Identification, characterization and engineering of plant cell wall degrading enzymes

• Grant number: 10/18850-2
• Support Opportunities: Research Projects - Thematic Grants
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Richard John Ward
• Grantee: Richard John Ward
• Host Institution: Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (FFCLRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Pesquisadores principais: Maria de Lourdes Teixeira de Moraes Polizeli
• Associated researchers: Arthur Henrique Cavalcante de Oliveira ; Luiz Alberto Beraldo de Moraes
• Associated research grant(s): 13/50892-5 - Development of strategies of immobilization and use of enzymes as biocatalysis for industrial applications: production of biodiesel and functional ingredients, AP.R
• Associated scholarship(s): 15/23200-0 - Production of enzymatic cocktails with potential in pulp biobleaching pulp for papermaking from lignocellulosic waste and secondary fibers, BP.MS; 15/18149-6 - Study of conformational and functional changes of the catalytic domain and of the CBM (Carbohydrate Binding Module) of a GH5 endoglucanase during catalysis, BP.PD; 14/07468-0 - Construction of a multifunctional enzyme feruloyl esterase/acetyl xylan esterase by rational design, BP.MS; + associated scholarships 14/06319-1 - Catalytic dynamics study of the endo-1,4-beta-xylanase from Bacillus subtilis by fluorescence correlation spectroscopy, BP.MS; 13/14454-3 - Bioprospection of pectinase supplementation aimed enzyme cocktails to the degradation of plant biomass, BP.PD; 12/24147-8 - Rational design of multifunctional enzymes for depolimerization of lignocelulosic material, BP.DR; 12/01066-2 - Design of multifunctional enzymes that combine the hydrolysis of B-glucans and Xylans to evaluate the conformation of these polysaccharides in plant cell walls, BP.PD; 11/23842-1 - Directed evolution and biochemical characterization of the 1,3-1,4-B-Glucanase from Bacillus subtilis, BP.IC; 11/00302-1 - Modulation of the selectivity of the xylanase A of Bacillus subtilis by directed evolution to optimize hydrolysis of the xylan from sugar-cane., BP.PD - associated scholarships

Abstract

Lignocellulose is composed of three principal plant cell wall polymers (cellulose, hemicellulose and lignin) in a closely knit compound matrix, which promotes stability and renders the component polysaccharides insoluble. Lignocellulose is therefore refractory to plant cell wall degrading enzymes that can be used in biorefinary applications, such as biobleaching and the production of fermentable sugars from plant cell-wall biomass. As a consequence of lignocellulose complexity, individual enzymes modify the constituent polymers inefficiently, and biorefinary applications will require mixtures of enzymes with different specificities. The current proposal aims to identify, characterize and engineer the catalytic properties of enzymes with the goal of developing a portfolio of biocatalysts that may be combined to form enzymatic cocktails for the biorefinary of lignocellulose. The overall strategy will focus on the characterization of enzymes identified in genomic sequencing projects of the fungi Aspergillus niveus, Aspergillus japonicus and Chaetomium thermophilum and the bacteria Bacillus subtilis and Clostridium thermocellum, using a battery of microbiological and biochemical techniques coupled with molecular structural biology approaches. This initial targeting and biochemical characterization effort will be undertaken in parallel with more sophisticated analyses which will identify in finer detail the exact enzymatic modifications of the plant cell wall polymers. These activities will provide information for a vigorous protein engineering program using rational protein design and directed evolution strategies which will aim to improve the catalytic properties of selected enzymes for biorefinary applications in the cellulose pulp and paper industries and in the development of biofuel technologies. (AU)

Scientific publications

(21)

(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)

FURTADO, G. P.; RIBEIRO, L. F.; LOURENZONI, M. R.; WARD, R. J.. A designed bifunctional laccase/beta-1,3-1,4-glucanase enzyme shows synergistic sugar release from milled sugarcane bagasse. PROTEIN ENGINEERING DESIGN & SELECTION, v. 26, n. 1, p. 15-23, JAN 2013. (10/18850-2, 07/01623-0)

FONSECA-MALDONADO, RAQUEL; VIEIRA, DAVI SERRADELLA; ALPONTI, JULIANA SANCHEZ; BONNEIL, ERIC; THIBAULT, PIERRE; WARD, RICHARD JOHN. Engineering the Pattern of Protein Glycosylation Modulates the Thermostability of a GH11 Xylanase. Journal of Biological Chemistry, v. 288, n. 35, p. 25522-25534, AUG 30 2013. (10/18850-2, 08/57908-6)

RIBEIRO, LUCAS FERREIRA; NICHOLES, NATHAN; TULLMAN, JENNIFER; COSTA RIBEIRO, LILIANE FRAGA; FUZO, CARLOS ALESSANDRO; VIEIRA, DAVI SERRADELLA; FURTADO, GILVAN PESSOA; OSTERMEIER, MARC; WARD, RICHARD JOHN. Insertion of a xylanase in xylose binding protein results in a xylose-stimulated xylanase. BIOTECHNOLOGY FOR BIOFUELS, v. 8, AUG 15 2015. (10/18850-2)

RIBEIRO, LILIANE F. C.; DE LUCAS, ROSYMAR C.; VITCOSQUE, GABRIELA L.; RIBEIRO, LUCAS F.; WARD, RICHARD J.; RUBIO, MARCELO V.; DAMASIO, ANDRE R. L.; SQUINA, FABIO M.; GREGORY, REBECCA C.; WALTON, PAUL H.; et al. A novel thermostable xylanase GH10 from Malbranchea pulchella expressed in Aspergillus nidulans with potential applications in biotechnology. BIOTECHNOLOGY FOR BIOFUELS, v. 7, JUL 29 2014. (10/18850-2, 12/12859-3, 13/18910-3, 10/07133-8, 10/10184-3, 12/02017-5, 08/57908-6)

SILVA, SERGIO BERGAMACHI; PINHEIRO, MATHEUS PINTO; FUZO, CARLOS ALESSANDRO; SILVA, SAMUEL REGHIM; FERREIRA, TATIANE LOPES; LOURENZONI, MARCOS ROBERTO; NONATO, M. CRISTINA; VIEIRA, DAVI SERRADELLA; WARD, RICHARD JOHN. The role of local residue environmental changes in thermostable mutants of the GH11 xylanase from Bacillus subtilis. International Journal of Biological Macromolecules, v. 97, p. 574-584, APR 2017. (10/18850-2, 12/01066-2)

FURTADO, GILVAN P.; SANTOS, CAMILA R.; CORDEIRO, ROSA L.; RIBEIRO, LUCAS F.; DE MORAES, LUIZ A. B.; DAMASIO, ANDR R. L.; POLIZELI, MARIA DE LOURDES T. M.; LOURENZONI, MARCOS R.; MURAKAMI, MARIO T.; WARD, RICHARD J.. Enhanced xyloglucan-specific endo-beta-1,4-glucanase efficiency in an engineered CBM44-XegA chimera. Applied Microbiology and Biotechnology, v. 99, n. 12, p. 5095-5107, JUN 2015. (10/18850-2, 12/20549-4, 13/18910-3, 13/13309-0, 14/07135-1, 10/07133-8)

FURTADO, GILVAN PESSOA; LOURENZONI, MARCOS ROBERTO; FUZO, CARLOS ALESSANDRO; FONSECA-MALDONADO, RAQUEL; GUAZZARONI, MARIA-EUGENIA; RIBEIRO, LUCAS FERREIRA; WARD, RICHARD J.. Engineering the affinity of a family 11 carbohydrate binding module to improve binding of branched over unbranched polysaccharides. International Journal of Biological Macromolecules, v. 120, p. 8-pg., 2018-12-01. (10/18850-2)

CARNEIRO, LARA A. B. C.; YU, LI; DUPREE, PAUL; WARD, RICHARD J.. Characterization of a beta-galactosidase from Bacillus subtilis with transgalactosylation activity. International Journal of Biological Macromolecules, v. 120, p. 9-pg., 2018-12-01. (12/24147-8, 10/18850-2)

CARNEIRO, LARA A. B. C.; WARD, RICHARD J.. Functionalization of paramagnetic nanoparticles for protein immobilization and purification. Analytical Biochemistry, v. 540, p. 45-51, JAN 1 2018. (10/18850-2, 16/15708-7, 12/24147-8)

MELEIRO, LUANA PARRAS; SANTOS SALGADO, JOSE CARLOS; MALDONADO, RAQUEL FONSECA; CARLI, SIBELI; BERALDO MORAES, LUIZ ALBERTO; WARD, RICHARD JOHN; JORGE, JOAO ATILIO; MELO FURRIEL, ROSA PRAZERES. Engineering the GH1 beta-glucosidase from Humicola insolens: Insights on the stimulation of activity by glucose and xylose. PLoS One, v. 12, n. 11, NOV 16 2017. (10/18850-2, 16/17582-0, 14/14415-0)

FURTADO, GILVAN PESSOA; LOURENZONI, MARCOS ROBERTO; FUZO, CARLOS ALESSANDRO; FONSECA-MALDONADO, RAQUEL; GUAZZARONI, MARIA-EUGENIA; RIBEIRO, LUCAS FERREIRA; WARD, RICHARD J.. Engineering the affinity of a family 11 carbohydrate binding module to improve binding of branched over unbranched polysaccharides. International Journal of Biological Macromolecules, v. 120, n. B, p. 2509-2516, DEC 2018. (10/18850-2)

CARNEIRO, LARA A. B. C.; YU, LI; DUPREE, PAUL; WARD, RICHARD J.. Characterization of a beta-galactosidase from Bacillus subtilis with transgalactosylation activity. International Journal of Biological Macromolecules, v. 120, n. A, p. 279-287, DEC 2018. (12/24147-8, 10/18850-2)

GUAZZARONI, MARIA-EUGENIA; SILVA-ROCHA, RAFAEL; WARD, RICHARD JOHN. Synthetic biology approaches to improve biocatalyst identification in metagenomic library screening. MICROBIAL BIOTECHNOLOGY, v. 8, n. 1, p. 52-64, JAN 2015. (13/04125-2, 10/18850-2)

ALPONTI, JULIANA SANCHEZ; MALDONADO, RAQUEL FONSECA; WARD, RICHARD J.. Thermostabilization of Bacillus subtilis GH11 xylanase by surface charge engineering. International Journal of Biological Macromolecules, v. 87, p. 522-528, JUN 2016. (10/18850-2)

RIBEIRO, LUCAS FERREIRA; TULLMAN, JENNIFER; NICHOLES, NATHAN; BERGAMACHI SILVA, SERGIO RUSCHI; VIEIRA, DAVI SERRADELLA; OSTERMEIER, MARC; WARD, RICHARD JOHN. A xylose-stimulated xylanase-xylose binding protein chimera created by random nonhomologous recombination. BIOTECHNOLOGY FOR BIOFUELS, v. 9, JUN 6 2016. (10/18850-2, 10/10184-3, 10/07133-8)

RULLER, ROBERTO; ALPONTI, JULIANA; DELIBERTO, LAILA APARECIDA; ZANPHORLIN, LETICIA MARIA; MACHADO, CARLA BOTELHO; WARD, RICHARD JOHN. Concommitant adaptation of a GH11 xylanase by directed evolution to create an alkali-tolerant/thermophilic enzyme. PROTEIN ENGINEERING DESIGN & SELECTION, v. 27, n. 8, p. 255-262, AUG 2014. (10/18850-2, 08/05181-5)

FONSECA-MALDONADO, RAQUEL; RIBEIRO, LUCAS F.; FURTADO, GILVAN P.; ARRUDA, LETICIA M.; MELEIRO, LUANA P.; ALPONTI, JULIANA S.; BOTELHO-MACHADO, CARLA; VIEIRA, DAVI S.; BONNEIL, ERIC; MELO FURRIEL, ROSA DOS PRAZERES; et al. Synergistic action of co-expressed xylanase/laccase mixtures against milled sugar cane bagasse. Process Biochemistry, v. 49, n. 7, p. 1152-1161, JUL 2014. (10/18850-2, 08/05181-5, 10/07133-8, 07/01623-0)

RIBEIRO, LUCAS F.; BRESSAN, FABIANA; FURTADO, GILVAN P.; MEIRELES, FLAVIO; WARD, RICHARD J.. D-Xylose detection in Escherichia coli by a xylose binding protein-dependent response. Journal of Biotechnology, v. 168, n. 4, p. 440-445, DEC 2013. (10/18850-2, 10/07133-8)

FONSECA-MALDONADO, RAQUEL; MELEIRO, LUANA P.; MENDES, LUIS F. S.; ALVES, LUANA F.; CARLI, SIBELI; MORERO, LUCAS D.; BASSO, LUIS G. M.; COSTA-FILHO, ANTONIO J.; WARD, RICHARD J.. Lignocellulose binding of a Cel5A-RtCBM11 chimera with enhanced beta-glucanase activity monitored by electron paramagnetic resonance. BIOTECHNOLOGY FOR BIOFUELS, v. 10, NOV 14 2017. (10/18850-2, 14/50884-5, 15/18149-6, 15/18390-5)

MACHADO, CARLA BOTELHO; CITADINI, ANA PAULA; GOLDBECK, ROSANA; DE LIMA, EVANDRO ANTONIO; FIGUEIREDO, FERNANDA LOPES; DA SILVA, TONY MARCIO; HOFFMAM, ZAIRA BRUNA; DE SOUSA, AMANDA SILVA; SQUINA, FABIO MARCIO; TEIXEIRA DE MORAES POLIZELI, MARIA DE LOURDES; et al. Increased biomass saccharification by supplementation of a commercial enzyme cocktail with endo-arabinanase from Bacillus licheniformis. Biotechnology Letters, v. 37, n. 7, p. 1455-1462, JUL 2015. (10/18850-2, 13/14454-3)

GUAZZARONI, MARIA-EUGENIA; SILVA-ROCHA, RAFAEL; WARD, RICHARD JOHN. Synthetic biology approaches to improve biocatalyst identification in metagenomic library screening. MICROBIAL BIOTECHNOLOGY, v. 8, n. 1, p. 13-pg., 2015-01-01. (13/04125-2, 10/18850-2)