Molecular cloning, overexpression, purification and crystallographic analysis of a GH43-xylosidase from Bacillus licheniformis

Autor(es)

Diogo, Jose Alberto ; Zanphorlin, Leticia Maria ; Sato, Helia Harumi ; Murakami, Mario Tyago ; Ruller, Roberto

• Tipo de documento: Artigo Científico
• Fonte: ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS; v. 71, p. 4-pg., 2015-08-01.

Resumo

-Xylosidases (EC 3.2.1.37) catalyze the hydrolysis of short xylooligosaccharides into xylose, which is an essential step in the complete depolymerization of xylan, the major hemicellulosic polysaccharide of plant cell walls, and has great biotechnological relevance for the production of lignocellulose-based biofuels and the paper industry. In this study, a GH43 -xylosidase identified from the bacterium Bacillus licheniformis (BlXylA) was cloned into the the pET-28a bacterial expression vector, recombinantly overexpressed in Escherichia coli BL21(DE3) cells and purified to homogeneity by metal-affinity and size-exclusion chromatography. The protein was crystallized in the presence of the organic solvent 2-methyl-2,4-pentanediol and a single crystal diffracted to 2.49 angstrom resolution. The X-ray diffraction data were indexed in the monoclinic space group C2, with unit-cell parameters a = 152.82, b = 41.9, c = 71.79 angstrom, = 91.7 degrees. Structural characterization of this enzyme will contribute to a better understanding of the structural requirements for xylooligosaccharide specificity within the GH43 family. (AU)

• Processo FAPESP: 13/10443-7 - Bioprospecção e construção de hemicelulases para a produção de xilose a partir de hidrolisado de licor de pentoses e sua utilização na produção de xilitol via fermentativa
• Beneficiário: José Alberto Diogo
• Modalidade de apoio: Bolsas no Brasil - Mestrado
• Processo FAPESP: 13/13309-0 - Estudos do comportamento funcional e estrutural de enzimas evolutivamente especializadas na degradação de biomassa vegetal com potenciais aplicações biotecnológicas
• Beneficiário: Mário Tyago Murakami
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 14/07135-1 - EMU concedido no processo 2013/13309-0 - aquisição do sistema de eletroforese capilar com detector de fluorescência - Beckman Coulter
• Beneficiário: Mário Tyago Murakami
• Modalidade de apoio: Auxílio à Pesquisa - Programa Equipamentos Multiusuários