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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.

Background Arabinoxylan is an abundant polysaccharide in industrially relevant biomasses such as sugarcane, corn stover and grasses. However, the arabinofuranosyl di-substitutions that decorate the xylan backbone are recalcitrant to most known arabinofuranosidases (Abfs). Results In this work, we identified a novel GH51 Abf (XacAbf51) that forms trimers in solution and can cope efficiently with both mono- and di-substitutions at terminal or internal xylopyranosyl units of arabinoxylan. Using mass spectrometry, the kinetic parameters of the hydrolysis of 33-α-l-arabinofuranosyl-xylotetraose and 23,33-di-α-l-arabinofuranosyl-xylotetraose by XacAbf51 were determined, demonstrating the capacity of this enzyme to cleave arabinofuranosyl linkages of internal mono- and di-substituted xylopyranosyl units. Complementation studies of fungal enzyme cocktails with XacAbf51 revealed an increase of up to 20% in the release of reducing sugars from pretreated sugarcane bagasse, showing the biotechnological potential of a generalist GH51 in biomass saccharification. To elucidate the structural basis for the recognition of internal di-substitutions, the crystal structure of XacAbf51 was determined unveiling the existence of a pocket strategically arranged near to the − 1 subsite that can accommodate a second arabinofuranosyl decoration, a feature not described for any other GH51 Abf structurally characterized so far. Conclusions In summary, this study reports the first kinetic characterization of internal di-substitution release by a GH51 Abf, provides the structural basis for this activity and reveals a promising candidate for industrial processes involving plant cell wall depolymerization.

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.

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.

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.

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.

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.