are delineated. Of all potentially deactivating factors of protein stability, temperature is the best studied. A brief outline of the strategies and approaches for the design of proteins to meet the desirable properties such as increased thermal stability are presented whereas the structural features responsible for stability of triosephosphate isomerase (TIM)-barrel fold is outlined under a separate section. Subsequently a short introduction of family 10 (GH10) xylanases, which has the ubiquitous TIM-barrel fold and their classifications are presented. A section is dedicated to describe various thermostable GH10 xylanases, their structural features responsible for stability, and current and potential biotechnological applications. At the end, the scope of the present work is detailed. Chapter 2| Crystallization, Data Collection and Data processing of recombinant BSX (RBSX) and its different variants: Chapter 2 presents the purification of recombinant xylanase from Bacillus sp. NG-27 (RBSX), its N-terminal variants (V1A, V1L), and aromatic variants (F4W, F4A, W6A, and Y343A). The expression and purification of RBSX and its variants were carried out at the laboratory of our collaborator Prof. V. S. Reddy, Plant Transformation Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India. Initial crystallization trials were screened by hanging drop vapor diffusion method and micro-batch diffusion method using crystallization-screening kits (Crystal Screen and Crystal Screen 2) from Hampton Research, USA and a laboratory made screen, which was based on reported crystallization condition of native BSX. After a few rounds of trials and optimization of the crystallization condition, diffraction quality rod shaped crystals of recombinant BSX (RBSX) were obtained within ten days, when 2-µl protein solution (10 g/ml) was mixed with 2-µl reservoir solution composed of 0.12 M MgCl2, 0.1 M NaCl, 0.1M Tris-HCl pH 8.5 and 15% PEG 8000. Subsequently, crystal was used for X-ray data collection and it diffracted X-rays to better than 2.2 Å at the home source at cryo-temperature (100 K). RBSX crystals belong to orthorhombic space group P212121 with unit cell parameters a = 54.77 Å, b = 75.65 Å, c = 179.91 Å and α = β = γ =90°. A three-dimensional screening grid was prepared based on crystallization condition of RBSX by carefully varying salt concentration (NaCl and MgCl2 from 10mM to 300mM in the interval of 10mM), different PEG variants (PEG 1000, PEG 3350, PEG 4000, PEG 8000, and PEG 10000) in the range of 5% to 20%. Tris-HCl buffer of pH 8.0 and of pH 8.5 was used in the concentration range of 0.05M and 0.1M respectively. Rod shaped crystals were obtained using hanging drop vapor diffusion method from the condition of 0.1M NaCl, 80mM MgCl2, 0.05M Tris-HCl pH 8.5 and 18 % PEG 8000 and 0.1M NaCl, 60mM MgCl2, 0.1M Tris-HCl pH 8.5 and 16 % PEG 8000 for V1L mutant and V1A mutant respectively. The diffracting crystals of F4A mutant were obtained from the condition of 0.1M NaCl, 140mM MgCl2, 0.05M Tris-HCl pH 8.5 and 15% PEG 8000 by using hanging drop vapor diffusion method. On the other hand, F4W and Y343A, crystals were grown by micro-batch diffusion method containing 1.1-μ1 ratio of protein and crystallization solution of 0.1M NaCl, 120mM MgCl2, 0.1M Tris-HCl pH 8.5 and 18% PEG 8000 and 0.1M NaCl, 150mM MgCl2, 0.1M Tris-HCl pH 8.5 and 15 % PEG 6000 respectively . W6A mutant crystals were grown by hanging drop vapor diffusion method of 0.1M NaCl, 160mM MgCl2, 0.05M Tris-HCl pH 8.5 and 20% PEG 8000. All the crystals were obtained at 20 °C-22 °C in 5-10 days, and were used for diffraction experiments (details in the table below). Table 1 Protein Space a b c α β γ X-ray source PDB group (Å) (Å) (Å) (°) (°) (°) ID RBSX P212121 54.77 75.65 176.91 90 90 90 Home-source 4QCE V1A C2 73.57 80.12 69.90 90 110.81 90 Home-source 4QCF V1L P212121 54.88 76.58 176.73 90 90 90 Synchrotron 4QDM F4W P212121 55.27 77.32 176.75 90 90 90 Home- source 5EB8 F4A P212121 52.62 67.71 181.54 90 90 90 Home- source 5EFF W6A P212121 54.99 76.60 181.54 90 90 90 Synchrotron 5EFD Y343A C2 73.86 80.11 69.21 90 111.19 90 Home- source 5EBA The quality of all dataset was assessed by SFCHECK. The data sets were found appropriate and useful for structure determination as discussed in Chapter 3. Chapter 3 | Molecular Replacement, Model Building, Refinement, validation of recombinant xylanase (RBSX), and different mutant structures: Chapter 3 details the application of molecular replacement method to the structure solution of RBSX structure, N-terminal and aromatic mutants of RBSX, the course of iterative model building and the refinement carried out and the quality of the final protein structure models. The structure solution for all the structures was obtained by the molecular replacement (MR) method with the program PHASER-MR in the PHENIX package using a search model of native-enzyme (2F8Q). The asymmetric unit of RBSX, V1L, F4A, F4W, W6A crystals was expected to contain two molecules whereas V1A and Y343A crystal was expected to contain one molecule as indicated by Matthews’s coefficient calculation. The final round of refinement was carried out with restrained refinement with TLS parameters for all the structures. The most essential refinement statistics of the final model of RBSX, V1A, and V1L mutant structures are given in Table 2 whereas the same for aromatic mutant structures, F4A, F4W, W6A, and Y343A are given in Table 3. Table 2 Refinement Statistics RBSX V1A V1L Resolution (Å) 27.7-2.32 26.8-2.26 40.2-1.96 Rwork / Rfree (%) 17.9/22.7 17.4/22.5 15.2/19.0 Average B-factors (Å2) Protein 21.6 26.3 13.9 Ligand/ion 15.6 26.4 18.74 Water 20.6 27.2 23.2 RMSD Bond distance (Å) 0.007 0.005 0.019 Bond angles (◦) 1.123 0.955 1.802 Luzzati coordinate 0.279 0.269 0.175 error (Å) Working set Table 3 Refinement Statistics F4A F4W W6A Y343A Resolution (Å) 18.15-2.23 18.97-2.22 32.1-1.67 34.03-2.30 Rwork / Rfree (%) 17.8/24.0 16.8/21.0 15.68/18.58 17.8/23.0 Average B-factors (Å2) Protein 13.1 19.5 16.7 26.8 Ligand/ion 14.2 20.0 21.4 26.1 Water 11.5 28.9 25.9 30.7 RMSD Bond distance (Å) 0.0144 0.0088 0.0139 0.0063 Bond angles (◦) 1.593 1.228 1.5995 1.0951 Luzzati coordinate 0.261 0.252 0.176 0.293 error (Å) Working set Chapter 4 | Mutations at the extreme N-terminus modulate thermostability of RBSX: Implications of interactions between termini for stability This chapter details the structural analysis of RBSX and its various extreme N-terminus mutations in relation to their different thermostability scale. Although several factors have been attributed to thermostability, the stabilization strategies used by proteins are still enigmatic. Studies on a RBSX, which has the ubiquitous (β/α)8-TIM (Triosephosphate isomerase) barrel fold showed that just a single mutation, Valine1→Leucine (V1L), though not part of any secondary structural element, markedly enhanced the stability from 70 °C to 75 °C without loss of catalytic activity. Conversely, substitution of Valine1→Alanine (V1A) at the same position decreased the stability of the enzyme from 70 °C to 68 °C. To gain structural insights as to how a single extreme N-terminus mutation can markedly influence the thermostability of the enzyme, the candidate has determined the crystal structure of RBSX and two mutants. Based on computational analysis of their crystal structures including residue interaction network, a link was established between N- to C-terminal contacts and RBSX thermostability. The study reveals that augmenting N- to C-terminal non-covalent interactions is associated with the enhancement of the stability of the enzyme. Perhaps, for the first time, the study provides a network perspective of N-terminal to C-terminal interactions and shows that the stabilizing interactions are not restricted to terminal regions but propagate to different parts of the protein structure. In addition, several lines of evidence were discussed that point to support the structural coupling between the chain termini and implications of stability changes in different proteins. It is proposed that the strategy of mutations at the termini could be exploited with a view to modulate stability without compromising on enzymatic activity, or in general, protein function, in diverse folds where N- and C-termini are in close proximity. Chapter 5 | Role of long-range aromatic cluster in the structural stability of RBSX Chapter 5 describes the different aromatic mutant crystal structures of RBSX namely F4W, F4A, W6A, and Y343A and the structural comparison with the RBSX crystal structure. Systematic studies of different alanine mutations (F4A, W6A, and Y343A) to disrupt this aromatic cluster showed that substitution of Phe4, Trp6, and Y343 by alanine drastically decreased the stability of recombinant BSX (RBSX). It was observed that substitution of Phe4 by Ala (F4A) decreased the RBSX stability by ~5 °C whereas substitutions of Trp6 by Ala (W6A) and Tyr343 by Ala (Y343A) markedly decreased the stability of the enzyme by ~10 °C. On the other hand, substitution of Phe4 by Trp (F4W) did not result any change in its thermal unfolding pattern of the enzyme. We observed that the mutated amino acid residues (Phe4, Trp6, and Tyr343) in the RBSX structure are part of an ‘aromatic-clique’. An aromatic-clique is defined as a cluster of aromatic residues in which each residue interacts with all other residues within the cluster through aromatic interactions. The study reveals that the decreased stability shown by F4A, W6A, and Y343A mutants resulted from cumulative effects in the loss of aromatic interactions and disruption of aromatic-clique, and reduced van der Waal interactions. In addition, the work exemplifies the importance of interactions between N-terminal and C-terminal through aromatic contacts or packing in folding and stability of the TIM-barrel fold protein. The structure based multiple sequence alignment of RBSX with other GH10 xylanase from Bacillus organisms revealed that aromatic-clique of interest is fully conserved in B. halodurans (BHX) and Bacillus firmus (BFX) xylanases, which are thermostable in nature, like RBSX. On the other hand, this aromatic-clique is not conserved in the GH10 xylanases from Bacillus N137, Bacillus alcalophilus, which are reported as thermo-labile in nature. Furthermore, analysis of available crystal structures of different thermostable xylanases from GH10 family showed the prevalence of aromatic-clique that may be playing a critical role in their structure-stability and folding. Lastly, a comprehensive analysis of homologous pairs of proteins from (hyper)thermophilic and mesophilic organisms was carried out and observed the high occurrence of aromatic-cliques in the thermophilic proteins in comparison to their mesophilic homologs. These results highlight an additional source of stability in thermophilic proteins, which can arise due to the prevalence of aromatic-cliques. The findings reported in the thesis provide important lessons for engineering xylanases for industrial applications. The strategy of mutations based on clustering of aromatic pairs in the form of ‘aromatic-clique’ may be effectively applied to other enzymes and provides new insights for engineers to design proteins for biotechnological applications. Chapter 6 | Tryptophan occludes surface pocket: Implications for protein stability Chapter 6 describes the structural feature of a partially exposed tryptophan residue, which effectively occludes a surface pocket and plays a critical role in RBSX thermo-stabilization. As a part our long-standing interest in the structural analysis of thermostable proteins, it was observed that just a single mutation, W6A of a recombinant xylanase (RBSX) from Bacillus sp. NG-27 decreased the stability from 70 °C to 60 °C. To gain structural insights into how a single mutation W6A can remarkably influence the thermostability of the enzyme, we determined the crystal structure of W6A mutant and compared the same with the crystal structure of RBSX. We serendipitously observed that substitution of Trp6 by alanine (W6A) in the protein results a small surface pocket, which was shielded by the bulky side-chain of Trp6 in the native structure. Inspection of the molecular structure of native protein structure revealed that side chain of Trp6 occludes the surface pocket, sterically impeding entry of solvent molecules including water. We demonstrated the experimental evidence depicting how a partially exposed tryptophan, which was shielding a surface pocket (tryptophan-shield), can directly influence the backbone solvation, and modulate the stability of the enzyme. Furthermore, computational analysis of high-resolution structures of hyperthermophilic proteins reveals that bulky and aromatic indole side-chain of tryptophan effectively occludes surface pockets in several hyperthermophilic proteins. The study provides a strong evidence that partially exposed tryptophan side-chain is recruited in hyperthermophilic proteins for occluding potential surface pockets to provide backbone solvent shielding and local stabilization. Chapter 7 | Summary and future direction Chapter 7 summaries the important findings of the present study from the crystal structure and computational analysis of a recombinant xylanase (RBSX) and its various N-terminal and C-terminal mutants and also outlines the future direction of the work. Appendix A details SFCHECK output for the processed data for all the structures reported in the thesis. Appendix B Reprints of the publications