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Quantification of lignin–carbohydrate linkages with high-resolution NMR spectroscopy

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Abstract

A quantitative approach to characterize lignin–carbohydrate complex (LCC) linkages using a combination of quantitative 13C NMR and HSQC 2D NMR techniques has been developed. Crude milled wood lignin (MWLc), LCC extracted from MWLc with acetic acid (LCC-AcOH) and cellulolytic enzyme lignin (CEL) preparations were isolated from loblolly pine (Pinus taeda) and white birch (Betula pendula) woods and characterized using this methodology on a routine 300 MHz NMR spectrometer and on a 950 MHz spectrometer equipped with a cryogenic probe. Structural variations in the pine and birch LCC preparations of different types (MWL, CEL and LCC-AcOH) were elucidated. The use of the high field NMR spectrometer equipped with the cryogenic probe resulted in a remarkable improvement in the resolution of the LCC signals and, therefore, is of primary importance for an accurate quantification of LCC linkages. The preparations investigated showed the presence of different amounts of benzyl ether, γ-ester and phenyl glycoside LCC bonds. Benzyl ester moieties were not detected. Pine LCC-AcOH and birch MWLc preparations were preferable for the analysis of phenyl glycoside and ester LCC linkages in pine and birch, correspondingly, whereas CEL preparations were the best to study benzyl ether LCC structures. The data obtained indicate that pinewood contains higher amounts of benzyl ether LCC linkages, but lower amounts of phenyl glycoside and γ-ester LCC moieties as compared to birch wood.

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Abbreviations

LCC:

Lignin–carbohydrate complex

HSQC:

Heteronuclear single quantum coherence

2D NMR:

Two-dimensional nuclear magnetic resonance (spectroscopy)

HMBC:

Hetero multinuclear quantum coherence

CEL:

Cellulolytic enzyme lignin

MWL:

Milled wood lignin

Rha:

Rhamnan

Ara:

Arabinan

Xyl:

Xylan

Man:

Mannan

Gal:

Galactan

Glc:

Glucan

AcOH:

Acetic acid

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Acknowledgments

The authors would like to thank Mr. Kevin Knagge (DHMRI) for acquiring NMR spectra on the 950 MHz NMR spectrometer and Dr. Clemens Anklin (Bruker BioSpin) for very valuable suggestions in the optimization of NMR conditions.

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Authors and Affiliations

  1. Department of Wood and Paper Science, North Carolina State University, Box 8005, Raleigh, NC, 27695, USA

    Mikhail Balakshin, Ewellyn Capanema, Hou-min Chang & Hasan Jameel

  2. Department of Biochemistry, North Carolina State University, Box 8005, Raleigh, NC, 27695, USA

    Hanna Gracz

  3. Currently at Lignol Innovations Ltd, Unit 101-4705 Wayburne Drive, Burnaby, BC, V5G 3L1, Canada

    Mikhail Balakshin

Authors
  1. Mikhail Balakshin
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  2. Ewellyn Capanema
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  3. Hanna Gracz
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  4. Hou-min Chang
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  5. Hasan Jameel
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Correspondence to Mikhail Balakshin.

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Balakshin, M., Capanema, E., Gracz, H. et al. Quantification of lignin–carbohydrate linkages with high-resolution NMR spectroscopy. Planta 233, 1097–1110 (2011). https://doi.org/10.1007/s00425-011-1359-2

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