Advertisement

Planta

, Volume 233, Issue 6, pp 1097–1110 | Cite as

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

  • Mikhail Balakshin
  • Ewellyn Capanema
  • Hanna Gracz
  • Hou-min Chang
  • Hasan Jameel
Original Article

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.

Keywords

Lignin–carbohydrate complex Benzyl ether LCC linkages Phenyl glycoside linkages Ester LCC linkages Quantitative HSQC NMR technique 

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

Notes

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.

References

  1. Balakshin MYu, Evtuguin DV, Pascoal Neto C, Silva AMS, Domingues PM, Amado FML (2001) Studies on lignin and lignin-carbohydrate complex by application of advanced spectroscopic techniques. In: Proceedings of the 11th ISWPC, Nice, 2001, vol 1, pp 103–110Google Scholar
  2. Balakshin MYu, Capanema EA, Chen C-L, Gracz H (2003) Elucidation of the structures of residual and dissolved pine kraft lignin using an HMQC technique. J Agric Food Chem 51:6116–6127PubMedCrossRefGoogle Scholar
  3. Balakshin MYu, Capanema EA, Chang H-M (2007a) A fraction of MWL with high concentration of lignin–carbohydrate linkages: isolation and analysis with 2D NMR spectroscopic techniques. Holzforschung 61:1–7CrossRefGoogle Scholar
  4. Balakshin MYu, Capanema EA, Chang H-M, Jameel H (2007b) Structural variations in pine and birch lignin–carbohydrate complex preparations. In: Proceedings of 14th International Symposium Wood Fibre Pulping Chem. CD-ROM. Durban, South Africa, June 25–28Google Scholar
  5. Balakshin MYu, Capanema EA, Chang H-m (2008) Recent advances in isolation and analysis of lignins and lignin–carbohydrate complexes. In: Hu T (ed) Characterization of Lignocellulosics. Blackwell, Oxford, pp 148–166CrossRefGoogle Scholar
  6. Bjorkman A (1956) Studies on finely divided wood. Part I. Extraction of lignin with neutral solvents. Svensk Papperstidn 59:477–485Google Scholar
  7. Capanema EA, Balakshin MYu, Kadla JF (2004) A comprehensive approach for quantitative lignin characterization by NMR spectroscopy. J Agric Food Chem 52:1850–1860PubMedCrossRefGoogle Scholar
  8. Capanema EA, Balakshin MYu, Kadla JF (2005) Comprehensive NMR studies on hardwood MWL. J Agric Food Chem 53:9639–9649PubMedCrossRefGoogle Scholar
  9. Capanema EA, Balakshin MYu, Katahira R, Chang H-m, Jameel H (2007) Structural variations in hardwood lignins. In: Proceedings of 14th international symposium on wood fibre pulping chemistry. CD-ROM. Durban, South Africa, June 25–28Google Scholar
  10. Chang H-M, Cowling EB, Brown W, Adler E, Miksche G (1975) Comparative studies on cellulolytic enzyme lignin and milled lignin of sweetgum and spruce. Holzforschung 29:153–159CrossRefGoogle Scholar
  11. Enoki A, Yaku F, Koshijima T (1983) Synthesis of LCC model compounds and their chemical and enzymatic stabilities. Holzfoschung 37:135–141CrossRefGoogle Scholar
  12. Eriksson Ö, Goring DAI, Lindgren BO (1980) Structural studies on the chemical bonds between lignins and carbohydrates in spruce wood. J Wood Sci Technol 14:267–279CrossRefGoogle Scholar
  13. Fengel D, Wegener G (1984) Wood: chemistry ultrastructure reactions. Walter de Gruyrer, BerlinGoogle Scholar
  14. Freudenberg K (1968) In: Freudenberg K, Neish AC (eds) Constitution and biosynthesis of lignin. Springer, Berlin, pp 93–94Google Scholar
  15. Fujimoto A, Matsumoto Y, Meshitsuka G, Chang H-m (2005) Quantitative evaluation of milling effects on lignin structure during the isolation process of milled wood lignin. J Wood Sci 51:89–91CrossRefGoogle Scholar
  16. Heikkinen S, Toikka MM, Karhunen T, Kilpelainen IA (2003) Quantitative 2D HSQC (Q-HSQC) via suppression of J-dependence of polarization transfer in NMR spectroscopy: Application to wood lignin. J Am Chem Soc 125:4362–4367PubMedCrossRefGoogle Scholar
  17. Helm RF (2000) Lignin-polysaccharide interaction in woody plants. In: Glasser WG, Northey RA, Schultz TP (eds) Lignin: historical, biological, and material perspectives ACS symposium series 742, Washington, DC, pp 161–171Google Scholar
  18. Hu Z, Yeh TF, Chang H-M, Matsumoto Y, Kadla JF (2006) Elucidation of the structure of cellulolitic enzyme lignin. Holzforschung 60:389–397CrossRefGoogle Scholar
  19. Ibarra D, Chavez MI, Rencoret J, Del Rio JC, Gutierrez A, Romero J, Camarero S, Martinez MJ, Jumenez-Barbero J, Martinez AT (2007) Lignin modification during Eucalyptus globulus kraft pulping followed by totally chlorine-free bleaching: a two-dimensional nuclear magnetic resonance, Fourier transform infrared, and pyrolysis-gas chromatography/mass spectrometry study. J Agric Food Chem 55:3477–3490PubMedCrossRefGoogle Scholar
  20. Juhást T, Szengyel Z, Réczey K, Siika-Aho M, Viikari L (2005) Characterization of cellulases and hemicellulases produced by Trichoderma reesei on various carbon sources. Process Biochem 40:3519–3525CrossRefGoogle Scholar
  21. Karlsson O, Ikeda T, Kishimoto T, Magara K, Matsumoto Y, Hosoya S (2004) Isolation of lignin–carbohydrate bonds in wood. Model experiments and preliminary application to pine wood. J Wood Sci 50:142–150CrossRefGoogle Scholar
  22. Koshijima T, Watanabe T (2003) Association between lignin and carbohydrates in wood and other plant tissues. Springer, BerlinGoogle Scholar
  23. Lawoko M, Henriksson G, Gellerstedt G (2005) Structural differences between the lignin carbohydrate complexes in wood and in chemical pulps. Biomacromolecules 6:3467–3473PubMedCrossRefGoogle Scholar
  24. Li K, Helm RF (1995) Synthesis and rearrangement reactions of ester-linked lignin–carbohydrate model compounds. J Agric Food Chem 43:2098–2103CrossRefGoogle Scholar
  25. Martínez AT, Rencoret J, Marques G, Gutiérrez A, Ibarra D, Jiménez-Barbero J, Del Rio JC (2008) Monolignol acylation and lignin structure in some nonwoody plants: a 2D NMR study. Phytochemistry 69:2831–2843PubMedCrossRefGoogle Scholar
  26. Minor JL (1982) Chemical linkage of pine polysaccharides to lignin. J Wood Chem Technol 2:1–16CrossRefGoogle Scholar
  27. Obst J (1982) Frequency and alkali resistance of lignin–carbohydrate bonds in wood. Tappi 65:109–112Google Scholar
  28. Ralph J, Lundquist K, Brunow G, Lu F, Kim H, Schatz PF, Marita JM, Hatfield R, Ralph SA, Christensen JH, Boerjan W (2004) Lignins: natural polymers from oxidative coupling of 4-hydroxyphenylpropanoids. Phytochem Rev 3:29–60CrossRefGoogle Scholar
  29. Ralph J, Akiyama T, Kim H, Lu F, Schatz PF, Marita JM, Ralph SA, Reddy MSS, Chen F, Dixon RA (2006) Effects of coumarate 3-hydroxylase down-regulation on lignin structure. J Biol Chem 281:8843–8853PubMedCrossRefGoogle Scholar
  30. Terashima N, Ralph SA, Landucci LL (1996) New facile synthesis of monolignol glycosides; p-glucocoumaryl alcohol, coniferin and syringin. Holzforschung 50:151–155CrossRefGoogle Scholar
  31. Toikka M, Brunow G (1999) Lignin–carbohydrate model compounds. Reactivity of methyl 3-O- (α-l-arabinofuranosyl)-β-d-xylopyranoside and methyl β-d-xylopyranoside towards a β-O-4-quinone methide. J Chem Soc Perkin Trans 1:1877–1883CrossRefGoogle Scholar
  32. Toikka M, Sipila J, Teleman A, Brunow G (1998) Lignin–carbohydrate model compounds. Formation of lignin-methyl arabinoside and lignin-methyl galactoside benzyl ethers via quinine methide intermediates. J Chem Soc Perkin Trans 1:3813–3818CrossRefGoogle Scholar
  33. Tokimatsu T, Umezawa T, Shimada M (1996) Synthesis of four diastereomeric lignin carbohydrate complexes (LCC) model compounds composed of a β-O-4 lignin model linked to methyl-β-d-glucose. Holzforschung 50:156–160CrossRefGoogle Scholar
  34. Wang Z, Yokoyama T, Chang H-M, Matsumoto Y (2009) Dissolution of beech and spruce milled woods in LiCl/DMSO. J Agric Food Chem 57:6167–6170PubMedCrossRefGoogle Scholar
  35. Watanabe T (1989) Structural studies on the covalent bonds between lignin and carbohydrate in lignin–carbohydrate complexes by selective oxidation of the lignin with 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone. Wood Res 76:59–123Google Scholar
  36. Yelle DJ, Ralph J, Frihart CR (2008) Characterization of nonderivatized plant cell walls using high-resolution solution-state NMR spectroscopy. Magn Res Chem 46:508–517CrossRefGoogle Scholar
  37. Zhang L, Gellerstedt G (2000) Achieving quantitative assignment of lignin structure by combining 13C and HSQC NMR techniques. In: Proceedings of the 6th European workshop on lignocellulosics and pulp, Bordeaux, France, September 3–6, pp 7–10Google Scholar
  38. Zhang L, Gellerstedt G (2007) Quantitative 2D HSQC NMR determination of polymer structures by selecting suitable internal standard reference. Magn Res Chem 45:37–45CrossRefGoogle Scholar
  39. Zhang L, Gellerstedt G, Lu F, Ralph J (2006) NMR studies on the occurrence of spirodienone structures in lignins. J Wood Chem Technol 26:65–79CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Mikhail Balakshin
    • 1
    • 3
  • Ewellyn Capanema
    • 1
  • Hanna Gracz
    • 2
  • Hou-min Chang
    • 1
  • Hasan Jameel
    • 1
  1. 1.Department of Wood and Paper ScienceNorth Carolina State UniversityRaleighUSA
  2. 2.Department of BiochemistryNorth Carolina State UniversityRaleighUSA
  3. 3.Currently at Lignol Innovations LtdBurnabyCanada

Personalised recommendations