A designed bifunctional laccase/beta-1,3-1,4-glucanase enzyme shows synergistic sugar release from milled sugarcane bagasse

Autor(es)

Furtado, G. P. ^[1] ; Ribeiro, L. F. ^{[1, 2]} ; Lourenzoni, M. R. ^{[3, 4]} ; Ward, R. J. ^{[3, 5]}

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Dept Bioquim & Imunol, FMRP USP, Sao Paulo - Brazil

^[2] Verdartis Desenvolvimento Biotecnol Ltda ME, Sao Paulo - Brazil

^[3] Univ Sao Paulo, Dept Quim, FFCLRP USP, Sao Paulo - Brazil

^[4] UNIDADE Ceara, Fundacao Oswaldo Cruz, Fortaleza, Ceara - Brazil

^[5] CTBE CNPEM, Lab Nacl Ciencia & Tecnol Bioetanol, Sao Paulo - Brazil

• Tipo de documento: Artigo Científico
• Fonte: PROTEIN ENGINEERING DESIGN & SELECTION; v. 26, n. 1, p. 15-23, JAN 2013.
• Citações Web of Science: 18

Resumo

A bifunctional enzyme has been created by fusing two Bacillus subtilis enzymes: the -1,31,4-glucanase (BglS, EC 3.2.1.73) that hydrolyzes plant cell wall -glucans and the copper-dependent oxidase laccase (CotA, EC 1.10.3.2) that catalyzes the oxidation of aromatic compounds with simultaneous reduction of oxygen to water. The chimeric laccase/-1,31,4-glucanase was created by insertion fusion of the bglS and cotA genes, and expressed in Escherichia coli. The affinity-purified recombinant chimeric enzyme showed both laccase and glucanase activities, with a maximum laccase activity at pH 4.5 and 75C that showed a V-max 30 higher than observed for the parental laccase. The maximum glucanase activity in the chimeric enzyme was at pH 6.0 and 50C, with a slight reduction in V-max by approximate to 10 compared with the parental glucanase. A decreased K-M resulted in an overall increase in the K-cat/K-M value for the glucanase activity of the chimeric enzyme. The hydrolytic activity of the chimera was 20 higher against natural milled sugarcane bagasse as compared with equimolar mixtures of the separate parental enzymes. Molecular dynamics simulations indicated the approximation of the two catalytic domains in the chimeric enzyme, and the formation of an inter-domain interface may underlie the improved catalytic function. (AU)

• Processo FAPESP: 10/18850-2 - Identificação, caracterização e engenharia de enzimas que degradam a parede celular das plantas
• Beneficiário: Richard John Ward
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 07/01623-0 - Desenho racional, construção e caracterização de uma enzima bifuncional (lacase-celulase) para uso na conversão de biomassa em biocombustível
• Beneficiário: Gilvan Pessoa Furtado
• Modalidade de apoio: Bolsas no Brasil - Mestrado