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.

Understanding Manganese Peroxidase-Catalyzed Conversion of the Lignin Structure and Its Application for the Polymerization of Kraft Lignin

“…The reduction in signal intensity of the guaiacyl unit suggests the deprotonation of the benzenic ring in KL and polymerization, which aligns with the results obtained from SEC analysis (Table ). Similar trends of involving decreased or vanished aromatic proton signals in NMR spectra have been noted in laccase-catalyzed polymerization of lignosulfonates , and kraft lignin. ,,, In these studies, the researchers attributed this phenomenon to the creation of polymerized structures through α-5′, 5-5′, and 4-O-5′ linkages. Hence, it is possible that similar things happened in our study.…”

“…The reaction proceeded for 12 h, after which an additional 0.25 mM H 2 O 2 was added and the reaction was allowed to continue for another 12 h. Subsequently, the pH of the reaction mixture was adjusted to 2.5 using hydrochloric acid, resulting in the formation of a precipitate comprising the reaction products. This precipitate was collected by centrifugation at 10,000 × g for 10 min and subjected to three washes with acidified water (pH 2.5). , The resulting sample was then lyophilized. A control experiment was also conducted following the same procedure but omitting Cs MnP.…”

“…This precipitate was collected by centrifugation at 10,000 × g for 10 min and subjected to three washes with acidified water (pH 2.5). 7,20 The resulting sample was then lyophilized. A control experiment was also conducted following the same procedure but omitting CsMnP.…”

“…Researchers have demonstrated that polymerizing KL enables the synthesis of lignin-based biomaterials, such as hydrogels and dispersants. , Presently, chemical treatments, such as oxidative treatment, are employed to modify the chemical properties of KL by capitalizing on its high phenolic content . However, these chemical methods often require harsh reaction temperatures, which may not always be economically feasible . As an alternative approach, researchers are investigating biocatalytic pathways involving the use of natural enzymes to achieve the desired structural modification of KL.…”

Physicochemical Insights into Enzymatic Polymerization of Lignosulfonates

“…Thus, to be able to further analyze the insoluble pLS polymers, e.g., by NMR or MALDI-TOF, a suitable solvent must be found beforehand. Several studies handled this limitation by using additional solid-state analyses, such as NMR or MALDI-TOF to verify the findings made by the liquid methods. ,, Anyhow, for the pLS samples investigated, even these solid-state methods, especially MALDI-TOF, turned out to be inappropriate and thus were not considered for the structural characterization of pLS.…”