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Integrated bioprocess strategies may facilitate ethanol production from both C6 and C5 fractions of lignocellulosic feedstocks. We propose a new process concept, SSICF, where sugarcane bagasse is hydrolyzed simultaneously with xylose isomerization and the co-fermentation of C6 and C5 sugars. A commercial cocktail was supplemented with our multi-enzymatic system composed of three recombinant enzymes immobilized in Feroxyhyte magnetic nanoparticles: β-glucosidase, β-xylosidase and xylose isomerase. SSICF was performed using non-GMO Saccharomyces at pH 6.0 and 35 °C for 72 h in a synthetic medium containing cellobiose and xylose, and another medium containing pretreated sugarcane bagasse (PSB). The results of ethanol global yields in SSICF were 77.67% and 73.24% for the synthetic medium and PSB, respectively. In a nutshell, this is the first report of a successful proof-of-concept of SSICF with four rounds of enzyme recycling and a non-GMO yeast, an innovative process with high potential for industrial use.

Isomerases are enzymes that induce physical changes in a molecule without affecting the original molecular formula. Among this class of enzymes, xylose isomerases (XIs) are the most studied to date, partly due to their extensive application in industrial processes to produce high-fructose corn sirups. In recent years, the need for sustainable initiatives has triggered efforts to improve the biobased economy through the use of renewable raw materials. In this context, D-xylose usage is crucial as it is the second-most abundant sugar in nature. The application of XIs in biotransforming xylose, enabling downstream metabolism in several microorganisms, is a smart strategy for ensuring a low-carbon footprint and producing several value-added biochemicals with broad industrial applications such as in the food, cosmetics, pharmaceutical, and polymer industries. Considering recent advancements that have expanded the range of applications of XIs, this review provides a comprehensive and concise overview of XIs, from their primary sources to the biochemical and structural features that influence their mechanisms of action. This comprehensive review may help address the challenges involved in XI applications in different industries and facilitate the exploitation of xylose bioprocesses.

Made available in DSpace on 2021-06-25T15:07:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-06-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Understanding the aspects that contribute to improving proteins' biochemical properties is of high relevance for protein engineering. Properties such as the catalytic rate, thermal stability, and thermal resistance are crucial for applying enzymes in the industry. Different interactions can influence those biochemical properties of an enzyme. Among them, the surface charge-charge interactions have been a target of particular attention. In this study, we employ the Tanford-Kirkwood solvent accessibility model using the Monte Carlo algorithm (TKSA-MC) to predict possible interactions that could improve stability and catalytic rate of a WT xylanase (XynA(WT)) and its M6 xylanase (XynA(M6)) mutant. The modeling prediction indicates that mutating from a lysine in position 99 to a glutamic acid (K99E) favors the native state stabilization in both xylanases. Our lab results showed that mutated xylanases had their thermotolerance and catalytic rate increased, which conferred higher processivity of delignified sugarcane bagasse. The TKSA-MC approach employed here is presented as an efficient computational-based design strategy that can be applied to improve the thermal resistance of enzymes with industrial and biotechnological applications. Brazilian Ctr Res Energy & Mat, Brazilian Biorenewables Natl Lab, Campinas, SP, Brazil Rice Univ, Ctr Theoret Biol Phys, Houston, TX USA Sao Paulo State Univ, Inst Biosci Letters & Exact Sci, Dept Phys, Sao Jose Do Rio Preto, SP, Brazil Brazilian Ctr Res Energy & Mat, Brazilian Biosci Natl Lab, Campinas, SP, Brazil Univ Fed Triangulo Mineiro, Inst Exact Sci Nat & Educ, Theoret Biophys Lab, Uberaba, MG, Brazil Univ Fed Mato Grosso do Sul, Inst Biosci, Microorganisms & Gen Biochem Lab, Campo Grande, MS, Brazil Sao Paulo State Univ, Inst Biosci Letters & Exact Sci, Dept Phys, Sao Jose Do Rio Preto, SP, Brazil FAPESP: 2016/13998-8 FAPESP: 2017/09662-7 CNPq: 141985/2013-5 FAPESP: 2017/14253-9 FAPESP: 2018/11614-3 FAPESP: 2014/06862-7 FAPESP: 2016/19766-1 FAPESP: 2019/22540-3 CNPq: 429829/2016-7

Cold-adapted endo-β-1,4-glucanases hold great potential for industrial processes requiring high activity at mild temperatures such as in food processing and extraction of bioactive compounds from plants. Here, we identified and explored the specificity, mode of action, kinetic behavior, molecular structure and biotechnological application of a novel endo-β-1,4-glucanase (XacCel8) from the phytopathogen Xanthomonas citri subsp. citri. This enzyme belongs to an uncharacterized phylogenetic branch of the glycoside hydrolase family 8 (GH8) and specifically cleaves internal β-1,4-linkages of cellulose and mixed-linkage β-glucans releasing short cello-oligosaccharides ranging from cellobiose to cellohexaose. XacCel8 acts in near-neutral pHs and in a broad temperature range (10–50 °C), which are distinguishing features from conventional thermophilic β-1,4-glucanases. Interestingly, XacCel8 was greatly stimulated by cobalt ions, which conferred higher conformational stability and boosted the enzyme turnover number. The potential application of XacCel8 was demonstrated in the caffeine extraction from guarana seeds, which improved the yield by 2.5 g/kg compared to the traditional hydroethanolic method (HEM), indicating to be an effective additive in this industrial process. Therefore, XacCel8 is a metal-stimulated and cold-adapted endo-β-1,4-glucanase that could be applied in a diverse range of biotechnological processes under mild conditions such as caffeine extraction from guarana seeds.

Orientador: Carlos Henrique Inácio Ramos Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química Resumo: Chaperonas moleculares e proteínas de choque térmico (Heat shock protein, Hsp) atuam contra a agregação e o enovelamento incorreto de proteínas, que são os agentes causais de doenças neurodegenerativas, como por exemplo, Alzheimer e Parkinson. A Hsp90 é uma das mais importantes chaperonas moleculares, considerada essencial para a viabilidade celular em eucariotos, pois está associada com a maturação de proteínas atuantes na sinalização e ciclo celular. Além disso, foi demonstrado que a Hsp90 está envolvida na estabilização do fenótipo tumoral de diversos tipos de câncer, destacando a sua importância biomédica. A interação com co-chaperonas, proteínas auxiliares das chaperonas, permite que a Hsp90 atue como uma proteína "hub", ou seja, um ponto central de regulação de diversas proteínas. Muitas dessas co-chaperonas possuem um ou mais domínios do tipo TPR (do inglês, tetratricopeptide repeat) que interagem com o C-terminal da Hsp90. No presente projeto de doutorado, investigamos as características estruturais e termodinâmicas da interação entre o domínio C-terminal da Hsp90 (C-Hsp90) e a co-chaperona TPR Tom70 humana, utilizando técnicas de reação-cruzada acoplada à espectrometria de massas (LC-MS/MS), calorimetria de titulação isotérmica (ITC), espalhamento de raios-X à baixos ângulos (SAXS) e modelagem molecular. Os resultados de LC-MS/MS e ITC evidenciaram novas regiões na interação do complexo C-Hsp90/Tom70 que envolve a hélice A7 presente na Tom70 e experimentos de SAXS revelaram a estrutura em baixa resolução das proteínas C-Hsp90, Tom70 e do complexo C-Hsp90/Tom70. Além disso, investigamos o efeito do celastrol, um composto com potencial atividade anti-câncer, na conformação e na função da Hsp90. Na presença do composto, a Hsp90 sofre um processo de oligomerização e a natureza dos oligômeros foi determinada por ferramentas bioquímicas e biofísicas, tais como espalhamento dinâmico de luz (DLS), cromatografia de exclusão molecular analítica acoplada a espalhamento de luz em multiângulos (SEC-MALS) e eletroforese em gel nativo. Interessantemente, a oligomerização induzida pelo celastrol não afetou a atividade de proteção da Hsp90 contra a agregação protéica e a capacidade de ligação as co-chaperonas com enovelamento tipo TPR. Este é o primeiro trabalho a apontar um possível mecanismo para a ação do celastrol sobre a Hsp90. Coletivamente, nossos resultados e descobertas contribuem para uma melhor compreensão dos mecanismos moleculares relacionados à interação entre chaperonas e co-chaperonas, bem como, chaperonas e potenciais ligantes. Abstract: Molecular chaperones and heat shock proteins (Hsp) act against protein aggregation and misfolding, which are the causal agents of neurodegenerative diseases such as Alzheimer and Parkinson. Hsp90 is one of the most important molecular chaperones, considered essential for cell viability in eukaryotes, since it is associated with the maturation of proteins involved in cell cycle and signaling. In addition, it was demonstrated that Hsp90 is implicated in the stabilization of the tumor phenotype of various types of cancer, highlighting its biomedical importance. The interaction with co-chaperones, auxiliary proteins of chaperones, allows that Hsp90 acts as a hub, being a central point for regulation of several other proteins. Many of these co-chaperones have one or more TPR domains that interact with the C-terminus of Hsp90. In this PhD project, we investigated structural and thermodynamic characteristics of the interaction between the C-terminus domain of Hsp90 (C-Hsp90) and the TPR co-chaperone human Tom70, using techniques of cross-linking coupled with mass spectrometry (LC-MS/MS), isothermal titration calorimetry (ITC), small angle X-ray scattering (SAXS) and molecular modeling. The results of LC-MS/MS and ITC revealed new regions involved in the interaction of the C-Hsp90 with Tom70, which encompasses the A7 helix from Tom70, and SAXS experiments unveiled the low resolution structure of the proteins C-Hsp90, Tom70 and the C-Hsp90/Tom70 complex. In addition, we investigated the effect of celastrol, a compound with a potential anti-cancer activity, on the conformation and function of Hsp90. In the presence of celastrol, Hsp90 undergoes oligomerization and the nature of the oligomers was determined by biochemical and biophysical tools such as dynamic light scattering (DLS), size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) and native gel electrophoresis. Interestingly, the celastrol-induced oligomerization did not affect the protective activities of Hsp90 against protein aggregation or the capacity to bind TPR co-chaperones. This is the first study to point out a possible mechanism for the action of celastrol on Hsp90. Collectively, our findings contribute to a better understanding of the molecular mechanisms associated to the interaction between chaperones and co-chaperones, as well as chaperones and potential ligands Doutorado Química Orgânica Doutora em Ciências

Trichoderma genus fungi present great potential for the production of carbohydrate-active enzymes (CAZYmes), including glycoside hydrolase (GH) family members. From a renewability perspective, CAZYmes can be biotechnologically exploited to convert plant biomass into free sugars for the production of advanced biofuels and other high-value chemicals. GH54 is an attractive enzyme family for biotechnological applications because many GH54 enzymes are bifunctional. Thus, GH54 enzymes are interesting targets in the search for new enzymes for use in industrial processes such as plant biomass conversion. Herein, a novel metal-dependent GH54 arabinofuranosidase (ThABF) from the cellulolytic fungus Trichoderma harzianum was identified and biochemically characterized. Initial in silico searches were performed to identify the GH54 sequence. Next, the gene was cloned and heterologously overexpressed in Escherichia coli. The recombinant protein was purified, and the enzyme’s biochemical and biophysical properties were assessed. GH54 members show wide functional diversity and specifically remove plant cell substitutions including arabinose and galactose in the presence of a metallic cofactor. Plant cell wall substitution has a major impact on lignocellulosic substrate conversion into high-value chemicals. These results expand the known functional diversity of the GH54 family, showing the potential of a novel arabinofuranosidase for plant biomass degradation.

Xyloglucans are highly substituted and recalcitrant polysaccharides found in the primary cell walls of vascular plants, acting as a barrier against pathogens. Here, we reveal that the diverse and economically relevant Xanthomonas bacteria are endowed with a xyloglucan depolymerization machinery that is linked to pathogenesis. Using the citrus canker pathogen as a model organism, we show that this system encompasses distinctive glycoside hydrolases, a modular xyloglucan acetylesterase and specific membrane transporters, demonstrating that plant-associated bacteria employ distinct molecular strategies from commensal gut bacteria to cope with xyloglucans. Notably, the sugars released by this system elicit the expression of several key virulence factors, including the type III secretion system, a membrane-embedded apparatus to deliver effector proteins into the host cells. Together, these findings shed light on the molecular mechanisms underpinning the intricate enzymatic machinery of Xanthomonas to depolymerize xyloglucans and uncover a role for this system in signaling pathways driving pathogenesis., Xyloglucans are polysaccharides found in plant cell walls. Here, the authors describe the xyloglucan depolymerization machinery of phytopathogenic Xanthomonas bacteria, and show that sugars released by this system induce the expression of key virulence factors driving pathogenesis.

Made available in DSpace on 2020-12-10T17:28:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-05-01 Background: Enzymatic isomerization is a promising strategy to solve the problem of xylose fermentation and, consequently, to leverage the production of advanced biofuels and biochemicals. In a previous work, our research group discovered a new strain of Streptomyces with great biotechnological potential due to its ability to produce a broad arsenal of enzymes related to lignocellulose degradation. Methods: We applied a multidisciplinary approach involving enzyme kinetics, biophysical methods, small angle X-ray scattering and X-ray crystallography to investigate two novel xylose isomerases, XylAlF1 and XylA2F1, from this strain. Results: We showed that while XylAlF1 prefers to act at lower temperatures and relatively lower pH, XylA2F1 is extremely stable at higher temperatures and presents a higher turnover number. Structural analysis revealed that XylA1F1 exhibits unique properties in the active site not observed in classical XylAs from classes I and II nor in its ortholog XylA2F1. It encompasses the natural substitutions, M86A and T93K, that create an extra room for substrate accommodation and narrow the active-site entrance, respectively. Such modifications may contribute to the functional differentiation of these enzymes. Conclusions: We have characterized two novel xylose isomerases that display distinct functional behavior and harbor unprecedented amino-acid substitutions in the catalytic interface. General significance: Our findings contribute to a better understanding of the functional and structural aspects of xylose isomerases, which might be instrumental for the valorization of the hemicellulosic fraction of vegetal biomass. Brazilian Ctr Res Energy & Mat CNPEM, Brazilian Biorenewables Natl Lab LNBR, Campinas, SP, Brazil Univ Estadual Campinas, Inst Biol, Campinas, SP, Brazil Sao Paulo State Univ, Inst Biosci Letters & Exact Sci, Sao Jose Do Rio Preto, SP, Brazil Univ Estadual Campinas, Inst Chem, Campinas, SP, Brazil Univ Fed Mato Grosso do Sul, Campo Grande, MS, Brazil Sao Paulo State Univ, Inst Biosci Letters & Exact Sci, Sao Jose Do Rio Preto, SP, Brazil

The classical microbial strategy for depolymerization of β-mannan polysaccharides involves the synergistic action of at least two enzymes, endo-1,4-β-mannanases and β-mannosidases. In this work, we describe the first exo-β-mannanase from the GH2 family, isolated from Xanthomonas axonopodis pv. citri (XacMan2A), which can efficiently hydrolyze both manno-oligosaccharides and β-mannan into mannose. It represents a valuable process simplification in the microbial carbon uptake that could be of potential industrial interest. Biochemical assays revealed a progressive increase in the hydrolysis rates from mannobiose to mannohexaose, which distinguishes XacMan2A from the known GH2 β-mannosidases. Crystallographic analysis indicates that the active-site topology of XacMan2A underwent profound structural changes at the positive-subsite region, by the removal of the physical barrier canonically observed in GH2 β-mannosidases, generating a more open and accessible active site with additional productive positive subsites. Besides that, XacMan2A contains two residue substitutions in relation to typical GH2 β-mannosidases, Gly(439) and Gly(556), which alter the active site volume and are essential to its mode of action. Interestingly, the only other mechanistically characterized mannose-releasing exo-β-mannanase so far is from the GH5 family, and its mode of action was attributed to the emergence of a blocking loop at the negative-subsite region of a cleft-like active site, whereas in XacMan2A, the same activity can be explained by the removal of steric barriers at the positive-subsite region in an originally pocket-like active site. Therefore, the GH2 exo-β-mannanase represents a distinct molecular route to this rare activity, expanding our knowledge about functional convergence mechanisms in carbohydrate-active enzymes.

// Danielly C. Ferraz da Costa 1, 2 , Nathali P.C. Campos 2, 3 , Ronimara A. Santos 1 , Francisca Hildemagna Guedes-da-Silva 2, 3 , Mafalda Maria D.C. Martins-Dinis 2, 3 , Leticia Zanphorlin 4 , Carlos Ramos 4 , Luciana P. Rangel 2, 5 and Jerson L. Silva 2, 3 1 Departamento de Nutricao Basica e Experimental, Instituto de Nutricao, Universidade do Estado do Rio de Janeiro, Rio de Janeiro 20550-013, RJ, Brazil 2 Instituto Nacional de Ciencia e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-902, RJ, Brazil 3 Programa de Biologia Estrutural, Instituto de Bioquimica Medica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-902, RJ, Brazil 4 Instituto de Quimica, Universidade de Campinas, Campinas 13083-970, SP, Brazil 5 Departamento de Analises Clinicas e Toxicologicas, Faculdade de Farmacia, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-902, RJ, Brazil Correspondence to: Jerson L. Silva, email: jerson@bioqmed.ufrj.br Keywords: amyloid aggregation; p53; cancer; aggregation; resveratrol Received: June 22, 2017 Accepted: June 04, 2018 Published: June 26, 2018 ABSTRACT One potential target for cancer therapeutics is the tumor suppressor p53, which is mutated in more than 50% of malignant tumors. Loss of function (LoF), dominant negative (DN) and gain of function (GoF) mutations in p53 are associated with amyloid aggregation. We tested the potential of resveratrol, a naturally occurring polyphenol, to interact and prevent the aggregation of wild-type and mutant p53 in vitro using fluorescence spectroscopy techniques and in human breast cancer cells (MDA-MB-231, HCC-70 and MCF-7) using immunofluorescence co-localization assays. Based on our data, an interaction occurs between resveratrol and the wild-type p53 core domain (p53C). In addition, resveratrol and its derivatives pterostilbene and piceatannol inhibit mutant p53C aggregation in vitro . Additionally, resveratrol reduces mutant p53 protein aggregation in MDA-MB-231 and HCC-70 cells but not in the wild-type p53 cell line MCF-7. To verify the effects of resveratrol on tumorigenicity, cell proliferation and cell migration assays were performed using MDA-MB-231 cells. Resveratrol significantly reduced the proliferative and migratory capabilities of these cells. Our study provides evidence that resveratrol directly modulates p53, enhancing our understanding of the mechanisms involved in p53 aggregation and its potential as a therapeutic strategy for cancer treatment.

Nucleoside diphosphate kinases (NDKs) are key enzymes in the purine-salvage pathway of trypanosomatids and have been associated with the maintenance of host-cell integrity for the benefit of the parasite, being potential targets for rational drug discovery and design. The NDK from Leishmania major (LmNDK) and mutants were expressed and purified to homogeneity. Thermal shift assays were employed to identify potential inhibitors for LmNDK. Calorimetric experiments, site-directed mutagenesis and molecular docking analysis were performed to validate the interaction and to evaluate the structural basis of ligand recognition. Furthermore, the anti-leishmanial activity of the newly identified and validated compound was tested in vitro against different Leishmania species. The molecule SU11652, a Sunitinib analog, was identified as a potential inhibitor for LmNDK and structural studies indicated that this molecule binds to the active site of LmNDK in a similar conformation to nucleotides, mimicking natural substrates. Isothermal titration calorimetry experiments combined with site-directed mutagenesis revealed that the residues H50 and H117, considered essential for catalysis, play an important role in ligand binding. In vitro cell studies showed that SU11652 had similar efficacy to Amphotericin b against some Leishmania species. Together, our results indicate the pyrrole-indolinone SU11652 as a promising scaffold for the rational design of new drugs targeting the enzyme NDK from Leishmania parasites.

Bovine papillomatosis is an infectious disease that is caused by bovine papillomavirus (BPV), which results in important economic losses. However, no BPV vaccines or effective treatment methods are commercially available to date. Moreover, the absence of papillomavirus replication in vitro makes the use of recombinant protein a promising candidate for vaccine formulations. Hence, we developed an integrated study on the L1 capsid protein of BPV-1, obtained from a bacterial expression system, regarding its purification, biosafety, thermostability and immunogenicity. The results indicated an absence of genotoxicity of the purified recombinant L1 protein, β-sheet prevalence of secondary structure folding, protein stability under high temperatures as well as the presence of capsomeres and VLPs. In addition, preliminary experimental vaccination of calves showed the production of specific antibodies against BPV-1 L1.

β-glucosidases play a critical role among the enzymes in enzymatic cocktails designed for plant biomass deconstruction. By catalysing the breakdown of β-1, 4-glycosidic linkages, β-glucosidases produce free fermentable glucose and alleviate the inhibition of other cellulases by cellobiose during saccharification. Despite this benefit, most characterised fungal β-glucosidases show weak activity at high glucose concentrations, limiting enzymatic hydrolysis of plant biomass in industrial settings. In this study, structural analyses combined with site-directed mutagenesis efficiently improved the functional properties of a GH1 β-glucosidase highly expressed by Trichoderma harzianum (ThBgl) under biomass degradation conditions. The tailored enzyme displayed high glucose tolerance levels, confirming that glucose tolerance can be achieved by the substitution of two amino acids that act as gatekeepers, changing active-site accessibility and preventing product inhibition. Furthermore, the enhanced efficiency of the engineered enzyme in terms of the amount of glucose released and ethanol yield was confirmed by saccharification and simultaneous saccharification and fermentation experiments using a wide range of plant biomass feedstocks. Our results not only experimentally confirm the structural basis of glucose tolerance in GH1 β-glucosidases but also demonstrate a strategy to improve technologies for bioethanol production based on enzymatic hydrolysis.

Made available in DSpace on 2018-11-26T17:55:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-09-02 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Canadian Institutes of Health Research INBEB (Instituto Nacional de Biologia Estrutural e Bioimagem) To accomplish its crucial role, mitochondria require proteins that are produced in the cytosol, delivered by cytosolic Hsp90, and translocated to its interior by the translocase outer membrane (TOM) complex. Hsp90 is a dimeric molecular chaperone and its function is modulated by its interaction with a large variety of co-chaperones expressed within the cell. An important family of co-chaperones is characterized by the presence of one TPR (tetratricopeptide repeat) domain, which binds to the C-terminal MEEVD motif of Hsp90. These include Tom70, an important component of the TOM complex. Despite a wealth of studies conducted on the relevance of Tom 70 center dot Hsp90 complex formation, there is a dearth of information regarding the exact molecular mode of interaction. To help fill this void, we have employed a combined experimental strategy consisting of cross-linking/mass spectrometry to investigate binding of the C-terminal Hsp90 domain to the cytosolic domain of Tom70. This approach has identified a novel region of contact between C-Hsp90 and Tom70, a finding that is confirmed by probing the corresponding peptides derived from cross-linking experiments via isothermal titration calorimetry and mitochondrial import assays. The data generated in this study are combined to input constraints for a molecular model of the Hsp90/Tom70 interaction, which has been validated by small angle x-ray scattering, hydrogen/deuterium exchange, and mass spectrometry. The resultant model suggests that only one of the MEEVD motifs within dimeric Hsp90 contacts Tom70. Collectively, our findings provide significant insight on the mechanisms by which preproteins interact with Hsp90 and are translocated via Tom70 to the mitochondria. Univ Campinas UNICAMP, Inst Chem, BR-13083970 Campinas, SP, Brazil McGill Univ, Dept Biochem, Grp Rech Axe Struct Prot, Montreal, PQ H3G 0B1, Canada State Univ Sao Paulo, Coll Agr & Vet Sci, BR-14884900 Sao Paulo, Brazil Rutgers State Univ, Dept Chem & Chem Biol, Piscataway, NJ 08854 USA Univ Sao Paulo, Inst Fis, BR-05508090 Sao Paulo, SP, Brazil State Univ Sao Paulo, Coll Agr & Vet Sci, BR-14884900 Sao Paulo, Brazil FAPESP: 2012/50161-8 Canadian Institutes of Health Research: MOP-130332

Xylanases catalyze the hydrolysis of β-1,4-linked xylosyl moieties from xylan chains, one of the most abundant hemicellulosic polysaccharides found in plant cell walls. These enzymes can exist either as single catalytic domains or as modular proteins composed of one or more carbohydrate-binding modules (CBMs) appended to the catalytic core. However, the molecular mechanisms governing the synergistic effects between catalytic domains and their CBMs are not fully understood. Thus, the goal of this study was to evaluate the functional effects of the fusion of a CBM belonging to family 6, which exhibits high affinity to xylan, with the GH11 xylanase from Bacillus subtilis, which does not have a CBM in its wild-type form. The wild-type enzyme (BsXyl11) and the chimeric protein (BsXyl11-CBM6) were heterologously produced in Escherichia coli and purified to homogeneity for biochemical characterization. The molecular fusion did not alter the pH and temperature dependence, but kinetic data revealed an increase of 65% in the catalytic efficiency of the chimeric enzyme. Furthermore, the BsXyl11-CBM6 chimera was used to supplement the commercial cocktail Accellerase® 1500 and improved the reducing sugar release by 17% from pretreated sugarcane bagasse. These results indicate that CBM6 can be used as a molecular tool to enhance the catalytic performance of endo-xylanases (GH11) and provide a new strategy for the development of optimized biocatalysts for biotechnological applications.

Agradecimentos: This study was funded by Fundação de Amparo do Estado de São Paulo FAPESP (2012/50161-8, 2014/17264-3 and 2015/15822-1), CNPq (305018/2015-9 and 306943/2015-8) and CAPES (88887.125517/2016-00). We thank the National Laboratory of Synchrotron Light (Campinas, SP, Brazil) and its staff for the use of SAXS beam line facilities. We acknowledge the Protein Analysis Facility (Center for Integrative Genomics, Faculty of Biology and Medicine, University of Lausanne, Switzerland) for cross-linking MS data Abstract: Proteostasis is dependent on the Hsp70/Hsp90 system (the two chaperones and their co-chaperones). Of these, Hop (Hsp70/Hsp90 organizing protein), also known as Sti1, forms an important scaffold to simultaneously binding to both Hsp70 and Hsp90. Hop/Sti1 has been implicated in several disease states, for instance cancer and transmissible spongiform encephalopathies. Therefore, human and yeast homologous have been better studied and information on plant homologous is still limited, even though plants are continuously exposed to environmental stress. Particularly important is the study of crops that are relevant for agriculture, such as Sorghum bicolor, a C4 grass that is among the five most important cereals and is considered as a bioenergy feedstock. To increase the knowledge on plant chaperones, the hop putative gene for Sorghum bicolor was cloned and the biophysical and structural characterization of the protein was done by cross-linking coupled to mass spectroscopy, small angle X-ray scattering and structural modeling. Additionally, the binding to a peptide EEVD motif, which is present in both Hsp70 and Hsp90, was studied by isothermal titration calorimetry and hydrogen/deuterium exchange and the interaction pattern structurally modeled. The results indicate SbHop as a highly flexible, mainly alpha-helical monomer consisting of nine tetratricopeptide repeat domains, of which one confers high affinity binding to Hsp90 through a conserved carboxylate clamp. Moreover, the present insights into the conserved interactions formed between Hop and Hsp90 can help to design strategies for potential therapeutic approaches for the diseases in which Hop has been implicated CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQ COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPES FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP Fechado

Rational design is an important tool for sculpting functional and stability properties of proteins and its potential can be much magnified when combined with in vitro and natural evolutionary diversity. Herein, we report the structure-guided design of a xylose-releasing exo-β-1,4-xylanase from an inactive member of glycoside hydrolase family 43 (GH43). Structural analysis revealed a nonconserved substitution (Lys247 ) that results in the disruption of the hydrogen bond network that supports catalysis. The mutation of this residue to a conserved serine restored the catalytic activity and crystal structure elucidation of the mutant confirmed the recovery of the proper orientation of the catalytically relevant histidine. Interestingly, the tailored enzyme can cleave both xylooligosaccharides and xylan, releasing xylose as the main product, being the first xylose-releasing exo-β-1,4-xylanase reported in the GH43 family. This enzyme presents a unique active-site topology when compared with closely related β-xylosidases, which is the absence of a hydrophobic barrier at the positive-subsite region, allowing the accommodation of long substrates. Therefore, the combination of rational design for catalytic activation along with naturally occurring differences in the substrate binding interface led to the discovery of a novel activity within the GH43 family. In addition, these results demonstrate the importance of solvation of the β-propeller hollow for GH43 catalytic function and expand our mechanistic understanding about the diverse modes of action of GH43 members, a key and polyspecific carbohydrate-active enzyme family abundant in most plant cell-wall-degrading microorganisms.

Made available in DSpace on 2019-10-06T15:19:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Lignocellulosic materials are abundant, renewable and are emerging as valuable substrates for many industrial applications such as the production of second-generation biofuels, green chemicals and pharmaceuticals. However, the recalcitrance and the complexity of cell wall polysaccharides require multiple enzymes for their complete conversion to oligo- and monosaccharides. The endoglucanases from GH45 family are a small and relatively poorly studied group of enzymes with potential industrial application. The present study reports cloning, heterologous expression and functional characterization of two GH45 endoglucanases from mesophilic fungi Gloeophyllum trabeum (GtGH45) and thermophilic fungi Myceliophthora thermophila (MtGH45), which belong to subfamilies GH45C and GH45A, respectively. Both enzymes have optimal pH 5.0 and melting temperatures (Tm) of 66.0 °C and 80.9 °C, respectively, as estimated from circular dichroism experiments. The recombinant proteins also exhibited different mode of action when incubated with oligosaccharides ranging from cellotriose to cellohexaose, generating mainly cellobiose and cellotriose (MtGH45) or glucose and cellobiose (GtGH45). The MtGH45 did not show activity against oligosaccharides smaller than cellopentaose while the enzyme GtGH45 was able to depolymerize cellotriose, however with lower efficiency when compared to larger oligosaccharides. Furthermore, both GHs45 were stable up to 70 °C for 24 h and useful to enhance initial glucan hydrolysis rates during saccharification of sugarcane pith by a mixture of cellulolytic enzymes. Recombinant GHs45 from diverging subfamilies stand out for differences in substrate specificity appearing as new tools for preparation of enzyme cocktails used in cellulose hydrolysis. Departamento de Biotecnologia Escola de Engenharia de Lorena Universidade de São Paulo Laboratório Nacional de Ciência e Tecnologia do Bioetanol Centro Nacional de Pesquisa em Energia e Materiais Departamento de Física e Ciências Aplicadas Instituto de Física de São Carlos Universidade de São Paulo Universidade Estadual Paulista (UNESP) Instituto de Biociências Letras e Ciências Exatas Universidade Estadual Paulista (UNESP) Instituto de Biociências Letras e Ciências Exatas

Galactanases (endo-β-1,4-galactanases—EC 3.2.1.89) catalyze the hydrolysis of β-1,4 galactosidic bonds in arabinogalactan and galactan side chains found in type I rhamnogalacturan. The aim of this work was to understand the catalytic function, biophysical properties, and use of a recombinant GH53 endo-beta-1,4-galactanase for commercial cocktail supplementation. The nucleotide sequence of the endo-β-1,4-galactanase from Bacillus licheniformis CBMAI 1609 (Bl1609Gal) was cloned and expressed in Escherichia coli, and the biochemical and biophysical properties of the enzyme were characterized. The optimum pH range and temperature of Bl1609Gal activity were 6.5–8 and 40 °C, respectively. Furthermore, Bl1609Gal showed remarkable pH stability, retaining more than 75 % activity even after 24 h of incubation at pH 4–10. The enzyme was thermostable, retaining nearly 100 % activity after 1-h incubation at pH 7.0 at 25–45 °C. The enzymatic efficiency (K cat /K m ) against potato galactan under optimum conditions was 241.2 s−1 mg−1 mL. Capillary zone electrophoresis demonstrated that the pattern of galactan hydrolysis by Bl1609Gal was consistent with that of endogalactanases. Supplementation of the commercial cocktail ACCELLERASE®1500 with recombinant Bl1609Gal increased hydrolysis of pretreated sugarcane bagasse by 25 %.

A novel GH1 β-glucosidase (EaBgl1A) from a bacterium isolated from Antarctica soil samples was recombinantly overexpressed in Escherichia coli cells and characterized. The enzyme showed unusual pH dependence with maximum activity at neutral pH and retention of high catalytic activity in the pH range 6 to 9, indicating a catalytic machinery compatible with alkaline conditions. EaBgl1A is also a cold-adapted enzyme, exhibiting activity in the temperature range from 10 to 40°C with optimal activity at 30°C, which allows its application in industrial processes using low temperatures. Kinetic characterization revealed an enzymatic turnover (Kcat) of 6.92s(-1) (cellobiose) and 32.98s(-1) (pNPG) and a high tolerance for product inhibition, which is an extremely desirable feature for biotechnological purposes. Interestingly, the enzyme was stimulated by up to 200 mM glucose, whereas the commercial cocktails tested were found fully inhibited at this concentration. These properties indicate EaBgl1A as a promising biocatalyst for biotechnological applications where low temperatures are required.

2S albumins, the seed storage proteins, are the primary sources of carbon and nitrogen and are involved in plant defense. The mature form of Moringa oleifera (M. oleifera), a chitin binding protein isoform 3-1 (mMo-CBP3-1) a thermostable antifungal, antibacterial, flocculating 2S albumin is widely used for the treatment of water and is potentially interesting for the development of both antifungal drugs and transgenic crops. The crystal structure of mMo-CBP3-1 determined at 1.7 Å resolution demonstrated that it is comprised of two proteolytically processed α-helical chains, stabilized by four disulfide bridges that is stable, resistant to pH changes and has a melting temperature (TM) of approximately 98 °C. The surface arginines and the polyglutamine motif are the key structural factors for the observed flocculating, antibacterial and antifungal activities. This represents the first crystal structure of a 2S albumin and the model of the pro-protein indicates the structural changes that occur upon formation of mMo-CBP3-1 and determines the structural motif and charge distribution patterns for the diverse observed activities.

β-Xylosidases (EC 3.2.1.37) catalyze the hydrolysis of short xylooligosaccharides into xylose, which is an essential step in the complete depolymerization of xylan, the major hemicellulosic polysaccharide of plant cell walls, and has great biotechnological relevance for the production of lignocellulose-based biofuels and the paper industry. In this study, a GH43 β-xylosidase identified from the bacteriumBacillus licheniformis(BlXylA) was cloned into the the pET-28a bacterial expression vector, recombinantly overexpressed inEscherichia coliBL21(DE3) cells and purified to homogeneity by metal-affinity and size-exclusion chromatography. The protein was crystallized in the presence of the organic solvent 2-methyl-2,4-pentanediol and a single crystal diffracted to 2.49 Å resolution. The X-ray diffraction data were indexed in the monoclinic space groupC2, with unit-cell parametersa= 152.82,b= 41.9,c= 71.79 Å, β = 91.7°. Structural characterization of this enzyme will contribute to a better understanding of the structural requirements for xylooligosaccharide specificity within the GH43 family.

The Amazon region holds most of the biological richness of Brazil. Despite their ecological and biotechnological importance, studies related to microorganisms from this region are limited. Metagenomics leads to exciting discoveries, mainly regarding non-cultivable microorganisms. Herein, we report the discovery of a novel β-glucosidase (glycoside hydrolase family 1) gene from a metagenome from Lake Poraque in the Amazon region. The gene encodes a protein of 52.9 kDa, named AmBgl-LP, which was recombinantly expressed in Escherichia coli and biochemically and structurally characterized. Although AmBgl-LP hydrolyzed the synthetic substrate p-nitrophenyl-β- d -glucopyranoside (pNPβG) and the natural substrate cellobiose, it showed higher specificity for pNPβG (kcat/Km = 6 s−1·mM−1) than cellobiose (kcat/Km = 0.6 s−1·mM−1). AmBgl-LP showed maximum activity at 40 °C and pH 6.0 when pNPβG was used as the substrate. Glucose is a competitive inhibitor of AmBgl-LP, presenting a Ki of 14 mM. X-ray crystallography and Small Angle X-ray Scattering were used to determine the AmBgl-LP three-dimensional structure and its oligomeric state. Interestingly, despite sharing similar active site architecture with other structurally characterized GH1 family members which are monomeric, AmBgl-LP forms stable dimers in solution. The identification of new GH1 members by metagenomics might extend our understanding of the molecular mechanisms and diversity of these enzymes, besides enabling us to survey their industrial applications.

Proteostasis is dependent on the Hsp70/Hsp90 system (the two chaperones and their co-chaperones). Of these, Hop (Hsp70/Hsp90 organizing protein), also known as Sti1, forms an important scaffold to simultaneously binding to both Hsp70 and Hsp90. Hop/Sti1 has been implicated in several disease states, for instance cancer and transmissible spongiform encephalopathies. Therefore, human and yeast homologous have been better studied and information on plant homologous is still limited, even though plants are continuously exposed to environmental stress. Particularly important is the study of crops that are relevant for agriculture, such as Sorghum bicolor, a C4 grass that is among the five most important cereals and is considered as a bioenergy feedstock. To increase the knowledge on plant chaperones, the hop putative gene for Sorghum bicolor was cloned and the biophysical and structural characterization of the protein was done by cross-linking coupled to mass spectroscopy, small angle X-ray scattering and structural modeling. Additionally, the binding to a peptide EEVD motif, which is present in both Hsp70 and Hsp90, was studied by isothermal titration calorimetry and hydrogen/deuterium exchange and the interaction pattern structurally modeled. The results indicate SbHop as a highly flexible, mainly alpha-helical monomer consisting of nine tetratricopeptide repeat domains, of which one confers high affinity binding to Hsp90 through a conserved carboxylate clamp. Moreover, the present insights into the conserved interactions formed between Hop and Hsp90 can help to design strategies for potential therapeutic approaches for the diseases in which Hop has been implicated.

Xanthomonas pathogens attack a variety of economically relevant plants, and their xylan CUT system (carbohydrate utilization with TonB-dependent outer membrane transporter system) contains two major xylanase-related genes, xynA and xynB, which influence biofilm formation and virulence by molecular mechanisms that are still elusive. Herein, we demonstrated that XynA is a rare reducing end xylose-releasing exo-oligoxylanase and not an endo-β-1,4-xylanase as predicted. Structural analysis revealed that an insertion in the β7-α7 loop induces dimerization and promotes a physical barrier at the +2 subsite conferring this unique mode of action within the GH10 family. A single mutation that impaired dimerization became XynA active against xylan, and high endolytic activity was achieved when this loop was tailored to match a canonical sequence of endo-β-1,4-xylanases, supporting our mechanistic model. On the other hand, the divergent XynB proved to be a classical endo-β-1,4-xylanase, despite the low sequence similarity to characterized GH10 xylanases. Interestingly, this enzyme contains a calcium ion bound nearby to the glycone-binding region, which is required for catalytic activity and structural stability. These results shed light on the molecular basis for xylan degradation by Xanthomonas and suggest how these enzymes synergistically assist infection and pathogenesis. Our findings indicate that XynB contributes to breach the plant cell wall barrier, providing nutrients and facilitating the translocation of effector molecules, whereas the exo-oligoxylanase XynA possibly participates in the suppression of oligosaccharide-induced immune responses.

Background Hsp90 is a molecular chaperone essential for cell viability in eukaryotes that is associated with the maturation of proteins involved in important cell functions and implicated in the stabilization of the tumor phenotype of various cancers, making this chaperone a notably interesting therapeutic target. Celastrol is a plant-derived pentacyclic triterpenoid compound with potent antioxidant, anti-inflammatory and anticancer activities; however, celastrol's action mode is still elusive. Results In this work, we investigated the effect of celastrol on the conformational and functional aspects of Hsp90α. Interestingly, celastrol appeared to target Hsp90α directly as the compound induced the oligomerization of the chaperone via the C-terminal domain as demonstrated by experiments using a deletion mutant. The nature of the oligomers was investigated by biophysical tools demonstrating that a two-fold excess of celastrol induced the formation of a decameric Hsp90α bound throughout the C-terminal domain. When bound, celastrol destabilized the C-terminal domain. Surprisingly, standard chaperone functional investigations demonstrated that neither the in vitro chaperone activity of protecting against aggregation nor the ability to bind a TPR co-chaperone, which binds to the C-terminus of Hsp90α, were affected by celastrol. Conclusion Celastrol interferes with specific biological functions of Hsp90α. Our results suggest a model in which celastrol binds directly to the C-terminal domain of Hsp90α causing oligomerization. However, the ability to protect against protein aggregation (supported by our results) and to bind to TPR co-chaperones are not affected by celastrol. Therefore celastrol may act primarily by inducing specific oligomerization that affects some, but not all, of the functions of Hsp90α. General significance To the best of our knowledge, this study is the first work to use multiple probes to investigate the effect that celastrol has on the stability and oligomerization of Hsp90α and on the binding of this chaperone to Tom70. This work provides a novel mechanism by which celastrol binds Hsp90α.

As part of an ongoing directed evolution program, the catalytic performance of the Xylanase A from Bacillus subtilis (XynA), which presents temperature and pH optima of 50°C and 6.0, respectively, has been enhanced to create a highly thermostable and alkali-tolerant enzyme. A library of random XynA mutants generated by error-prone polymerase chain reaction was screened by halo formation on agar containing xylan at pH 8.0. Two mutants showing higher catalytic activity at elevated pH in relation to the wild-type XynA were selected, and pooled with a further 5 XynA variants selected by screening thermostable XynA obtained from a previous directed evolution study for activity at alkaline pH. This pool of variants was used as a template for a further round of error-prone polymerase chain reaction and DNase fragment shuffling, with screening at pH 12.0 at 55°C. Selected mutants were subjected to further DNase shuffling, and a final round of screening at pH 12.0 and 80°C. A XynA variant containing eight mutations was isolated (Q7H/G13R/S22P/S31Y/T44A/I51V/I107L/S179C) that presented a temperature optimum of 80°C, a 3-fold increase in the specific activity compared with the wild-type enzyme at pH 8.0, and a 50% loss of activity (t50) of 60 min at 80°C (wild type

Snake venom metalloproteinases (SVMPs) belonging to P-I class are able to hydrolyze extracellular matrix proteins and coagulation factors triggering local and systemic reactions by multiple molecular mechanisms that are not fully understood. BmooMPα-I, a P-I class SMVP from Bothrops moojeni venom, was active upon neuro- and vaso-active peptides including angiotensin I, bradykinin, neurotensin, oxytocin and substance P. Interestingly, BmooMPα-I showed a strong bias towards hydrolysis after proline residues, which is unusual for most of characterized peptidases. Moreover, the enzyme showed kininogenase activity similar to that observed in plasma and cells by kallikrein. FRET peptide assays indicated a relative promiscuity at its S2–S′2 subsites, with proline determining the scissile bond. This unusual post-proline cleaving activity was confirmed by the efficient hydrolysis of the synthetic combinatorial library MCA-GXXPXXQ-EDDnp, described as resistant for canonical peptidases, only after Pro residues. Structural analysis of the tripeptide LPL complexed with BmooMPα-I, generated by molecular dynamics simulations, assisted in defining the subsites and provided the structural basis for subsite preferences such as the restriction of basic residues at the S2 subsite due to repulsive electrostatic effects and the steric impediment for large aliphatic or aromatic side chains at the S1 subsite. These new functional and structural findings provided a further understanding of the molecular mechanisms governing the physiological effects of this important class of enzymes in envenomation process.

Dynamic high pressure (DHP) has been investigated as an innovative suitable method to induce protein modifications. This work evaluated the effect of DHP (up to three passes at 100, 150 and 200MPa, with an inlet temperature of 20°C) on functional and structural properties of bovine serum albumin (BSA). Results indicated that DHP process applied up to an energy limit of 100MPa increased the protein foaming capacity (FC) (p0.05 - increase up to 63% after 1 pass at 100MPa) and the utilization of multiple passes at high pressure promoted a reduction in this property (p0.05 - reduction up to 31.6% after 3 passes at 200MPa). Similar results were observed for sulfhydryl group, indicating an influence of free thiol groups on FC. Complementarily, DHP process promoted an increase of proteins particles size, suggesting a new rearrangement of their conformational structure. DHP did not affect tryptophan microenvironment in BSA; however, this process induced the rearrangement of secondary structure elements. In the first cycle, the pressure increase resulted in a loss of secondary structure, while in the second and third cycles the DHP process resulted in the gain of secondary structure elements. These results indicated that the second and third passes triggered a molecular rearrangement of the protein structure, giving rise to a novel and more stable conformational state. This conclusion was also supported by thermal unfolding studies (melting temperature reduction from 67.5 to 54.6°C after 1 pass at 200MPa), in which the additional cycles of DHP caused the occurrence of an initial denaturation at high temperatures, compared to the first cycle.

Carbohydrate-binding modules (CBMs) are appended to glycoside hydrolases and can contribute to the degradation of complex recalcitrant substrates such as the plant cell wall. For application in bioethanol production, novel enzymes with high catalytic activity against recalcitrant lignocellulosic material are being explored and developed. In this work, we report the functional and structural study of CBM_E1, which was discovered through a metagenomics approach and is the founding member of a novel CBM family, CBM81. CBM_E1, which is linked to an endoglucanase, displayed affinity for mixed linked β1,3-β1,4-glucans, xyloglucan, Avicel, and cellooligosaccharides. The crystal structure of CBM_E1 in complex with cellopentaose displayed a canonical β-sandwich fold comprising two β-sheets. The planar ligand binding site, observed in a parallel orientation with the β-strands, is a typical feature of type A CBMs, although the expected affinity for bacterial crystalline cellulose was not detected. Conversely, the binding to soluble glucans was enthalpically driven, which is typical of type B modules. These unique properties of CBM_E1 are at the interface between type A and type B CBMs.

Made available in DSpace on 2018-12-11T17:29:26Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-12-01 The GH10 endo-xylanase from Thermoascus aurantiacus CBMAI 756 (XynA) is industrially attractive due to its considerable thermostability and high specific activity. Considering the possibility of a further improvement in thermostability, eleven mutants were created in the present study via site-directed mutagenesis using XynA as a template. XynA and its mutants were successfully overexpressed in Escherichia coli Rosetta-gami DE3 and purified, exhibiting maximum xylanolytic activity at pH 5 and 65 °C. Three of the eleven mutants, Q158R, H209N, and N257D, demonstrated increased thermostability relative to the wild type at 70 °C and 75 °C.Q158R and N257D were stable in the pH range 5.0–10.0, while WT and H209N were stable from pH 8–10. CD analysis demonstrated that the WT and the three mutant enzymes were expressed in a folded form. H209N was the most thermostable mutant, showing a Tm of 71.3 °C. Molecular dynamics modeling analyses suggest that the increase in H209N thermostability may beattributed to a higher number of short helices and salt bridges, which displayed a positive charge in the catalytic core, stabilizing its tertiary structure. UNESP (São Paulo State University – Júlio de Mesquita Filho) Biochemistry and Applied Microbiology Laboratory University of Brasília Department of Cell Biology – Brasília, Distrito Federal CTBE (Brazilian Bioethanol Science and Technology LaboratoryatNational Center for Research in Energy and Materials) Bioenergy Research Unit National Center for Agricultural Utilization Research USDA – ARS 1815 N. University St UNESP (São Paulo State University – Júlio de Mesquita Filho) Biochemistry and Applied Microbiology Laboratory

Background The conversion of biomass-derived sugars via enzymatic hydrolysis for biofuel production is a challenge. Therefore, the search for microorganisms and key enzymes that increase the efficiency of the saccharification of cellulosic substrates remains an important and high-priority area of study. Trichoderma harzianum is an important fungus known for producing high levels of cellulolytic enzymes that can be used for cellulosic ethanol production. In this context, β-glucosidases, which act synergistically with cellobiohydrolases and endo-β-1,4-glucanases in the saccharification process, are potential biocatalysts for the conversion of plant biomass to free glucose residues. Results In the present study, we used RNA-Seq and genomic data to identify the major β-glucosidase expressed by T. harzianum under biomass degradation conditions. We mapped and quantified the expression of all of the β-glucosidases from glycoside hydrolase families 1 and 3, and we identified the enzyme with the highest expression under these conditions. The target gene was cloned and heterologously expressed in Escherichia coli, and the recombinant protein (rThBgl) was purified with high yields. rThBgl was characterized using a comprehensive set of biochemical, spectroscopic, and hydrodynamic techniques. Finally, we determined the crystallographic structure of the recombinant protein at a resolution of 2.6 Å. Conclusions Using a rational approach, we investigated the biochemical characteristics and determined the three-dimensional protein structure of a β-glucosidase that is highly expressed by T. harzianum under biomass degradation conditions. The methodology described in this manuscript will be useful for the bio-prospection of key enzymes, including cellulases and other accessory enzymes, for the development and/or improvement of enzymatic cocktails designed to produce ethanol from plant biomass. Electronic supplementary material The online version of this article (doi:10.1186/s13068-016-0487-0) contains supplementary material, which is available to authorized users.

Thermophilic fungi produce thermostable enzymes which have a number of applications, mainly in biotechnological processes. In this work, we describe the characterization of a protease produced in solidstate (SSF) and submerged (SmF) fermentations by a newly isolated thermophilic fungus identified as a putative new species in the genus Myceliophthora. Enzyme-production rate was evaluated for both fermentation processes, and in SSF, using a medium composed of a mixture of wheat bran and casein, the proteolytic output was 4.5-fold larger than that obtained in SmF. Additionally, the peak of proteolytic activity was obtained after 3 days for SSF whereas for SmF it was after 4 days. The crude enzyme obtained by both SSF and SmF displayed similar optimum temperature at 50 degrees C, but the optimum pH shifted from 7 (SmF) to 9(SSF). The alkaline protease produced through solid-state fermentation (SSF), was immobilized on beads of calcium alginate, allowing comparative analyses of free and immobilized proteases to be carried out. It was observed that both optimum temperature and thermal stability of the immobilized enzyme were higher than for the free enzyme. Moreover, the immobilized enzyme showed considerable stability for up to 7 reuses.

Protease production was carried out in solid state fermentation. The enzyme was purified through precipitation with ethanol at 72% followed by chromatographies in columns of Sephadex G75 and Sephacryl S100. It was purified 80-fold and exhibited recovery of total activity of 0.4%. SDS-PAGE analysis indicated an estimated molecular mass of 24.5 kDa and the N-terminal sequence of the first 22 residues was APYSGYQCSMQLCLTCALMNCA. Purified protease was only inhibited by EDTA (96.7%) and stimulated by Fe(2+) revealing to be a metalloprotease activated by iron. Optimum pH was 5.5, optimum temperature was 75 degrees C, and it was thermostable at 65 degrees C for 1 h maintaining more than 70% of original activity. Through enzyme kinetic studies, protease better hydrolyzed casein than azocasein. The screening of fluorescence resonance energy transfer (FRET) peptide series derived from Abz-KLXSSKQ-EDDnp revealed that the enzyme exhibited preference for Arg in P(1) (k(cat)/K(m) = 30.1 mM(-1) s(-1)).

Psychrophilic enzymes evolved from a plethora of structural scaffolds via multiple molecular pathways. Elucidating their adaptive strategies is instrumental to understand how life can thrive in cold ecosystems and to tailor enzymes for biotechnological applications at low temperatures. In this work, we used X-ray crystallography, in solution studies and molecular dynamics simulations to reveal the structural basis for cold adaptation of the GH1 β-glucosidase from Exiguobacterium antarcticum B7. We discovered that the selective pressure of low temperatures favored mutations that redesigned the protein surface, reduced the number of salt bridges, exposed more hydrophobic regions to the solvent and gave rise to a tetrameric arrangement not found in mesophilic and thermophilic homologues. As a result, some solvent-exposed regions became more flexible in the cold-adapted tetramer, likely contributing to enhance enzymatic activity at cold environments. The tetramer stabilizes the native conformation of the enzyme, leading to a 10-fold higher activity compared to the disassembled monomers. According to phylogenetic analysis, diverse adaptive strategies to cold environments emerged in the GH1 family, being tetramerization an alternative, not a rule. These findings reveal a novel strategy for enzyme cold adaptation and provide a framework for the semi-rational engineering of β-glucosidases aiming at cold industrial processes.

Exfoliative toxins are serine proteases secreted by Staphylococcus aureus that are associated with toxin-mediated staphylococcal syndromes. To date, four different serotypes of exfoliative toxins have been identified and 3 of them (ETA, ETB, and ETD) are linked to human infection. Among these toxins, only the ETD structure remained unknown, limiting our understanding of the structural determinants for the functional differentiation between these toxins. We recently identified an ETD-like protein associated to S. aureus strains involved in mild mastitis in sheep. The crystal structure of this ETD-like protein was determined at 1.95 Å resolution and the structural analysis provide insights into the oligomerization, stability and specificity and enabled a comprehensive structural comparison with ETA and ETB. Despite the highly conserved molecular architecture, significant differences in the composition of the loops and in both the N- and C-terminal α-helices seem to define ETD-like specificity. Molecular dynamics simulations indicate that these regions defining ET specificity present different degrees of flexibility and may undergo conformational changes upon substrate recognition and binding. DLS and AUC experiments indicated that the ETD-like is monomeric in solution whereas it is present as a dimer in the asymmetric unit indicating that oligomerization is not related to functional differentiation among these toxins. Differential scanning calorimetry and circular dichroism assays demonstrated an endothermic transition centered at 52 °C, and an exothermic aggregation in temperatures up to 64 °C. All these together provide insights about the mode of action of a toxin often secreted in syndromes that are not associated with either ETA or ETB.

Cellulose synthesis in bacteria is a complex process involving the concerted action of several enzymes whose genes are often organized in operons. This process influences many fundamental physiological aspects such as bacteria and host interaction, biofilm formation, among others. Although it might sound contradictory, the participation of cellulose-degrading enzymes is critical to this process. The presence of endoglucanases from family 8 of glycosyl hydrolases (GH8) in bacterial cellulose synthase (Bcs) complex has been described in different bacteria, including the model organism Komagataeibacter xylinus; however, their role in this process is not completely understood. In this study, we describe the biochemical characterization and three-dimensional structure of a novel GH8 member from Raoultella ornithinolytica, named AfmE1, which was previously identified by our group from the metagenomic analysis of the giant snail Achatina fulica. Our results demonstrated that AfmE1 is an endo-β-1,4-glucanase, with maximum activity in acidic to neutral pH over a wide temperature range. This enzyme cleaves cello-oligosaccharides with a degree of polymerization ≥ 5 and presents six glucosyl-binding subsites. The structural comparison of AfmE1 with other GH8 endoglucanases showed significant structural dissimilarities in the catalytic cleft, particularly in the subsite +3, which correlate with different functional mechanisms, such as the recognition of substrate molecules having different arrangements and crystallinities. Together, these findings provide new insights into molecular and structural features of evolutionarily conserved endoglucanases from the bacterial cellulose biosynthetic machinery.

p53 has an essential role in preventing cancer development by inducing cell cycle arrest and/or apoptosis in response to cellular stress. Mutations in the p53 gene are described in over 50% of all ...

Product inhibition of β-glucosidases (BGs) by glucose is considered to be a limiting step in enzymatic technologies for plant-biomass saccharification. Remarkably, some β-glucosidases belonging to the GH1 family exhibit unusual properties, being tolerant to, or even stimulated by, high glucose concentrations. However, the structural basis for the glucose tolerance and stimulation of BGs is still elusive. To address this issue, the first crystal structure of a fungal β-glucosidase stimulated by glucose was solved in native and glucose-complexed forms, revealing that the shape and electrostatic properties of the entrance to the active site, including the +2 subsite, determine glucose tolerance. The aromatic Trp168 and the aliphatic Leu173 are conserved in glucose-tolerant GH1 enzymes and contribute to relieving enzyme inhibition by imposing constraints at the +2 subsite that limit the access of glucose to the −1 subsite. The GH1 family β-glucosidases are tenfold to 1000-fold more glucose tolerant than GH3 BGs, and comparative structural analysis shows a clear correlation between active-site accessibility and glucose tolerance. The active site of GH1 BGs is located in a deep and narrow cavity, which is in contrast to the shallow pocket in the GH3 family BGs. These findings shed light on the molecular basis for glucose tolerance and indicate that GH1 BGs are more suitable than GH3 BGs for biotechnological applications involving plant cell-wall saccharification.

Snake venom serine proteinases (SVSPs) are hemostatically active toxins that perturb the maintenance and regulation of both the blood coagulation cascade and fibrinolytic feedback system at specific points, and hence, are widely used as tools in pharmacological and clinical diagnosis. The crystal structure of a thrombin-like enzyme (TLE) from Bothrops jararacussu venom (Jararacussin-I) was determined at 2.48 A resolution. This is the first crystal structure of a TLE and allows structural comparisons with both the Agkistrodon contortrix contortrix Protein C Activator and the Trimeresurus stejnegeri plasminogen activator. Despite the highly conserved overall fold, significant differences in the amino acid compositions and three-dimensional conformations of the loops surrounding the active site significantly alter the molecular topography and charge distribution profile of the catalytic interface. In contrast to other SVSPs, the catalytic interface of Jararacussin-I is highly negatively charged, which contributes to its unique macromolecular selectivity.

This work reports the purification of a novel alkaline protease enzyme from a putative new thermophilic fungus Myceliophthora sp. The molecular weight of the enzyme was determined as 28.2kDa by using MALDI-TOF MS and it was inhibited by PMSF indicating it is a serine-protease. The optimum pH and temperature were 9.0 and 40–45°C, respectively. Mg was the only tested cation able to promote an increase of the protease activity. The N-terminal sequence of the purified protease (GVVGVC) presented identity and homology when compared to other proteases from fungi. This study also provides biochemical information about substrate specificity using fluorescence resonance energy transfer (FRET) peptide families derived from Abz-KLRSSKQ-EDDnp. The results showed that Abz-KLISSKQ-EDDnp is the best substrate among those tested for the purified protease (kcat/Km=1275.3mM−1s−1). Also, the specificity data suggest that subsites S1, S2, S3 and S1′, S2′, S3′, in general, present a preference for hydrophobic residues with the exception of Glu in P3, His in P2′ and Arg in P3′. The highest values for the specificity constant kcat/Km were obtained for P1, P2 and P2′.

Em virtude da crescente demanda por energia e de problemas ambientais causados pelo consumo de combustíveis fósseis, tem-se aumentado a necessidade de produção de combustíveis renováveis. O etanol de segunda geração (2G) surgiu como uma alternativa promissora aos combustíveis fósseis devido ao uso de biomassa vegetal como matéria-prima. Para que haja o processo de hidrólise enzimática da biomassa vegetal e obtenção de açúcares fermentáveis é fundamental o uso de diversas enzimas, tais como endo-&#945-1,5-L-arabinanases e &#946-xilosidases. Dessa forma, este trabalho teve por objetivo caracterizar as enzimas glicosil hidrolases endo-&#945-1,5-L-arabinanase e &#946-xilosidase de Geobacillus sp. JS12 e Geobacillus thermodenitrificans, respectivamente. Os genes das enzimas putativas, endo-&#945-1,5-L-arabinanase (GeoARA) e &#946-xilosidase (GeoXIL) foram obtidos a partir de análises da base de dados CAZy e posteriormente sintetizados e clonados para a expressão das proteínas recombinantes em E. coli BL21. Análises de bioinformática foram realizadas da sequência de aminoácidos da GeoARA e GeoXIL. As enzimas foram purificadas por cromatografia de afinidade em coluna de níquel-sefarose e realizou-se a caracterização bioquímica e análises de termoestabilidade por dicroísmo circular. As enzimas GeoARA e GeoXIL foram agrupadas dentro da família de glicosil hidrolases 43 (GH43). A partir dos testes de atividade, notou-se que a GeoARA possui maior atividade em pH 7,0 e temperatura de 70 °C, enquanto a GeoXIL possui maior atividade em pH 5,0 e temperatura de 60 °C. Nos testes com íons metálicos e EDTA, ambas as enzimas foram estáveis. Verificou-se que a GeoARA possui uma boa estabilidade térmica em 70 °C, principalmente na presença do íon Co2+ após 48 horas, enquanto que a GeoXIL apresentou maior estabilidade a 50 °C por 4 horas. A GeoARA possui uma significativa atividade sobre a arabinana desramificada (226,67 U/mg) e eficiência catalítica de 53 mL/mg/s. Por sua vez, a GeoXIL apresentou atividade bifuncional de &#946-xilosidase e &#945- L-arabinofuranosidase, com maior atividade sobre o substrato p-nitrofenil-&#946-D-xilopiranosídeo. A atividade específica sobre o p-nitrofenil-&#946-D-xilopiranosídeo foi de 18,33 U/mg e eficiência catalítica de 20,21 /mM/s; comparável com outras &#946-xilosidases. A atividade em elevadas temperaturas, termoestabilidade e estabilidade a íons metálicos são características desejáveis a enzimas industriais. Nesse sentido, as enzimas GeoARA e GeoXIL apresentam características bioquímicas interessantes para aplicação na hidrólise enzimática de biomassa vegetal Due to the growing demand for energy and environmental problems caused by the consumption of fossil fuels, the need to produce renewable fuels has increased. Second generation ethanol (2G) has emerged as a promising alternative to fossil fuels due to the use of plant biomass as a feedstock. In order to enzymatic hydrolysis the plant biomass and obtain fermentable sugars, it is essential to use several enzymes, such as endo-&#945-1,5-L-arabinanases and &#946-xylosidases. Thus, this work aimed to characterize the glycosyl hydrolases enzymes endo-&#945-1,5-L-arabinanase and &#946-xylosidase from Geobacillus sp. JS12 and Geobacillus thermodenitrificans, respectively. The genes sequences corresponding to the putative endo-&#945-1,5-L-arabinanase (GeoARA) and &#946-xylosidase (GeoXIL) enzymes were recovered from analyzes of the CAZy database and later synthesized and cloned for the expression of recombinant proteins in E. coli BL21. Bioinformatics analysis was performed on the amino acid sequence of GeoARA and GeoXIL. The enzymes were purified by affinity chromatography on a nickel-sepharose column and biochemical characterization and thermostability analysis by circular dichroism were performed. The enzymes GeoARA and GeoXIL were grouped within the family of glycosyl hydrolases 43 (GH43). Through activity tests, it was found that GeoARA has greater activity at pH 7.0 and temperature of 70 °C while GeoXIL has greater activity at pH 5.0 and temperature of 60 °C. In tests with metal ions and EDTA, the enzymes were stable. It was found that GeoARA has a good thermal stability at 70 °C, mainly in the presence of the Co2+ ion after 48 hours, while GeoXIL showed greater stability at 50 °C for 4 hours. GeoARA has significant activity on debranched arabinan (226.67 U/mg) and catalytic efficiency of 53 mL/mg/s. In fact, GeoXIL showed bifunctional activity of &#946-xylosidase and &#945-L-arabinofuranosidase, with greater activity on the substrate p-nitrophenyl-&#946-D-xylopyranoside. The specific activity on p-nitrophenyl-&#946-D-xylopyranoside was 18.33 U/mg and catalytic efficiency of 20.21 /mM/s, comparable to other &#946-xylosidases. The activity at high temperatures, thermostability and stability to metal ions are desired characteristics for industrial enzymes. In this sense, the GeoARA and GeoXIL enzymes present interesting biochemical characteristics for application in the enzymatic hydrolysis of lignocellulosic biomass

Orientador: Ljubica Tasic Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química Resumo: Os objetivos dessa tese de doutorado abrangem aplicações da nanotecnologia durante a exploração do bagaço de laranja (BL) para: (1) obtenção de um bionanomaterial de alto valor agregado ¿ nanocelulose; e (2) aplicação de nanopartículas, desenhadas para o reaproveitamento de hidrolases, na hidrólise do BL. O BL é um subproduto agroindustrial obtido após da extração do suco, sendo que cerca de 10 milhões de toneladas desse resíduo são produzidos no Brasil cada ano. Para a obtenção da nanocelulose foram empregados dois métodos: enzimático e físico-químico. A via enzimática baseou-se na aplicação de enzimas isoladas da bactéria Xanthomonas axonopodis pv. citri, o biomaterial obtido apresentou diâmetro médio de 10 nm e 63% da cristalinidade. Já o processo físico-químico levou a obtenção da nanocelulose com diâmetro médio de 20 nm e uma maior cristalinidade de 73%. Ressalta-se que foram investigados resíduos de restaurante e de indústria ¿ Citrosuco (Matão, SP, Brasil), sendo que esta última biomassa caracterizou-se por um maior rendimento e um menor consumo energético durante a etapa de fibrilação. Por outro lado, visando a produção de etanol de segunda geração (etanol-2G) a partir do bagaço da laranja, em escala piloto, uma estratégia de imobilização covalente do complexo enzimático comercial de Trichoderma reesei (Celluclast® 1,5 L) foi desenvolvida. As enzimas foram imobilizadas na superfície inerte funcionalizada de nanopartículas ferromagnéticas (NPM). A técnica de imobilização aumentou a vida útil e estabilidade das enzimas, visando uma redução de custos da hidrólise do BL. As nanopartículas do tipo casca-caroço tiveram como casca: (a) (3-aminopropil)trietoxisilano (APTES) ou (b) quitosana. As atividades específicas da endoglucanase e da ?-glicosidase foram maiores quando estas ligadas nas NPMs com casca de APTES. Os dois tipos de NPMs armadas com as enzimas apresentaram atividades residuais de ao redor de 65% após seis ciclos de hidrólise, contudo, o rendimento e viabilidade relacionados à liberação de glicose foram melhores para o sistema recoberto com quitosana Abstract: The objectives of this doctoral thesis include applications of nanotechnology during the exploitation of orange bagasse (OB) to: (1) obtain a high added value bionanomaterial ¿ nanocellulose-; and (2) synhtesis and functionalization of nanoparticles, designed for reuse of hydrolases during the hydrolysis of OB. OB is an agro-industrial byproduct obtained after the extraction of the juice, with about 10 million tons of this residue being produced in Brazil each year. Two methods were used to obtain the nanocellulose: enzymatic and physicochemical. The enzymatic pathway was based on the application of enzymes isolated from the bacterium Xanthomonas axonopodis pv. citri and, the obtained biomaterial showed average diameter of 10 nm and 63% of crystallinity. The physico-chemical process led to the production of nanocellulose with an average diameter of 20 nm and a higher crystallinity of 73%. It¿s noteworthy that were investigated restaurant and industry waste - Citrosuco (Matão, SP, Brazil). On the other hand, a strategy of covalent immobilization of the commercial enzymatic complex of Trichoderma reesei (Celluclast® 1,5 L) was developed aiming the production of second generation ethanol from the orange bagasse on pilot scale. The enzymes were immobilized on the functionalized inert surface of ferromagnetic nanoparticles (MNPs). The immobilization technique increased the shelf life and stability of the enzymes, aiming at a reduction of costs associated to the hydrolysis of lignocellulosic biomass. The shell-like nanoparticles had as their shell: (a) (3-aminopropyl) triethoxysilane (APTES) or (b) chitosan. The specific activities of endoglucanase and ?-glycosidase were higher when bound in MNPs with APTES as shell. The two types of MNPs binded with the enzymes showed residual activities of around 65% after six cycles of hydrolysis; however, yield and viability related to glucose release were better for the chitosan-coated system Doutorado Química Orgânica Doutora em Ciências CAPES

Orientador: Matheus de Castro Fonseca Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: A Doença de Parkinson (DP) é a segunda doença neurodegenerativa mais comum atualmente, atingindo cerca de 10 milhões de pessoas no mundo. Compromete o sistema nervoso central de forma crônica e progressiva, causando danos e morte dos neurônios dopaminérgicos do mesencéfalo, especificamente da substância negra, acarretando diminuição de dopamina no organismo. O indivíduo portador dessa deficiência apresenta como principais sintomas a lentidão motora, rigidez nas articulações, desequilíbrio e tremores involuntários durante repouso. A principal hipótese para a morte dos neurônios é a presença de agregados proteicos denominados Corpos de Lewy (LB’s) compostos principalmente pela alfa-sinucleína (alfa-sin). Essa proteína está intimamente relacionada com mecanismos de transporte de vesícula nas células e se concentra principalmente nos terminais pré-sinápticos dos neurônios, uma região submetida a altos níveis e flutuações de cálcio (Ca2+). Além disso, proteínas da família Rab GTPases (Rab’s) tem relação intima com a alfa-sin, com destaque para a Rab8a. Estudos recentes demonstram que a desregulação da homeostase de Ca2+ em neurônios está intimamente relacionada com o desenvolvimento de doenças neurodegenerativas como a DP, mas os mecanismos envolvidos nesse processo ainda não foram elucidados. Neste trabalho, descrevemos que a perda da homeostase de Ca2+ em células neuronais SH-SY5Y promove a agregação da alfa-sin e faz com que a sua interação com a Rab8a seja intensificada. Além disso, estudos biofísicos in vitro utilizando a alfa-sin recombinante mostraram alterações estruturais da proteína de maneira dependente de sua concentração no meio, mas independente de sua interação com diferentes concentrações de Ca2+. Experimentos com a Rab8a recombinante in vitro, indicam que a interação entre alfa-sin e Rab8a ocorre em condições basais e é intensificada na presença de Ca2+, processo análogo ao observado no interior das células. Assim, os dados obtidos mostram relação direta do aumento de Ca2+ e o mau funcionamento de ambas as proteínas, indicando ser um dos possíveis mecanismos responsáveis para o desenvolvimento de DP Abstract: Parkinson's disease (PD) is the second most common neurodegenerative disease today, affecting about 10 million people worldwide. It affects the central nervous system in a chronic and progressive way, causing damage and death to the dopaminergic neurons of the midbrain, specifically the substantia nigra, leading to a huge decrease in dopamine in the body. The individual with this deficiency presents as main symptoms motor slowness, joint stiffness, unbalance, and involuntary tremors during rest. The main hypothesis for the death of neurons is the presence of protein aggregates called Lewy bodies (LB's), mainly composed of alpha-synuclein (alpha-sin). This protein is closely related to vesicle transport mechanisms in cells and is mainly concentrated in the presynaptic terminals of neurons, a region subjected to high levels and fluctuations of calcium (Ca2+). Moreover, the Rab GTPases proteins family (Rab's) are closely related to alpha-sin, especially Rab8a. Recent studies show that the dysregulation of Ca2+ homeostasis in neurons is closely related to the development of neurodegenerative diseases such as PD, but the mechanisms involved in this process have not yet been elucidated. In this work, we describe that disruption of Ca2+ homeostasis in SH-SY5Y neuronal cells promotes aggregation of alpha-sin and causes its interaction with Rab8a to be intensified. Furthermore, in vitro biophysical studies using recombinant alpha-sin showed structural changes of the protein in a manner dependent on its concentration in the medium but independent of its interaction with different Ca2+ concentrations. Experiments with recombinant Rab8a in vitro indicate that the interaction between alpha-sin and Rab8a occurs under basal conditions and is intensified in the presence of Ca2+, a process analogous to that observed inside the cells. Thus, the data obtained show a direct relationship between increased Ca2+ and the malfunction of both proteins, and this is one of the possible mechanisms responsible for the development of PD Mestrado Biologia Celular Mestre em Biologia Celular e Estrutural CNPQ 130646/2020-2 CAPES 001

A biomassa vegetal pode ser uma importante fonte de obtenção de diversos produtos a partir da desestruturação de suas frações por um vasto grupo de enzimas. No entanto, a geração de compostos de alto valor agregado a partir da biomassa lignocelulósica requer o desenvolvimento de novos sistemas enzimáticos. Pensando nisso, a prospecção e a caracterização de novas enzimas que estão presentes no secretoma de fungos degradadores da biomassa lignocelulósica tem sido fonte de pesquisa por pesquisadores do mundo todo. O objetivo deste trabalho foi prospectar, clonar e expressar de maneira heteróloga o gene codificante de uma enzima putativa do fungo termofílico T. terrestris em cepas do fungo filamentoso A. nidulans A773 e promover sua caracterização bioquímica e biofísica. O gene da enzima foi amplificado, clonado e inserido no vetor pEXPYR antes de ser inserido no sistema de expressão do A. nidulans. Os transformantes obtidos foram induzidos em meio mínimo de cultivo contendo 3% (m/v) de maltose e 1% (m/v) de glicose em meio estacionário para a produção, seguido da purificação da enzima. Estudos bioquímicos foram realizados para determinar o pH e a temperatura ótima de reação, bem como, a especificidade aos substratos e a determinação dos parâmetros cinéticos. A termoestabilidade da enzima também foi avaliada por estudo de dicroísmo circular (DC). Além disso, foi avaliado o efeito colaborativo entre uma xilanase GH10 e a enzima em estudo na hidrólise do bagaço de cana-de-açúcar pré-tratado. A enzima obtida por expressão heteróloga foi caracterizada como uma xilo-oligossacarídeo oxidase (XylO). Por meio da análise filogenética das sequências de aminoácidos entre a enzima expressa e outras enzimas oxidativas, a XylO foi classificada como pertencente a família das flavoproteínas e subfamília das BBE. A enzima TtXylO demonstrou ter especificidade em oligossacarídeos de C5 apresentando boa atividade enzimática em substratos complexos de xilana. A enzima possui pH ótimo de 5,5 e temperatura ótima de 25 ºC. As análises de DC indicaram temperatura de desnaturação de 62,7 ºC, caracterizando esta enzima como termofílica. Contudo, novos estudos ainda são necessários para avaliar os produtos gerados a partir da oxidação dos diferentes xilo-oligossacarídeos pela XylO e seu potencial uso na indústria. Plant biomass is an important source for generation of several products obtained from enzymatic cleavage of its fractions by a large group of enzymes. However, the generation of high value compounds from lignocellulosic biomass requires the development of new enzymatic systems. Considering that, prospection and characterization of enzymes present in the biomass-degrading fungi secretome has been a source of study by researchers around the world. The aim of this work was to prospect, clone and heterologously express a putative enzyme encoding gene from the thermophilic fungus T. terrestris in A. nidulans A773 strains and to promote its biochemical and biophysical characterization. The gene was amplified, cloned and inserted into the pEXPYR vector before being inserted into A. nidulans expression system. The transformants were induced by culture in minimal médium containing 3% (w/v) maltose and 1% (w/v) glucose by stationary culture for the production, followed by enzyme purification. Biochemical analyses were performed to determine optimum pH and temperature as well as the substrate specificities and kinetic parameters. The enzyme thermostability was also evaluated by circular dichroism (CD). In addition, the collaborative effect between the enzyme and a GH10 on hydrolys of pre-treated sugarcane bagasse was evaluated. The enzyme obtained by heterologous expression was characterized as a xylooligosaccharide oxidase (XylO). Phylogenetic analysis between amino acid sequences of expressed enzyme and other oxidative enzymes classified XylO as belonging to flavoproteins family and subfamily of BBE. TtXylO has been shown to have specificity on C5 oligosaccharides exhibiting good enzymatic activity on complex xylan substrates. The enzyme has an optimum pH of 5.5 and optimum temperature of 25 ºC. DC analyses showed melting temperature of 62.7 ºC, characterizing this enzyme as thermophilic. In general, further studies are still needed to evaluate the products generated from oxidation of xylooligosaccharides by XylO and their potential use in the industry.

Orientadores: Hélia Harumi Sato, Roberto Ruller Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos Resumo: A biomassa lignocelulósica é um importante recurso renovável que está prontamente disponível, sendo uma fonte de matéria-prima com alto potencial biotecnológico. Os polissacarídeos complexos que compõem lignocelulose podem ser convertidos em monossacarídeos fermentescíveis, com grande aplicabilidade em diversos bioprocessos industriais. A degradação dos materiais lignocelulósicos pode ser realizada por uma diversidade de vias enzimáticas complexas, onde é requerido um número considerável de enzimas ativas sobre carboidratos. Entre elas, as famílias das celulases e hemicelulases, além de atuarem na hidrólise dos materiais lignocelulósicos, possuem um uso versátil em setores industriais, tais como, nas áreas alimentícia, bebidas e de biocombustíveis. A tese teve como principais objetivos o delineamento de estratégias para a produção de enzimas e coquetéis enzimáticos eficientes para o uso na hidrólise da biomassa vegetal e, a aplicação de técnicas de imobilização para ampliar a utilização de enzimas em escala comercial. Inicialmente, a bioprospecção de novos micro-organismos secretores de enzimas atuantes na biomassa lignocelulósica foi realizada, e dentre as oitenta linhagens de Streptomyces testadas, duas linhagens (F1 e F7) se destacaram por apresentarem elevadas atividades celulolíticas e hemicelulolíticas. Uma abordagem genômica dessas linhagens possibilitou a identificação de 85 hidrolases glicosídicas (GHs) distribuídas em 33 famílias diferentes na linhagem F1, e 100 GHs dispostas em 44 famílias na linhagem F7. Além disso, os dados genômicos das linhagens F1 e F7 também indicaram a presença de genes relacionados à degradação da lignina. Ferramentas estatísticas também foram aplicadas e possibilitaram a ampliação da produção de GHs pela linhagem F1. Com a otimização, elevadas concentrações de GHs foram alcançadas com meio nutriente adicionado de 16,4 g L-1 de farelo de trigo e 10,0 g L-1 de caseína, onde se obteve 9,27 U mL -1 de xilanase e 0,22 U mL -1 de celulase. Para confirmar a diversidade de GHs expressas pela linhagem F1, uma análise por espectrometria de massa foi realizada e observou-se que quanto maior a complexidade da fonte de carbono utilizada, maior foi a gama de proteínas expressas, incluindo vários tipos de celulases e hemicelulases. A eficiência do extrato enzimático produzido pela linhagem F1 foi estudada para a sacarificação da biomassa vegetal e possibilitou um aumento significativo na liberação de açúcares quando adicionado ao extrato celulolítico comercial, indicando que as enzimas secretadas pela Streptomyces sp. F1 podem ser aplicadas para o melhoramento dos atuais coquetéis comerciais. Foi estudada também a criação de métodos de imobilização de enzimas em condições neutras de pH. Os novos suportes produzidos com a agarose foram utilizados para a imobilização de enzimas monoméricas e multiméricas de grande importância biotecnológica. Um estudo mais detalhado explorando os novos suportes e o uso de técnicas de pós-imobilização foi também proposto. O processo desenvolvido aplicando o polímero polietilenimina (PEI) possibilitou a formação de um excelente sistema para imobilizar e estabilizar a ?-glicosidase obtida de Exiguobacterium antarcticum. A ?-glicosidase imobilizada apresentou uma melhora em suas características, incluindo estabilidade térmica e de armazenamento. Além disso, a ?-glicosidase manteve sua atividade elevada mesmo após vários ciclos de hidrólise com celobiose como substrato Abstract: Lignocellulosic biomass is an important renewable resource that is readily available, being a source of raw material with high biotechnological potential. The complex polysaccharides that compose lignocellulose can be converted into fermentable monosaccharides, with great applicability in several industrial bioprocesses. The degradation of lignocellulosic materials can be accomplished by a variety of complex enzymatic pathways, where a considerable number of carbohydrate active enzymes are required. Among them, the families of cellulases and hemicellulases, besides acting in the hydrolysis of lignocellulosic materials, have a versatile use in industrial sectors, such as in food, beverages and biofuels. The main aims of this thesis were the design of efficient strategies for the production of enzymes and enzymatic cocktails for use in plant biomass hydrolysis and the application of immobilization techniques to increase the use of enzymes in a commercial scale. Initially, bioprospection of new enzyme secreting microorganisms active in lignocellulosic biomass was performed, and among the eighty Streptomyces strains tested, two strains (F1 and F7) were distinguished by their high cellulolytic and hemicellulolytic activities. A genomic approach of these strains allowed the identification of 85 glycoside hydrolases (GHs) distributed in 33 different families the strain F1, and 100 GHs arranged in 44 families the strain F7. In addition, the genomic data from strains F1 and F7 also indicated the presence of genes related to lignin degradation. Statistical tools were also applied and allowed the increase in GH production by strain F1. With the optimization, high concentrations of GHs were achieved with a nutrient medium containing 16.4 g L-1 of wheat bran and 10.0 g L-1 of casein, where 9.27 U mL-1 of xylanase and 0.22 U mL-1 of cellulase were obtained. To confirm the diversity of GHs expressed by the strain F1, an analysis using mass spectrometry technique was performed and it was observed that the greater the complexity of the carbon source used, the greater the range of proteins secreted, including several types of cellulases and hemicellulases. The efficiency of the enzymatic extract produced with strain F1 was studied for the saccharification of plant biomass and allowed a significant increase in sugar release when added to the commercial cellulolytic extract, indicating that the enzymes expressed by Streptomyces sp. F1 can be applied for the improvement of the current commercial cocktails. The creation of enzyme immobilization methods under neutral conditions of pH was also study. The new agarose supports were used for the immobilization of monomeric and multimeric enzymes of great industrial and biotechnological importance. A more detailed study exploring the new supports and the use of post-immobilization techniques with polymers and small molecules was also proposed. The process developed by applying the polymer polyethyleneimine (PEI) enabled the formation of an excellent system to stabilize the glucose-tolerant tetrameric ?-glycosidase obtained from Exiguobacterium antarcticum. The immobilized ?-glycosidase showed an improvement in its characteristics, with an increased activity, including thermal and storage stability. In addition, the ?-glycosidase maintained a high activity even after several cycles of hydrolysis applying cellobiose as substrate Doutorado Ciência de Alimentos Doutor em Ciência de Alimentos CNPQ 140610/2014-6