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Structure and function of enzymes and auxiliary proteins from Trichoderma, active in cell-wall hydrolysis

Grant number: 08/56255-9
Support Opportunities:Program for Research on Bioenergy (BIOEN) - Thematic Grants
Duration: November 01, 2009 - September 30, 2014
Field of knowledge:Biological Sciences - Biophysics - Molecular Biophysics
Convênio/Acordo: CNPq - Pronex
Principal Investigator:Igor Polikarpov
Grantee:Igor Polikarpov
Host Institution: Instituto de Física de São Carlos (IFSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil
Pesquisadores principais:
Munir Salomao Skaf ; Valtencir Zucolotto
Associated grant(s):10/18849-4 - Purification and characterization of glycoside hydrolases produced by the filamentous fungus Trichoderma harzianum, with perspective of applicability of these enzymes in cocktails for biomass hydrolysis and bioethanol production, AV.EXT
Associated scholarship(s):12/08402-8 - Expression, purification, cristalization and functional assays of the enzime beta-galactosidase from the arqueobacteria haloterrigena turkmenica, BP.IC
11/19100-0 - Functional and structural studies of enzyme class endoglucanases GHF45 aiming for application in biomass hydrolysis, BP.DD
11/20505-4 - Two important classes of glycosyl hydrolases: functional studies and structural analysis, BP.PD
+ associated scholarships 11/05712-3 - Structural and functional studies of beta-galactosidases from Xanthomonas campestris, BP.DD
10/20681-4 - Design and development of an enzyme reactor for production of ethanol from sugarcane bagasse-cane, BP.PD
10/18773-8 - Molecular cloning, expression, purification and structural characterization of endoglucanase from Trichoderma harzianum aiming the development of enzymatic blends for production of lignocellulosic ethanol, BP.DD
10/16542-9 - Studies of the cellulolytic enzymes with potential in the enzymatic transformation of the sugarcane biomass using Small Angle X-ray Scattering, BP.PD
10/11135-6 - Structural and morphological aspects of the cell-wall degradation and its biopolymers, BP.PD
10/08680-2 - Molecular aspectos of lignocellulosic biomass degradation: dynamics of enzymes and plant cell wall nanoarchitecture, BP.DR
09/13185-3 - Genomic structure analysis of regions including genes coding cellulolytic activity enzymes from Trichoderma harzianum, BP.MS
09/11536-3 - Biochemical and structural studies of the cellulolytic system of Trichoderma reesei and Phanerochaete chrysosporium, applied to improve the efficiency biomass degradation, BP.PD
09/14107-6 - Molecular dynamics simulations of hydrolases for saccharification of cellulose and related proteins, BP.DR
09/08233-9 - Molecular, structural and functional studies of the Cel12A from Gloeophyllum trabeum, an endo-1,4-²-glucanase from the family 12 of glycosyde hidrolases, BP.DD - associated scholarships

Abstract

Lignocellulosic biomass, such as sugarcane bagasse, holds a promise of environmentally friendly bioenergy production in Brazil. However, enzymatic hydrolysis, currently considered a method of choice in biomass saccharification, is hampered by considerable cell-wall recalcitrance. To make this technology sustainable and cost effective, our comprehension of cellulose enzymatic hydrolysis should be significantly improved. Here we propose to conduct systematic structure-functional studies of Trichoderma cellulases and auxiliary proteins active in cell-wall degradation using a combination of X-ray protein crystallography, biophysical and biochemical studies, molecular dynamics simulations, statistical coupling analysis aligned with the site-directed mutagenesis and enzymatic assays aiming to obtain in-depth comprehension of cellulose hydrolysis. We plan to contribute toward structural analysis of Trichoderma reesei endoglucanases by solving a crystal structure of endoglucanase II (Cel5A), main enzymatically active, but structurally uncharacterized endoglucanase of this important industrial fungus. Moreover, we will contribute toward our knowledge of Trichoderma cellulases molecular organization by solving X-ray structures of main Trichoderma harzianum endo- and exoglucanases (primarily focusing on Cel7A and Cel5A) and by comparing them with the correspondent T. reesei enzymes. We also aim to structurally characterize swollenins, non-hydrolytic proteins, shown to enhance cellulose hydrolysis catalyzed by celulases, and to study thermodynamically its interactions with cellulose. In addition, we will construct chimeric enzymes by fusing of swollenin with the cellulases and will study enzymatic properties of such chimeras. Furthermore, we will conduct systematic molecular dynamics studies of the cellulases and swollenin, and investigate their flexibility by hydrogen deuterium exchange followed by massspectrometry. Finally, we will use all these acquired knowledge to modify the proteins using site-directed mutagenesis aiming to better comprehend molecular basis of their function and to produce enzymes and their mixtures with enhanced hydrolytic properties. (AU)

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Scientific publications (37)
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
LIMA, MARISA A.; LAVORENTE, GABRIELA B.; DA SILVA, HANA K. P.; BRAGATTO, JULIANO; REZENDE, CAMILA A.; BERNARDINELLI, OIGRES D.; DEAZEVEDO, EDUARDO R.; GOMEZ, LEONARDO D.; MCQUEEN-MASON, SIMON J.; LABATE, CARLOS A.; et al. Effects of pretreatment on morphology, chemical composition and enzymatic digestibility of eucalyptus bark: a potentially valuable source of fermentable sugars for biofuel production - part 1. BIOTECHNOLOGY FOR BIOFUELS, v. 6, . (10/11135-6, 08/56255-9, 10/08370-3, 10/52362-5, 09/18354-8)
FLORINDO, RENATA N.; SOUZA, VALQUIRIA P.; MANZINE, LIVIA R.; CAMILO, CESAR M.; MARANA, SANDRO R.; POLIKARPOV, IGOR; NASCIMENTO, ALESSANDRO S.. Structural and biochemical characterization of a GH3 beta-glucosidase from the probiotic bacteria Bifidobacterium adolescentis. Biochimie, v. 148, p. 107-115, . (14/19439-5, 08/56255-9, 14/06565-2)
GODOY, ANDRE S.; DE LIMA, MARIANA Z. T.; CAMILO, CESAR M.; POLIKARPOV, IGOR. Crystal structure of a putative exo-beta-1,3-galactanase from Bifidobacterium bifidum S17. ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS, v. 72, n. 4, p. 288-293, . (09/52840-7, 11/20505-4, 08/56255-9, 09/05328-9, 11/05712-3)
NASCIMENTO, ALESSANDRO S.; MUNIZ, JOAO RENATO C.; APARICIO, RICARDO; GOLUBEV, ALEXANDER M.; POLIKARPOV, IGOR. Insights into the structure and function of fungal beta-mannosidases from glycoside hydrolase family 2 based on multiple crystal structures of the Trichoderma harzianum enzyme. FEBS Journal, v. 281, n. 18, SI, p. 4165-4178, . (08/56255-9, 09/52840-7)
MELLO, BRUNO LUAN; POLIKARPOV, IGOR. Family 1 carbohydrate binding-modules enhance saccharification rates. AMB EXPRESS, v. 4, . (08/56255-9, 10/52362-5, 09/05349-6)
PELLEGRINI, VANESSA O. A.; SERPA, VIVIANE ISABEL; GODOY, ANDRE S.; CAMILO, CESAR M.; BERNARDES, AMANDA; REZENDE, CAMILA A.; PEREIRA JUNIOR, NEI; CAIRO, JOAO PAULO L. FRANCO; SQUINA, FABIO M.; POLIKARPOV, IGOR. Recombinant Trichoderma harzianum endoglucanase I (Cel7B) is a highly acidic and promiscuous carbohydrate-active enzyme. Applied Microbiology and Biotechnology, v. 99, n. 22, p. 9591-9604, . (09/52840-7, 10/18773-8, 08/56255-9, 11/20977-3, 09/05328-9, 11/05712-3)
CAMILO, CESAR M.; POLIKARPOV, IGOR. High-throughput cloning, expression and purification of glycoside hydrolases using Ligation-Independent Cloning (LIC). Protein Expression and Purification, v. 99, p. 35-42, . (10/08680-2, 09/54035-4, 08/56255-9, 09/11536-3)
LIMA, MARISA A.; GOMEZ, LEONARDO D.; STEELE-KING, CLARE G.; SIMISTER, RACHAEL; BERNARDINELLI, OIGRES D.; CARVALHO, MARCELO A.; REZENDE, CAMILA A.; LABATE, CARLOS A.; DEAZEVEDO, EDUARDO R.; MCQUEEN-MASON, SIMON J.; et al. Evaluating the composition and processing potential of novel sources of Brazilian biomass for sustainable biorenewables production. BIOTECHNOLOGY FOR BIOFUELS, v. 7, . (10/11135-6, 08/56255-9, 10/08370-3, 10/52362-5, 09/18354-8)
DOS REIS, CAIO VINICIUS; BERNARDES, AMANDA; POLIKARPOV, IGOR. Expression, purification, crystallization and preliminary X-ray diffraction analysis of Bifidobacterium adolescentis xylose isomerase. ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS, v. 69, n. 5, p. 588-591, . (08/56255-9, 09/52840-7)
BROGNARO, HEVILA; ALMEIDA, VITOR MEDEIROS; DE ARAUJO, EVANDRO ARES; PIYADOV, VASILY; MORIM SANTOS, MARIA AUXILIADORA; MARANA, SANDRO ROBERTO; POLIKARPOV, IGOR. Biochemical Characterization and Low-Resolution SAXS Molecular Envelope of GH1 beta-Glycosidase from Saccharophagus degradans. MOLECULAR BIOTECHNOLOGY, v. 58, n. 12, p. 777-788, . (08/56255-9)
SKAF, MUNIR SALOMAO; POLIKARPOV, IGOR; STANKOVIC, IVANA M.. A linker of the proline-threonine repeating motif sequence is bimodal. Journal of Molecular Modeling, v. 26, n. 7, . (08/56255-9, 10/08680-2, 09/54035-4)
GODOY, ANDRE S.; DE LIMA, MARIANA Z. T.; CAMILO, CESAR M.; POLIKARPOV, IGOR. Crystal structure of a putative exo-beta-1,3-galactanase from Bifidobacterium bifidum S17. ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS, v. 72, p. 6-pg., . (11/05712-3, 09/52840-7, 11/20505-4, 08/56255-9, 09/05328-9)
TEXTOR, LARISSA C.; SANTOS, JADEMILSON C.; HIDALGO CUADRADO, NAZARET; ROIG, MANUEL G.; ZHADAN, GALINA G.; SHNYROV, VALERY L.; POLIKARPOV, IGOR. Purification, crystallization and preliminary crystallographic analysis of peroxidase from the palm tree Chamaerops excelsa. ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS, v. 67, p. 4-pg., . (09/52840-7, 08/56255-9, 09/14333-6)
LIBERATO, MARCELO VIZONA; GENEROSO, WESLEY CARDOSO; MALAGO, WILSON, JR.; HENRIQUE-SILVA, FLAVIO; POLIKARPOV, IGOR. Crystallization and preliminary X-ray diffraction analysis of endoglucanase III from Trichoderma harzianum. ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS, v. 68, p. 4-pg., . (09/52840-7, 08/56255-9, 09/14333-6)
ROSSETO, FLAVIO R.; PUHL, ANA C.; ANDRADE, MAXUEL O.; POLIKARPOV, IGOR. Crystallization and preliminary diffraction analysis of the catalytic domain of major extracellular endoglucanase from Xanthomonas campestris pv. campestris. ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS, v. 69, p. 4-pg., . (10/52362-5, 09/05349-6, 08/56255-9, 07/08706-9)
DOS REIS, CAIO VINICIUS; BERNARDES, AMANDA; POLIKARPOV, IGOR. Expression, purification, crystallization and preliminary X-ray diffraction analysis of Bifidobacterium adolescentis xylose isomerase. ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS, v. 69, p. 4-pg., . (09/52840-7, 08/56255-9)
BERNARDINELLI, OIGRES DANIEL; LIMA, MARISA APARECIDA; REZENDE, CAMILA ALVES; POLIKARPOV, IGOR; DEAZEVEDO, EDUARDO RIBEIRO. Quantitative C-13 MultiCP solid-state NMR as a tool for evaluation of cellulose crystallinity index measured directly inside sugarcane biomass. BIOTECHNOLOGY FOR BIOFUELS, v. 8, . (10/11135-6, 08/56255-9, 10/08370-3, 10/52362-5, 09/18354-8)
DOS SANTOS, JADEMILSON CELESTINO; BERNARDES, AMANDA; GIAMPIETRO, LETIZIA; AMMAZZALORSO, ALESSANDRA; DE FILIPPIS, BARBARA; AMOROSO, ROSA; POLIKARPOV, IGOR. Different binding and recognition modes of GL479, a dual agonist of Peroxisome Proliferator-Activated Receptor alpha/gamma. Journal of Structural Biology, v. 191, n. 3, p. 332-340, . (09/14333-6, 08/56255-9)
FLORINDO, RENATA N.; SOUZA, VALQUIRIA P.; MUTTI, HEMILY S.; CAMILO, CESAR; MANZINE, LIVIA REGINA; MARANA, SANDRO R.; POLIKARPOV, IGOR; NASCIMENTO, ALESSANDRO S.. Structural insights into beta-glucosidase transglycosylation based on biochemical, structural and computational analysis of two GH1 enzymes from Trichoderma harzianum. NEW BIOTECHNOLOGY, v. 40, n. B, p. 218-227, . (14/19439-5, 08/56255-9, 14/06565-2)
LIMA, MARISA A.; OLIVEIRA-NETO, MARIO; KADOWAKI, MARCO ANTONIO S.; ROSSETO, FLAVIO R.; PRATES, ERICA T.; SQUINA, FABIO M.; LEME, ADRIANA F. P.; SKAF, MUNIR S.; POLIKARPOV, IGOR. Aspergillus niger beta-Glucosidase Has a Cellulase-like Tadpole Molecular Shape INSIGHTS INTO GLYCOSIDE HYDROLASE FAMILY 3 (GH3) beta-GLUCOSIDASE STRUCTURE AND FUNCTION. Journal of Biological Chemistry, v. 288, n. 46, p. 32991-33005, . (09/54035-4, 10/16542-9, 08/56255-9)
ROSSETO, FLAVIO R.; PUHL, ANA C.; ANDRADE, MAXUEL O.; POLIKARPOV, IGOR. Crystallization and preliminary diffraction analysis of the catalytic domain of major extracellular endoglucanase from Xanthomonas campestris pv. campestris. ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS, v. 69, n. 2, p. 137-140, . (08/56255-9, 10/52362-5, 07/08706-9, 09/05349-6)
DE ARAUJO, EVANDRO ARES; TOMAZINI, JR., ATILIO; SEIKI KADOWAKI, MARCO ANTONIO; MURAKAMI, MARIO TYAGO; POLIKARPOV, IGOR. Crystallization and preliminary X-ray diffraction analysis of a new xyloglucanase from Xanthomonas campestris pv. campestris. ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS, v. 69, n. 6, p. 676-678, . (09/05349-6, 10/52362-5, 07/08706-9, 08/56255-9)
TOMAZINI JUNIOR, ATILIO; DOLCE, LUCIANO GRACIANI; DE OLIVEIRA NETO, MARIO; POLIKARPOV, IGOR. Xanthomonas campestris expansin-like X domain is a structurally disordered beta-sheet macromolecule capable of synergistically enhancing enzymatic efficiency of cellulose hydrolysis. Biotechnology Letters, v. 37, n. 12, p. 2419-2426, . (08/56255-9, 10/52362-5, 07/08706-9, 09/05349-6)
PRATES, ERICA T.; STANKOVIC, IVANA; SILVEIRA, RODRIGO L.; LIBERATO, MARCELO V.; HENRIQUE-SILVA, FLAVIO; PEREIRA, JR., NEI; POLIKARPOV, IGOR; SKAF, MUNIR S.. X-ray Structure and Molecular Dynamics Simulations of Endoglucanase 3 from Trichoderma harzianum: Structural Organization and Substrate Recognition by Endoglucanases That Lack Cellulose Binding Module. PLoS One, v. 8, n. 3, . (09/54035-4, 08/56255-9, 10/08680-2)
LIMA, LEONARDO H. F.; SERPA, VIVIANE I.; ROSSETO, FLAVIO R.; SARTORI, GERALDO RODRIGUES; DE OLIVEIRA NETO, MARIO; MARTINEZ, LEANDRO; POLIKARPOV, IGOR. Small-angle X-ray scattering and structural modeling of full-length: cellobiohydrolase I from Trichoderma harzianum. Cellulose, v. 20, n. 4, p. 1573-1585, . (09/52840-7, 07/08706-9, 08/56255-9, 08/05637-9, 10/16947-9)
ROSSETO, FLAVIO RODOLFO; MANZINE, LIVIA REGINA; NETO, MARIO DE OLIVEIRA; POLIKARPOV, IGOR. Biophysical and biochemical studies of a major endoglucanase secreted by Xanthomonas campestris pv. campestris.. Enzyme and Microbial Technology, v. 91, p. 1-7, . (10/08370-3, 09/54035-4, 10/16542-9, 08/56255-9)
GODOY, ANDRE SCHUTZER; CAMILO, CESAR MOISES; KADOWAKI, MARCO ANTONIO; MUNIZ, HELOISA DOS S.; SANTO, MELISSA ESPIRITO; MURAKAMI, MARIO TYAGO; NASCIMENTO, ALESSANDRO S.; POLIKARPOV, IGOR. Crystal structure of beta 1 -> 6-galactosidase from Bifidobacterium bifidum S17: trimeric architecture, molecular determinants of the enzymatic activity and its inhibition by alpha-galactose. FEBS Journal, v. 283, n. 22, p. 4097-4112, . (09/52840-7, 11/20505-4, 08/56255-9, 09/05328-9, 14/06565-2, 11/05712-3)
LIBERATO, MARCELO V.; SILVEIRA, RODRIGO L.; PRATES, ERICA T.; DE ARAUJO, EVANDRO A.; PELLEGRINI, VANESSA O. A.; CAMILO, CESAR M.; KADOWAKI, MARCO A.; NETO, MARIO DE O.; POPOV, ALEXANDER; SKAF, MUNIR S.; et al. Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism. SCIENTIFIC REPORTS, v. 6, . (13/08293-7, 09/52840-7, 10/18773-8, 13/15582-5, 08/56255-9, 14/10448-1)
TSUCHIDA, JEFFERSON ESQUINA; REZENDE, CAMILA ALVES; DE OLIVEIRA-SILVA, RODRIGO; LIMA, MARISA APARECIDA; D'EURYDICE, MARCEL NOGUEIRA; POLIKARPOV, IGOR; BONAGAMBA, TITO JOSE. Nuclear magnetic resonance investigation of water accessibility in cellulose of pretreated sugarcane bagasse. BIOTECHNOLOGY FOR BIOFUELS, v. 7, . (09/54880-6, 10/11135-6, 08/56255-9, 12/22119-7, 10/08370-3, 09/18354-8)
MIOTTO, LIS SCHWARTZ; DE REZENDE, CAMILA ALVES; BERNARDES, AMANDA; SERPA, VIVIANE ISABEL; TSANG, ADRIAN; POLIKARPOV, IGOR. The Characterization of the Endoglucanase Cel12A from Gloeophyllum trabeum Reveals an Enzyme Highly Active on beta-Glucan. PLoS One, v. 9, n. 9, . (09/52840-7, 08/56255-9, 12/22802-9, 10/52362-5, 09/08233-9)
BERNARDES, AMANDA; TEXTOR, LARISSA C.; SANTOS, JADEMILSON C.; CUADRADO, NAZARET HIDALGO; KOSTETSKY, EDUARD YA.; ROIG, MANUEL G.; BAVRO, VASSILIY N.; MUNIZ, JOAO R. C.; SHNYROV, VALERY L.; POLIKARPOV, IGOR. Crystal structure analysis of peroxidase from the palm tree Chamaerops excelsa. Biochimie, v. 111, p. 58-69, . (12/22802-9, 10/52362-5, 08/56255-9, 09/05349-6)
GODOY, ANDRE SCHUTZER; CAMILO, CESAR MOISES; KADOWAKI, MARCO ANTONIO; MUNIZ, HELOISA DOS S.; SANTO, MELISSA ESPIRITO; MURAKAMI, MARIO TYAGO; NASCIMENTO, ALESSANDRO S.; POLIKARPOV, IGOR. Crystal structure of beta 1 -> 6-galactosidase from Bifidobacterium bifidum S17: trimeric architecture, molecular determinants of the enzymatic activity and its inhibition by alpha-galactose. FEBS Journal, v. 283, n. 22, p. 16-pg., . (11/05712-3, 09/52840-7, 11/20505-4, 14/06565-2, 08/56255-9, 09/05328-9)
FLORINDO, RENATA N.; SOUZA, VALQUIRIA P.; MUTTI, HEMILY S.; CAMILO, CESAR; MANZINE, LIVIA REGINA; MARANA, SANDRO R.; POLIKARPOV, IGOR; NASCIMENTO, ALESSANDRO S.. Structural insights into beta-glucosidase transglycosylation based on biochemical, structural and computational analysis of two GH1 enzymes from Trichoderma harzianum. NEW BIOTECHNOLOGY, v. 40, p. 10-pg., . (14/19439-5, 08/56255-9, 14/06565-2)
NASCIMENTO, ALESSANDRO S.; MUNIZ, JOAO RENATO C.; APARICIO, RICARDO; GOLUBEV, ALEXANDER M.; POLIKARPOV, IGOR. Insights into the structure and function of fungal beta-mannosidases from glycoside hydrolase family 2 based on multiple crystal structures of the Trichoderma harzianum enzyme. FEBS Journal, v. 281, n. 18, p. 14-pg., . (09/52840-7, 08/56255-9)
DE ARAUJO, EVANDRO ARES; TOMAZINI, ATILIO, JR.; SEIKI KADOWAKI, MARCO ANTONIO; MURAKAMI, MARIO TYAGO; POLIKARPOV, IGOR. Crystallization and preliminary X-ray diffraction analysis of a new xyloglucanase from Xanthomonas campestris pv. campestris. ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS, v. 69, p. 3-pg., . (09/05349-6, 10/52362-5, 08/56255-9, 07/08706-9)
COLUSSI, FRANCIELI; TEXTOR, LARISSA C.; SERPA, VIVIANE; MAEDA, ROBERTO NOBUYUKI; PEREIRA, NEI, JR.; POLIKARPOV, IGOR. Purification, crystallization and preliminary crystallographic analysis of the catalytic domain of the extracellular cellulase CBHI from Trichoderma harzianum. ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS, v. 66, p. 4-pg., . (09/05349-6, 08/56255-9, 07/08706-9)

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