Isolation And Characterization Of Lignin: Cell Wall Cross-linking In Grasses By Lignin And Ferulates

Lignin, one of the major components in the cell wall of vascular plants, is the second most important and abundant nature polymer after cellulose. Isolation of representative lignin preparations from plant cell walls has been and still is a challenging task for researchers to study lignin structures. It becomes more difficult to isolate grass lignins because of cross-linking between carbohydrates and lignin by ferulates and diferulates. However, how these cross-linkages are formed is still in debates.In chapters 2 and 3 of this thesis, two new methods for isolation of lignin from plant cell wall were proposed and the isolated lignins by using such methods were characterized with 31P-NMR and 2D HSQC NMR etc.In the first method (Chapter 2), attempts were made to enhance polysaccharide digestibility by crude cellulases in the isolation of cellulolytic enzyme lignin (CEL) by dissolution of ball-milled wood in dimethyl sulfoxide (DMSO) and N-methylimidazole (NMI) solvent system as a pretreatment step. Wood regenerated from the DMSO/NMI solution was hydrolyzed with cellulases for 48 h, removing 73.7% and 66.9% of the original carbohydrate for basswood and loblolly pine, respectively; only 61.7% and 49.2% were hydrolyzed by cellulases without pretreatment. The yields of CEL isolated from regenerated ball-milled wood samples were therefore higher than directly from ball-milled wood material, presumably via decreasing the crystallinity of cellulose. For basswood, the yields of lignin were 45.8% and 36.5% (based on Klason lignin); for Loblolly Pine, the yields were 35.3% and 30.5%. The isolated lignins were structurally examined using two-dimensional heteronuclear single quantum coherence (1H–13C HSQC) NMR methods which showed that the main structural characteristics of the lignin fractions obtained using these two methods are similar except for slightly higher amounts of carbohydrates in SCEL.The second method developed in this work is a rapid three-step sequential extraction-precipitation method. It was used to isolate lignins from eucalyptus and fast-growing poplar, Triploid of Populus tomentosa Carr. The ball-milled wood material was extracted with 96% dioxane, 50% dioxane, and 80% dioxane containing 1% NaOH at boiling temperature, respectively, consecutively resulting in solubilization of lignin and hemicelluloses. By precipitating such solutions into 95% aqueous ethanol, the hemicelluloses were removed. Lignins in the ethanol solutions were recovered via concentration and precipitation in acidified water. The overall yields of lignin and hemicelluloses from Eucalyptus were 11.2% and 7.5% (based on dry weight), respectively. And they were 11.4% and 14.8% for the poplar. The obtained lignin fractions were characterized by quantitative 31P-NMR and two-dimensional heteronuclear single-quantum coherence (13C–1H) NMR. The results showed that dioxane extraction under conditions used in this study did not result in any significant changes of the macromolecular structure of lignin. The obtained lignin fractions were fairly free of carbohydrate except for the 50% dioxane soluble fraction. Lignin fraction obtained from 96% dioxane extraction was found to have more phenolic hydroxyl and less aliphatic hydroxyl than the other two preparations.Five different cell wall fractions from sugarcane bagasse were prepared: Fraction a: Ball-milled sugarcane bagasse; Fraction b: The residue after crude cellulase enzymatic hydrolysis; Fraction c: The water-soluble parts after the enzymatic hydrolysis; Fraction d: the residues after enzymatic hydrolysis followed by 96% aqueous dioxane extraction; Fraction e: the 96% dioxane-soluble components following enzymatic hydrolysis.Saponification at room temperature was used to release hydroxycinnamic acids from the above five fractions. The released p-CA, FA as well as their dimers were characterized and quantified by GC-MS. As resulted, the predominant monomers were p-coumaric acid and ferulic acid. p-Coumaric acid was found in both cis and trans forms, but the trans form in every case predominated. The content of p-CA in ball-milled sugarcane bagasse was 3.6%, however ferulic acid was only 0.27%. Among these fractions, fraction b contains most of the p-CA and fraction c has more FA than any other fractions. The content of p-CA and FA was 3.91% and 0.29% in fraction b and fraction c, respectively, based on dry weight of ball-milled bagasse, which indicated that most of p-CA was esterified into lignin, and the alkaline-releasable FA was linked to carbohydrate components. Most FA derived dimers detected by GC-MS were FA-(β-O-4)-CA cross-coupled dimer and FA homo-coupled dimers with linkages of 8-8', 8-5', 5-5'. Small amount of sinapyl (SA)-(β-O-4)-FA and p-CA-(8-8)-p-CA were found in all five fractions.Ethyl ferulate, a model for feruloyl polysaccharide esters in grass cell walls, readily couples with coniferyl alcohol in an in vitro model system producing variety of cross-coupled dimers. Among of them the cross-linking dimers of CA-(β-5)-FA and FA-(8-5)-CA were fistly reported. Four dimers fromed between ferulate, coniferyl alcohol, coniferyl aldehyde (CAld) and sinapyl alcohol (SA) were synthesized for the fist time. CA-(β-5)-FA, SA-(β-5)-FA and FA-(8-5)-CA crosslinkages were detected from grass cell walls for the fist time.Radical coupling reactions between ethyl ferulate (Et-FA), a simple model for feruloyl polysaccharides in planta, and coniferyl alcohol (CA), a monolignol, were studied in order to better understand the polymer cross-coupling interactions among polysaccharides and monolignols or lignin, mediated by ferulate (FA), in plant cell walls. Cross-coupled FA/CA dimers produced in an aqueous buffer (pH 5.0) containing peroxidase/hydrogen peroxide were isolated and characterized by NMR. The total coupling products were characterized by 2D 13C–1H correlation (HSQC) NMR spectroscopy and GC-MS. Results from this study showed that ferulate readily cross-couples with coniferyl alcohol through free-radical coupling mechanisms producing a series of cross-coupled FA/CA dimers withβ–O–4'-,β–5'-/8–5'-, and 8–β'-linkages; the syntheses and isolation ofβ–5'- and 8–5'-cross-coupled dimers are reported here. The transformation from 8–β'-coupled FA/CA hydroxyl esters into lactones through intramolecular transesterification is demonstrated for the first time and mechanisms behind these transformations are discussed. The finding of bothβ–5'- and 8–5'-cross-coupled dimers in this study suggests that analogs of both may be present in plant cell walls. Finally it is suggested that ferulates in plants indeed react with monolignols through free radical mechanisms producing a more diverse array of cross-coupled dimers than previously reported.According to the radical coupling mechanism, the presumedβ-5'/8-5' dimers formed by ferulate,coniferyl alcohol,coniferyl aldehyde and sinapy alcohol may be present in plant cell wall. However, the methods for synthesis of these kinds of dimers were rarely reported. In this thesis, the possible existed cross-couplesβ-5'/8-5' dimers were synthesized. Among of them FA-(8-5)-CAld.,SA-(β-5)–CAld.,SA-(β-5)-CA and SA-(β-5)-FA were synthesized for the first time. However, CA-(β-5)-FA,CA-(β-5)-CA,CA-(β-5)-CAld.,FA-(8-5)-CA were prepared using new method because the published method for preparation of these dimes were tedious and low yields. Coniferyl alcohol-(β-O-4)- and-(β-8)-ferulate cross-coupled products have been found in grass cell walls by NaOH hydrolysis, whereas the presumed CA-(β-5)-FA and FA-(8-5)-CA cross-coupled products are missing. We envision that CA-(β-5)-FA would lose formaldehyde, and FA-(8-5)-CA would decarboxylate to the same product in the currently used NaOH hydrolysis. The product can also not be distinguished from the decarboxylated diferulate. Its true origin can therefore not be determined. A novel approch combining basic hydrolysis and hydrogenation allows the diferulate and the cross-coupled products to be differentiated. By applying this method, the CA-(β-5)-FA and FA-(8-5)-CA cross-coupled dimers have now been authenticated in several plant cell walls.It is well known that ferulate could be incorported into lignin polymerization in the growing grass cell wall. However, because of its small amount, it's difficult to detect it directly using NMR technology from plant cell wall. A mild base treatment to hydrolyze esters between ferulic acid and polysaccharides would help to release lignins containing ferulic acid with ether linkages. In this paper, two alkali lignin samples of corn and bagasse were isolated by mild base treatment (1N NaOH, room temperature) of dioxane-water extracted ball-milled cell wall. Ferulate-lignin cross-linkages produced fromβ-5- andβ-O-4-cross-coupling modes have been observed and identified in these two alkali lignin samples by 2D NMR experiments. Finding such structures in lignin isolates provides further evidence that ferulate-lignin cross-linking is achieved through free radical coupling reactions.