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(Referência obtida automaticamente do Web of Science, por meio da informação sobre o financiamento pela FAPESP e o número do processo correspondente, incluída na publicação pelos autores.)

Crystal structure and biochemical characterization of the recombinant ThBgl, a GH1 beta-glucosidase overexpressed in Trichoderma harzianum under biomass degradation conditions

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Autor(es):
Santos, Clelton A. [1] ; Zanphorlin, Let-Cia M. [2] ; Crucello, Aline [1] ; Tonoli, Celisa C. C. [3] ; Ruller, Roberto [2] ; Horta, Maria A. C. [1] ; Murakami, Mario T. [3] ; de Souza, Anete Pereira [4, 1]
Número total de Autores: 8
Afiliação do(s) autor(es):
[1] Univ Estadual Campinas, Ctr Biol Mol & Engn Genet, Campinas, SP - Brazil
[2] Ctr Nacl Pesquisa Energia & Mat, Lab Nacl Ciencia & Tecnol Bioetanol, Campinas, SP - Brazil
[3] Ctr Nacl Pesquisa Energia & Mat, Lab Nacl Biociencias, Campinas, SP - Brazil
[4] Univ Estadual Campinas, Inst Biol, Dept Biol Vegetal, Campinas, SP - Brazil
Número total de Afiliações: 4
Tipo de documento: Artigo Científico
Fonte: BIOTECHNOLOGY FOR BIOFUELS; v. 9, MAR 22 2016.
Citações Web of Science: 9
Resumo

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, beta-glucosidases, which act synergistically with cellobiohydrolases and endo-beta-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 beta-glucosidase expressed by T. harzianum under biomass degradation conditions. We mapped and quantified the expression of all of the beta-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 angstrom. Conclusions: Using a rational approach, we investigated the biochemical characteristics and determined the three-dimensional protein structure of a beta-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. (AU)

Processo FAPESP: 08/52197-4 - Genomic-assisted breeding of sugarcane: using molecular markers for understanding the genetic architecture of quantitative traits and to implement marker assisted selection
Beneficiário:Anete Pereira de Souza
Linha de fomento: Auxílio à Pesquisa - Programa BIOEN - Temático
Processo FAPESP: 14/09720-9 - Estudos do papel da fosforilação e da ligação de proteínas parceiras na regulação da função de miosinas V humanas
Beneficiário:Mário Tyago Murakami
Linha de fomento: Auxílio à Pesquisa - Regular
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
Linha de fomento: Auxílio à Pesquisa - Regular
Processo FAPESP: 14/18856-1 - Estudo da expressão gênica global e de regiões genômicas associadas ao controle da expressão de enzimas envolvidas na degradação de compostos lignocelulósicos por Trichoderma harzianum
Beneficiário:Maria Augusta Crivelente Horta
Linha de fomento: Bolsas no Brasil - Pós-Doutorado