Characterization and elimination of undesirable protein residues in plant cell wall materials for enhancing lignin analysis by solution-state nuclear magnetic resonance spectroscopy

Authors: KIM, HOON - University Of Wisconsin; PADMAKSHAN, DHARSHANA - University Of Wisconsin; LI, YANDING - University Of Wisconsin; RENCORET, JORGE - Instituto De Recursos Naturales Y Agrobiologia De Sevilla (IRNAS-CSIC); Hatfield, Ronald; RALPH, JOHN - University Of Wisconsin

Submitted to: Biomacromolecules
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/24/2017
Publication Date: 10/24/2017
Citation: Kim, H., Padmakshan, D., Li, Y., Rencoret, J., Hatfield, R.D., Ralph, J. 2017. Characterization and elimination of undesirable protein residues in plant cell wall materials for enhancing lignin analysis by solution-state nuclear magnetic resonance spectroscopy. Biomacromolecules. 18(12): 4184-4195. doi: 10.1021/acs.biomac.7b01223.
DOI: https://doi.org/10.1021/acs.biomac.7b01223

Interpretive Summary: The cell walls of forages represent a large supply of potential energy for ruminants such as dairy cows. Unfortunately, not all the components in the cell wall are easily digested, and some, such as lignin, are completely indigestible. In addition, lignin tends to protect significant portions of the cell wall’s digestible components such as carbohydrates and proteins. Accurate characterization of lignin within the cell wall is important to understanding its role in cell wall structure and its negative impacts on utilization. Recent work, using an analytical technique based on nuclear magnetic resonance (NMR) analysis of total cell walls, made it possible to “see” the composition and amounts of lignin. This work revealed that proteins within the cell wall can produce signals that are nearly identical to some lignin subunits (p-coumaroyl alcohol), leading to an overestimation of this component in lignin. Fortunately the protein contamination can be removed using proteases, providing a clearer picture of true lignin materials in the cell wall. Overestimation of this lignin component would lead to errors concerning how the lignin polymers are interacting with other cell wall components, providing structural strength, and impacting degradability by ruminants. These new findings allow a more accurate assessment of the total cell wall and improve our estimations of how forage cell walls can be utilized effectively by dairy cows.

Technical Abstract: Proteins exist in every plant cell wall. Certain protein residues interfere with lignin characterization and quantification. The current solution-state 2D-NMR technique (gel-NMR) for whole plant cell wall structural profiling provides detailed information regarding cell walls and proteins. However, certain components intrude on lignin structural analysis. Here, we report the structural characterization of the protein residue peaks in 2D gel-NMR spectra that we found from corn cob and kenaf samples, but note that the protein residues were ubiquitous and evident in spectra from various other plants and tissues, especially leaves, roots, and tissues from young plants. The correlations from protein amino acid residues from proteins were assigned and identified as tyrosine, phenylalanine, and tryptophan. One of the phenylalanine peaks was superimposed on the typical p-hydroxyphenyl (H-unit) structure of lignins, causing an overestimation of the H-units for many plants (or tissues). Similarly, protein residues in many species also provided incorrect values ''of lignin analyses by the Klason method. These protein residues were hard to remove, even with the extensive solvent washing methods that are used to isolate cell wall residues prior to running the NMR experiments. Protein contamination also occurred when preparing cellulolytic enzyme lignins (CELs and ELs), which are typically produced using crude cellulase treatments from virtually protein-free wood samples, leaving the same amino acid residues as those from naturally occurring protein residues in many cell wall samples. Even milled wood lignins (MWLs) also contain a fair amount of such residues. In this study, we used a protease to remove the protein residues from the ball-milled cell wall samples. It is still unlikely that we can completely remove all of the proteins from cell walls by treating with a protease; however, we were able to reveal H-unit structures in lignins more clearly from the 2D-NMR spectra, providing a better basis for their estimation. Additionally, our findings suggested that lignin amount can be overestimated by many assays, as previous studies have reported.