Purification and Partial Characterization of Lignin Peroxidase from <Emphasis Type="Italic">Acinetobacter calcoaceticus</Emphasis> NCIM 2890 and Its Application in Decolorization of Textile Dyes

Lignin peroxidase was purified (72-fold) from Acinetobacter calcoaceticus NCIM 2890. The purified lignin peroxidase (55–65 kDa) showed dimeric nature. The maximum enzyme activity was observed at pH 1.0, between a broad temperature range of 50 and 70°C, at H₂O₂ concentration (40 mM) and the substrate concentration (n-propanol, 100 mM). Purified lignin peroxidase was able to oxidize a variety of substrates including Mn²⁺, tryptophan, mimosine, l-Dopa, hydroquinone, xylidine, n-propanol, veratryl alcohol, and ten textile dyes of various groups indicating as a versatile peroxidase. Most of the dyes decolorized up to 90%. Tryptophan stabilizes the lignin peroxidase activity during decolorization of dyes.     

Pathways for degradation of lignin in bacteria and fungi

Lignin is a heterogeneous aromatic polymer found as 10-35% of lignocellulose, found in plant cell walls. The bio-conversion of plant lignocellulose to glucose is an important part of second generation biofuel production, but the resistance of lignin to breakdown is a major obstacle in this process, hence there is considerable interest in the microbial breakdown of lignin. White-rot fungi are known to break down lignin with the aid of extracellular peroxidase and laccase enzymes. There are also reports of bacteria that can degrade lignin, and recent work indicates that bacterial lignin breakdown may be more significant than previously thought. The review will discuss the enzymes for lignin breakdown in fungi and bacteria, and the catabolic pathways for breakdown of the β-aryl ether, biphenyl and other components of lignin in bacteria and fungi. The review will also discuss small molecule phenolic breakdown products from lignin that have been identified from lignin-degrading microbes, and includes a bioinformatic analysis of the occurrence of known lignin-degradation pathways in Gram-positive and Gram-negative bacteria.     

Oxidation capacity of laccases and peroxidases as reflected in experiments with methoxy-substituted benzyl alcohols

A set of methoxy-substituted benzyl alcohol (MBA) congeners were examined regarding susceptibility to oxidation by Trametes versicolor laccase, T. versicolor lignin peroxidase and horseradish peroxidase: 2,4,5-trimethoxybenzyl alcohol (DMBA), 3,4,5-TMBA, 2,3,4-TMBA, 2,5-dimethoxybenzyl alcohol (DMBA), 3,4-DMBA, and 2,3-DMBA. The corresponding methoxysubstituted benzaldehydes were strongly predominant as products on enzymic oxidation. This together with different reaction rates and redox potentials makes the MBAs suitable as substrates in the characterization of ligninolytic enzymes. For fungal laccase, the reaction rate order was: 2,4,5-TMBA≫2,5-DMBA>3,4-DMBA>3,4,5-TMBA∼2,3,4-TMBA∼2,3-DMBA. Horseradish peroxidase displayed a similar reactivity order. Oxidation of some of the MBAs with laccase and horseradish peroxidase was only observed when the reactions were carried out at low pH and with relatively high-substrate concentration. 3,4-DMBA (veratryl alcohol) was the best substrate for lignin peroxidase and the reaction rate order was: 3,4-DMBA>2,4,5-TMBA∼3,4,5-TMBA>2,5-DMBA>2,3,4-TMBA∼2,3-DMBA. The oxidation experiments with different MBAs elucidate the potential of the enzymes as oxidants in various applications.     

Production of oxychemicals from precipitated hardwood lignin

Lignin is a major byproduct in the biomass-to-ethanol process. The lignin produced from acid treatment of biomass has characteristics suitable for further conversion to organic chemicals. It is free of contaminants and has a relatively low molecular weight. In this study, catalytic oxidative conversion of the acid-soluble lignin precipitated from acid hydrolysates of hardwood was investigated. The process is based on aqueous alkaline oxidation of lignin with dissolved O₂ in the presence of Fe³⁺ and Cu²⁺ catalysts at moderate reaction temperatures (160–180°C). Aromatic aldehydes, ketones, and organic acids are found to be the primary products identifiable on extraction with ether. The combined weight yield of the total ether extractable products is about 20–25% of the initial lignin. The yield of the aldehydes (vanillin + syringaldehyde) is in the vicinity of 15% with an additional 3 to 4% of aromatic ketones. The yields of aldehydes plus ketones observed in this work far exceeded those obtainable from the conventional alkaline air oxidation of spent sulfite liquors. This article also provides comprehensive batch reaction data on conversion and product distribution.     

Effects of pH and Temperature on Recombinant Manganese Peroxidase Production and Stability

The enzyme manganese peroxidase (MnP) is produced by numerous white-rot fungi to overcome biomass recalcitrance caused by lignin. MnP acts directly on lignin and increases access of the woody structure to synergistic wood-degrading enzymes such as cellulases and xylanases. Recombinant MnP (rMnP) can be produced in the yeast Pichia pastoris αMnP1-1 in fed-batch fermentations. The effects of pH and temperature on recombinant manganese peroxidase (rMnP) production by P. pastoris αMnP1-1 were investigated in shake flask and fed-batch fermentations. The optimum pH and temperature for a standardized fed-batch fermentation process for rMnP production in P. pastoris αMnP1-1 were determined to be pH 6 and 30 °C, respectively. P. pastoris αMnP1-1 constitutively expresses the manganese peroxidase (mnp1) complementary DNA from Phanerochaete chrysosporium, and the rMnP has similar kinetic characteristics and pH activity and stability ranges as the wild-type MnP (wtMnP). Cultivation of P. chrysosporium mycelia in stationary flasks for production of heme peroxidases is commonly conducted at low pH (pH 4.2). However, shake flask and fed-batch fermentation experiments with P. pastoris αMnP1-1 demonstrated that rMnP production is highest at pH 6, with rMnP concentrations in the medium declining rapidly at pH less than 5.5, although cell growth rates were similar from pH 4–7. Investigations of the cause of low rMnP production at low pH were consistent with the hypothesis that intracellular proteases are released from dead and lysed yeast cells during the fermentation that are active against rMnP at pH less than 5.5.     

Construction and characterization of a manganese-binding site in cytochrome c peroxidase: towards a novel manganese peroxidase

Background: Manganese-binding sites are found in several heme peroxidases, namely manganese peroxidase (MnP), chloroperoxidase, and the cationic isozyme of peanut peroxidase. The Mn-binding site in MnP is of particular interest. Oxidation of Mn(II) to Mn(III) is a key step in the biodegradation of lignin, a complex phenylpropanoid polymer, as well as of many aromatic pollutants. Cytochrome c peroxidase (CcP), which is structurally homologous to MnP despite a poor sequence homology, does not bind manganese. Thus, engineering a Mn-binding site into CcP will allow us to elucidate principles behind designing metal-binding sites in proteins, to understand the structure and function of this class of Mn-binding centers, and to prepare novel enzymes that can degrade both lignin and other xenobiotic compounds.Results: Based on a comparison of the crystal structures of CcP and MnP, a site-directed triple mutant (GIy41→ Glu, Val45 →- GIu, His181 → Asp) of residues near the putative Mn-binding site in CcP was prepared and purified to homogeneity. Titrating MnSO₄ into freshly prepared mutant CcP resulted in electronic absorption spectral changes similar to those observed in MnP. The calculated apparent dissociation constant and the stoichiometry of Mn-binding of CcP were also similar to MnP. Titration with MnSO₄ resulted in the disappearance of specific paramagnetically shifted nuclear magnetic resonance spectroscopy signals assigned to residues close to the putative Mn-binding site in the mutant CcP. None of the spectral features were observed in wild-type CcP. In addition, the triple mutant was capable of oxidizing Mn(II) at least five times more efficiently than the native CcP.Conclusions: A Mn-binding site has been created in CcP and based on our spectroscopic studies the designed Mn-binding site is similar to the Mn-binding site in MnP. The results provide a basis for understanding the structure and function of the Mn-binding site and its role in different heme peroxidases.     

木质素酶及其化学模拟的研究进展

木质素酶（包括木质素过氧化物酶和锰木质素过氧化物酶）及其模拟物对木质素的催化降解是绿色化学的一个重要研究课题。本文综述了近年来有关木质素酶的分子结构，活性中心结构、催化木质降解机理及模型物的研究进展。     

钌碳耦合碱还原催化降解三倍体毛白杨

化石资源的过度消耗及因此产生的环境问题驱动了生物质资源各组分转化为燃料、化学品及材料的研究发展.木质素是自然界最丰富的可再生芳香碳氢资源,也是生物质中最难以实现高值化利用的组分.将木质素催化降解生成苯酚类单体化合物是其高值化利用的关键,因此开发新型催化体系对于提高当前生物精炼产业经济效益具有重要意义.本文利用钌碳耦合碱...     

Alkoxyl- and carbon-centered radicals as primary agents for degrading non-phenolic lignin-substructure model compounds

Lignin degradation by white-rot fungi proceeds via free radical reaction catalyzed by oxidative enzymes and metabolites. Basidiomycetes called selective white-rot fungi degrade both phenolic and non-phenolic lignin substructures without penetration of extracellular enzymes into the cell wall. Extracellular lipid peroxidation has been proposed as a possible ligninolytic mechanism, and radical species degrading the recalcitrant non-phenolic lignin substructures have been discussed. Reactions between the non-phenolic lignin model compounds and radicals produced from azo compounds in air have previously been analysed, and peroxyl radical (PR) is postulated to be responsible for lignin degradation (Kapich et al., FEBS Lett., 1999, 461, 115-119). However, because the thermolysis of azo compounds in air generates both a carbon-centred radical (CR) and a peroxyl radical (PR), we re-examined the reactivity of the three radicals alkoxyl radical (AR), CR and PR towards non-phenolic monomeric and dimeric lignin model compounds. The dimeric lignin model compound is degraded by CR produced by reaction of 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), which under N(2) atmosphere cleaves the α-β bond in 1-(4-ethoxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-1,3-propanediol to yield 4-ethoxy-3-methoxybenzaldehyde. However, it is not degraded by the PR produced by reaction of Ce(4+)/tert-BuOOH. In addition, it is degraded by AR produced by reaction of Ti(3+)/tert-BuOOH. PR and AR are generated in the presence and absence of veratryl alcohol, respectively. Rapid-flow ESR analysis of the radical species demonstrates that AR but not PR reacts with the lignin model compound. Thus, AR and CR are primary agents for the degradation of non-phenolic lignin substructures.     

Depolymerization and hydrodeoxygenation of lignin to aromatic hydrocarbons with a Ru catalyst on a variety of Nb-based supports

Efficient conversion of lignin to aromatic hydrocarbons via depolymerization and subsequent hydrodeoxygenation is important. Previously, we found that NbO_x species played a key role in the activation and cleavage of C–O bonds in lignin and its model compounds. In this study, commercial niobic acid (HY-340), niobium phosphate (NbPO-CBMM) and lab-made layered niobium oxide (Nb₂O₅-Layer) were chosen as supports to study the effect of Brönsted and Lewis acids on the activation of C–O bonds in lignin conversion. A variety of Ru-loaded, Nb-based catalysts with different Ru particle sizes were prepared and applied to the conversion of p-cresol. The results show that all the Ru/Nb-based catalysts produce high mole yields of C₇–C₉ hydrocarbons (82.3–99.1%). What's more, Ru/Nb₂O₅-Layer affords the best mole yield of C₇–C₉ hydrocarbons and selectivity for C₇–C₉ aromatic hydrocarbons, of up to 99.1% and 88.0%, respectively. Moreover, it was found that Lewis acid sites play important roles in the depolymerization of enzymatic lignin into phenolic monomers and the cleavage of the C–O bond of phenols. Additionally, the electronic state and particle size of Ru are significant factors which influence the selectivity for aromatic hydrocarbons. A partial positive charge on the metallic Ru surface and a smaller Ru particle size are beneficial in improving the selectivity for aromatic hydrocarbons.     

Abrasive Stripping Voltammetric Studies of Lignin and Lignin Model Compounds

Lignin is potentially a major renewable, nonfossil source of aromatic and cyclohexyl compounds. In this study, we have investigated the abrasive stripping voltammetry of lignin and four lignin model compounds in the room temperature ionic liquids (RTILs) [C₄mim][NTf₂], [N_6,2,2,2][NTf₂] and [C₄mim][OTf] (where [C₄mim]⁺=1‐butyl‐3‐methylimidazolium, [N_6,2,2,2]⁺=n‐hexyltriethylammonium, [NTf₂]^?=bis(trifluoromethanesulfonyl)imide and [OTf]^? =trifluoromethanesulfonate) on a gold macrodisk and in 0.1 M H₂SO₄ and 0.1 M NaOH on a boron‐doped diamond (BDD) macroelectrode, with the hope of using the voltammetry to fingerprint the functional groups within the lignin molecule. The use of RTILs on metal electrodes, or either acidic or basic media in combination with BDD electrodes allows solvent systems with wide electrochemical potential windows, useful for studying voltammetry which may be difficult to observe in systems where early breakdown of the solvent occurs.     

In vitro degradation of insoluble lignin in aqueous media by lignin peroxidase and manganese peroxidase

The abilities of lignin peroxidase (LIP) and manganese peroxidase (MNP) fromPhanerochaete chrysosporium to degrade an insoluble hardwood lignin in vitro in aqueous media were tested. Neither LIP nor MNP appreciably changed the mass or lignin content, although both produced small amounts of unique solubilized lignin fragments. Treatment with both LIP and MNP, however, decreased the mass by 11%, decreased the lignin content by 5.1% (4.2% as total weight), and solubilized unique lignin-derived molecules. These results suggest that LIP and MNP synergistically degrade high molecular weight insoluble lignin, but singly, neither enzyme is sufficient to effect lignin degradation.     

Peroxidase activity of new mixed‐valence cobalt complexes with ligands derived from pyridoxal

New mixed‐valence cobalt complexes with ligands derived from pyridoxal were synthesized and characterized, and their application as mimetics of the peroxidase enzyme was investigated. Single‐crystal X‐ray diffraction was used to analyze all complex structures in the solid state and their electrochemical behavior was investigated. A reactivity pattern was observed in the complex synthesis regarding the cobalt compounds from which analogous zwitterionic derivatives were obtained. The importance of these compounds lies in understanding their behavior in an oxidizing environment and evaluating whether they can activate hydrogen peroxide to oxidize phenolic compounds. In nature, enzymes called peroxidases, which efficiently oxidize phenolic compounds, trigger many reactions involving the activation of hydrogen peroxide to oxidize organic substrates. However, these enzymes present several disadvantages, including denaturation and elevated costs. Therefore, these limitations can be overcome by expanding research into the study of synthetic catalysts for the oxidation of phenolic compounds using hydrogen peroxide, which is a highly relevant field of bioinorganic chemistry.     

Lignin Peroxidase from Streptomyces viridosporus T7A: Enzyme Concentration Using Ultrafiltration

It is well known that lignin degradation is a key step in the natural process of biomass decay whereby oxidative enzymes such as laccases and high redox potential ligninolytic peroxidases and oxidases play a central role. More recently, the importance of these enzymes has increased because of their prospective industrial use for the degradation of the biomass lignin to increase the accessibility of the cellulose and hemicellulose moieties to be used as renewable material for the production of fuels and chemicals. These biocatalysts also present potential application on environmental biocatalysis for the degradation of xenobiotics and recalcitrant pollutants. However, the cost for these enzymes production, separation, and concentration must be low to permit its industrial use. This work studied the concentration of lignin peroxidase (LiP), produced by Streptomyces viridosporus T7A, by ultrafiltration, in a laboratory-stirred cell, loaded with polysulfone (PS) or cellulose acetate (CA) membranes with molecular weight cutoffs (MWCO) of 10, 20, and 50 KDa. Experiments were carried out at 25 °C and pH 7.0 in accordance to the enzyme stability profile. The best process conditions and enzyme yield were obtained using a PS membrane with 10 KDa MWCO, whereby it was observed a tenfold LiP activity increase, reaching 1,000 U/L and 90% enzyme activity upholding.     

Development of Ceria-Supported Ruthenium Catalysts Effective for Various Synthetic Reactions

Our recent results on organic transformations such as C–C bond formation via the activation of stable C–C or C–H bonds and aerobic oxidation of alcohols catalyzed by CeO₂-supported ruthenium are reviewed. A simple, recyclable heterogeneous Ru/CeO₂ catalyst showed excellent activity for sequential transfer-allylation/isomerization of homoallyl alcohols with aldehydes to saturated ketones via the C–C bond activation. While homogeneous ruthenium and rhodium complex catalysts require additives and/or pressurized CO, the reaction with Ru/CeO₂ smoothly proceeded in the absence of any additives. The Ru/CeO₂ catalyst also showed excellent activity for the addition of sp² C–H bonds of aromatic ketones to vinylsilanes. The Ru/CeO₂ catalyst realized the chelation-assisted arylation of stable aromatic C–H bonds with aryl chlorides. The activity of the catalyst was greatly improved by the PPh₃-modification under hydrogen atmosphere prior to the reactions. The catalyst acts heterogeneously without a significant leaching of ruthenium species, indicating that the Ru/CeO₂ catalyst has an advantage over homogeneous catalysts from practical and environmental points of view. The effects of chemical and physical properties of CeO₂ on the activity of CeO₂-supported noble metal catalysts were examined. Porous CeO₂ powders were prepared by the coagulation of solvothermally synthesized colloidal ceria nanoparticles, and the thus-prepared CeO₂ powders showed an oxygen migration ability far superior to the CeO₂ samples prepared by the usual precipitation method. The ruthenium catalysts supported on the former CeO₂ powders showed a high activity for the aerobic oxidation of benzyl alcohol. The effects of the pore structure of CeO₂ powders on the activity of the Ru/CeO₂ catalysts are also discussed.     

Advancements and Perspectives toward Lignin Valorization via O-Demethylation

Lignin represents the largest aromatic carbon resource in plants, holding significant promise as a renewable feedstock for bioaromatics and other cyclic hydrocarbons in the context of the circular bioeconomy. However, the methoxy groups of aryl methyl ethers, abundantly found in technical lignins and lignin-derived chemicals, limit their pertinent chemical reactivity and broader applicability. Unlocking the phenolic hydroxyl functionality through O-demethylation (ODM) has emerged as a valuable approach to mitigate this need and enables further applications. In this review, we provide a comprehensive summary of the progress in the valorization of technical lignin and lignin-derived chemicals via ODM, both catalytic and non-catalytic reactions. Furthermore, a detailed analysis of the properties and potential applications of the O-demethylated products is presented, accompanied by a systematic overview of available ODM reactions. This review primarily focuses on enhancing the phenolic hydroxyl content in lignin-derived species through ODM, showcasing its potential in the catalytic funneling of lignin and value-added applications. A comprehensive synopsis and future outlook are included in the concluding section of this review.     

Laser-induced breakdown spectroscopy for polymer identification

This study aims at differentiating several organic materials, particularly polymers, by laser induced breakdown spectroscopy. The goal is to apply this technique to the fields of polymer recycling and cultural heritage conservation. We worked with some usual polymers families: polyethylene (PE), polypropylene (PP), polyoxymethylene, (POM), poly(vinyl chloride), polytetrafluoroethylene, polyoxyethylene (POE), and polyamide for the aliphatic ones, and poly(butylene terephthalate), acrylonitrile–butadiene–styrene, polystyrene, and polycarbonate for the aromatic ones. The fourth harmonic of a Nd:YAG laser (266 nm) in ambient air at atmospheric pressure was used. A careful analysis of the C₂ Swan system (0,0) band in polymers containing no C–C (POM), few C–C (POE), or aromatic C–C linkages led us to the conclusion that the C₂ signal might be native, i.e., the result of direct ablation from the sample. With use of these results, aliphatic and aromatic polymers could be differentiated. Further data treatments, such as properly chosen line ratios, principal component analysis, and partial least squares regression, were evaluated. It was shown that many polymers could be separated, including PE and PP, despite their similar chemical structures.     

Biodegradation of aromatic compounds by the yeast-like fungus Trichosporon cutaneum

The biodegradation reactions of veratryl- and guaiacylpropane compounds modeling structural fragments of the lignin macromolecule by the yeast-like fungusTrichosporon cutaneum D-46 have been investigated. It has been shown that the enzyme complex ofT. cutaneum is capable of catalyzing a series of reactions of the biotransformation of arylpropane substrates, namely: oxidation, the cleavage of the C_α-C_β bond of the propane chain, demethylation, and decarboxylation. The action of the enzymes was shown most actively in the case of substrates having an unsubstituted phenolic hydroxy group. Institute of Organic Chemistry, Siberian Branch, Russian Academy of Sciences, Irkutsk. Institute of Microbiology, Belorussian Academy of Sciences, Minsk. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 558–564, September–October, 1992.     

Mechanistic studies on the effect of veratryl alcohol on the lignin peroxidase catalyzed oxidation of pyrogallol red in reversed micelles

The lignin peroxidase (LiP) catalyzed oxidation of pyrogallol red (PR) in the absence and presence of veratryl alcohol (3,4-dimethoxybenzyl alcohol, VA) was carried out in bis (2-ethylhexyl) sulfosuccinate sodium (AOT)/ polyoxyethylene lauryl ether (Brij30) reversed micelles to elucidate the role of VA. Results indicated that VA could accelerate the LiP catalyzed oxidation of PR, especially at low H₂O₂ concentrations. Unlike in bulk aqueous medium, the protection of LiP by VA in the present medium was relatively unsubstantial, even at high H₂O₂ concentrations. Analysis of data from a series of experiments showed that the enhancement of the PR oxidation caused by VA was mainly due to the indirect oxidation of PR by VA^+∙ from the LiP catalyzed oxidation of VA. It was also found that at the same protector concentration (40 μM), VA (the physiological substrate of LiP) was less effective than PR (a phenolic compound) in protecting LiP from the H₂O₂ derived inactivation. This novel phenomenon deserves further study.      

Diamond-like carbon films for polyethylene femoral parts: Raman and FT-IR spectroscopy before and after incubation in simulated body liquid

In artificial prosthetics for knee, hip, finger or shoulder joints, ultrahigh molecular weight polyethylene (UHMW-PE) is a significant material. Several attempts to reduce the wear rate of UHMW-PE, i.e. the application of suitable coatings, are in progress. A surface modification of polyethylene with wear-resistant hydrogenated diamond-like carbon is favourable, owing to the chemical similarity of polyethylene (–C–H₂–) _n and C:H or amorphous C:H (a–C:H) coatings with diamond-like properties. In the present study, the microstructure of a–C:H coatings on UHMW-PE substrates was investigated by Raman and Fourier transform infrared (FT-IR) spectroscopy. FT-IR spectroscopy shows very broad absorption lines, which point to the disorder and diversity of different symmetric, asymmetric aromatic, olefin sp ²-hybridized or sp ³-hybridized C–H groups in the amorphous diamond-like carbon coating. Following a long incubation of 12 months in a simulated body liquid, the structural investigations were repeated. Furthermore, fractured cross-sections and the wetting behaviour with polar liquids were examined. After incubation in simulated body liquid, Raman spectroscopy pointed to a reduction of the C–H bonds in the diamond-like carbon coatings. On the basis of these findings, one can conclude that hydrogenated diamond-like carbon is able to interact with salt solutions by substituting the hydrogen with appropriate ions.