Enzymatic cleavage of model lignin dimers depends on pH, enzyme, and bond type

Abstract

Lignin is composed of phenylpropanoid monomers linked by ether and carbon-carbon bonds to form a complex heterogeneous structure. Bond-specific studies of lignin-modifying enzymes (LMEs; e.g., laccases and peroxidases) are limited by the polymerization of model lignin substrates and repolymerization of cleavage products. Here we present a high throughput platform to screen LME activities on four tagged model lignin compounds that represent the β-O-4', β-β', 5-5', and 4-O-5' linkages in lignin. We utilized nanostructure-initiator mass spectrometry (NIMS) and model lignin compounds with tags containing perfluorinated and cationic moieties, which effectively limit polymerization and condensation of the substrates and their degrading products. Sub-microliter sample droplets were printed on the NIMS chip with a novel robotics method. This rapid platform enabled characterization of LMEs across a range of pH 3-10 and relative quantification of modified (typically oxidized), cleaved, and polymerized products. All tested enzymes oxidized the four substrates and cleaved the β-O-4' and β-β' substrates to monomeric products. We discovered that the active pH range depended on both the substrate and the enzyme type. This has important applications for biomass conversion to biofuels and bioproducts, where the relative percentages of different bond types in lignin varies depending on feedstock and chemical pretreatment methods.

Keywords: Laccase; Lignin; Mass spectrometry; Peroxidase.

Fig. 1

a Chemical structure of the NIMS tag. Asterisk indicates the attachment point for the model lignin compound. b Hypothetical structure of lignin (central structure in black) with common bond types highlighted by colored circles and colored bonds. The structure of the NIMS-tagged model lignin compounds (“LigNIMS substrates”) are shown by the colored structures. The hypothetical lignin structure is intended for illustrative purposes and is a simplified representation of true lignin structure (for example, most 5–5’ bonds are located in dibenzodioxocin structures, which are not pictured here, and relative proportions of bond types do not reflect natural ratios). c Workflow for rapid testing of enzymes with the LigNIMS substrates. i Enzymatic reactions were prepared in 96-well PCR plates via robotic liquid handlers and/or multichannel pipettes. ii Samples were acidified, then transferred to the NIMS chip with robotic liquid handlers by touching the NIMS chip surface with the pipette tips. iii Dried droplet arrays were analyzed by mass spectrometry imaging. iv Sample locations were determined via the OpenMSI Arrayed Analysis Toolkit (OMAAT) and ions were binned according to their m/z values to either “cleaved,” “unreacted,” “modified,” or “polymerized.” The relative ion intensities of these four categories were plotted. Figure created in part with ChemDraw (a-b) and biorender.com (c).

Fig. 2

Mass spectra of the synthesized LigNIMS compounds. The y-axis shows intensity (arbitrary units). The β-O-4’ substrate was synthesized with a different, previously designed tag structure. Measured m/z values for all four LigNIMS substrates are shown.

Fig. 3

Average relative proportions of products after a 3-hour incubation of 11 different enzymes at pH 3–10 with the a β-O-4’ substrate, b β-β substrate, c 5–5’ substrate, or d 4-O-5’ substrate. pH is shown on the x-axis and the enzyme is shown on the y-axis. The size of colored squares represents the relative proportion of ion intensities for unreacted substrate, modified substrate (typically an oxidized product), cleaved substrate, or polymerized substrate. All enzyme reactions with each substrate were repeated in three or four independent repeat experiments. HRP, horseradish peroxidase. Reg., regular Metzyme laccase L111. Alk., alkaliphilic Metzyme laccase L371.

Fig. 4

Oxidation and cleavage pathway for the β-β’ substrate. a Relative abundance of ions during incubation with T. versicolor laccase over a 90-minute incubation at pH 5. Error bars represent standard deviation of two biological replicates. All m/z values are shown as [M + H]⁺. b Proposed reaction pathway for β-β’ substrate hydroxylation followed by bond cleavage (based on previous studies^,).