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Crystal structure and biochemical characterization of the recombinant ThBgl, a GH1 ß-glucosidase overexpressed in Trichoderma harzianum under biomass degradation conditions.
Santos, Clelton A; Zanphorlin, Letícia M; Crucello, Aline; Tonoli, Celisa C C; Ruller, Roberto; Horta, Maria A C; Murakami, Mario T; de Souza, Anete Pereira.
Affiliation
  • Santos CA; Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Campinas, SP Brazil.
  • Zanphorlin LM; Laboratório Nacional de Ciência e Tecnologia do Bioetanol, Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP Brazil.
  • Crucello A; Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Campinas, SP Brazil.
  • Tonoli CCC; Laboratório Nacional de Biociências, Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP Brazil.
  • Ruller R; Laboratório Nacional de Ciência e Tecnologia do Bioetanol, Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP Brazil.
  • Horta MAC; Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Campinas, SP Brazil.
  • Murakami MT; Laboratório Nacional de Biociências, Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP Brazil.
  • de Souza AP; Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Campinas, SP Brazil.
Biotechnol Biofuels ; 9: 71, 2016.
Article in En | MEDLINE | ID: mdl-27006690
ABSTRACT

BACKGROUND:

The conversion of biomass-derived sugars via enzymatic hydrolysis for biofuel production is a challenge. Therefore, the search for microorganisms and key enzymes that increase the efficiency of the saccharification of cellulosic substrates remains an important and high-priority area of study. Trichoderma harzianum is an important fungus known for producing high levels of cellulolytic enzymes that can be used for cellulosic ethanol production. In this context, ß-glucosidases, which act synergistically with cellobiohydrolases and endo-ß-1,4-glucanases in the saccharification process, are potential biocatalysts for the conversion of plant biomass to free glucose residues.

RESULTS:

In the present study, we used RNA-Seq and genomic data to identify the major ß-glucosidase expressed by T. harzianum under biomass degradation conditions. We mapped and quantified the expression of all of the ß-glucosidases from glycoside hydrolase families 1 and 3, and we identified the enzyme with the highest expression under these conditions. The target gene was cloned and heterologously expressed in Escherichia coli, and the recombinant protein (rThBgl) was purified with high yields. rThBgl was characterized using a comprehensive set of biochemical, spectroscopic, and hydrodynamic techniques. Finally, we determined the crystallographic structure of the recombinant protein at a resolution of 2.6 Å.

CONCLUSIONS:

Using a rational approach, we investigated the biochemical characteristics and determined the three-dimensional protein structure of a ß-glucosidase that is highly expressed by T. harzianum under biomass degradation conditions. The methodology described in this manuscript will be useful for the bio-prospection of key enzymes, including cellulases and other accessory enzymes, for the development and/or improvement of enzymatic cocktails designed to produce ethanol from plant biomass.
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Full text: 1 Collection: 01-internacional Database: MEDLINE Type of study: Prognostic_studies Language: En Journal: Biotechnol Biofuels Year: 2016 Document type: Article

Full text: 1 Collection: 01-internacional Database: MEDLINE Type of study: Prognostic_studies Language: En Journal: Biotechnol Biofuels Year: 2016 Document type: Article