Effect of phenolic acids on manganese peroxidase-dependent peroxidation of linoleic acid and degradation of a non-phenolic lignin model compound

The influence of aromatic phenolic and non-phenolic acids on manganese peroxidase (MnP)-dependent peroxidation of linoleic acid, and oxidation of a non-phenolic lignin model compound (LMC) was studied. Phenolic compounds inhibited both the MnP-dependent lipid peroxidation (LPO) and non-phenolic LMC degradation in the system. The antioxidant activity of the aromatic compounds in the enzymatic system with MnP-dependent LPO depends on the presence of the phenolic hydroxyl groups attached to the aromatic ring structure, the methoxylation of the hydroxyl group in the ortho position in diphenolics, and number of carbon atoms in the side chain. Natural phenolic compounds inhibit the oxidation of non-phenolic lignin in the system based on MnP-mediated LPO, but do not prevent it. This result indicates that MnP-mediated LPO may play an important role in lignin degradation even in the presence of the phenolic antioxidant compounds, and supports the possibility of the involvement of LPO in the degradation of lignin in wood.     

Degradation of non-phenolic lignin by the white-rot fungus Pycnoporus cinnabarinus

High-molecular-weight lignin was methylated with diazomethane. The lignin (i.e., phenolic lignin) and methylated lignin (i.e., non-phenolic lignin) were mixed with fully bleached softwood pulp. Degradation of the lignin preparations by the white rot fungus Pycnoporus cinnabarinus was studied. After a 3-month incubation with the fungus, over 40% of the non-phenolic lignin and about 70% the phenolic lignin were degraded. The presence of phenolic hydroxyl groups in lignin greatly enhanced the degradation rate of lignin. This study reveals that P. cinnabarinus, an exclusively laccase-producing fungus, is capable of oxidatively degrading both phenolic and non-phenolic lignins. The ability of the fungus to degrade non-phenolic lignin suggests that a laccase/mediator system is involved in the complete degradation of lignin. After the fungal degradation of lignins, the content of carboxylic acids substantially increased for both phenolic and non-phenolic lignins.     

漆酶／介体体系降解黄麻纤维木质素的研究

为了研究漆酶／介体处理过程中,黄麻纤维木质素结构的变化,采用二氧六环水溶液抽提制取了黄麻纤维木质素,再用漆酶／介体对其处理,通过GPC、元素分析、酚羟基含量测定、红外光谱以及核磁共振氢谱分析了漆酶／介体处理后,黄麻纤维木质素结构的变化。结果表明：经漆酶／介体处理后,黄麻纤维木质素重均分子量和数均分子量减小,酚羟基、醇羟基以及甲氧基含量降低,羰基含量增加。     

Evaluating laccase-facilitated coupling of phenolic acids to high-yield kraft pulps

In an effort to alter the physical properties of high-yield kraft, fibers were treated at high consistency (20%) with laccase and syringic, vanillic, or 4-hydroxybenzoic acid. Treatment with laccase and 4-hydroxybenzoic acid resulted in a 20-point increase in kappa number and a 100% increase in bulk acid groups. ESCA analysis of the treated and untreated pulp revealed that the laccase-grafted fibers had a two-fold enrichment in acid groups, strongly suggesting a laccase-facilitated coupling of 4-hydroxybenzoic acid to the fiber surface. A model system consisting of lignin-coated cellulosic fibers was developed to determine changes to the lignin structure during laccase grafting. ³¹P NMR analysis of lignin from the model system revealed an increase in acid groups with a concomitant decrease in phenolic hydroxyl groups.     

Role of laccase in lignin degradation by white-rot fungi

Abstract Laccase is commonly found in white-rot fungi and catalyses the abstraction of one electron from the phenolic hydroxyl group to polymerize or depolymerize lignin model compounds. Laccase degrades both β-1 and β-O-4 dimers via C _α - C _β cleavage, C _α oxidation and alkyl-aryl cleavage. Also, aromatic ring cleavage may be detected following the action of laccase. Laccase can also oxidize non-phenolic compounds when primary mediators, such as 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate), are co-present. Laccase produces Mn(III) chelates which allow wood-decaying enzymes to penetrate wood cell walls. Laccase is considered to be capable of degrading lignin together with lignin peroxidase and manganese peroxidase.     

Incorporation of p-cresol into lignins via peroxidase-catalysed copolymerization in nonaqueous media

Lignin, the second most abundant biopolymer after cellulose, is a low value by-product of agricultural and wood conversion processes, including wood pulp manufacture. Copolymerization with phenols has the potential to convert by-product lignins to higher-value phenolic resins. In this initial investigation, we have studied the use of horseradish peroxidase (HRP) in aqueous dioxane to catalyse the grafting of p-cresol (p-methylphenol) onto milled wood lignin, kraft lignin, and a lignin selectively o-demethylated by a brown-rot fungus. Advantages of this system are (1) the mild reaction conditions employed and (2) the ability of HRP to function in the dioxane: water solutions which solubilize lignin. The reaction is monitored by gel permeation chromatography using a reaction system of [¹⁴C]-p-cresol with unlabeled lignins. We have found that optimal incorporation of cresol into high-molecular-weight polymer occurs at 50–70% dioxane in water under the conditions used; a maximum incorporation of ca. 4 mol% (e.g., p-cresol incorporated per C₉ lignin unit) was obtained. Blocking the phenolic hydroxyl groups of the lignin inhibits copolymerization, consistent with the proposed mechanism of phenoxy radical copolymerization for this reaction.     

Hydrogen bonding in lignin: a Fourier transform infrared model compound study

Hydrogen bonding plays an important role in the thermal and mechanical properties of biopolymers. To investigate hydrogen bond formation in lignin, an abundant natural polymer found in plants, Fourier transform infrared (FTIR) analysis of various lignin model compounds was performed. Four monomeric model compounds and one dimeric model compound were studied under various conditions. FTIR analysis revealed aliphatic hydroxyl groups form stronger hydrogen bonds than phenolic hydroxyl groups. Further, the dimeric biphenyl-type structure formed significantly stronger intermolecular hydrogen bonds as compared to the other monomeric model compounds. Results from the model compound studies were used to explain the observed complex hydrogen-bonding system present in both softwood and hardwood technical lignins. Together with chemical analysis, we discuss the difference in hydrogen bonding between hardwood and softwood lignin and the observed differences in the glass transition temperature.     

Enzymatic modification of kraft lignin through oxidative coupling with water-soluble phenols

The aromatic polymer lignin can be modified through promotion of oxidative coupling between phenolic groups on lignin and various phenols. The reaction is initiated by an oxidation of both components, e.g., by using the oxidoreductases laccase or peroxidase. Coupling between phenolic monomers and lignin has previously been studied by the use of radio-labeled phenols. In this study, incorporation of water-soluble phenols into kraft lignin, using laccase as catalyst, was investigated. Several phenols with carboxylic or sulfonic acid groups were used as markers for the incorporation. The modified lignin was isolated and the amount of phenol incorporated was characterized by means of titration, quantitative 1H-NMR, and quantitative 31P-NMR after modification with 2-chloro-4,4,5,5-tetramethyl-1,2,3-dioxaphospholane. Only a few of the phenols studied were found to be incorporated into lignin. When the phenol guaiacol sulfonate was incorporated into kraft lignin, the lignin became water-soluble at pH 2.4 and a low ionic strength due to the introduction of sulfonic acid groups. The content of sulfonic acid groups in the product was 0.5-0.6 mmol/g lignin. A lower amount of 4-hydroxyphenylacetic acid was incorporated under similar conditions.     

木质素酚醛树脂的研究进展

综述酚醛树脂发展史、种类、合成机理及进展,由于木质素分子中有酚羟基和醛基,因此使用木质素既可改善胶粘剂的性质,又可节约苯酚的用量,降低甲醛释放量,达到废物利用与保护环境的目的。重点论述了新型改性酚醛树脂的研究进展,尤其木质素改性酚醛树脂,并对新型酚醛树脂复合材料的发展方向和前景作了展望。     

Monitoring of hydroxyl groups in wood during heat treatment using NIR spectroscopy

This paper deals with the evaluation of thermally treated wood by near-infrared (NIR) spectroscopy. In the NIR second derivative spectrum, the absorption band at 6913 cm(-1) appeared with the procession of heat treatment, which conclusively assigned to the phenolic hydroxyl groups due to the lignin in comparison with the spectrum of acetylated spruce wood. As a result of the changes in the ratio of the areal integral calculated from spectral separation in the region of hydroxyl groups (7200-6100 cm(-1)) by the Gauss-Newton method, it was clear that the degradation of hydroxyl group in the cellulose started predominantly from the amorphous region and followed to semicrystalline and crystalline region. There was an obvious correlation between the weight decrement of wood and the decrement of hydroxyl groups in the cellulose by heat treatment.     

Degradation of benzyl ether bonds of lignin by ruminal microbes

We examined microbial activity in the rumen to cleave benzyl ether bonds of lignin model compounds that fluoresced when the bonds were cleaved. 4-Methylumbelliferone veratryl ether dimer was degraded completely within 8 h even in the presence of fungicidal antibiotics, but no significant degradation occurred with bactericidal antibiotics. Degradation of a phenolic beta-O-4 trimer incorporating 4-methylumbelliferone by a benzyl ether linkage was stimulated by ruminal microbes, although its corresponding non-phenolic model compound, 1-(4-ethoxy-3-methoxyphenyl)-1-O-(4-methylumbelliferyl)-2-(2-methoxyp henoxy)-3-propanol, was not degraded. A coniferyl dehydrogenation polymer bearing fluorescent beta-O-4 benzyl ether that contains both phenolic and non-phenolic benzyl ether bonds was partially degraded (about 20%) in 48 h. These results suggest that ruminal microbes decompose benzyl ether linkages of lignin polymers under anaerobic conditions.     

Do the extracellular enzymes cellobiose dehydrogenase and manganese peroxidase form a pathway in lignin biodegradation?

The extracellular enzyme manganese peroxidase is believed to degrade lignin by a hydrogen peroxide-dependent oxidation of Mn(II) to the reactive species Mn(III) that attacks the lignin. However, Mn(III) is not able to directly oxidise the non-phenolic lignin structures that predominate in native lignin. We show here that pretreatment of a non-phenolic lignin model compound with another extracellular fungal enzyme, cellobiose dehydrogenase, allows the manganese peroxidase system to oxidise this molecule. The mechanism behind this effect is demethoxylation and/or hydroxylation, i.e. conversion of a non-phenolic structure to a phenolic one, mediated by hydroxyl radicals generated by cellobiose dehydrogenase. This suggests that cellobiose dehydrogenase and manganese peroxidase may act in an extracellular pathway in fungal lignin biodegradation. Analytical techniques used in this paper are reverse-phase high-pressure liquid chromatography, gas chromatography connected to mass spectroscopy and UV-visible spectroscopy.     

Biodegradation of two tetrameric lignin model compounds by a mixed bacterial culture

Summary The ability of a mixed bacterial culture to decompose two tetrameric lignin model com-pounds as a sole source of carbon and energy was investigated. The mixed bacterial culture con-sisted mainly of Gram negative rods. The tetram-ers contained two types of lignin substructures, namely the most abundant β-O-4 ether structure in lignin and also the 5-5 biphenyl structure. The tetramer (MW 638) containing two phe-nolic hydroxyls was decomposed readily; after 13 days of incubation, all intermediate products formed were almost totally decomposed. The non-phenolic tetramer (MW 666) was decom-posed much more slowly; after 53 days of incuba-tion, 5% of the substrate was unchanged. When both tetramers were degraded simultaneously, the non-phenolic tetramer was decomposed similarly to the phenolic tetramer. Determination of molecular weights of cata-bolic products showed that the degradation of the non-phenolic tetramer had proceeded at least to dimer level. SKF 525A, inhibitor of cytochrome P-450, caused one catabolic product to accumulate in the culture medium. This indicates involvement of cy-tochrome P-450 in the degradation pathway of the model compounds used. We conclude that this mixed bacterial culture was able to degrade the lignin model compounds used and that free phenolic groups seem to in-crease the biodegradability significantly.     

Selective synthesis of 4-methoxyestrogen from 4-hydroxyestrogen.

The introduction of an oxygen atom into the C-6 position of 4-hydroxyestrogen allowed for the selective methylation of the two phenolic hydroxyl groups. When the 6-oxo derivative of 4-hydroxyestrone was benzylated in ethanol, only the 3-monobenzyl ether was obtained without formation of the 4-monobenzyl ether. Moreover, the 6-carbonyl group was further reduced to methylene almost quantitatively in the reaction of 4-acetoxy-6-oxoestrone 3-benzyl ether derivative with sodium borohydride. Therefore, 4-methoxyestrogen was synthesized by essentially combining these two reactions.     

Structural differences between the lignin-carbohydrate complexes present in wood and in chemical pulps

Lignin-carbohydrate complexes (LCCs) were prepared in quantitative yield from spruce wood and from the corresponding kraft and oxygen-delignified pulps and were separated into different fractions on the basis of their carbohydrate composition. To obtain an understanding of the differences in lignin structure and reactivity within the various LCC fractions, thioacidolysis in combination with gas chromatography was used to quantify the content of beta-O-4 structures in the lignin. Periodate oxidation followed by determination of methanol was used to quantify the phenolic hydroxyl groups. Furthermore, size exclusion chromatography (SEC) of the thioacidolysis fractions was used to monitor any differences between the original molecular size distribution and that after the delignification processes. Characteristic differences between the various LCC fractions were observed, clearly indicating that two different forms of lignin are present in the wood fiber wall. These forms are linked to glucomannan and xylan, respectively. On pulping, the different LCCs have different reactivities. The xylan-linked lignin is to a large extent degraded, whereas the glucomannan-linked lignin undergoes a partial condensation to form more high molecular mass material. The latter seems to be rather unchanged during a subsequent oxygen-delignification stage. On the basis of these findings, a modified arrangement of the fiber wall polymers is suggested.     

酚酮类对树干毕赤酵母乙醇发酵及脂肪酸组成的影响

木质素降解产物对微生物产生的抑制作用,是燃料乙醇生物炼制的主要瓶颈之一。本文以树干毕赤酵母为发酵菌株,研究木质素降解产物中3种酚酮类(4-羟基苯乙酮、4-羟基-3-甲氧基苯乙酮、4-羟基-3,5-二甲氧基苯乙酮)对其木糖乙醇发酵及酵母细胞脂肪酸组成的影响。采用高效液相色谱(HPLC)和气相色谱-质谱联用(GC/MS)技术对乙醇发酵性能和酵母细胞脂肪酸组成进行分析。研究结果表明,酚酮类物质对乙醇发酵呈现抑制作用且其分子量越低抑制作用越明显,当4-羟基苯乙酮浓度为1.50 g/L时,发酵24 h的木糖利用率、乙醇得率和乙醇浓度分别下降了42.47%、5.30%和9.76 g/L;培养基中存在酚酮类物质时,酵母细胞中的不饱和脂肪酸的比例上升,添加1.50 g/L的3种酚酮类物质后,树干毕赤酵母细胞不饱和脂肪酸和饱和脂肪酸的比例从原来的2.58分别上升到3.03、3.06和3.61,酵母细胞膜的流动性随之上升,不稳定性提高。因此,酚酮类物质能够降低酵母生长、提高不饱和脂肪酸的比例以及降低乙醇发酵能力,有效降低或去除木质素降解产物含量是提高木质纤维原料生物炼制的关键。     

Production of useful modified lignin polymers by bioconversion of lignocellulose with Streptomyces

Lignin degrading strains of Streptomyces were grown on lignocelluloses from a variety of plant sources. These actinomycetes readily degraded the lignin present in the residues and released a major portion of the lignin into the growth medium as a water soluble, modified polymer. The polymer, an acid precipitable polyphenolic lignin (APPL), was recovered from spent culture media by acid precipitation or dialysis/lyophilization. APPL's were shown to be mostly free of nonlignin components. As compared to native lignin they were more oxidized, were especially enriched in phenolic hydroxyl groups, and were significantly reduced in methoxyl groups. The yield of APPL from different lignocelluloses correlated with their biodegradability. Grasses such as corn stover were the optimal lignocellulose type for APPL production by Streptomyces. In contrast white-rot fungi produced only small amounts of APPL as they decomposed lignin. A solid state bioconversion process was developed using Streptomyces viridosporus T7A to produce APPL from corn stover lignocellulose in yields >or= 30% of the initial lignin present in the substrate. APPL produced by S. viridosporus was examined for its properties and possible use as an antioxidant. The APPL was shown to have good antioxidant properties after mild chemical treatment to reduce the alpha-carbonyl groups present in the APPL. Oxidation of the APPL with hydroxyl radical (OH(*)) further improved its antioxidant properties probably as the result of aromatic ring hydroxylation reactions. As compared with currently used commercial antioxidants, the modified APPL was thought to be competitive when economics of production was considered. Native lignin on the other hand was shown to exhibit no antioxidant properties, even after reduction and/or oxidation.     

Potentiality of lignin from the Kraft pulping process for removal of trace nickel from wastewater: effect of demineralisation

An industrial raw Kraft lignin was investigated to ascertain its potential use for removal of trace Ni(II) ion from wastewater by using dilute solutions (0.34-1.7 mM) as models. The effect of demineralisation on its metal sorption ability was examined by employing acid pre-treated samples. Under fixed pre-established equilibrium conditions, the raw lignin exhibited a lower effectiveness than the demineralised one, with the latter attaining an almost complete removal of Ni(II) ions. For both lignins, sorption kinetics was properly described by a pseudo-second order rate model. Equilibrium isotherms were also determined and adequately represented by conventional two-parameter models. The higher nickel sorption capacity for the demineralised lignin compared to the raw sample was consistent with enhancements in the negative magnitude of zeta potential, sodium sorption capacity, and content of phenolic hydroxyl groups occasioned by the acid pre-treatment. Accordingly, demineralisation appears as a readily convenient strategy to improve the behaviour of industrial Kraft lignin for potential use as a biosorbent of trace nickel from polluted water.     

Synthesis of dihydrofuran-fused perhydrophenanthrenes having a phenolic hydroxyl group as a novel anti-Alzheimer's disease agent

As a part of our research program on developing novel anti-Alzheimer's disease medicines, several dihydrofuran-fused perhydrophenanthrenes (DFs) possessing a phenolic hydroxyl group were found to exhibit potent dendritic and axonal regeneration activities. Introduction of a methoxy group into the perhydrophenanthrene skeleton was successfully achieved via a PhI(OAc)(2)-mediated phenolic oxidation of a benzocyclobutene nucleus and subsequent tandem intramolecular electrocyclic reactions based on o-quinodimethane chemistry. We could reveal that a new methoxy derivative having a phenolic hydroxyl group exerted the most significant effects on the dendritic and axonal extensions in the damaged neurons, among DFs examined in this study.     

Hydroxyl radical generation by an extracellular low-molecular-weight substance and phenol oxidase activity during wood degradation by the white-rot basidiomycete Trametes versicolor

One-electron oxidation activity, as measured by ethylene generation from 2-keto-4-thiomethylbutyric acid, phenol oxidase activity, and the generation of hydroxyl radical were examined in cultures of the lignin-degrading white-rot basidiomycete fungus, Trametes (Coriolus) versicolor. The activity levels of specific lignin-degrading enzymes and cellulases, as well as the rate of wood degradation, also were examined. The fungus secreted a low-molecular-weight substance (M(r) 1000-5000) that catalyzed a redox reaction between molecular oxygen and an electron donor, to produce the hydroxyl radical via hydrogen peroxide. During wood decay, T. versicolor also produced significant amounts of laccase and lignin peroxidase, carboxymethyl cellulase, and Avicelase. The roles of the hydroxyl radical, phenol oxidases, and cellulases in wood degradation by white-rot fungi are discussed. That the hydroxyl radical produced by the low-molecular-weight substance secreted by T. versicolor results in new phenolic substructures on the lignin polymer, making it susceptible to attack by laccase or manganese peroxidase is suggested.