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Rational design, construction and characterization of a bifunctional enzyme (laccase-cellulase) for biomass conversion to biofuel

Grant number: 07/01623-0
Support type:Scholarships in Brazil - Master
Effective date (Start): September 01, 2007
Effective date (End): August 31, 2009
Field of knowledge:Biological Sciences - Biochemistry - Chemistry of Macromolecules
Principal researcher:Richard John Ward
Grantee:Gilvan Pessoa Furtado
Home Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil


The application of enzymes in industrial processos has growm considerably in recent decades. The benefits of the use of enzymes include the elimination of the use of high temperatures, organic solvents and extreemes of pH, whilst simultaneously increasing the specificity of the reactions and purifty of the product.The use of ethanol as a biofuel has attracted wide attention, and enzymes may be applied to the conversion of abundant biomass, such as lignocellulose, into fermentable sugars for the production of biofuels. In this process, two enzymes have been identified as candidates fro further development, cellulases which hydrolyse celulose to produce reducing sugars, and laccases which oxidize phenolic compounds present in lignin that may inhibit the metabolism of microorganisms used in the subsequent fermenation steps.The current project has the ojective to create a novel bifunctional cellulase-laccase enzyme by the fusion of the Beg1 and CotA genes from Bacillus subtilis. A model of the bifunctional enzyme has been created by rational design techniques, and a strategy for teh construction of the hybrid enzyme has been elaborated using the technique of overlap PCR. The hybrid gene will be cloned and expressed in E. coli. After purifaction of the hybrid enzyme, its activity will be evaluated and cinetic parameters such as optimuum pH, Vmax and Km will be determined.

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Scientific publications
(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, . (07/01623-0, 10/18850-2)
FURTADO, GILVAN PESSOA; RIBEIRO, LUCAS FERREIRA; SANTOS, CAMILA RAMOS; TONOLI, CELISA CALDANA; DE SOUZA, ANGELICA RODRIGUES; OLIVEIRA, RENATA ROCHA; MURAKAMI, MARIO TYAGO; WARD, RICHARD JOHN. Biochemical and structural characterization of a beta-1,3-1,4-glucanase from Bacillus subtilis 168. Process Biochemistry, v. 46, n. 5, p. 1202-1206, . (07/01615-8, 10/51890-8, 08/57908-6, 07/01623-0)
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, . (08/05181-5, 07/01623-0, 10/18850-2, 10/07133-8)

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