1-26

19 Feb 2024

Synthetic methods for ¹³C-labeled monolignol glycosides (coniferin, syringin and p-glucocoumaryl alcohol), with the label on the α-, β-, or γ-carbon of the side chain or 4-, 5-, 3-, or 1-carbon in the aromatic ring, are described. Synthetic procedures are given for dehydrogenation polymers (DHPs) from the coniferin and ¹³C-enriched cell wall-DHPs using isolated tree soft parts. Experimental procedures are also described for the ¹³C-enrichment of specific carbon atoms of cell wall lignin by feeding the ¹³C-labeled coniferin to ginkgo tree shoot. A beginner’s guide to these chemical experiments is included.

1-9

01 Aug 2023

White-rot fungi play important roles in the global carbon cycle by efficiently degrading lignin and polysaccharides from lignocellulose. Over the years, extensive efforts have been made to elucidate the mechanisms underlying lignin degradation by white-rot fungi. One of them is a molecular genetics approach, which includes genetic modification to alter wood-degrading abilities or metabolism, with the aim of producing commercially valuable chemicals from unutilized lignocellulosic resources. Molecular genetic studies have been conducted on several species, including Pleurotus ostreatus, Phanerochaete sordida, and Phlebia sp., for which a genetic transformation system has been developed. However, the techniques and methodologies available for these fungi are limited, posing a serious bottleneck. Here, we describe recent studies that have developed powerful and effective techniques and methodologies, removing the restrictions of molecular genetics studies on lignin degradation by white-rot fungi.

10-17

03 Aug 2023

Hydroxybenzaldehydes (HBAs) are ubiquitous in reaction mixtures obtained from various types of lignin degradation, particularly oxidative degradation. This article reviews the formation behaviors, pathways, and mechanisms of HBAs during alkaline nitrobenzene and alkaline aerobic oxidations of lignin. The former is a well-known analytical method for lignin, while the latter is of industrial importance from the viewpoint of the production of bio-based aromatics.

1-8

17 May 2022

To detect natural laccase mediator, the extracellular culture fluid of Trametes versicolor laccase production medium was fractionated using degradation of anthracene which can not be degraded with laccase alone. As a result, 4-hydroxybenzoic acid (HBA) was isolated from the culture fluid, and it was clear that HBA acted as a natural mediator and the degradation efficiency of anthracene by laccase greatly increased. The laccase-HBA system could also oxidized the nonphenolic β-O-4 lignin model dimer to pruduce the Cα-oxidation, β-ether cleavage and aromatic ring cleavage products. The oxidation mechanisms of the laccase-HBA system are very similar to those for lignin peroxidase and laccase-1-hydroxybenzotriazol (HBT) system.

9-14

26 Aug 2022

The β–5 linkage is an intermolecular linkage in lignin. Sphingobium sp. strain SYK-6 can assimilate various lignin-derived dimers, including a β–5 dimer, dehydrodiconiferyl alcohol. In SYK-6, the hydroxyl group at Cγ of the B-ring side chain of dehydrodiconiferyl alcohol is oxidized to generate the γ-carboxylic compound, DCA-C. Then, the hydroxyl group at Cγ of the A-ring side chain of DCA-C is oxidized to the carboxyl group to generate the dicarboxylic compound, DCA-CC. The carboxylic group at Cγ of the A-ring side chain of DCA-CC is decarboxylated, and the accompanying spontaneous ether cleavage of the coumaran ring produces a stilbene-type compound, DCA-S. The conversions of DCA-C and DCA-CC are catalyzed by enantiospecific oxidases (PhcC and PhcD) and enantiospecific decarboxylases (PhcF and PhcG), respectively. DCA-S is subjected to cleavage of the interphenyl double bond by lignostilbene α,β-dioxygenase to generate 5-formylferulate and vanillin. Among the eight lignostilbene α,β-dioxygenase genes, vanillate-induced lsdD plays a critical role in cleaving DCA-S. The formyl group of 5-formylferulate is oxidized, and the resultant carboxylic group is subsequently decarboxylated to produce ferulate. Finally, ferulate and vanillin are further catabolized via a previously characterized pathway.

15-25

28 December 2022

A non-phenolic β-O-4-type lignin model compound, veratrylglycerol-β-guaiacyl ether (VG), was treated with H₂O₂ at high pH levels in our previous study. Veratraldehyde (Vald) and veratric acid (Vacid), which are non-phenolic analogues of the most common lignin-based fine chemicals, vanillin and vanillic acid, respectively, were obtained with a high total yield of 77% based on the amount of degraded VG, although only 22% of VG was degraded and hence the total yield of Vald and Vacid was only 17% based on the initial amount of VG. This study optimized the alkaline H₂O₂ treatment to enhance the degradation of VG and yields of Vald and Vacid. The degradation of VG and total yield of Vald and Vacid were enhanced from 22% to 54% and from 17% to 39%, respectively, by optimizing the conditions as follows: 1.96 mmol of H₂O₂ were added stepwise 400 times with an interval of 15 s in a reaction solution containing 1.0 mmol/L VG, 3.0 mol/L NaOH, 1.08 mmol/L FeCl₃, and 3.24 mmol/L mannitol at 90℃. Doubling the reaction period and employing twice the amount of H₂O₂ (3.92 mmol) in the absence of mannitol further enhanced the degradation of VG to 61% and the total yield to 44%.

1-8

23 Feb 2021

Non-phenolic C₆-C₂- and C₆-C₃-type lignin model compounds with the β-O-4 bond were treated in tert-butyl alcohol (tBuOH) or dimethyl sulfoxide (DMSO) containing potassium tert-butoxide (KOtBu) or various other bases under mild conditions (at a base concentration of 0.5 mol/L and 30ºC) to examine how the reactions differ between these systems. The β-O-4 bond cleavage in KOtBu/tBuOH was slower than that in KOtBu/DMSO owing to the greater solvation of tBuO¯ in tBuOH than in DMSO. The β-O-4 bond cleavage of the erythro isomer of the C₆-C₃-type compound was slower than that of the threo isomer in all the reactions. This is explained by the preferred formation of a strong hydrogen bond between the α-hydroxy and the dissociated γ-alkoxy groups (or vice versa) of theformer, which interferes with the cleavage. The rates of the β-O-4 bond cleavages in DMSO were in the order of the systems containing: NaOtBu > KOtBu >> LiOtBu, which seems to relate to whether each base dissolves as an ion pair or free ions in DMSO. Those in DMSO were in the order of the systems containing KOtBu >> potassium hydride (KH) >> potassium iso-propoxide > potassium ethoxide, which is consistent with their basicities except for KH.

9-18

20 May 2021

Aerobic oxidation of lignin in reaction media containing Bu₄NOH facilitates efficient production of vanillin (4-hydroxy-3-methoxybenzaldehyde) and vanillic acid (4-hydroxy-3-methoxybenzoic acid). This study presents production of these compounds from polyethylene glycol (PEG)-modified glycol lignin (PEG-lignin) from Japanese cedar (Cryptomeria japonica). A Bu₄NOH-based reaction medium was prepared by the addition of NaOH(s) to 1.25 mol/L Bu₄NOH aq. Degradation of the PEG-lignin (14 mg) in 2.0 mL of the medium at 120 ^oC under O₂ in a sealed test tube produced vanillin and vanillic acid with 9.1 and 2.6 wt% yields, respectively, based on the Klason lignin amount of the PEG-lignin. The total yield of vanillin and vanillic acid reached 79.6 % of that in the corresponding alkaline nitrobenzene oxidation (14.7 wt%), indicating high performance of the medium. Bench-scale aerobic oxidation of the PEG-lignin (100 g /1.0 L) under O₂ pressurized at 0.7 MPa gave the products with their maximum yields similar to those in the above lab-scale experiment. Further increase in the O₂ pressure to 2.5 MPa significantly shortened the reaction time to achieve the maximum product yield. This pressure increase did not affect the vanillin yield, but suppressed the formation of O₂-sensitive vanillic acid, by which selective vanillin production was achieved.

19-24

12 July 2021

Nitrobenzene oxidation of lignin has long been one of the typical lignin chemical degradation methods. Previously, we reported a high-throughput and microscale protocol for the method. However, the reactor used in the protocol was available only in Japan and not supported outside Japan. In this study, we prepared an alternative protocol of alkaline nitrobenzene oxidation using an alternative reactor, which is available in many countries. Using the new protocol almost the same product yields as the previous protocol were obtained using a stable-isotope-dilution method. In addition, ethylvanillin, which is readily and commercially available, was also found to be usable as an alternative internal standard.

1-10

28 May 2020

MnO₂ can potentially suppress the degradation of carbohydrates in oxygen delignification, because MnO₂ catalyzes the decomposition of H₂O₂ to H₂O and O₂ and possibly that of organic peroxides to alcohols and O₂ without the formation of any active oxygen species, which degrade carbohydrates. The addition of MnO₂ actually suppressed the degradation of a carbohydrate model compound, methyl β-D-glucopyranoside, when reacted with active oxygen species generated from reactions between a phenolic lignin model compound, vanillyl alcohol, and O₂ under oxygen delignification conditions. However, the addition of MnO₂ did not have any meaningful effect when hardwood unbleached kraft pulp was oxygen-delignified. The addition surprisingly had a deleterious effect on pulp viscosity when MnO₂ was generated in situ in pulp fibers. This deleterious effect would result from a phenomenon whereby the oxidation of Mn²⁺ to MnO₂ was not complete in the in situ generation and Mn³⁺-related species, along others, were generated. In contrast, substitution of the latter half of oxygen delignification with a MnO₂ oxidation stage at a pH of 2 substantially suppressed the degradation of carbohydrates, compared to the common oxygen delignification without substitution.

11-19

28 May 2020

The antioxidant properties of technical lignins have been extensively investigated but there are still gaps of knowledge that should be filled to facilitate the practical applications of lignins as antioxidants (AOs). In the present investigation, we compared the short-term (60 min) and long-term (48h) AO performance of lignins with different contents of functional groups and lignin model compounds (LMCs) with different aromatic ring substituents in the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH*) antioxidant assay. We found some LMCs to quickly expend their AO capacity while others started off slowly but after 48h had consumed more DPPH* per phenolic hydroxyl group. Reaction time was also a factor in the relative AO performance of lignins. For softwood lignins, a higher phenolic hydroxyl content was associated with increased DPPH* reactivity. CatLignin, a thermally treated lignin rich in phenolic units and especially catechol groups, consumed more than twice as much DPPH* than any other lignin during 48h (over two mol/lignin unit of 180 g/mol). CatLignin also had the lowest 60 min half-maximal effective concentration (EC50). In polypropylene, lignins provided better UV protection than commercial primary antioxidants applied at similar loadings. Similarly, the better performance of lignin over commercial AOs was observed against thermal oxidation.

20-28

28 May 2020

Dehydrogenation polymers (DHPs) were prepared by laccase from Rhus vernicifera and horseradish peroxidase (HRP). The enzymatic ability of oxidation and polymerization was compared between these enzymes. Laccase showed higher enzyme activity against syringaldazine than ABTS, while HRP exhibited lower enzyme activity against syringaldazine than 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid). These enzymes had various enzyme activity against these substrates. DHPs from sinapyl alcohol (SA) were hardly produced by each enzyme, whereas DHPs from coniferyl alcohol (CA) were produced by both enzymes. The laccase oxidized sinapyl alcohol faster than coniferyl alcohol. The nuclear magnetic resonance (NMR) analysis demonstrated that acetylated-DHP (Ac-DHP) from CA by laccase contained β-5 and β-β linkages, but not β-O-4 linkage. On the other hand, Ac-DHP from CA by HRP carried all these three linkages.

29

17 August 2020

30-41

14 September 2020

Lignocellulose biomass is indispensable for establishing sustainable societies. Trees and large-sized grasses are the major sources of lignocellulose biomass. Moreover, large-sized grasses greatly surpass trees in terms of lignocellulose biomass productivity. With an overall aim to improve lignocellulose usability, it is vital to improve lignin content and simplify lignin structures in biomass plants via lignin metabolic engineering. In this mini review, recent studies of lignin metabolic engineering of grass biomass plants mainly from the authors’ research group are summarized, which includes characterization of lignocellulose properties of large-sized grass biomass plants and the augmentation of lignin content and simplification of lignin structures in grasses.

42-50

12 January 2021

The mulberry cultivar with unusual red-colored wood, “Sekizaisou,” was discovered in the bushland of Okushiri Island, Hokkaido, Japan, around a century ago. The leaves of this cultivar were used as feed for sericulture on the island for a short duration from 1916. Although propagules of Sekizaisou have been preserved by sequential vegetative propagation in several public research institutes in Japan, Sekizaisou is believed to have already become extinct on the island. Recently, a point mutation in the first exon of the CINNAMYL ALCOHOL DEHYDROGENASE 1 locus in Sekizaisou was identified to be responsible for the change in the color of the wood and the structural alteration of lignin. In this study, we performed genotyping of the allele in nearly 600 mulberry individuals grown naturally on Okushiri Island for the rediscovery of Sekizaisou and its wild relatives. A simple protocol for the detection of the mutant allele using polymerase chain reaction followed by direct Sanger sequencing was applied. Although no individuals with the mutant allele were identified in the present study, our results will provide an insight into the flow of the mutant gene in the natural mulberry population.