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23 Congress of the International Union for Biochemistry and Molecular Biology
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44 Annual Meeting of the Brazilian Society for Biochemistry and Molecular Biology
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Foz do Iguaçu, PR, Brazil, August 24 to 28 , 2015
Formulation of an enzymatic cocktail and biochemical characterization of
lignocellulolytic and amylolytic systems for depolymerization of agroindustrial residues
Scarcella, A.S.A.1; Vici, A.C.2; Santos, C.B.2; Monteiro, L.M.O.1; Pereira, M.G.2;
Almeida, P. Z.1; Segato, F.3; Ribeiro, L.F.C.4; Vitcosque, G.L.1;Polizeli, M.L.T.M.2
1
Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão
Preto, Universidade de São Paulo, Ribeirão Preto, Brazil; 2 Departamento de
Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto,
Universidade de São Paulo, Ribeirão Preto, Brazil; 3 Escola de Engenharia de
Lorena, Universidade de São Paulo, Brazil; 4 University of Maryland, Baltimore
County, USA.
Second generation ethanol is produced from lignocellulosic biomass, requiring a
variety of enzymes synergistically working to complete hydrolysis. Thus, this study
aimed to characterize enzymes that hydrolyze agro-industrial residues. The
enzymes were: laccase, β-glucosidase and glucoamylase produced by Trametes
versicolor, Aspergillus sp. and A. brasiliensis, respectively; xylanase (GH10) from
Malbranchea pulchella; xyloglucanase (GH12) and endo-β-1,4-glucanase (GH12)
from A. terreus; cellobiohydrolase I (GH7) from A. niveus all expressed in A.
nidulans A773. Parameters analyzed for each enzyme were optimum temperature
(40 to 95°C) and pH (3.0-8.0), influence of buffer system in pH 5.0 (50 mM sodium
acetate, 50 mM sodium citrate and 50 mM ammonium acetate), effect of salts,
EDTA and β-mercaptoethanol and enzymatic stability of the cocktail. The best
conditions were: laccase at 55°C and pH 4.0-4.5; xylanase from 70 to 80°C and
pH 5.0-5.5; xyloglucanase at 65°C and pH 5.0-5.5; endo-β-1,4-glucanase from 45
to 55ºC and pH 5.0-5.5; cellobiohydrolase I from 55 to 65°C and pH 5.0; βglucosidase at 55ºC and pH 4.0-4.5 and glucoamylase at 70ºC and pH 4.0-4.5.
Endoglucanase, cellobiohydrolase and laccase were stable over 2, 6 and 4 h,
respectively, under the conditions tested for application of the cocktail. The
remaining enzymes were stable for at least 24 h. Xylanase and glucoamylase
were activated in the presence of salts, EDTA, and β-mercaptoethanol, whereas
the laccase was inhibited. β-glucosidase was inhibited only in the presence of
NiSO4.6H2O. Venn diagram showed that cellobiohydrolase and xyloglucanase
were inhibited in the presence of HgCl2; endoglucanase and xyloglucanase were
inhibited when exposed to MgCl2.6H2O, CuSO4.5H2O, NH4Cl and EDTA. Thus, the
best conditions for the application of this cocktail were 55°C, 50 mM sodium citrate
buffer, pH 5.0. Considering the conditions of this cocktail, which contains a laccase
and some hydrolases, it has great potential for ethanol and other biotechnological
applications.
Key words: characterization, enzyme cocktail, lignocellulosic biomass
Acknowledgements: FAPESP
Brazilian Society for Biochemistry and
Molecular Biology (SBBq)