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Background: Trichoderma reesei CE16 acetyl esterase (AcE) is a component of the plant cell wall degrading system of the fungus. The enzyme behaves as an exo-acting deacetylase removing acetyl groups from non-reducing end sugar residues., Methods: In this work we demonstrate this exo-deacetylating activity on natural acetylated xylooligosaccharides using MALDI ToF MS., Results: The combined action of GH10 xylanase and acetylxylan esterases (AcXEs) leads to formation of neutral and acidic xylooligosaccharides with a few resistant acetyl groups mainly at their non-reducing ends. We show here that these acetyl groups serve as targets for TrCE16 AcE. The most prominent target is the 3-O-acetyl group at the non-reducing terminal Xylp residues of linear neutral xylooligosaccharides or on aldouronic acids carrying MeGlcA at the non-reducing terminus. Deacetylation of the non-reducing end sugar may involve migration of acetyl groups to position 4, which also serves as substrate of the TrCE16 esterase., Conclusion: Concerted action of CtGH10 xylanase, an AcXE and TrCE16 AcE resulted in close to complete deacetylation of neutral xylooligosaccharides, whereas substitution with MeGlcA prevents removal of acetyl groups from only a small fraction of the aldouronic acids. Experiments with diacetyl derivatives of methyl β-d-xylopyranoside confirmed that the best substrate of TrCE16 AcE is 3-O-acetylated Xylp residue followed by 4-O-acetylated Xylp residue with a free vicinal hydroxyl group., General Significance: This study shows that CE16 acetyl esterases are crucial enzymes to achieve complete deacetylation and, consequently, complete the saccharification of acetylated xylans by xylanases, which is an important task of current biotechnology., (© 2013.)

Background: Substitutions on the xylan main chain are widely accepted to limit plant cell wall degradability and acetylations are considered as one of the most important obstacles. Hence, understanding the modes of action of a range of acetylxylan esterases (AcXEs) is of ample importance not only to increase the understanding of the enzymology of plant decay/bioremediation but also to enable efficient bioconversion of plant biomass., Methods: In this study, the modes of action of acetylxylan esterases (AcXEs) belonging to carbohydrate esterase (CE) families 1, 4, 5 and 6 on xylooligosaccharides generated from hardwood acetyl glucuronoxylan were compared using MALDI ToF MS. Supporting data were obtained by following enzymatic deacetylation by (1)H NMR spectroscopy., Conclusions: None of the used enzymes were capable of complete deacetylation, except from linear xylooligosaccharides which were completely deacetylated by some of the esterases in the presence of endoxylanase. A clear difference was observed between the performance of the serine-type esterases of CE families 1, 5 and 6, and the aspartate-metalloesterases of family CE4. The difference is mainly due to the inability of CE4 AcXEs to catalyze deacetylation of 2,3-di-O-acetylated xylopyranosyl residues. Complete deacetylation of a hardwood acetyl glucuronoxylan requires additional deacetylating enzyme(s)., General Significance: The results contribute to the understanding of microbial degradation of plant biomass and outline the way to achieve complete saccharification of plant hemicelluloses which did not undergo alkaline pretreatment., (© 2013.)