title: Characterization of Clostridium thermocellum (B8) secretome and purified cellulosomes for lignocellulosic biomass degradation.
abstract: The main goal of the present study was a complete proteomic characterization of total proteins eluted from residual substrate-bound proteins (RSBP), and cellulosomes secreted by Clostridium thermocellum B8 during growth in the presence of microcrystalline cellulose as a carbon source. The second goal was to evaluate their potential use as enzymatic blends for hydrolyzing agro-industrial residues to produce fermentable sugars. Protein identification through LC-MS/MS mass spectrometry showed that the RSBP sample, in addition to cellulosomal proteins, contains a wide variety of proteins, including those without a well-characterized role in plant cell wall degradation. The RSBP subsample defined as purified cellulosomes (PC) consists mainly of glycoside hydrolases grouped in families 5, 8, 9, 10 and 48. Dynamic light scattering, DLS, analysis of PC resulted in two protein peaks (pi1 and pi2) presenting molecular masses in agreement with those previously described for cellulosomes and polycellulosomes. These peaks weren't detected after PC treatment with 1.0% Tween. PC and RSBP presented maximal activities at temperatures ranging from 60° to 70°C and at pH 5.0. RSBP retained almost all of its activity after incubation at 50, 60 and 70°C and PC showed remarkable thermostability at 50 and 60°C. RSBP holocellullolytic activities were inhibited by phenolic compounds, while PC showed either increasing activity or a lesser degree of inhibition. RSBP and PC hydrolyze sugar cane straw, cotton waste and microcrystalline cellulose, liberating a diversity of saccharides; however, the highest concentration of released sugar was obtained for assays carried out using PC as an enzymatic blend and after ten days at 50°C.
authors.affiliation: Enzymology Laboratory, Department of Cellular Biology, University of Brasília, Brasilia, DF, Brazil.
authors.forename: Karen O
authors.initials: KO
authors.lastname: Osiro
authors.affiliation: Enzymology Laboratory, Department of Cellular Biology, University of Brasília, Brasilia, DF, Brazil.
authors.forename: Brenda R
authors.initials: BR
authors.lastname: de Camargo
authors.affiliation: Enzymology Laboratory, Department of Cellular Biology, University of Brasília, Brasilia, DF, Brazil.
authors.forename: Rachel
authors.initials: R
authors.lastname: Satomi
authors.affiliation: Enzymology Laboratory, Department of Cellular Biology, University of Brasília, Brasilia, DF, Brazil.
authors.forename: Pedro Ricardo V
authors.initials: PR
authors.lastname: Hamann
authors.affiliation: Enzymology Laboratory, Department of Cellular Biology, University of Brasília, Brasilia, DF, Brazil.
authors.forename: Jéssica Pinheiro
authors.initials: JP
authors.lastname: Silva
authors.affiliation: Biochemistry and Protein Chemistry Laboratory, Department of Cellular Biology, University of Brasília, Brasília, DF, Brazil.
authors.forename: Marcelo Valle
authors.initials: MV
authors.lastname: de Sousa
authors.affiliation: Embrapa-Agroenergy, Parque Estação Biológica, s/n, Brasília, Brazil.
authors.forename: Betania F
authors.initials: BF
authors.lastname: Quirino
authors.affiliation: Enzymology Laboratory, Department of Cellular Biology, University of Brasília, Brasilia, DF, Brazil.
authors.forename: Elaine N
authors.initials: EN
authors.lastname: Aquino
authors.affiliation: Enzymology Laboratory, Department of Cellular Biology, University of Brasília, Brasilia, DF, Brazil.
authors.forename: Carlos R
authors.initials: CR
authors.lastname: Felix
authors.affiliation: Embrapa Genetic Resources and Biotechnology, Laboratory of Synthetic Biology, Brasília, DF, Brazil.
authors.forename: André Melro
authors.initials: AM
authors.lastname: Murad
authors.affiliation: Enzymology Laboratory, Department of Cellular Biology, University of Brasília, Brasilia, DF, Brazil. Electronic address: enoronha@unb.br.
authors.forename: Eliane F
authors.initials: EF
authors.lastname: Noronha
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D056804:Biofuels; D018533:Biomass; D001709:Biotechnology; D044603:Cellulosomes / Q000378:metabolism; D048013:Acetivibrio thermocellus / Q000201:enzymology / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006868:Hydrolysis; D008031:Lignin / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism; D040901:Proteomics; D053719:Tandem Mass Spectrometry
chemical_list: D001426:Bacterial Proteins; D056804:Biofuels; D020543:Proteome; C036909:lignocellulose; D008031:Lignin; D006026:Glycoside Hydrolases
keywords: C. thermocellum; Cellulosome; Holocellulases; Lignocellulosic biomass