Lignin-carbohydrate Complex Structures Research Articles

Synthesis of a natural core substrate with lignin-xylan cross-linkage for unveiling the productive kinetic parameters of glucuronoyl esterase

Lignin-carbohydrate complexes (LCCs) present a considerable hurdle to the economic utilization of lignocellulosic biomass. Glucuronoyl esterase (GE) is an LCC-degrading enzyme that catalyzes the cleavage of the cross-linkages between lignin and xylan in LCCs. Benzyl-d-glucuronate (Bn-GlcA), a commercially available substrate, is widely used to evaluate GE activity assays. However, since Bn-GlcA lacks the structural backbone of naturally occurring LCCs, the mechanisms underlying the activity of GEs and their diversity in the structure-activity relationship are not fully understood. Herein, we provided a synthesis scheme for designing 1,23-α-d-(6-benzyl-4-O-methyl-glucuronyl)-1,4-β-d-xylotriose (Bn-MeGlcA3Xyl3) as a natural core substrate bearing cross-linkage between lignin and glucuronoxylan. A well-defined and yet more realistic synthetic substrate was successfully synthesized via a key step of the benzyl esterification of 4-O-methyl-glucuronyl-1,4-β-d-xylotriose (MeGlcA3Xyl3), a minimized fragment of glucuronoxylan enzymatically digested by β-1,4-xylanase. To the best of our knowledge, this is the first report of the productive GE kinetic analysis of the fungal Pestalotiopsis sp. AN-7 using this substrate. Kinetic parameters, i.e., the Km, Vmax, and kcat of Bn-MeGlcA3Xyl3, were 0.43 mM, 55.5 μmol·min-1·mg-1, and 35.8 s-1, respectively. On the other hand, as reported to date, the productive kinetic parameters for Bn-GlcA were not obtained because of its excessively high Km value (>16 mM). The substantial variance in the enzymatic activity of GE regarding substrate-binding affinity between Bn-MeGlcA3Xyl3 and Bn-GlcA was also demonstrated using in silico docking simulation. These results suggested that the extended xylan fragment is a key structural determinant affecting GE substrate recognition. Furthermore, the presence of a natural xylan backbone allows for evaluating the enzyme activity of xylan-degrading enzymes. Accordingly, the synthesized substrate with the natural core structure of LCC allowed us to unveil the productive kinetic parameters of GEs, serving as a versatile substrate for further elucidating the cascade reaction of GE and xylan-degrading enzymes.

Differential Studies on the Structure of Lignin-Carbohydrate Complexes (LCC) in Alkali-Extracted Plant Hemicelluloses.

Hemicellulose extracted by alkali treatment is of interest because of the advantages of its intact sugar structure and high degree of polymerization. However, the hemicellulose extracted by alkali treatment contained more lignin fragments and the presence of a lignin-carbohydrate complex (LCC), which affected the isolation and purification of hemicellulose and its comprehensive utilization. Therefore, the evaluation of the LCC structure of different types of lignocellulosic resources is of great significance. In this study, the LCC structures of hardwoods and Gramineae were enriched in alkaline systems. Information on the composition, structural proportions, and connection patterns of LCC samples was discussed. The similarities and differences between the LCC structures of different units of raw materials were comparatively studied. The results indicated that the monosaccharide fractions were higher in the LCC of Gramineae compared to hardwoods. The composition of the lignin fraction was dominated by G and S units. The phenyl glycosidic (PhGlc) bond is the predominant LCC linkage under alkali-stabilized conditions. In addition, Gramineae PhGlc types are more numerous compared to hardwoods. The results of the study provide insights into the differences in the chemical composition and structural features of LCC in different plants and provide important guidance for the optimization of the process of purifying hemicellulose.

Structural Characterization of Lignin-Carbohydrate Complexes (LCCs) and Their Biotransformation by Intestinal Microbiota In Vitro.

Lignin-carbohydrate complexes (LCCs) have recently emerged as natural products with pharmaceutical and nutraceutical potential. Here, we compared the structure of LCCs from ginkgo (GK, gymnosperms), wheat straw (WST, monocotyledons), and aspen white poplar (AW, dicotyledons). We also investigated the biotransformation of LCCs by intestinal microbiota in vitro. We found that human intestinal microbiota could use LCCs as a carbon source for growth, breaking resistant cross-linkages in LCCs to generate a plethora of short-chain fatty acids (SCFAs) and aromatic compounds with putative beneficial effects on human health. The yield of SCFAs reached 1837.8 ± 44.1 μmol/g using AW LCCs as a carbon source. The biomass of intestinal microbiota increased the fastest using GK LCCs. The greatest amounts of phenolics were present at 4 h in a WST LCCs fermentation system. Many phenolic acids with potential bioactivity were obtained after 24 h fermentation using each LCCs, such as ferulic acid.

Enrichment and Identification of Lignin–Carbohydrate Complexes in Softwood Extract

Lignin–carbohydrate complexes (LCCs) are hybrid structures containing covalently linked moieties of lignin and carbohydrates. The structure and behavior of LCCs affect both industrial processes and practical applications of lignocellulosic biomass. However, the identification of phenylglycoside, benzylether, and gamma (γ)-ester LCC bonds in lignocellulosic biomass is limited due to their relatively low abundance compared to plain carbohydrate and lignin structures. Herein, we enriched the LCC bonds in softwood galactoglucomannan (GGM)-rich extract fractionated by (1) a solvent (ethanol), (2) enzymes, and (3) physical techniques. Two-dimensional nuclear magnetic resonance (NMR) spectroscopy analysis was used to identify the LCC bonds. Phenylglycoside and benzylether bonds were concentrated in the ethanol-soluble GGM fractions. A benzylether bond was concentrated into GGM fractions containing larger molecules (>500 Da) through physical techniques. The γ-ester bond was identified in all studied GGM fractions, which is explained by its stability and possible presence in residual xylan. In summary, we demonstrated the potential of the suggested techniques to enrich LCC bonds in softwood extract and improve LCC identification. Such techniques may also enable further studies on the structure and functionality of LCC bonds and open new prospects in the engineering of biomolecules.

The influence of lignin content and structure on hemicellulose alkaline extraction for non-wood and hardwood lignocellulosic biomass

The extractability of hemicellulose from different lignocellulosics depends on the source of biomass. Differences in hemicellulose extractability are believed to be due to plant-specific hemicellulose arrangement alongside lignin within the cell wall. In this research, six biomasses were used to probe hemicellulose alkaline extractability as a function of the native lignin within the biomasses. Quantitative 2D-HSQC and 13C NMR analysis were performed to determine the S/G (S: syringyl, G: guaiacyl) and lignin-carbohydrate complex (LCC) linkages of milled wood lignin isolated from these biomasses. A strong negative correlation was observed between total lignin content and hemicellulose extractability, demonstrating that a greater presence of lignin in the original material results in lower xylan solubilization. In addition, a correlation between S/G of lignin and xylan dissolution was found within a group of hardwoods and within a group of non-woods. This suggests that monomeric constituency also influences xylan’s propensity for dissolution in 10% NaOH. Although there is some uncertainty in the quantification of LCC linkages, both non-woods and hardwoods exhibited negative correlations between alkaline-stable LCC linkages content and xylan extractability. This suggests that alkaline-stable LCC structures are associated with a decrease in the alkaline extractability of hemicellulose.

Determination of Lignin-Carbohydrate Complexes Structure of Wheat Straw using Carbon-13 Isotope as a Tracer

To maximize the use of wheat straw as a feedstock for biofuels and other biorefinery products, the structure of lignin-carbohydrate complexes (LCCs) was characterized by injection of 13C isotope-labeled xylose into living wheat straw. Afterwards, lignin-carbohydrate complexes were extracted from the harvested straw by the Bjorkman method. The extracted LCCs were chemically characterized by Fourier transform-infrared spectroscopy (FT-IR), sugar composition, molecular weight analysis, 13C-NMR, and HSQC. The results showed that LCCs in wheat straw were particularly enriched with xylan and exhibited narrow polydispersity (Mw/Mn < 1.5). NMR analysis showed that the lignin was linked with the carbohydrates through γ-ester, phenyl glycoside, and benzyl ether bonds. In addition to S, G, and H lignin units, p-coumarate and ferulate were also in the LCCs. The substructures in lignin were β-O-4', β-β', and β-5'. Quantitative data analysis of 13C-NMR combined with HSQC showed that the lignin in the LCCs of wheat straw contained guaiacyl (G), syringyl (S), and p-hydroxyphenyl (H) units in the proportion of 5:4:1 (S:G:H). The main lignin substructure, β-O-4', comprised 71.64% of the isolated lignin. The total LCC linkages (the sum of phenyl glycoside, γ-ester and benzyl ether bonds) were 210.86 /100C9 in 13C-LCC, which was dominated by phenyl glycoside linkages, followed by γ-ester bonds and minor amounts of benzyl ether bonds. Lignin and xylan in the LCCs of wheat straw were mainly linked by benzyl ether bonds and phenyl glycoside linkages.

Unveiling the structure and ultrastructure of lignin carbohydrate complexes in softwoods

Lignocellulose-based polymers are presently being investigated for potential as raw materials for polymer industry. However, covalent bonds between carbohydrates and lignin, so called lignin carbohydrate complexes (LCCs) present one of the factors impeding clean fractionation of the polymers. Understanding the chemical structure of LCCs in relation to the cell wall ultra-structure therefore provides relevant insight for technical fractionation. In the present work, new mild analytical protocols for LCCs were developed and fractions thereof subjected to detailed structural/ultra-structural characterization. When combined with size exclusion chromatography, the application of mono-component ploysaccharidases proved to be crucial for the identification of LCC moieties and provided indirect evidence for existence of lignin-carbohydrate bonds. The carbohydrate composition of the LCC moieties was essential to determination of the ultra-structural origin of the LCC types. Analytical 31P NMR of the LCCs and of their thioacidolysis products was useful in unveiling lignin structure in LCCs. A complete structure/ultra-structure relationship of LCCs in softwood was determined.

Quantification of lignin–carbohydrate linkages with high-resolution NMR spectroscopy

A quantitative approach to characterize lignin-carbohydrate complex (LCC) linkages using a combination of quantitative ¹³C NMR and HSQC 2D NMR techniques has been developed. Crude milled wood lignin (MWLc), LCC extracted from MWLc with acetic acid (LCC-AcOH) and cellulolytic enzyme lignin (CEL) preparations were isolated from loblolly pine (Pinus taeda) and white birch (Betula pendula) woods and characterized using this methodology on a routine 300 MHz NMR spectrometer and on a 950 MHz spectrometer equipped with a cryogenic probe. Structural variations in the pine and birch LCC preparations of different types (MWL, CEL and LCC-AcOH) were elucidated. The use of the high field NMR spectrometer equipped with the cryogenic probe resulted in a remarkable improvement in the resolution of the LCC signals and, therefore, is of primary importance for an accurate quantification of LCC linkages. The preparations investigated showed the presence of different amounts of benzyl ether, γ-ester and phenyl glycoside LCC bonds. Benzyl ester moieties were not detected. Pine LCC-AcOH and birch MWLc preparations were preferable for the analysis of phenyl glycoside and ester LCC linkages in pine and birch, correspondingly, whereas CEL preparations were the best to study benzyl ether LCC structures. The data obtained indicate that pinewood contains higher amounts of benzyl ether LCC linkages, but lower amounts of phenyl glycoside and γ-ester LCC moieties as compared to birch wood.

Native Lignin Structure of Miscanthus x giganteus and Its Changes during Acetic and Formic Acid Fractionation

Milled wood lignin (MWL) and acetic and formic acid lignin (AL and FL) from Miscanthus x giganteus bark were produced, respectively, before and after organosolv fractionations under optimal conditions, in terms of organic and hydrochloric acid concentrations, liquid/wood ratio, and reaction time. In order to study the M. x giganteus native lignin structure and its modifications during the fractionation process, the lignins were studied by two-dimensional heteronuclear single quantum coherence (2D-(HSQC)), (13)C- and (31)P nuclear magnetic resonance (NMR) spectroscopy, Fourier transform infrared (FTIR), size-exclusion chromatography (SEC) both before and after thioacidolysis, and elemental analysis. In addition, chemical composition analysis was performed on ash, Klason lignin, and carbohydrate content. The analyses demonstrated that M. x giganteus native lignin (MWL) is highly acylated at the C(gamma) of the lignin side chain (46%), possibly with p-coumarate and/or acetate groups. This is newsworthy since several earlier studies showed that acylation at the gamma-carbon commonly occurs in C(3) and CAM grasses, whereas M. x giganteus is a C(4) grass. Furthermore, M. x giganteus showed a low S/G ratio (0.7) and a predominance of beta-O-4' linkages (up to 93% of all linkages). AL and FL lose part of these linkages during organosolv fractionation (up to 21 and 32%, respectively). The p-coumarate groups resist fractionation processes and are still present in high quantities in AL and FL. During the fractionation process, lignin is acetylated (acetic acid process) and condensed, with the G units condensing more than S units. M. x giganteus MWL contains a high content of carbohydrates (22.8%), suggesting that it is a lignin-carbohydrate complex (LCC). AL and FL showed low carbohydrate contents because of the breaking down of the LCC structures. AL and FL have high molecular weights and low polydispersities, and are high in phenolic content, qualities that make these suitable for different applications. These results suggest that refinement of M. x giganteus via organosolv processes could potentially turn this grass into a valuable source of both fiber and lignin.

Analysis of the structure of lignin-carbohydrate complexes by the specific13C tracer method

In the present study the specifically13C-enriched lignin precursors of biosynthesis (i.e., coniferin-[side chainα−13C], coniferin-[side chain-β−13C] and coniferin-[side chain-γ−13C]) were synthesized and administered exogeneously to ginkgo shoots (Ginkgo biloba L.) to obtain13C-enriched lignin-carbohydrate complexes (LCCs). The specifically13C-enriched LCCs were isolated from the newly formed xylem of ginkgo shoots administered with the13C-enriched precursors and degraded by enzymes. Lignin-rich fractions, so called enzyme-degraded LCCs (EDLCCs), were obtained. By determining their13C-NMR spectra, information related to the chemical structure of lignin building units and linkages between phenylpropane units of lignin and carbohydrates were obtained. It was found that these precursors were incorporated in natural lignin successfully. Three lignin-carbohydrates linkages (i.e., ether type, ester type, ketal type) were found at the Cα-position of the side chain of phenylpropane units in ginkgo LCC. No lignin-carbohydrate bond at the Cβ- or Cγ-position of the lignin side chain was observed in the13C-NMR spectra of the13C-enriched LCCs. This fact indicates that a specific13C tracer technique can be useful in NMR study of the chemical structure of LCCs.