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

Author(s)

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

• Document type: Journal article
• Source: ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS; v. 71, p. 4-pg., 2015-08-01.

Abstract

-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)

• FAPESP's process: 13/10443-7 - Bioprospecting and construction of hemicellulases for the production of xylose from hydrolyzate licor pentoses and their use in fermentative xylitol production
• Grantee: José Alberto Diogo
• Support Opportunities: Scholarships in Brazil - Master
• FAPESP's process: 13/13309-0 - Studies of the structural and functional behavior of enzymes evolutionarily specialized in the degradation of plant biomass with potential biotechnological applications
• Grantee: Mário Tyago Murakami
• Support Opportunities: Regular Research Grants
• FAPESP's process: 14/07135-1 - Multi-user equipament approved in grant 2013/13309-0: Beckman Coulter capillary electrophoresis systems with LIF detection system
• Grantee: Mário Tyago Murakami
• Support Opportunities: Multi-user Equipment Program