title: Crystal structure and biochemical characterization of the recombinant ThBgl, a GH1 β-glucosidase overexpressed in Trichoderma harzianum under biomass degradation conditions.
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, β-glucosidases, which act synergistically with cellobiohydrolases and endo-β-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 β-glucosidase expressed by T. harzianum under biomass degradation conditions. We mapped and quantified the expression of all of the β-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 β-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.
authors.affiliation: Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Campinas, SP Brazil.
authors.forename: Clelton A
authors.initials: CA
authors.lastname: Santos
authors.affiliation: Laboratório Nacional de Ciência e Tecnologia do Bioetanol, Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP Brazil.
authors.forename: Letícia M
authors.initials: LM
authors.lastname: Zanphorlin
authors.affiliation: Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Campinas, SP Brazil.
authors.forename: Aline
authors.initials: A
authors.lastname: Crucello
authors.affiliation: Laboratório Nacional de Biociências, Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP Brazil.
authors.forename: Celisa C C
authors.initials: CCC
authors.lastname: Tonoli
authors.affiliation: Laboratório Nacional de Ciência e Tecnologia do Bioetanol, Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP Brazil.
authors.forename: Roberto
authors.initials: R
authors.lastname: Ruller
authors.affiliation: Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Campinas, SP Brazil.
authors.forename: Maria A C
authors.initials: MAC
authors.lastname: Horta
authors.affiliation: Laboratório Nacional de Biociências, Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP Brazil.
authors.forename: Mario T
authors.initials: MT
authors.lastname: Murakami
authors.affiliation: Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, Campinas, SP Brazil.|Departamento de Biologia Vegetal, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, SP Brazil.
authors.forename: Anete Pereira
authors.identifier: 0000-0003-3831-9829
authors.initials: AP
authors.lastname: de Souza
keywords: Biomass degradation; Enzyme prospection; Overexpression; Trichoderma harzianum; β-Glucosidase