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Action of different types of endoxylanases on eucalyptus xylan in situ

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Most studies of the mode of action of industrially important endoxylanases have been done on alkali extracted-plant xylan. In just few cases, the native form of the polysaccharide, acetylated xylan, was used as a substrate. In this work action of xylanases belonging to three glycoside hydrolase families, GH10, GH11, and GH30 was investigated on acetylglucuronoxylan directly in hardwood cell walls. Powdered eucalyptus wood was used as xylanase substrate. Enzyme-generated fragments were characterized by TLC, MALDI ToF MS, and NMR spectroscopy. All three xylanases generated from eucalyptus wood powder acetylated xylooligosaccharides. Those released by GH10 enzyme were the shortest, and those released by GH30 xylanase were of the largest diversity. For GH30 xylanase the 4-O-methyl-D-glucuronic acid (MeGlcA) side residues function as substrate specificity determinants regardless the acetylation of the neighboring hydroxyl group. Much simpler xylooligosaccharide patterns were observed when xylanases were applied in combination with carbohydrate esterase family 6 acetylxylan esterase. In the presence of the esterase, all aldouronic acids remained 3-O-acetylated on the xylopyranosyl (Xylp) residue substituted with MeGlcA. The 3-O-acetyl group, in contrast to the acetyl groups of otherwise unsubstituted Xylp residues, does not affect the mode of action of endoxylanases, but contributes to recalcitrance of the acidic xylan fragments. The results confirm importance of acetylxylan esterases in microbial degradation of acetylated hardwood glucuronoxylan. They also point to still unresolved question of efficient enzymatic removal of the 3-O-acetyl group on MeGlcA-substituted Xylp residues negatively affecting the saccharification yields.

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Xylp :

β-D-xylopyranosyl residue


4-O-methyl-D-glucuronic acid


Glycoside hydrolase


Carbohydrate esterase

Xyln :

β-1,4-xylooligosaccharide of n Xylp residues

MeGlcAiXyln :

Aldouronic acid containing one residue of MeGlcA linked to Xylp residue marked in Xyln with i which is the number of Xylp residue counted from the reducing end (i would be equal to 1 if MeGlcA would be linked to the reducing-end Xyl, 2 if MeGlcA would be linked to the second Xylp residue from the reducing end, etc.

Ac3MeGlcA3Xyl3 :

Aldotetraouronic acid in which both MeGlcA and the acetyl group are linked to non-reducing-end Xylp residue of Xyl3

Ac3MeGlcA3Xyl4 :

Aldopentaouronic acid in which both MeGlcA and the acetyl group are linked to penultimate Xylp residue from the non-reducing-end of Xyl4

MeGlcA3Xyl3Ac2 2,3 :

Aldotetraouronic acid MeGlcA3Xyl3 doubly acetylated at the middle and non-reducing-end Xylp residues


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The authors are thankful to Dr. Andre L. Ferraz (University of Sao Paulo, Lorena, Brazil) for supplying eucalyptus wood powder. Dr. Iveta Uhliariková (Institute of Chemistry, Slovak Academy of Sciences) is gratefully acknowledged for 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. 2/0016/18. 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.


This study was funded by Slovak Research and Development Agency (grant number APVV-0602-12) and Scientific Grant Agency (grant number 2/0016/18).

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Correspondence to Peter Biely.

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Puchart, V., Fraňová, L., Mørkeberg Krogh, K.B.R. et al. Action of different types of endoxylanases on eucalyptus xylan in situ. Appl Microbiol Biotechnol 102, 1725–1736 (2018).

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