Rational design of a novel bifunctional laccase-xylanase enzyme from Bacillus subtilis for potential application in paper and cellulose industry

• Grant number: 07/01615-8
• Support Opportunities: Scholarships in Brazil - Master
• Effective date (Start): February 01, 2008
• Effective date (End): August 31, 2009
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Richard John Ward
• Grantee: Lucas Ferreira Ribeiro
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil

Abstract

Conventional cellulose pulp bleaching processes use chemical reagents that create a significant environmental impact, which has gained the paper and celulose industry the reputation of being a major source of fluvial and groundwater pollution. In the present project, we propose to create a novel bifunctional enzyme combining xylanase (EC 3.2.1.8) and laccase (EC 1.10.3.2) activities in a single polypeptide. These two catalytic properties act against the principal components of wood fibres (xylan and lignin respectively) that complicate the bleaching process of the Kraft pulp. The use of enzymes in the pre-treatment of the pulp can reduce the quantity of chemicals that are necessary in the overall bleaching process.A chimeric gene which encodes the laccase from Bacilus subtilis (CotA) and the xylanase A (XylA) from the same organism will be fused using the technique of overlap PCR in sucgha manner that the xylanase coding sequence is inserted within the laccase coding region. With this construct we aim to avoid potential interferance between the active sites of the fused enzymes, to create separate and functional domains, and to maintain the stability of the final protein. The two genes will initially be cloned and joined using the pGEM-T vector, after which the bifunctional enzyme will be expressed in E. coli using the pET28a(+) vector. After affinity purification, the catalytic activities of the bifunctional enzyme will be evaluated, and the utility of the new enzyme will be assessed in the bleaching of Kraft pulp on the laboratory scale.

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, 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, MAY 2011. (07/01615-8, 07/01623-0, 08/57908-6, 10/51890-8)