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

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]}

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

• 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: 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
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• 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
• Modalidade de apoio: Auxílio à Pesquisa - Programa BIOEN - Temático
• 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
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• 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
• Modalidade de apoio: Auxílio à Pesquisa - Regular