Publications by authors named "Igor Polikarpov"

196 Publications

Polymer ultrastructure governs AA9 lytic polysaccharide monooxygenases functionalization and deconstruction efficacy on cellulose nano-crystals.

Bioresour Technol 2021 Nov 18:126375. Epub 2021 Nov 18.

Photobiocatalysis Unit - CPBL, and Biomass Transformation Lab - BTL, École Interfacultaire de Bioingénieurs, Université Libre de Bruxelles, Belgium. Electronic address:

Lytic Polysaccharide MonoOxygenases display great variability towards cellulose ultrastructure while performing oxidative functionalization of the polymers. Aiming at employing AA9-LPMOs for isolation of cellulose nano-crystals (CNCs), the ratio between functionalization/crystalline degradation became a crucial parameter. Here are reported the constraints posed by the substrate ultrastructure on the activity of seven different AA9 LPMOs representative of various regioselectivity and substrate affinity: TtAA9E, TaAA9A, PcAA9D, MtAA9A, MtAA9D, MtAA9I-CBM and MtAA9J. The substrate crystallinity and dry matter loading greatly affected the seven AA9s in an enzyme-specific manner, impacting their efficiency for CNCs functionalization purposes. X-ray diffraction pattern analyses were used to assess the cracking efficacy of the enzymatic treatment to de-crystallize CNCs, revealing that those AA9s with minor efficiency in releasing oligosaccharides resulted instead the most disruptive towards the crystal lattice and in reducing the particle sizes. These non-catalytic effects were found comparable with the one caused by the expansin BsEXLX1 enzyme.
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http://dx.doi.org/10.1016/j.biortech.2021.126375DOI Listing
November 2021

SAXSMoW 3.0: New advances in the determination of the molecular weight of proteins in dilute solutions from SAXS intensity data on a relative scale.

Protein Sci 2022 01 18;31(1):251-258. Epub 2021 Nov 18.

Sao Carlos Institute of Physics, University of Sao Paulo, São Paulo, Brazil.

SAXSMoW (SAXS Molecular Weight) is an online platform widely used over the past few years for determination of molecular weights of proteins in dilute solutions. The scattering intensity retrieved from small-angle X-ray scattering (SAXS) raw data is the sole input to SAXSMoW for determination of molecular weights of proteins in liquid. The current updated SAXSMoW version 3.0 determines the linear dependence of the true protein volume on their apparent protein volume, based on SAXS curves calculated for 67,000 protein structures selected from the Protein Data Bank. SAXSMoW 3.0 was tested against 43 experimental SAXS scattering curves from proteins with known molecular weights. Our results demonstrate that most of the molecular weights determined for the nonglycosylated and also for the glycosylated proteins are in good agreement with their expected molecular weights. Additionally, the average discrepancies between the calculated molecular weights and their nominal values for glycosylated proteins are similar to those for nonglycosylated ones.
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http://dx.doi.org/10.1002/pro.4227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8740845PMC
January 2022

Paludibacter propionicigenes GH10 xylanase as a tool for enzymatic xylooligosaccharides production from heteroxylans.

Carbohydr Polym 2022 Jan 21;275:118684. Epub 2021 Sep 21.

Instituto de Física de São Carlos, Universidade de São Paulo, Avenida Trabalhador São-carlense 400, 13566-590 São Carlos, SP, Brazil. Electronic address:

Bioconversion of lignocellulosic biomass into value-added products relies on polysaccharides depolymerization by carbohydrate active enzymes. This work reports biochemical characterization of Paludibacter propionicigenes xylanase from GH10 (PpXyn10A) and its application for enzymatic xylooligosaccharides (XOS) production from commercial heteroxylans and liquor of hydrothermally pretreated corn cobs (PCC). PpXyn10A is tolerant to ethanol and NaCl, and releases xylobiose (X2) and xylotriose (X3) as the main hydrolytic products. The conversion rate of complex substrates into short XOS was approximately 30% for glucuronoxylan and 8.8% for rye arabinoxylan, after only 4 h; while for PCC, PpXyn10A greatly increased unbranched XOS yields. B. adolescentis fermentation with XOS from beechwood glucuronoxylan produced mainly acetic and lactic acids. Structural analysis shows that while the glycone region of PpXyn10A active site is well preserved, the aglycone region has aromatic interactions in the +2 subsite that may explain why PpXyn10A does not release xylose.
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http://dx.doi.org/10.1016/j.carbpol.2021.118684DOI Listing
January 2022

Recent advances in the enzymatic production and applications of xylooligosaccharides.

World J Microbiol Biotechnol 2021 Sep 6;37(10):169. Epub 2021 Sep 6.

São Carlos Institute of Physics, University of São Paulo, 400 Trabalhador São-carlense Ave, São Carlos, SP, 13566-590, Brazil.

The majority of lignocellulosic biomass on the planet originates from plant cell walls, which are complex structures build up mainly by cellulose, hemicellulose and lignin. The largest part of hemicellulose, xylan, is a polymer with a β-(1→4)-linked xylose residues backbone decorated with α-D-glucopyranosyl uronic acids and/or L-arabinofuranose residues. Xylan is the second most abundant biopolymer in nature, which can be sustainably and efficiently degraded into decorated and undecorated xylooligosaccharides (XOS) using combinations of thermochemical pretreatments and enzymatic hydrolyses, that have broad applications in the food, feed, pharmaceutical and cosmetic industries. Endo-xylanases from different complex carbohydrate-active enzyme (CAZyme) families can be used to cleave the backbone of arabino(glucurono)xylans and xylooligosaccharides and degrade them into short XOS. It has been shown that XOS with a low degree of polymerization have enhanced prebiotic effects conferring health benefits to humans and animals. In this review we describe recent advances in the enzymatic production of XOS from lignocellulosic biomass arabino- and glucuronoxylans and their applications as food and feed additives and health-promoting ingredients. Comparative advantages of xylanases from different CAZy families in XOS production are discussed and potential health benefits of different XOS are presented.
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http://dx.doi.org/10.1007/s11274-021-03139-7DOI Listing
September 2021

Differences in Gluco and Galacto Substrate-Binding Interactions in a Dual 6Pβ-Glucosidase/6Pβ-Galactosidase Glycoside Hydrolase 1 Enzyme from .

J Chem Inf Model 2021 09 23;61(9):4554-4570. Epub 2021 Aug 23.

São Carlos Institute of Physics, University of São Paulo, São Carlos 13566-590, Brazil.

Bacterial glycoside hydrolase 1 (GH1) enzymes with 6-phospho-β-galactosidase and 6-phospho-β-glucosidase activities have the important task of releasing phosphorylated and nonphosphorylated monosaccharides into the cytoplasm. Curiously, dual 6-phospho-β-galactosidase/6-phospho-β-glucosidase (dual-phospho) enzymes have broad specificity and are able to hydrolyze galacto- and gluco-derived substrates. This study investigates the structure and substrate specificity of a GH family 1 enzyme from , hereafter known as BglC. The enzyme structure has been solved, and sequence analysis, molecular dynamics simulations, and binding free energy calculations offered evidence of dual-phospho activity. Both test ligands -nitrophenyl-β-d-galactoside-6-phosphate (PNP6Pgal) and -nitrophenyl-β-d-glucoside-6-phosphate (PNP6Pglc) demonstrated strong binding to BglC although the pose and interactions of the PNP6Pglc triplicates were slightly more consistent. Interestingly, known specificity-inducing residues, Gln23 and Trp433, bind strongly to the ligand O3 hydroxyl group in the PNP6Pgal-BglC complex and to the ligand O4 hydroxyl group in the PNP6Pglc-BglC complex. Additionally, the BglC-His124 residue is a major contributor of hydrogen bonds to the PNP6Pgal O3 hydroxyl group but does not form any hydrogen bonds with PNP6Pglc. On the other hand, BglC residues Tyr173, Tyr301, Gln302, and Thr321 form hydrogen bonds with PNP6Pglc but not PNP6Pgal. These findings provide important details of the broad specificity of dual-phospho activity GH1 enzymes.
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http://dx.doi.org/10.1021/acs.jcim.1c00413DOI Listing
September 2021

Phytoplankton bloom detection during the COVID-19 lockdown with remote sensing data: Using Copernicus Sentinel-3 for north-western Arabian/Persian Gulf case study.

Mar Pollut Bull 2021 Oct 28;171:112734. Epub 2021 Jul 28.

Crisis Decision Support Program, Kuwait Institute for Scientific Research, P.O. Box 24885, 13109 Safat, Kuwait.

To examine whether a country-wide COVID-19 lockdown affected phytoplankton development, variability of chlorophyll-a concentrations in the north-western Arabian/Persian Gulf (Kuwait Bay) was investigated using remote sensing instruments Sentinel OLCI between 2018 and 2020 and compared to available in situ collected data. Satellite-retrieved chlorophyll concentrations considerably increased in inshore waters of Kuwait Bay, 1-2 months following the initiation of the 24/7 curfew. The extremely high concentrations of dissolved inorganic nutrients, especially ammonia, and coincided phytoplankton bloom were revealed in June-July 2020 by opportunity field sampling, supporting the satellite-derived bloom detection. Remote sensing operational monitoring with high spatial resolution sensors provides an exceptional opportunity for emergency analysis and decision making in conditions of natural or anthropogenic crises, which forces the development of regional remote sensing algorithms for the shallow marine environment of the Gulf.
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http://dx.doi.org/10.1016/j.marpolbul.2021.112734DOI Listing
October 2021

Unlocking the structural features for the xylobiohydrolase activity of an unusual GH11 member identified in a compost-derived consortium.

Biotechnol Bioeng 2021 10 14;118(10):4052-4064. Epub 2021 Jul 14.

Grupo de Biotecnologia Molecular, Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, São Paulo, Brazil.

The heteropolysaccharide xylan is a valuable source of sustainable chemicals and materials from renewable biomass sources. A complete hydrolysis of this major hemicellulose component requires a diverse set of enzymes including endo-β-1,4-xylanases, β-xylosidases, acetylxylan esterases, α-l-arabinofuranosidases, and α-glucuronidases. Notably, the most studied xylanases from glycoside hydrolase family 11 (GH11) have exclusively been endo-β-1,4- and β-1,3-xylanases. However, a recent analysis of a metatranscriptome library from a microbial lignocellulose community revealed GH11 enzymes capable of releasing solely xylobiose from xylan. Although initial biochemical studies clearly indicated their xylobiohydrolase mode of action, the structural features that drive this new activity still remained unclear. It was also not clear whether the enzymes acted on the reducing or nonreducing end of the substrate. Here, we solved the crystal structure of MetXyn11 in the apo and xylobiose-bound forms. The structure of MetXyn11 revealed the molecular features that explain the observed pattern on xylooligosaccharides released by this nonreducing end xylobiohydrolase.
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http://dx.doi.org/10.1002/bit.27880DOI Listing
October 2021

Xyloglucan processing machinery in Xanthomonas pathogens and its role in the transcriptional activation of virulence factors.

Nat Commun 2021 06 30;12(1):4049. Epub 2021 Jun 30.

Brazilian Biorenewables National Laboratory (LNBR), Brazilian Center for Research in Energy and Materials (CNPEM), Campinas, São Paulo, Brazil.

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.
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http://dx.doi.org/10.1038/s41467-021-24277-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8245568PMC
June 2021

Impact of cellulose properties on enzymatic degradation by bacterial GH48 enzymes: Structural and mechanistic insights from processive Bacillus licheniformis Cel48B cellulase.

Carbohydr Polym 2021 Jul 9;264:118059. Epub 2021 Apr 9.

São Carlos Institute of Physics, University of São Paulo (USP), São Carlos 13560-970, São Paulo, Brazil. Electronic address:

Processive cellulases are highly efficient molecular engines involved in the cellulose breakdown process. However, the mechanism that processive bacterial enzymes utilize to recruit and retain cellulose strands in the catalytic site remains poorly understood. Here, integrated enzymatic assays, protein crystallography and computational approaches were combined to study the enzymatic properties of the processive BlCel48B cellulase from Bacillus licheniformis. Hydrolytic efficiency, substrate binding affinity, cleavage patterns, and the apparent processivity of bacterial BlCel48B are significantly impacted by the cellulose size and its surface morphology. BlCel48B crystallographic structure was solved with ligands spanning -5 to -2 and +1 to +2 subsites. Statistical coupling analysis and molecular dynamics show that co-evolved residues on active site are critical for stabilizing ligands in the catalytic tunnel. Our results provide mechanistic insights into BlCel48B molecular-level determinants of activity, substrate binding, and processivity on insoluble cellulose, thus shedding light on structure-activity correlations of GH48 family members in general.
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http://dx.doi.org/10.1016/j.carbpol.2021.118059DOI Listing
July 2021

Structural and molecular dynamics investigations of ligand stabilization via secondary binding site interactions in GH11 xylanase.

Comput Struct Biotechnol J 2021 7;19:1557-1566. Epub 2021 Mar 7.

Instituto de Física de São Carlos, Universidade de São Paulo, Avenida Trabalhador São-carlense 400, 13566-590 São Carlos, SP, Brazil.

Glycoside hydrolases (GHs) are essential for plant biomass deconstruction. GH11 family consist of endo-β-1,4-xylanases which hydrolyze xylan, the second most abundant cell wall biopolymer after cellulose, into small bioavailable oligomers. Structural requirements for enzymatic mechanism of xylan hydrolysis is well described for GH11 members. However, over the last years, it has been discovered that some enzymes from GH11 family have a secondary binding sites (SBS), which modulate the enzymes activities, but mechanistic details of the molecular communication between the active site and SBS of the enzymes remain a conundrum. In the present work we structurally characterized GH11 xylanase from (Xyn11B), a microorganism of agricultural importance, using protein crystallography and molecular dynamics simulations. The Xyn11B structure was solved to 2.5 Å resolution and different substrates (xylo-oligosaccharides from X3 to X6), were modelled in its active and SBS sites. Molecular Dynamics (MD) simulations revealed an important role of SBS in the activity and conformational mobility of Xyn11B, demonstrating that binding of the reaction products to the SBS of the enzyme stabilizes the N-terminal region and, consequently, the active site. Furthermore, MD simulations showed that the longer the ligand, the better is the stabilization within active site, and the positive subsites contribute less to the stabilization of the substrates than the negative ones. These findings provide rationale for the observed enzyme kinetics, shedding light on the conformational modulation of the GH11 enzymes via their SBS mediated by the positive molecular feedback loop which involve the products of the enzymatic reaction.
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http://dx.doi.org/10.1016/j.csbj.2021.03.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7994722PMC
March 2021

Light-stimulated T. thermophilus two-domain LPMO9H: Low-resolution SAXS model and synergy with cellulases.

Carbohydr Polym 2021 May 15;260:117814. Epub 2021 Feb 15.

São Carlos Institute of Physics, University of São Paulo, Av. Trabalhador São-carlense 400, São Carlos, São Paulo, Brazil. Electronic address:

Lytic polysaccharide monooxygenases (LPMOs), monocopper enzymes that oxidatively cleave recalcitrant polysaccharides, have important biotechnological applications. Thermothelomyces thermophilus is a rich source of biomass-active enzymes, including many members from auxiliary activities family 9 LPMOs. Here, we report biochemical and structural characterization of recombinant TtLPMO9H which oxidizes cellulose at the C1 and C4 positions and shows enhanced activity in light-driven catalysis assays. TtLPMO9H also shows activity against xyloglucan. The addition of TtLPMO9H to endoglucanases from four different glucoside hydrolase families (GH5, GH12, GH45 and GH7) revealed that the product formation was remarkably increased when TtLPMO9H was combined with GH7 endoglucanase. Finally, we determind the first low resolution small-angle X-ray scattering model of the two-domain TtLPMO9H in solution that shows relative positions of its two functional domains and a conformation of the linker peptide, which can be relevant for the catalytic oxidation of cellulose and xyloglucan.
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http://dx.doi.org/10.1016/j.carbpol.2021.117814DOI Listing
May 2021

Insights into the dual cleavage activity of the GH16 laminarinase enzyme class on β-1,3 and β-1,4 glycosidic bonds.

J Biol Chem 2021 Jan-Jun;296:100385. Epub 2021 Feb 5.

Programa de Processos Tecnológicos e Ambientais, Universidade de Sorocaba, Sorocaba, São Paulo, Brazil. Electronic address:

Glycoside hydrolases (GHs) are involved in the degradation of a wide diversity of carbohydrates and present several biotechnological applications. Many GH families are composed of enzymes with a single well-defined specificity. In contrast, enzymes from the GH16 family can act on a range of different polysaccharides, including β-glucans and galactans. SCLam, a GH16 member derived from a soil metagenome, an endo-β-1,3(4)-glucanase (EC 3.2.1.6), can cleave both β-1,3 and β-1,4 glycosidic bonds in glucans, such as laminarin, barley β-glucan, and cello-oligosaccharides. A similar cleavage pattern was previously reported for other GH16 family members. However, the molecular mechanisms for this dual cleavage activity on (1,3)- and (1,4)-β-D-glycosidic bonds by laminarinases have not been elucidated. In this sense, we determined the X-ray structure of a presumably inactive form of SCLam cocrystallized with different oligosaccharides. The solved structures revealed general bound products that are formed owing to residual activities of hydrolysis and transglycosylation. Biochemical and biophysical analyses and molecular dynamics simulations help to rationalize differences in activity toward different substrates. Our results depicted a bulky aromatic residue near the catalytic site critical to select the preferable configuration of glycosidic bonds in the binding cleft. Altogether, these data contribute to understanding the structural basis of recognition and hydrolysis of β-1,3 and β-1,4 glycosidic linkages of the laminarinase enzyme class, which is valuable for future studies on the GH16 family members and applications related to biomass conversion into feedstocks and bioproducts.
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http://dx.doi.org/10.1016/j.jbc.2021.100385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7961093PMC
September 2021

Production of prebiotic xylooligosaccharides from arabino- and glucuronoxylan using a two-domain Jonesia denitrificans xylanase from GH10 family.

Enzyme Microb Technol 2021 Mar 12;144:109743. Epub 2021 Jan 12.

Instituto de Física de São Carlos, Universidade de São Paulo, Avenida Trabalhador São-carlense 400, 13566-590, São Carlos, SP, Brazil. Electronic address:

Development of a more environmentally sustainable society is based on the maximum use of renewable carbon sources and their valorization of environmentally-friendly green technologies. This includes a thorough use of plant biomass and agricultural residues for the production of value-added bioproducts. Xylan is the second most abundant biopolymer in nature which can be sustainable converted into pentoses and xylooligosaccharides, that have wide applications in the food, feed, pharmaceutical, and cosmetic industry. Within the scope of present study, we biochemically characterized two-domain GH10 xylanase from Jonesia denitrificans (JdXyn10A) and evaluated its applicability for production of xylooligosaccharides (XOS). JdXyn10A has a specific activity of 84 ± 2 U/mg and 65 ± 5 U/mg when acting on beechwood glucuronoxylan and rye arabinoxylan, respectively. The enzyme is stable in a wide pH range and is tolerant to high concentrations of NaCl and ethanol. Interestingly, the profile of products released by the enzyme is predominant in xylobiose and xylotriose, with a very low fraction of xylose which is desirable for XOS production. The efficiencies of enzymatic conversion of beechwood glucuronoxylan and rye arabinoxylan are 47.67 % and 26.01 %, respectively, after 6 h of enzymatic hydrolysis only. Structural comparison between the JdXyn10A homology model and the structure from its homologous that while the glycone region of its active site is well preserved, the aglycone region presents structural differences in the +2 subsite that may explain why JdXyn10A does not release xylose.
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http://dx.doi.org/10.1016/j.enzmictec.2021.109743DOI Listing
March 2021

Comparative analysis of two recombinant LPMOs from Aspergillus fumigatus and their effects on sugarcane bagasse saccharification.

Enzyme Microb Technol 2021 Mar 13;144:109746. Epub 2021 Jan 13.

Department of Biotechnology, Lorena School of Engineering, University of São Paulo, Lorena, SP, Brazil. Electronic address:

Lytic polysaccharide monooxygenases (LPMOs) have been introduced into industrial cocktails used for biomass saccharification due to their capacity to boost enzymatic conversion of recalcitrant cellulose. The genome of the thermotolerant ascomycete Aspergillus fumigatus encodes 7 genes for LPMOs that belong to auxiliary activity family 9 (AA9). Here, we cloned, successfully expressed and performed biochemical evaluation of two CBM-less A. fumigatus LPMOs (AfAA9A and AfAA9B). A high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) analysis demonstrated that AfAA9A and AfAA9B are able to oxide cellulose at C1 and C1/C4 positions, respectively. Synergic effects of LPMOs (separately and in combination) with cellulases were investigated. Supplementation of Celluclast 1.5 L with a low concentration of AfAA9B improved in 20 % the saccharification of sugarcane bagasse pretreated by steam explosion (SEB), while AfAA9A did not improvethe saccharification. Analysis of the hydrolyzed biomass by confocal laser scanning microscopy (CLSM) showed the LPMOs are promoting lignin oxidation in the lignocellulosic material. This study complements the available results concerning the utilization of LPMOs in the enzymatic saccharification of lignocellulosic biomass.
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http://dx.doi.org/10.1016/j.enzmictec.2021.109746DOI Listing
March 2021

Cellulose nanofibers production using a set of recombinant enzymes.

Carbohydr Polym 2021 Mar 15;256:117510. Epub 2020 Dec 15.

São Carlos Institute of Physics, University of São Paulo, Av. Trabalhador Sãocarlense, 400, São Carlos, SP, 13566-590, Brazil. Electronic address:

Cellulose nanofibers (CNF) are renewable and biodegradable nanomaterials with attractive barrier, mechanical and surface properties. In this work, three different recombinant enzymes: an endoglucanase, a xylanase and a lytic polysaccharide monooxygenase, were combined to enhance cellulose fibrillation and to produce CNF from sugarcane bagasse (SCB). Prior to the enzymatic catalysis, SCB was chemically pretreated by sodium chlorite and KOH, while defibrillation was accomplished via sonication. We obtained much longer (μm scale length) and more thermostable (resisting up to 260 °C) CNFs as compared to the CNFs prepared by TEMPO-mediated oxidation. Our results showed that a cooperative action of the set of hydrolytic and oxidative enzymes can be used as a "green" treatment prior to the sonication step to produce nanofibrillated cellulose with advanced properties.
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http://dx.doi.org/10.1016/j.carbpol.2020.117510DOI Listing
March 2021

Multienzyme Cellulose Films as Sustainable and Self-Degradable Hydrogen Peroxide-Producing Material.

Biomacromolecules 2020 12 17;21(12):5315-5322. Epub 2020 Nov 17.

Department of Chemistry, University of Bath, Bath BA2 7AY, U.K.

The use of hydrogen peroxide-releasing enzymes as a component to produce alternative and sustainable antimicrobial materials has aroused interest in the scientific community. However, the preparation of such materials requires an effective enzyme binding method that often involves the use of expensive and toxic chemicals. Here, we describe the development of an enzyme-based hydrogen peroxide-producing regenerated cellulose film (RCF) in which a cellobiohydrolase (CBHI) and a cellobiose dehydrogenase (CDHA) were efficiently adsorbed, 90.38 ± 2.2 and 82.40 ± 5.7%, respectively, without making use of cross-linkers. The enzyme adsorption kinetics and binding isotherm experiments showed high affinity of the proteins possessing cellulose-binding modules for RCF, suggesting that binding on regenerated cellulose via specific interactions can be an alternative method for enzyme immobilization. Resistance to compression and porosity at a micrometer scale were found to be tunable by changing cellulose concentration prior to film regeneration. The self-degradation process, triggered by stacking CBHI and CDHA (previously immobilized onto separate RCF), produced 0.15 nmol/min·cm of HO. Moreover, the production of HO was sustained for at least 24 h reaching a concentration of ∼2 mM. The activity of CDHA immobilized on RCF was not affected by reuse for at least 3 days (1 cycle/day), suggesting that no significant enzyme leakage occurred in that timeframe. In the material herein designed, cellulose (regenerated from a 1-ethyl-3-methylimidazolium acetate/dimethyl sulfoxide (DMSO) solution) serves both as support and substrate for the immobilized enzymes. The sequential reaction led to the production of HO at a micromolar-millimolar level revealing the potential use of the material as a self-degradable antimicrobial agent.
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http://dx.doi.org/10.1021/acs.biomac.0c01393DOI Listing
December 2020

X-ray Structure, Bioinformatics Analysis, and Substrate Specificity of a 6-Phospho-β-glucosidase Glycoside Hydrolase 1 Enzyme from .

J Chem Inf Model 2020 12 9;60(12):6392-6407. Epub 2020 Nov 9.

São Carlos Institute of Physics, University of São Paulo, São Carlos 13566-590, Brazil.

In bacteria, mono- and disaccharides are phosphorylated during the uptake processes through the vastly spread transport system phosphoenolpyruvate-dependent phosphotransferase. As an initial step in the phosphorylated disaccharide metabolism pathway, 6-phospho-β-glucosidases and 6-phospho-β-galactosidases play a crucial role by releasing phosphorylated and nonphosphorylated monosaccharides. However, structural determinants for the specificity of these enzymes still need to be clarified. Here, an X-ray structure of a glycoside hydrolase family 1 enzyme from , hereafter known as BglH, was determined at 2.2 Å resolution, and its substrate specificity was investigated. The sequence of BglH was compared to the sequences of 58 other GH1 enzymes using sequence alignments, sequence identity calculations, phylogenetic analysis, and motif discovery. Through these various analyses, BglH was found to have sequence features characteristic of the 6-phospho-β-glucosidase activity enzymes. Motif and structural observations highlighted the importance of loop L8 in 6-phospho-β-glucosidase activity enzymes. To further affirm enzyme specificity, molecular docking and molecular dynamics simulations were performed using the crystallographic structure of BglH. Docking was carried out with a 6-phospho-β-glucosidase enzyme activity positive and negative control ligand, followed by 400 ns of MD simulations. The positive and negative control ligands were PNP6Pglc and PNP6Pgal, respectively. PNP6Pglc maintained favorable interactions within the active site until the end of the MD simulation, while PNP6Pgal exhibited instability. The favorable binding of substrate stabilized the loops that surround the active site. Binding free energy calculations showed that the PNP6Pglc complex had a substantially lower binding energy compared to the PNP6Pgal complex. Altogether, the findings of this study suggest that BglH possesses 6-phospho-β-glucosidase enzymatic activity and revealed sequence and structural differences between bacterial GH1 enzymes of various activities.
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http://dx.doi.org/10.1021/acs.jcim.0c00759DOI Listing
December 2020

Transformation of xylan into value-added biocommodities using Thermobacillus composti GH10 xylanase.

Carbohydr Polym 2020 Nov 3;247:116714. Epub 2020 Jul 3.

Instituto de Física de São Carlos, Universidade de São Paulo, Avenida Trabalhador São-carlense 400, 13566-590, São Carlos, SP, Brazil. Electronic address:

Enzymatic transformation of xylans into renewable fuels and value-added products is mediated by xylanases. Here we describe the biochemical and X-ray structural characterization of Thermobacillus composti GH10 xylanase (TcXyn10A) at 2.1 Å resolution aiming to unravel details of its recognition of glucurono- and arabinoxylan at a molecular level. TcXyn10A improves the efficiency of pretreated lignocellulosic biomass hydrolysis by a commercial enzyme cocktail causing a 15.35 % increase in xylose release and 4.38 % glucose release after 24 h of reaction. The enzyme releases predominantly xylobiose and xylotriose, as well as MeGlcA3 × 3 (from beechwood glucuronoxylan) and a range of decorated xylooligosaccharides (XOS) from rye arabinoxylan, with Ara2 × 2 being the major product. The enzyme liberates XOS with the yields of 29.09 % for beechwood glucuronoxylan and 16.98 % for rye arabinoxylan. Finally, TcXyn10A has a high thermal stability, halotolerance, and resistance to ethanol, biochemical properties that can be desirable for a number of industrial applications.
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http://dx.doi.org/10.1016/j.carbpol.2020.116714DOI Listing
November 2020

Functional characterization of a novel thermophilic exo-arabinanase from Thermothielavioides terrestris.

Appl Microbiol Biotechnol 2020 Oct 19;104(19):8309-8326. Epub 2020 Aug 19.

Department of Biotechnology, Lorena School of Engineering, University of São Paulo, Lorena, SP, Brazil.

Arabinanases from glycoside hydrolase family GH93 are enzymes with exo-activity that hydrolyze the α-1,5 bonds between arabinose residues present on arabinan. Currently, several initiatives aiming to use byproducts rich in arabinan such as pectin and sugar beet pulp as raw material to produce various compounds of interest are being developed. However, it is necessary to use robust enzymes that have an optimal performance under pH and temperature conditions used in the industrial processes. In this work, the first GH93 from the thermophilic fungus Thermothielavioides terrestris (Abn93T) was heterologously expressed in Aspergillus nidulans, purified and biochemically characterized. The enzyme is a thermophilic glycoprotein (optimum activity at 70 °C) with prolonged stability in acid pHs (4.0 to 6.5). The presence of glycosylation affected slightly the hydrolytic capacity of the enzyme, which was further increased by 34% in the presence of 1 mM CoCl. Small-angle X-ray scattering results show that Abn93T is a globular-like-shaped protein with a slight bulge at one end. The hydrolytic mechanism of the enzyme was elucidated using capillary zone electrophoresis and molecular docking calculations. Abn93T has an ability to produce (in synergism with arabinofuranosidases) arabinose and arabinobiose from sugar beet arabinan, which can be explored as fermentable sugars and prebiotics. KEY POINTS: • Thermophilic exo-arabinanase from family GH93 • Molecular basis of arabinan depolymerization.
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http://dx.doi.org/10.1007/s00253-020-10806-6DOI Listing
October 2020

Low-resolution molecular shape, biochemical characterization and emulsification properties of a halotolerant esterase from Bacillus licheniformis.

Eur Biophys J 2020 Sep 18;49(6):435-447. Epub 2020 Jul 18.

São Carlos Institute of Physics, University of São Paulo, São Carlos, SP, Brazil.

Bacterial esterases are highly versatile enzymes, currently widely used in detergents, biosurfactants, bioemulsifiers and as biocatalysts in paper and food industries. Present work describes heterologous expression, purification, and biophysical and biochemical characterization of a halotolerant esterase from Bacillus licheniformis (BlEstA). BlEstA preferentially cleaves pNP-octanoate and both activity and stability of the enzyme increased in the presence of 2 M NaCl, and also with several organic solvents (ethanol, methanol and DMSO). Furthermore, BlEstA has considerable emulsifying properties, particularly with olive oil as substrate. Our studies also show that the enzyme is monomeric in solution and its small-angle X-ray scattering low-resolution molecular envelope fits well its high-resolution homology model.
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http://dx.doi.org/10.1007/s00249-020-01448-7DOI Listing
September 2020

Enzymatic versatility and thermostability of a new aryl-alcohol oxidase from Thermothelomyces thermophilus M77.

Biochim Biophys Acta Gen Subj 2020 10 10;1864(10):129681. Epub 2020 Jul 10.

São Carlos Institute of Physics, University of São Paulo, Av. Trabalhador São-carlense, 400, São Carlos, SP 13566-590, Brazil. Electronic address:

Background Fungal aryl-alcohol oxidases (AAOx) are extracellular flavoenzymes that belong to glucose-methanol-choline oxidoreductase family and are responsible for the selective conversion of primary aromatic alcohols into aldehydes and aromatic aldehydes to their corresponding acids, with concomitant production of hydrogen peroxide (HO) as by-product. The HO can be provided to lignin degradation pathway, a biotechnological property explored in biofuel production. In the thermophilic fungus Thermothelomyces thermophilus (formerly Myceliophthora thermophila), just one AAOx was identified in the exo-proteome. Methods The glycosylated and non-refolded crystal structure of an AAOx from T. thermophilus at 2.6 Å resolution was elucidated by X-ray crystallography combined with small-angle X-ray scattering (SAXS) studies. Moreover, biochemical analyses were carried out to shed light on enzyme substrate specificity and thermostability. Results This flavoenzyme harbors a flavin adenine dinucleotide as a cofactor and is able to oxidize aromatic substrates and 5-HMF. Our results also show that the enzyme has similar oxidation rates for bulky or simple aromatic substrates such as cinnamyl and veratryl alcohols. Moreover, the crystal structure of MtAAOx reveals an open active site, which might explain observed specificity of the enzyme. Conclusions MtAAOx shows previously undescribed structural differences such as a fully accessible catalytic tunnel, heavy glycosylation and Ca binding site providing evidences for thermostability and activity of the enzymes from AA3_2 subfamily. General significance Structural and biochemical analyses of MtAAOx could be important for comprehension of aryl-alcohol oxidases structure-function relationships and provide additional molecular tools to be used in future biotechnological applications.
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http://dx.doi.org/10.1016/j.bbagen.2020.129681DOI Listing
October 2020

A linker of the proline-threonine repeating motif sequence is bimodal.

J Mol Model 2020 Jun 19;26(7):178. Epub 2020 Jun 19.

Institute of Chemistry, University of Campinas - UNICAMP, P.O. Box 6154, Campinas, SP, 130820864, Brazil.

The linker of the endoglucanase from Xanthomonas campestris pv. campestris ((PT)) has a specific sequence, a repeating proline-threonine motif. In order to understand its role, it has been compared to a regular sequence linker, in this work-the cellobiohydrolase 2 from Trichoderma reesei (CBH2). Elastic properties of the two linkers have been estimated by calculating free energy profile along the linker length from an enhanced sampling molecular dynamics simulation. The (PT) exhibits more pronounced elastic behaviour than CBH2. The PT repeating motif results in a two-mode energy profile which could be very useful in the enzyme motions along the substrate during hydrolytic catalysis.
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http://dx.doi.org/10.1007/s00894-020-04434-0DOI Listing
June 2020

Essential Metabolic Routes as a Way to ESKAPE From Antibiotic Resistance.

Front Public Health 2020 28;8:26. Epub 2020 Feb 28.

São Carlos Institute of Physics, University of São Paulo, São Carlos, Brazil.

Antibiotic resistance is a worldwide concern that requires a concerted action from physicians, patients, governmental agencies, and academia to prevent infections and the spread of resistance, track resistant bacteria, improve the use of current antibiotics, and develop new antibiotics. Despite the efforts spent so far, the current antibiotics in the market are restricted to only five general targets/pathways highlighting the need for basic research focusing on the discovery and evaluation of new potential targets. Here we interrogate two biosynthetic pathways as potentially druggable pathways in bacteria. The biosynthesis pathway for thiamine (vitamin B1), absent in humans, but found in many bacteria, including organisms in the group of the ESKAPE pathogens (, and sp.) and the biosynthesis pathway for pyridoxal 5'-phosphate and its vitamers (vitamin B6), found in . Using current genomic data, we discuss the possibilities of inhibition of enzymes in the pathway and review the current state of the art in the scientific literature.
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http://dx.doi.org/10.3389/fpubh.2020.00026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7093009PMC
May 2021

Evaluating the potential area and environmental key factors for Sargassum bed restoration in highly turbid waters of the northwestern Arabian Gulf.

Mar Pollut Bull 2020 Apr 12;153:110949. Epub 2020 Feb 12.

Environment and Life Sciences Research Center, Kuwait Institute for Scientific Research, Salmiya 22017, Kuwait.

This study evaluates the potential area and the key environmental factors supporting Sargassum bed restoration (SBR) in the highly turbid northwestern Arabian Gulf where rapid coastal development impinges on the marine ecosystem functioning. Water depth was a primary environmental factor governing the distribution of the subtidal macroalgae beds in these turbid waters. The relationship between Sargassum coverage and water depth measured by an echo sounder indicated optimal water depths where the maximum coverage was observed. The availability of stable hard substrate was another key factor introducing heterogeneity in Sargassum coverage. Potential area for the SBR estimated based on the optimal depths was 4.26 km, whereas only 50% of the potential area (2.19 km) was currently vegetated due to the absence of hard substrate. The outcomes of this study offer beneficial information toward implementation of the SBR as a part of mitigation measures in future coastal development plans in the region.
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http://dx.doi.org/10.1016/j.marpolbul.2020.110949DOI Listing
April 2020

Structural insights into the hydrolysis pattern and molecular dynamics simulations of GH45 subfamily a endoglucanase from Neurospora crassa OR74A.

Biochimie 2019 Oct 28;165:275-284. Epub 2019 Aug 28.

São Carlos Institute of Physics, University of São Paulo, São Carlos, São Paulo, Brazil. Electronic address:

Glycoside hydrolase (GH) family 45 is one of the smallest and poorly studied endoglucanase family with a broad biotechnological application ranging from treatment of textiles to conversion of complex cell wall polysaccharides into simple oligo- and monosaccharides. In a present study, GH45 cellulase from Neurospora crassa OR74A (NcCel45A) was characterized both biochemically and structurally. HPLC analysis of the hydrolytic products confirmed the endo-β(1,4) mode of action of the enzyme. Moreover, such pattern revealed that NcCel45A cannot hydrolyze efficiently oligosaccharides with a degree of polymerization smaller than six. The crystal structure of NcCel45A catalytic domain in the apo-form was determined at 1.9 Å resolution and the structure of the enzyme bound to cellobiose was solved and refined to 1.8 Å resolution. Comparative structural analyses and molecular dynamics simulations show that the enzyme dynamics is affected by substrate binding. Taken together, MD simulations and statistical coupling analysis revealed previously unknown correlation of a loop 6 with the breakdown of cellulose substrates by GH45.
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http://dx.doi.org/10.1016/j.biochi.2019.08.016DOI Listing
October 2019

Biochemical characterization and low-resolution SAXS shape of a novel GH11 exo-1,4-β-xylanase identified in a microbial consortium.

Appl Microbiol Biotechnol 2019 Oct 12;103(19):8035-8049. Epub 2019 Aug 12.

Instituto de Física de São Carlos, Universidade de São Paulo, Avenida Trabalhador São-carlense 400, São Carlos, SP, 13566-590, Brazil.

Biotechnologies that aim to produce renewable fuels, chemicals, and bioproducts from residual ligno(hemi)cellulosic biomass mostly rely on enzymatic depolymerization of plant cell walls (PCW). This process requires an arsenal of diverse enzymes, including xylanases, which synergistically act on the hemicellulose, reducing the long and complex xylan chains to oligomers and simple sugars. Thus, xylanases play a crucial role in PCW depolymerization. Until recently, the largest xylanase family, glycoside hydrolase family 11 (GH11) has been exclusively represented by endo-catalytic β-1,4- and β-1,3-xylanases. Analysis of a metatranscriptome library from a microbial lignocellulose community resulted in the identification of an unusual exo-acting GH11 β-1,4-xylanase (MetXyn11). Detailed characterization has been performed on recombinant MetXyn11 including determination of its low-resolution small-angle X-ray scattering (SAXS) molecular envelope in solution. Our results reveal that MetXyn11 is a monomeric globular enzyme that liberates xylobiose from heteroxylans as the only product. MetXyn11 has an optimal activity in a pH range from 6 to 9 and an optimal temperature of 50 °C. The enzyme maintained above 65% of its original activity in the pH range 5 to 6 after being incubated for 72 h at 50 °C. Addition of the enzyme to a commercial enzymatic cocktail (CelicCtec3) promoted a significant increase of enzymatic hydrolysis yields of hydrothermally pretreated sugarcane bagasse (16% after 24 h of hydrolysis).
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http://dx.doi.org/10.1007/s00253-019-10033-8DOI Listing
October 2019

A novel thermostable GH5 β-xylosidase from Thermogemmatispora sp. T81.

N Biotechnol 2019 Nov 9;53:57-64. Epub 2019 Jul 9.

Sao Carlos Institute of Physics, University of Sao Paulo, São Carlos, São Paulo, Brazil. Electronic address:

A glycoside hydrolase family 5 (GH5) subfamily 22 gene, designated T81Xyl5_22A, was identified in the genome of the aerobic thermophilic bacterium, Thermogemmatispora sp. T81 (locus A4R35_07040). The gene was cloned and heterologously expressed in Escherichia coli and the gene product characterized biochemically. The recombinant enzyme had an optimal catalytic activity at pH5.0 and 65 °C, and was active against beechwood xylan and rye arabinoxylan. It yielded only xylose molecules as products of beechwood xylan hydrolysis, indicating that it is a GH5 family β-d-xylosidase. Using 4-nitrophenyl β-d-xylopyranoside (pNPX) as a substrate, the K, Vmax, k and k/K kinetic parameters were determined as 0.25 ± 0.03 mM, 889.47 ± 28.54 U/mg, 39.20 s and 156.8 mM s, respectively. Small-angle X-ray scattering (SAXS) data enabled reconstruction of the enzyme's low-resolution molecular envelope and revealed that it formed dimers in solution. As far as we are aware, this is the first description of a thermostable bacterial GH5 family β-d-xylosidase.
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http://dx.doi.org/10.1016/j.nbt.2019.07.002DOI Listing
November 2019

The structure of the extended E2 DNA-binding domain of the bovine papillomavirus-1.

Proteins 2020 01 1;88(1):106-112. Epub 2019 Aug 1.

Laboratório de Cristalografia, Physics Department, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.

Bovine papillomavirus proteins were extensively studied as a prototype for the human papillomavirus. Here, the crystal structure of the extended E2 DNA-binding domain of the dominant transcription regulator from the bovine papillomavirus strain 1 is described in the space group P3 21. We found two protein functional dimers packed in the asymmetric unit. This new protein arrangement inside the crystal led to the reduction of the mobility of a previously unobserved loop directly involved in the protein-DNA interaction, which was then modeled for the first time.
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http://dx.doi.org/10.1002/prot.25773DOI Listing
January 2020

Crystallographic structure and molecular dynamics simulations of the major endoglucanase from Xanthomonas campestris pv. campestris shed light on its oligosaccharide products release pattern.

Int J Biol Macromol 2019 Sep 16;136:493-502. Epub 2019 Jun 16.

Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil. Electronic address:

Cellulases are essential enzymatic components for the transformation of plant biomass into fuels, renewable materials and green chemicals. Here, we determined the crystal structure, pattern of hydrolysis products release, and conducted molecular dynamics simulations of the major endoglucanase from the Xanthomonas campestris pv. campestris (XccCel5A). XccCel5A has a TIM barrel fold with the catalytic site centrally placed in a binding groove surrounded by aromatic side chains. Molecular dynamics simulations show that productive position of the substrate is secured by a network of hydrogen bonds in the four main subsites, which differ in details from homologous structures. Capillary zone electrophoresis and computational studies reveal XccCel5A can act both as endoglucanase and licheninase, but there are preferable arrangements of substrate regarding β-1,3 and β-1,4 bonds within the binding cleft which are related to the enzymatic efficiency.
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http://dx.doi.org/10.1016/j.ijbiomac.2019.06.107DOI Listing
September 2019

Structure and dynamics of Trichoderma harzianum Cel7B suggest molecular architecture adaptations required for a wide spectrum of activities on plant cell wall polysaccharides.

Biochim Biophys Acta Gen Subj 2019 06 19;1863(6):1015-1026. Epub 2019 Mar 19.

Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador Sãocarlense, 400, São Carlos SP 13566-590, Brazil. Electronic address:

Cellulases from glycoside hydrolase family 7 (GH7) play crucial roles in plant lignocellulose deconstruction by fungi, but structural information available for GH7 fungal endoglucanases is limited when compared to the number of known sequences in the family. Here, we report the X-ray structure of the glycosylated catalytic domain (CD) of Trichoderma harzianum endoglucanase, ThCel7B, solved and refined at 2.9 Å resolution. Additionally, our extensive molecular dynamics simulations of this enzyme in complex with a variety of oligosaccharides provide a better understanding of its promiscuous hydrolytic activities on plant cell wall polysaccharides. The simulations demonstrate the importance of the hydrogen bond between substrate O2 hydroxyl in the subsite -1 and a side chain of catalytic Glu196 which renders ThCel7B capable to catalytically cleave cello and xylooligosaccharides, but not mannooligosaccharides. Moreover, detailed structural analyses and MD simulations revealed an additional binding pocket, suitable for accommodation of oligosaccharide decorations and/or substrates with mixed glycoside bonds that abuts onto the binding cleft close to subsite +2.
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http://dx.doi.org/10.1016/j.bbagen.2019.03.013DOI Listing
June 2019
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