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20 páginas.-- 7 figuras.-- 6 tablas.-- 37 referencias.--  Artículo en PrensaEucalypt feedstock was pretreated with Myceliophthora thermophila laccase and methyl syringate, as mediator, in a multistage sequence consisting of successive enzymatic and alkaline peroxide stages, directly on the ground wood. Lignin modification was studied by two-dimensional (2D) nuclear magnetic resonance (NMR) of wood (at the gel state) after each stage of the sequence. Decrease of guaiacyl and syringyl lignin units (with preferential removal of the former) and aliphatic (mainly β-O-4′-linked) side chains was observed along the sequence, without a substantial change in polysaccharide cross-signals along the sequence, was observed. However, the most noticeable modification was the formation of Cα-oxidized syringyl units in the enzymatic stages, which were partially removed in the alkaline extractions. Further insight was attained by 2D NMR of the cellulolytic-enzyme lignins, isolated from the pretreated samples, and the filtrates from the different stages. Additionally, a comparison of eucalypt delignification with the high redox potential laccase from Pycnoporus cinnabarinus in the presence of the synthetic mediator 1-hydroxybenzotriazole was carried out. Wood delignification already occurred at the first cycle of the pretreatment with P. cinnabarinus laccase and 1-hydroxybenzotriazole, which directly correlated with increases in glucose (~10 %) yields after enzymatic hydrolysis. In contrast, the first cycle of pretreatment with M. thermophila laccase and methyl syringate did not produce any delignification or saccharification improvement. However, at the end of the sequence, similar delignification (~50 %) of eucalypt feedstock and increases (~30 %) in glucose yields were attained in both laccase-mediator pretreatments. Finally, the influence of lignin content and the ratio of its syringyl and guaiacyl units on recalcitrance of eucalypt feedstock to enzymatic saccharification is discussed.This study was funded by the INDOX (KBBE-2013-7-613549) EU-project and the LIGNOCELL (AGL2011-25379) and HIPOP (BIO2011-2669) Spanish MICINN projects (co-financed by FEDER funds). J. Rencoret acknowledges the CSIC JAE-Doc contract of the program “Junta para la Ampliación de Estudios” co-financed by the European Social Fund (ESF). H. Lund and L. Kalum from Novozymes (Bagsvaerd, Denmark) are acknowledged for the cellulases (Celluclast and Novozym 188) and the M. thermophila laccase, Beldem (Andenne, Belgium) for the P. cinnabarinus laccase and J. Romero (ENCE, Spain) for the eucalypt wood samples. Manuel Angulo (CITIUS, University of Seville) is acknowledged for providing technical assistance in the NMR analyses.Peer reviewe

In-Depth 2D NMR Study of Lignin Modification During Pretreatment of Eucalyptus Wood with Laccase and Mediators