title: Development of a chimeric hemicellulase to enhance the xylose production and thermotolerance.
abstract: Xylan is an abundant plant cell wall polysaccharide and its reduction to xylose units for subsequent biotechnological applications requires a combination of distinct hemicellulases and auxiliary enzymes, mainly endo-xylanases and ß-xylosidases. In the present work, a bifunctional enzyme consisting of a GH11 endo-1,4-β-xylanase fused to a GH43 β-xylosidase, both from Bacillus subtilis, was designed taking into account the quaternary arrangement and accessibility to the substrate. The parental enzymes and the resulting chimera were successfully expressed in Escherichia coli, purified and characterized. Interestingly, the substrate cleavage rate was altered by the molecular fusion improving at least 3-fold the xylose production using specific substrates as beechwood xylan and hemicelluloses from pretreated biomass. Moreover, the chimeric enzyme showed higher thermotolerance with a positive shift of the optimum temperature from 35 to 50 °C for xylosidase activity. This improvement in the thermal stability was also observed by circular dichroism unfolding studies, which seems to be related to a gain of stability of the β-xylosidase domain. These results demonstrate the superior functional and stability properties of the chimeric enzyme in comparison to individual parental domains, suggesting the molecular fusion as a promising strategy for enhancing enzyme cocktails aiming at lignocellulose hydrolysis.
authors: Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, SP, Brazil; Laboratório Nacional de Biociências (LNBio), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, SP, Brazil.
authors: José A
authors: JA
authors: Diogo
authors: Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, SP, Brazil; Laboratório Nacional de Biociências (LNBio), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, SP, Brazil.
authors: Zaira B
authors: ZB
authors: Hoffmam
authors: Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, SP, Brazil.
authors: Letícia M
authors: LM
authors: Zanphorlin
authors: Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, SP, Brazil.
authors: Junio
authors: J
authors: Cota
authors: Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, SP, Brazil.
authors: Carla B
authors: CB
authors: Machado
authors: Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, SP, Brazil.
authors: Lúcia D
authors: LD
authors: Wolf
authors: Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, SP, Brazil.
authors: Fabio
authors: F
authors: Squina
authors: Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, SP, Brazil.
authors: André R L
authors: AR
authors: Damásio
authors: Laboratório Nacional de Biociências (LNBio), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, SP, Brazil.
authors: Mario T
authors: MT
authors: Murakami
authors: Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, SP, Brazil. Electronic address: roberto.ruller@bioetanol.org.br.
authors: Roberto
authors: R
authors: Ruller
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018533:Biomass; D001709:Biotechnology; D002942:Circular Dichroism; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D008031:Lignin / Q000378:metabolism; D008958:Models, Molecular; D011134:Polysaccharides / Q000378:metabolism; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014990:Xylans / Q000378:metabolism; D014994:Xylose / Q000096:biosynthesis*; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D014990:Xylans; C036909:lignocellulose; C007916:hemicellulose; D008031:Lignin; D014994:Xylose; D006026:Glycoside Hydrolases; D014995:Xylosidases; C023305:hemicellulase; C026579:exo-1,4-beta-D-xylosidase; D043364:Endo-1,4-beta Xylanases
keywords: Bacillus subtilis; Bifunctional chimera; Xylanase; Xylosidase