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.
Keywords: 4-O-methyl-d-glucuronic acid or 4-O-methyl-d-glucuronosyl; AcE; AcXE; Acetyl esterase; Acetyl glucuronoxylan; CE; Carbohydrate esterase family; HexXyl(x)Ac(y); MALDI ToF MS; MeGlcA; MeGlcAXyl(x)Ac(y); MeXylp; NMR; Positional specificity; Xyl(2)–Xyl(7); Xyl(x)Ac(y); Xylp; acetyl esterase; acetylated aldouronic acid containing one MeGlcA, x xylose residues and y acetyl groups; acetylated β-1,4-xylooligosaccharide containing x xylose residues and y acetyl groups; acetylxylan esterase; carbohydrate esterase; d-xylopyranose or d-xylopyranosyl; methyl β-d-xylopyranoside; oligosaccharide containing one hexopyranose residue of unknown nature, x xylose residues and y acetyl groups; β-1,4-xylobiose–β-1,4-xyloheptaose.
© 2013.