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Redistribution of acetyl groups on the non-reducing end xylopyranosyl residues and their removal by xylan deacetylases

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Monoacetylated xylosyl residues of the main hardwood hemicellulose acetylglucuronoxylan undergo acetyl group migration between positions 2 and 3, and predominantly to position 4 of the non-reducing end xylopyranosyl (NRE-Xylp) residues which are amplified by saccharifying enzymes. On monoacetylated non-reducing end xylopyranosyl (NRE-Xylp) residues of xylooligosaccharides the acetyl group migrates predominantly to position 4 and hinders their hydrolysis by β-xylosidase.


Acetyl migration on the NRE-Xylp residues and their enzymatic deacetylation by various xylan deacetylases was followed by 1H-NMR spectroscopy and TLC.


A 5-min heat treatment of 4-nitrophenyl 3-O-acetyl-β-D-xylopyranoside was sufficient to establish equilibrium between monoacetate derivatives acetylated at positions 2, 3 and 4. Rapid acetyl migration along the NRE-Xylp ring at elevated temperature was confirmed in derivatives of methyl β-1,4-xylotrioside (Xyl3Me) monoacetylated solely on the NRE-Xylp residue, the analogues of naturally occurring acetylated xylooligosaccharides. The Xyl3Me monoacetates were used as substrates to study regioselectivity of the NRE-Xylp residue deacetylation by various acetylxylan esterases (AcXEs) of distinct carbohydrate esterase (CE) families. CE1, CE4 and CE6 AcXEs hydrolyzed considerably faster the 2″-O-acetyl derivative than the 3″-O-acetyl derivative. In contrast, the CE16 acetyl esterase preferred to attack the ester bond at position 3 followed by position 4.


Redistribution of acetyl group on the NRE-Xylp residues is extremely rapid at elevated temperature and includes the formation of 4-acetate. Regioselectivity of AcXEs and CE16 acetyl esterase on these monoacetates is complementary.

General significance

The formation of all isomers of acetylated xylosyl residues must be taken into account after a long-term incubation of acetylxylan and acetylated xylooligosaccharides solutions or upon their treatment at elevated temperatures. This phenomenon emphasizes requirement of both types of xylan deacetylases to enable a rapid saccharification of xylooligosaccharides by glycoside hydrolases.

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Carbohydrate esterase


Acetylxylan esterase


Acetyl esterase


Methyl β-D-xylopyranosyl-1,4-β-D-xylopyranosyl-1,4-β-D-xylopyranoside, methyl β-D-xylotrioside

Xylp :



4-O-methyl-α-D-glucuronic acid


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Mrs. Mária Cziszárová and Dr. Iveta Uhliariková are gratefully acknowledged for their excellent technical assistance and NMR spectroscopy measurements. This work was supported by the Slovak Research and Development Agency under the contract No. APVV-0602-12, and by Scientific Grant Agency under the contract No. 2/0037/14. This work was also supported by the Research & Development Operation Programme funded by the European Regional Development Fund (ITMS 26220120054) and by SP Grant 2003SP200280203.

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Correspondence to Peter Biely.

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• Heating accelerates Ac group migration at non-reducing end of xylooligosaccharides

• Ac group migrates also to position 4, originally involved in glycosidic linkage

• Enzymatic deacetylation of non-reducing end Xylp residues was studied by NMR and TLC

• Acetylxylan esterases and CE16 acetyl esterase are complementary in their action

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Puchart, V., Biely, P. Redistribution of acetyl groups on the non-reducing end xylopyranosyl residues and their removal by xylan deacetylases. Appl Microbiol Biotechnol 99, 3865–3873 (2015).

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