NMR Study on Laccase Polymerization of Kraft Lignin Using Different Enzymes Source

Abstract

The usage of laccases is a sustainable and environmentally friendly approach to modifying the Kraft lignin structure for use in certain applications. However, the inherent structure of Kraft lignin, as well as that resulting from laccase modification, still presents challenges for fundamental comprehension and successful lignin valorization. In this study, bacterial and fungal laccases were employed to modify eucalypt Kraft lignin. To evaluate the type and range of the chemical and structural changes of laccase-treated lignins, different NMR techniques, including solution ¹H and 2D NMR (heteronuclear single quantum correlation (HSQC)), and solid-state ¹³C NMR, were applied. Size exclusion chromatography and infrared spectroscopy were also used. Interestingly, HSQC analysis showed substantial changes in the oxygenated aliphatic region of lignins, showing an almost complete absence of signals corresponding to side-chains due to laccase depolymerization. Simultaneously, a significant loss of aromatic signals was observed by HSQC and ¹H NMR, which was attributed to a deprotonation of the lignin benzenic rings due to polymerization/condensation by laccase reactions. Then, condensed structures, such as α-5', 5-5', and 4-O-5', were detected by HSQC and ¹³C NMR, supporting the increment in molecular weight, as well as the phenolic content reduction determined in lignins.

Keywords: Kraft lignin; NMR characterization; eucalypt; laccase; polymerization.

Figure 1

HSQC 2D-NMR spectra of untreated Kraft lignin. (a) whole spectrum, δ_C/δ_H 0.0–150.0/0.0–9.0; (b) aliphatic oxygenated region, δ_C/δ_H 45.0–95.0/2.5–6.0 ppm; (c) aromatic region, δ_C/δ_H 90.0–150.0/5.0–9.0 ppm.

Figure 2

Main lignin and carbohydrate substructures identified in aliphatic oxygenated region of untreated and laccase-treated Kraft lignins with MtL and SiLA laccases. A, β-O-4′ alkyl-aryl ether; AG, aryl-glycerol; B, resinols; B′, epiresinols; B′′, diaresinol; C, α-5′; E, spirodienones; F, Ar–CHOH–COOH; I, cinnamyl alcohol end-groups; X, xylopyranose (R, OH).

Figure 3

Main lignin substructures identified in aromatic region of untreated and laccase-treated Kraft lignins with MtL and SiLA laccases. G, guaiacyl unit; G′, vanillin; G″, acetovanillona; H, p-hydroxyphenyl unit; S′, syringaldehyde (R=H) or acetosyringone (R=CH₃); S_1–1′, 3,5-tetramethoxy-para-diphenol; G_1–1′, 3-dimethoxy-para-diphenol; S₁-G_1′/G_5′; SB1, stilbene-β-1′; SB5, stilbene-β-5′.

Figure 4

HSQC 2D-NMR spectra of laccase-treated Kraft lignins. Whole spectrum, δ_C/δ_H 0.0–150.0/0.0–9.0, for SiLA-KL1 (a) and MtL-KL1 (d); aliphatic oxygenated region, δ_C/δ_H 45.0–95.0/2.5–6.0 ppm, for SiLA-KL1 (b) and MtL-KL1 (e); aromatic region, δ_C/δ_H 90.0–150.0/5.0–9.0 ppm, for SiLA-KL1 (c) and MtL-KL1 (f).

Figure 5

HSQC 2D-NMR spectra of laccase-treated Kraft lignins. Whole spectrum, δ_C/δ_H 0.0–150.0/0.0–9.0, for SiLA-KL2 (a) and MtL-KL2 (d); aliphatic oxygenated region, δ_C/δ_H 45.0–95.0/2.5–6.0 ppm, for SiLA-KL2 (b) and MtL-KL2 (e); aromatic region, δ_C/δ_H 90.0–150.0/5.0–9.0 ppm, for SiLA-KL2 (c) and MtL-KL2 (f).

Figure 6

¹³C NMR spectra, δ_C 0.0–250.0 ppm, of untreated Kraft lignin (a) and the resulting treated lignins with SiLA ((b), SiLA-KL1; (d), SiLA-KL2) and MtL ((c), MtL-KL1; (e), MtL-KL2) laccases.