Versatile Peroxidase Research Articles

Improvement of versatile peroxidase activity and stability by a cholinium-based ionic liquid

Deep eutectic solvents (DESs) have been introduced as green solvents and less expensive alternatives for ionic liquids. In the current study, effects of a cholinium based DES, Choline chloride:Glycerol (ChCl:G), on function and structure of versatile peroxidase (VP) were studied. Functional analysis of VP in the presence of various concentrations of ChCl:G revealed lower Km values at lower concentrations and improved activity in neutral pH. According to our results, VP presented higher exposure of hydrophobic patches and formed protein assemblies as revealed by extrinsic fluorescence, fluorescence lifetime, and anisotropy measurements. Circular dichroism and UV–vis spectroscopies demonstrated secondary structural changes and some rearrangements in the heme region. Moreover, ChCl:G seemed to protect VP from thermal denaturation probably by forming a protective layer around VP molecules. In contrast to experimental studies on hydrated ChCl:G, molecular dynamic (MD) simulations of VP in a box of pure ChCl:G suggested minor conformational changes. However, MD studies confirmed the rearrangements of the heme region in the presence of ChCl:G compared to water. According to the results, the authors concluded that lower concentration of ChCl:G could improve VP's activity and kinetic due to inducing some conformational changes, particularly in the heme vicinity, and also by stabilizing the intermediate complex during catalysis.

The Role of Ligninolytic Enzymes Laccase and a Versatile Peroxidase of the White‐Rot Fungus Lentinus tigrinus in Biotransformation of Soil Humic Matter: Comparative In Vivo Study

AbstractSoil organic matter (SOM) turnover by ligninolytic fungi is a large‐scale process that controls organic carbon geochemistry in terrestrial ecosystems. However, the role of certain oxidative enzymes (e.g., laccase) in humus degradation remains unclear, as well as the molecular structure and recalcitrance of SOM components. In order to address these questions, the degradation of forest soil humic acid (HA) in the presence of laccase and a versatile peroxidase (VP) has been studied in the liquid culture of Lentinus tigrinus. Contrary to the evolving views on humus structure, we have found that alkali‐extractable and acid‐insoluble constituents of SOM (HA) contain true macromolecular components, stable in the presence of 0.1% sodium dodecyl sulfate but degradable/resynthesizable by oxidative enzymes acting on covalent linkages. The HA degradation in the presence of laccase (high N medium) occurs at slower initial rate than in the presence of VP (low N medium). However, each of the enzymes caused about 60% color loss and almost complete degradation of HA into smaller molecules (gel‐filtration data) within 2 weeks of cultivation. Depolymerization of HA in the culture liquid in the presence of laccase was accompanied by polymerization of degradation products on mycelium. Our results show that (1) humus macromolecules are not stable to oxidative enzymes once desorbed from the mineral phase, (2) laccase of Lentinus tigrinus is comparable by its degradation potential to VP, and (3) interfacial secondary synthesis reactions occur during humus decay in the presence of laccase. Our results highlight the important role of laccases in SOM sequestration in soils.

Activities of Versatile Peroxidase in Cultures of Clonostachys rosea f. catenulata and Clonostachys rosea f. rosea during Biotransformation of Alkali Lignin

The aim of this study was the evaluation of activities of versatile peroxidase (VP) in cultures of Clonostachys sp. (Gliocladium sp.) strains during biotransformation of 0.2% alkali lignin (AL). The principal component analysis (PCA) method was applied to determine the main factors and correlation between biotransformation of 0.2% AL, activity of VPs, pH value, and Clonostachys sp. strains. The biotransformation of 0.2% AL in cultures of microscopic fungi included decolorization (medium lightening) and colorization (darkening of the medium). The versatile peroxidase synthesized by the microscopic fungi tested showed activity for oxidation of Mn(II) and guaiacol, but the activity for oxidation of guaiacol in the presence of Mn(II) was significantly higher. The PCA analysis indicated a strong correlation between biotransformation of 0.2% AL and pH and between oxidation of Mn(II), guaiacol without and in the presence of Mn(II) ions, strains, and pH.

Integrative visual omics of the white-rot fungus Polyporus brumalis exposes the biotechnological potential of its oxidative enzymes for delignifying raw plant biomass

BackgroundPlant biomass conversion for green chemistry and bio-energy is a current challenge for a modern sustainable bioeconomy. The complex polyaromatic lignin polymers in raw biomass feedstocks (i.e., agriculture and forestry by-products) are major obstacles for biomass conversions. White-rot fungi are wood decayers able to degrade all polymers from lignocellulosic biomass including cellulose, hemicelluloses, and lignin. The white-rot fungus Polyporus brumalis efficiently breaks down lignin and is regarded as having a high potential for the initial treatment of plant biomass in its conversion to bio-energy. Here, we describe the extraordinary ability of P. brumalis for lignin degradation using its enzymatic arsenal to break down wheat straw, a lignocellulosic substrate that is considered as a biomass feedstock worldwide.ResultsWe performed integrative multi-omics analyses by combining data from the fungal genome, transcriptomes, and secretomes. We found that the fungus possessed an unexpectedly large set of genes coding for Class II peroxidases involved in lignin degradation (19 genes) and GMC oxidoreductases/dehydrogenases involved in generating the hydrogen peroxide required for lignin peroxidase activity and promoting redox cycling of the fungal enzymes involved in oxidative cleavage of lignocellulose polymers (36 genes). The examination of interrelated multi-omics patterns revealed that eleven Class II Peroxidases were secreted by the fungus during fermentation and eight of them where tightly co-regulated with redox cycling enzymatic partners.ConclusionAs a peculiar feature of P. brumalis, we observed gene family extension, up-regulation and secretion of an abundant set of versatile peroxidases and manganese peroxidases, compared with other Polyporales species. The orchestrated secretion of an abundant set of these delignifying enzymes and redox cycling enzymatic partners could contribute to the delignification capabilities of the fungus. Our findings highlight the diversity of wood decay mechanisms present in Polyporales and the potentiality of further exploring this taxonomic order for enzymatic functions of biotechnological interest.

Structure-based Engineering of a Plant-Fungal Hybrid Peroxidase for Enhanced Temperature and pH Tolerance

In an age of ever-increasing biotechnological and industrial demand for new and specialized biocatalysts, rational protein engineering offers a directapproach to enzyme design and innovation. Heme peroxidases, as indispensable oxidative biocatalysts, provide a relatively mild alternative to the traditional harsh, and often toxic, chemical catalysts, and subsequently, have found widespread application throughout industry. However, the potential forthese enzymes is far greater than their present use, as processes are currently restricted to the more stable, but less catalytically powerful, subset of peroxidases. Here we describe the structure-guided,rational engineering of a plant-fungal hybrid peroxidase built to overcome the application barrier of these high-reduction potential peroxidases. This engineered enzyme has the catalytic versatility and oxidative ability of a high-reduction potential versatile peroxidase, with enhanced temperature and pH tolerance similar to that of a highly stable plant peroxidase.

One-Pot Enzymatic Production of Lignin-Composites

A novel and efficient one-pot system for green production of artificial lignin bio-composites has been developed. Monolignols such as sinapyl (SA) and coniferyl (CA) alcohols were linked together with caffeic acid (CafAc) affording a polymeric network similar with natural lignin. The interaction of the dissolved SA/CA with CafAc already bound on a solid support (SC2/SC6-CafAc) allowed the attachment of the polymeric product direct on the support surface (SC2/SC6-CafAc-L1 and SC2/SC6-CafAc-L2, from CA and SA, respectively). Accordingly, this procedure offers the advantage of a simultaneous polymer production and deposition. Chemically, oxi-copolymerization of phenolic derivatives (SA/CA and CAfAc) was performed with H2O2 as oxidation reagent using peroxidase enzyme (2-1B mutant of versatile peroxidase from Pleurotus eryngii) as catalyst. The system performance reached a maximum of conversion for SA and CA of 71.1 and 49.8%, respectively. The conversion is affected by the system polarity as resulted from the addition of a co-solvent (e.g., MeOH, EtOH, or THF). The chemical structure, morphology, and properties of the bio-composites surface were investigated using different techniques, e.g., FTIR, TPD-NH3, TGA, contact angle, and SEM. Thus, it was demonstrated that the SA monolignol favored bio-composites with a dense polymeric surface, high acidity, and low hydrophobicity, while CA allowed the production of thinner polymeric layers with high hydrophobicity.

Enzyme Activities of Two Recombinant Heme-Containing Peroxidases, TvDyP1 and TvVP2, Identified from the Secretome of Trametes versicolor.

Trametesversicolor is a wood-inhabiting agaricomycete known for its ability to cause strong white-rot decay on hardwood and for its high tolerance of phenolic compounds. The goal of the present work was to gain insights into the molecular biology and biochemistry of the heme-including class II and dye-decolorizing peroxidases secreted by this fungus. Proteomic analysis of the secretome of T. versicolor BRFM 1218 grown on oak wood revealed a set of 200 secreted proteins, among which were the dye-decolorizing peroxidase TvDyP1 and the versatile peroxidase TvVP2. Both peroxidases were heterologously produced in Escherichia coli, biochemically characterized, and tested for the ability to oxidize complex substrates. Both peroxidases were found to be active against several substrates under acidic conditions, and TvDyP1 was very stable over a relatively large pH range of 2.0 to 6.0, while TvVP2 was more stable at pH 5.0 to 6.0 only. The thermostability of both enzymes was also tested, and TvDyP1 was globally found to be more stable than TvVP2. After 180 min of incubation at temperatures ranging from 30 to 50°C, the activity of TvVP2 drastically decreased, with 10 to 30% of the initial activity retained. Under the same conditions, TvDyP1 retained 20 to 80% of its enzyme activity. The two proteins were catalytically characterized, and TvVP2 was shown to accept a wider range of reducing substrates than TvDyP1. Furthermore, both enzymes were found to be active against two flavonoids, quercetin and catechin, found in oak wood, with TvVP2 displaying more rapid oxidation of the two compounds. They were tested for the ability to decolorize five industrial dyes, and TvVP2 presented a greater ability to oxidize and decolorize the dye substrates than TvDyP1.IMPORTANCETrametesversicolor is a wood-inhabiting agaricomycete known for its ability to cause strong white-rot decay on hardwood and for its high tolerance of phenolic compounds. Among white-rot fungi, the basidiomycete T. versicolor has been extensively studied for its ability to degrade wood, specifically lignin, thanks to an extracellular oxidative enzymatic system. The corresponding oxidative system was previously studied in several works for classical lignin and manganese peroxidases, and in this study, two new components of the oxidative system of T. versicolor, one dye-decolorizing peroxidase and one versatile peroxidase, were biochemically characterized in depth and compared to other fungal peroxidases.

Elimination of Isoxazolyl-Penicillins antibiotics in waters by the ligninolytic native Colombian strain Leptosphaerulina sp. considerations on biodegradation process and antimicrobial activity removal

In this work, Leptosphaerulina sp. (a Colombian native fungus) significantly removed three Isoxazolyl-Penicillin antibiotics (IP): oxacillin (OXA, 16000 μg L−1), cloxacillin (CLX, 17500 μg L−1) and dicloxacillin (DCX, 19000 μg L−1) from water. The biological treatment was performed at pH 5.6, 28 °C, and 160 rpm for 15 days. The biotransformation process and lack of toxicity of the final solutions (antibacterial activity (AA) and cytotoxicity) were tested. The role of enzymes in IP removal was analysed through in vitro studies with enzymatic extracts (crude and pre-purified) from Leptosphaerulina sp., commercial enzymes and enzymatic inhibitors. Furthermore, the applicability of mycoremediation process to a complex matrix (simulated hospital wastewater) was evaluated. IP were considerably abated by the fungus, OXA was the fastest degraded (day 6), followed by CLX (day 7) and DCX (day 8). Antibiotics biodegradation was associated to laccase and versatile peroxidase action. Assays using commercial enzymes (i.e. laccase from Trametes versicolor and horseradish peroxidase) and inhibitors (EDTA, NaCl, sodium acetate, manganese (II) ions) confirmed the significant role of enzymatic transformation. Whereas, biomass sorption was not an important process in the antibiotics elimination. Evaluation of AA against Staphylococcus aureus ATCC 6538 revealed that Leptosphaerulina sp. also eliminated the AA. In addition, the cytotoxicity assay (MTT) on the HepG2 cell line demonstrated that the IP final solutions were non-toxic. Finally, Leptosphaerulina sp. eliminated OXA and its AA from synthetic hospital wastewater at 6 days. All these results evidenced the potential of Leptosphaerulina sp. mycoremediation as a novel environmentally friendly process for the removal of IP from aqueous systems.

The degradation of three-ringed polycyclic aromatic hydrocarbons by wood-inhabiting fungus Pleurotus ostreatus and soil-inhabiting fungus Agaricus bisporus

The degradation of two isomeric three-ringed polycyclic aromatic hydrocarbons by the white rot fungus Pleurotus ostreatus D1 and the litter-decomposing fungus Agaricus bisporus F-8 was studied. Despite some differences, the degradation of phenanthrene and anthracene followed the same scheme, forming quinone metabolites at the first stage. The further fate of these metabolites was determined by the composition of the ligninolytic enzyme complexes of the fungi. The quinone metabolites of phenanthrene and anthracene produced in the presence of only laccase were observed to accumulate, whereas those formed in presence of laccase and versatile peroxidase were metabolized further to form products that were further included in basal metabolism (e.g. phthalic acid). Laccase can catalyze the initial attack on the PAH molecule, which leads to the formation of quinones, and that peroxidase ensures their further oxidation, which eventually leads to PAH mineralization.A. bisporus, which produced only laccase, metabolized phenanthrene and anthracene to give the corresponding quinones as the dominant metabolites. No products of further utilization of these compounds were detected. Thus, the fungi's affiliation with different ecophysiological groups and their cultivation conditions affect the composition and dynamics of production of the ligninolytic enzyme complex and the completeness of PAH utilization.

Lignin-Degrading Abilities of Novel Autochthonous Fungal Isolates Trametes hirsuta F13 and Stereum gausapatum F28

SUMMARYThe aim of this research is to isolate and identify fungi with high lignin-degrading abilities that are autochthonous to southern Serbian region. Two novel fungal isolates identified as Trametes hirsuta F13 and Stereum gausapatum F28 were selected to assess their ligninolytic enzyme activities and the efficiency of lignin removal from beech wood sawdust. Obtained results show that both isolates are good sources of industrially valuable enzymes with a potential for application in various biotechnological and industrial processes. Both isolates showed laccase, manganese-dependent peroxidase, and versatile peroxidase activities, while only S. gausapatum F28 had lignin peroxidase activity. This is the first record of the ability of S. gausapatum species to produce lignin peroxidase. T. hirsuta F13 showed higher laccase activity than S. gausapatum F28, while S. gausapatum F28 had higher manganese peroxidase activity. Also, T. hirsuta F13 exhibited much higher laccase activity under submerged cultivation conditions than solid-state cultivation conditions, which is rare for fungi. This is important for industrial processes since the submerged fermentation is a dominant technique in industry. The test of the efficiency of lignin removal showed that both isolates are efficient lignin decomposers. After five weeks of incubation on beech wood sawdust, the total lignin losses were 33.84% with T. hirsuta F13 and 28.8% with S. gausapatum F28.

Lessons learned from the treatment of organosolv pulp with ligninolytic enzymes and chemical delignification agents

Although organosolv pretreatment allows extensive delignification of beech wood, the residual lignin present in the pulp may hinder the subsequent hydrolysis of the cellulose into fermentable sugars. With the purpose of increasing sugar production from cellulose hydrolysis, enzymatic and chemical oxidation stages were applied to the pulp previously to a stage of enzymatic hydrolysis with cellulases. Neither lignin content was reduced, nor sugar yield was improved after the versatile peroxidase treatment. On the other hand, laccase oxidation caused an increase in total lignin and kappa number and did not influence digestibility. Similarly, the chemical oxidation with H2O2 had also a negligible impact on the sugar yield. Only a significant removal of the lignin content in fibers was attained after ethanol wash, also confirmed by FT-IR analysis, which allowed increasing cellulose digestibility by 8.4%, and reducing the phenol content of the hydrolysate by 45.5%. Although the improvement of cellulose digestibility was lower than expected, this work provides valuable lessons for practical use on the opportunities of organosolv pulp.

A role for small secreted proteins (SSPs) in a saprophytic fungal lifestyle: Ligninolytic enzyme regulation in Pleurotus ostreatus

Small secreted proteins (SSPs), along with lignocellulose degrading enzymes, are integral components of the secretome of Pleurotus ostreatus, a white rot fungus. In this study, we identified 3 genes (ssp1, 2 and 3) encoding proteins that are annotated as SSPs and that exhibited of ~4,500- fold expression, 24 hr following exposure to the toxic compound 5-hydroxymethylfurfural (HMF). Homologues to genes encoding these SSPs are present in the genomes of other basidiomycete fungi, however the role of SSPs is not yet understood. SSPs, aryl-alcohol oxidases (AAO) and the intracellular aryl-alcohol dehydrogenases (AAD) were also produced after exposure to other aryl-alcohols, known substrates and inducers of AAOs, and during idiophase (after the onset of secondary metabolism). A knockdown strain of ssp1 exhibited reduced production of AAO-and AAD-encoding genes after HMF exposure. Conversely, a strain overexpressing ssp1 exhibited elevated expression of genes encoding AAOs and ADD, resulting in a 3-fold increase in enzymatic activity of AAOs, as well as increased expression and protein abundance of versatile peroxidase 1, which directly degrades lignin. We propose that in addition to symbionts and pathogens, SSPs also have roles in saprophytes and function in P. ostreatus as components of the ligninolytic system.

Rational design of Pleurotus eryngii versatile ligninolytic peroxidase for enhanced pH and thermal stability through structure-based protein engineering.

Versatile peroxidase (VP) from Pleurotus eryngii is a high redox potential peroxidase. It has aroused great biotechnological interest due to its ability to oxidize a wide range of substrates, but its application is still limited due to low pH and thermal stability. Since CiP (Coprinopsis cinerea peroxidase) and PNP (peanut peroxidase) exhibited higher pH and thermal stability than VP, several motifs, which might contribute to their pH and thermal stability, were identified through structure and sequence alignment. Six VP variants incorporating the beneficial motifs were designed and constructed. Most variants were nearly completely inactivated except V1 (Variant 1) and V4. V1 showed comparable activity to WT VP against ABTS, while V4 exhibited reduced activity. V1 displayed improved pH stability than WT VP, at pH 3.0 in particular, whereas the pH stability of V4 did not change a lot. The thermal stabilities of V1 and V4 were enhanced with T50 raised by 3°C. The results demonstrated that variants containing the beneficial motifs of CiP and PNP conferred VP with improved pH and thermal stability.

Polymerization of coniferyl alcohol by Mn3+ -mediated (enzymatic) oxidation: Effects of H2 O2 concentration, aqueous organic solvents, and pH.

The objective of this study was to evaluate the ability of one versatile peroxidase and the biocatalytically generated complex Mn(III)-malonate to polymerize coniferyl alcohol (CA) to obtain dehydrogenation polymers (DHPs) and to characterize how closely the structures of the formed DHPs resemble native lignin. Hydrogen peroxide was used as oxidant and Mn2+ as mediator. Based on the yields of the polymerized product, it was concluded that the enzymatic reaction should be performed in aqueous solution without organic solvents at 4.5 ≤ pH ≤ 6.0 and with 0.75 ≤ H2 O2 :CA ratio ≤ 1. The results obtained from the Mn3+ -malonate-mediated polymerization showed that the yield was almost 100%. Reaction conditions had, however, effect on the structures of the formed DHPs, as detected by size exclusion chromatography and pyrolysis-GC/MS. It can be concluded that from the structural point of view, the optimal pH for DHP formation using the presently studied system was 3 or 4.5. Low H2 O2 /CA ratio was beneficial to avoid oxidative side reactions. However, the high frequency of β-β linkages in all cases points to dimer formation between monomeric CA rather than endwise polymerization. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:81-90, 2018.

A novel and efficient fungal delignification strategy based on versatile peroxidase for lignocellulose bioconversion

BackgroundThe selective lignin-degrading white-rot fungi are regarded to be the best lignin degraders and have been widely used for reducing the saccharification recalcitrance of lignocellulose. However, the biological delignification and conversion of lignocellulose in biorefinery is still limited. It is necessary to develop novel and more efficient bio-delignification systems.ResultsPhysisporinus vitreus relies on a new versatile peroxidase (VP)-based delignification strategy to remove enzymatic recalcitrance of corn stover efficiently, so that saccharification of corn stover was significantly enhanced to 349.1 mg/g biomass (yield of glucose) and 91.5% (hydrolysis yield of cellulose) at 28 days, as high as levels reached by thermochemical treatment. Analysis of the lignin structure using pyrolysis–gas chromatography–mass spectrometry (Py–GC/MS) showed that the total abundance of lignin-derived compounds decreased by 54.0% and revealed a notable demethylation during lignin degradation by P. vitreus. Monomeric and dimeric lignin model compounds were used to confirm the ligninolytic capabilities of extracellular ligninases secreted by P. vitreus. The laccase (Lac) from P. vitreus could not oxidize nonphenolic lignin compounds and polymerized β-O-4 and 5-5′ dimers to precipitate which had a negative effect on the enzymatic hydrolysis of corn stover in vitro. However, the VP from P. vitreus could oxidize both phenolic and nonphenolic lignin model compounds as well as break the β-O-4 and 5-5′ dimers into monomeric compounds, which were measured by high-performance liquid chromatography–electrospray ionization–mass spectrometry (LC–ESI–MS). Moreover, we showed that addition of purified VP in vitro improved the enzymatic hydrolysis of corn stover by 14.1%.ConclusionsFrom the highly efficient system of enzymatic recalcitrance removal by new white-rot fungus, we identified a new delignification strategy based on VP which could oxidize both phenolic and nonphenolic lignin units and break different linkages in lignin. In addition, this is the first evidence that VP could break 5-5′ linkage efficiently in vitro. Moreover, VP improved the enzymatic hydrolysis of corn stover in vitro. The remarkable lignin-degradative potential makes VP attractive for biotechnological applications.

The making of versatile peroxidase by directed evolution

Versatile peroxidase (VP) secreted by white-rot fungi is involved in the degradation of lignin within land ecosystems. With a broad substrate scope and minor requirements, VP is an extremely attractive blueprint to be designed by the directed evolution tool-box. In recent years, improved VP variants have been generated that meet industrial requirements in terms of improved heterologous functional expression and activity, as well as stability at high temperatures or in the presence of strong inhibitors. This review describes the making of VP by directed evolution, addressing the most important findings and challenges that were faced, along with the future prospects for this research field.

Laccase enzyme detoxifies hydrolysates and improves biogas production from hemp straw and miscanthus

The impact of various phenolic compounds, vanillic acid, ferulic acid, p-coumaric acid and 4-hydroxybenzoic acid on anaerobic digestion of lignocellulosic biomass (hemp straw and miscanthus) was studied. Such phenolic compounds have been known to inhibit biogas production during anaerobic digestion. The different phenolic compounds were added in various concentrations: 0, 100, 500, 1000 and 2000mg/L. A difference in inhibition of biomethane production between the phenolic compounds was noted. Hydrolysis rate, during anaerobic digestion of miscanthus was inhibited up to 50% by vanillic acid, while vanillic acid had no influence on the initial rate of biogas production during the anaerobic digestion of hemp straw. Miscanthus has a higher lignin concentration (12–30g/100gDM) making it less accessible for degradation, and in combination with phenolic compounds released after harsh pretreatments, it can cause severe inhibition levels during the anaerobic digestion, lowering biogas production. To counter the inhibition, lignin degrading enzymes can be used to remove or degrade the inhibitory phenolic compounds. The interaction of laccase and versatile peroxidase individually with the different phenolic compounds was studied to have insight in the polymerization of inhibitory compounds or breakdown of lignocellulose. Hemp straw and miscanthus were incubated with 0, 100 and 500mg/L of the different phenolic compounds for 0, 6 and 24h and pretreated with the lignin degrading enzymes. A laccase pretreatment successfully detoxified the substrate, while versatile peroxidase however was inhibited by 100mg/L of each of the individual phenolic compounds. Finally a combination of enzymatic detoxification and subsequent biogas production showed that a decrease in phenolic compounds by laccase treatment can considerably lower the inhibition levels of the biogas production.

Insights into the Mechanism of Lignocellulose Degradation by Versatile Peroxidases

Lignocelluloses are imperative structural components of plant cell wall and are profusely found in agricultural crop residues. The structural heterogeneity and recalcitrance of lignin limit the accessibility of cell wall carbohydrates for constructive exploitation. During the past decades, diverse lignin degrading enzymes were characterized to facilitate the utilization of lignocellulosic biomass for technological applications. Versatile peroxidases are unique among ligninolytic enzymes for their remarkably high redox potential and ability to oxidize lignin without the requisite of redox mediators. The hybrid structural architecture of this enzyme bearing functional features of lignin peroxidase and manganese peroxidase demonstrates its versatility in aromatics oxidation. This review summarizes the distinctive structural aspects of fungal versatile peroxidase in correlation to its oxidation of aromatic substrates besides emphasizing on the catalytic environment conducive for substrate oxidation. This review also focuses on the general strategies employed for production of this enzyme, its molecular framework, potential biotechnological applications of versatile peroxidase and prospects on enhancing the production of enzyme. Finally, the significance of this enzyme in improving the nutritive value of crop residues to promote ruminal productivity is highlighted.

Enzymatic Degradation of Lignin in Soil: A Review

Lignin is a major component of soil organic matter and also a rich source of carbon dioxide in soils. However, because of its complex structure and recalcitrant nature, lignin degradation is a major challenge. Efforts have been made from time to time to understand the lignin polymeric structure better and develop simpler, economical, and bio-friendly methods of degradation. Certain enzymes from specialized bacteria and fungi have been identified by researchers that can metabolize lignin and enable utilization of lignin-derived carbon sources. In this review, we attempt to provide an overview of the complexity of lignin’s polymeric structure, its distribution in forest soils, and its chemical nature. Herein, we focus on lignin biodegradation by various microorganism, fungi and bacteria present in plant biomass and soils that are capable of producing ligninolytic enzymes such as lignin peroxidase (LiP), manganese peroxidase (MnP), versatile peroxidase (VP), and dye-decolorizing peroxidase (DyP). The relevant and recent reports have been included in this review.

Synergetic integration of laccase and versatile peroxidase with magnetic silica microspheres towards remediation of biorefinery wastewater.

In this study, a tailor-made biocatalyst consisting of a co-immobilized lignolytic enzyme cascade on multi-functionalized magnetic silica microspheres (MSMS) was developed. Physical adsorption was the most promising strategy for the synthesis of individual immobilized laccase (IL), immobilized versatile peroxidase (IP), as well as co-immobilized laccase (Lac) and versatile peroxidase (VP) with an enzyme activity recovery of about 79, 93, 27, and 27.5%, respectively. Similarly, the biocatalytic load of 116, 183, 23.6, and 31U/g was obtained for IL, IP, and co-immobilized Lac and VP, respectively. The co-immobilized enzyme system exhibited better pH stability than the free and individual immobilized system by retaining more than 100% residual activity at pH7.0 after a 150-h incubation; whereas, the thermal stability and kinetics of the co-immobilized biocatalyst were not much improved. IL and IP could be recycled for 10cycles after which they retained 31 and 44% of their initial activities. Co-immobilized Lac and VP were reused for ten consecutive cycles at the end of which Lac activity was depleted, and 37% of VP activity was left. Free enzymes, IL, IP, co-immobilized Lac, and VP were applied to biorefinery wastewater (BRW) in a batch study to investigate the transformation of phenolic contaminants over a period of 5days. The major classes of phenolic constituents in terms of their order of removal in a Lac-VP system was phenol >2-chlorophenol > trichlorophenol > dichlorophenol > cresols > dimethylphenol >2 methyl- 4, 6-dinitrophenol > 4-nitrophenol > tetrachlorophenols > pentachlorophenol. The free enzymes and individually immobilized enzymes resulted in 80% dephenolization in 5days. By contrast, the co-immobilized biocatalyst provided rapid dephenolization yielding the same 80% removal within 24h and 96% removal of phenols in 60h after which the system stabilized, which is the major advantage of the co-immobilized biocatalyst. ᅟ Graphical abstract.