Abstract
One of the most abundant enzymes found in nature is peroxidases where it is present in plants, animals microbes. Peroxidases catalyze the oxidation of various organic and inorganic substrates in the presence of hydrogen peroxide as electron acceptor. Among peroxidases lignin peroxidase plays an important role in biodegradation where it possesses a high redox potential for the oxidation of non-phenolic structures which constitute up to 90% of lignin. There are several important applications with regards to lignin peroxidases such as delignification of feedstock for ethanol production; textile effluent treatment and dye decolorization; coal depolymerization and degradation of other xenobiotics; and as melanin oxidizes for the use as cosmetic lightning agents. When considering the structure, lignin peroxidase folds to form a globular shape which is segregated into proximal and distal domains by the heme in the protein. There are three mode of action of lignin peroxidase proposed: direct oxidation pathway, indirect oxidation pathway, and reduction pathway. The catalytic activity of lignin peroxide to oxidize lignin and other high redox potential compounds has been attributed to its exposed tryptophan residue (Trp171) which forms a tryptophanyl radical on the surface of the enzyme through long-range electron transfer to the heme. Immobilization of lignin peroxide reduces some disadvantages of the use of enzymes, mainly the cost factor because immobilised enzymes can be easily reused. Different obstacles prevent lignin peroxidases from being used in industrial and environmental applications including low stability towards their natural oxidizing-substrate hydrogen peroxide. Protein engineering provides a solution where it enhances the stability with regards to hydrogen peroxide.
Introduction to lignin
Lignocellulose is a major constituent of the bio mass of and is composed of mainly three types of polymers: Cellulose (40-50%), Hemicellulose (25-30%), and Lignin (15-20%). Other than these there are other extractives such as pectin and tannins. These three polymers are highly attached to each other by non-covalent forces as well as covalent forces.1, 2
Cellulose & hemicelluloses are known as macromolecules which produced from different sugar units. Cellulose is the most abundant polysaccharide on earth as well as in lignocellulose network. The networks in lignocelluloses such as cellulose-hemicellulose or lignin molecules are mainly joined by hydrogen bonds. In addition to that, hemicelluloses and lignin have chemical bonds between galactose residues, arabinose residues on the side chains of hemicellulose molecules and lignin, which results a small amount of carbohydrate in the final mixture of the process of lignin isolation.1, 2
Figure 1: Schematic representation of the Lignin structural units3
Lignin is a macromolecule which has a three-dimensional structure. Constituents of lignin are p-Coumaryl alcohol, Coniferyl alcohol & Sinapyl alcohol (Figure 1). These three phenols polymerize and form the amorphous hetero polymer which is synthesized by the generation of free radicals.4, 3
Polymer is made of phenyl propane units linked by several types of linkages. Basically, these sub units are linked by C-C and aryl-ether linkages with arylglycerol-β-aryl ether. It acts as “glue” & combines cellulose and hemicelluloses. Lignin is insoluble in water and provides the structural support, impermeability and resistance against microbial attack and oxidative stress.3
Classes and natural sources of peroxidases
One of the most abundant enzymes found in nature is peroxidases where it is present in plants, animals microbes. Peroxidases catalyze the oxidation of various organic and inorganic substrates in the presence of hydrogen peroxide as electron acceptor. There are different classifications to peroxidases. One classification divides peroxidases considering the presence or absence of a heme group.5
• Heme peroxidases: contain a protoporphyrin IX (heme) as prosthetic group
• Non-heme peroxidases: lack protoporphyrin IX (heme) as prosthetic group.
Furthermore, the heme peroxidases can be further divided phylogenetically (i.e. evolutionary development and diversification of a species or group of organisms, or of a feature of an organism) into two superfamilies and three families. However, this classification does not provide any relationship between the two superfamilies of heme peroxidases and the three families of heme peroxidase found. The two superfamilies and the three families of peroxidases are as follows:5, 6
• Two superfamilies:
¬ Peroxidase-cyclooxygenase superfamily
¬ Peroxidase-catalase superfamily
• Three families:
¬ Di-heme peroxidases
¬ DyP-type peroxidases (DyPPrx)
¬ Haloperoxidases (HalPrx)
The peroxidase-cyclooxygenase superfamily includes members from all domains of life where its main function is to catalyze halide oxidation. Some enzymes which are included in peroxidase-cyclooxygenase superfamily are involved in the innate immune system. This function in the innate immune system is seen in mammalian peroxidases and several peroxidases of bacterial origin. In the latter case where bacterial peroxidases are involved in the innate immune system, it is proposed to be involved in unspecific defense mechanisms.5
The peroxidase-catalase superfamily may be further subdivided into three classes as follows:5, 7
¬ Class I intracellular peroxidases: examples for this class of peroxidases and their function is given below.
⎫ Yeast cytochrome c peroxidase (CcP): this enzyme is involved in protecting against toxic peroxide in the electron transport chain.
⎫ Ascorbate peroxidase (APx): this enzyme is associated with the removal of hydrogen peroxide in the chloroplast and cytosol of higher plants
⎫ Bacterial catalase-peroxidase (KatG): this enzyme is exhibit hybrid catalytic activities of both peroxidase and catalase.
¬ Class II secretory fungal peroxidases: examples for this class of peroxidases are involved in lignin degradation.
⎫ Lignin peroxidase (LiP): this enzyme possesses a high redox potential for the oxidation of non-phenolic structures which constitute up to 90% of lignin. Lignin peroxidase is also able to oxidize a wide range of aromatic compounds, hence, the reason why it is able to enzymatically degrade lignin.
⎫ Manganese peroxidase (MnP): in the mechanism of action, first manganese peroxide binds to Mn2+ via two or three residues corresponding to Glu-35, Glu-39 and Asp-175 of Phanerochaete chrysosporium MnP 1. Then catalytic oxidation of Mn2+ to Mn3+ occurs. Then Mn3+ which is highly reactive, oxidizes a wide range of phenolic substrates including lignin phenolic structures.
⎫ Versatile peroxidase (VP): this enzyme has a unique molecular architecture with different oxidation-active sites present in the enzyme which results in its dual activity of both manganese peroxide and lignin peroxidase. This ability of versatile peroxidase to oxidize high redox potential compounds is due to an exposed catalytic tryptophan which forms a radical on the surface of the enzyme through a long-range electron transfer to the heme.
¬ Class III secretory plant peroxidase: example for this class of peroxidases is horseradish peroxidase (HRP). This enzyme is involved in cell wall biosynthesis, Indole-3-acetic acid catabolism and oxidation of poisonous compounds.
Application of lignin peroxidase bio-catalysis
a) Delignification of feedstock for ethanol production: Ethanol is a good alternative to fossil fuel. The use of lignocellulosic biomass as cheap source of feedstock for production of ethanol has drawn attention since it is renewable and eco-friendly. Delignification of lignocellulose is a must to convert lignocellulose to ethanol. Biological method of delignification has drawbacks such as long incubation period, less recovered products. Lignocellulolytic enzyme system has been suggested as an effective treatment strategy. The suggested lignocellulolytic enzyme system includes lignin peroxidase which has reaction specificity and high product yield since it does not utilize products as source of energy. This makes the system a very promising model for industrial application.5, 8
b) Textile effluent treatment and dye decolorization: The textile industry consumes synthetic dyes significantly. Furthermore, these dyes are major sources of environmental pollution. These synthetic dyes can be categorized by their structural difference into azo, diazo, acidic, basic, reactive, disperse, metal-complex, anthraquinone-based dyes, etc. During the textile dyeing process about 10–15% of dyes are lost in water. Subsequent release as effluent into various environment has been estimated to amount to about 2–20%. Furthermore, many of these dyes and their degradation products have been declared toxic. Hence, their presence in the environment is a major concern. As a result, various methods for dye decolorization and treatment of textile effluents have been developed. These methods in general are not effective, costly, and they generate a great amount of sludge which may eventually create secondary pollution problem. Decolorization of industrial waste by oxidative enzymes from bacteria and fungi have been reported with positive outcomes. The enzymes oxidize phenolic compounds to aryl-oxy radicals creating insoluble complexes. Other mechanisms of action of these enzymes include polymerization of contaminants and copolymerization with other nontoxic substrates to promote easy removal of the contaminants by other purification methods such as sedimentation, filtration, and adsorption. This indicates the potential of lignin peroxidase in textile and other industrial effluent treatment.5. 8
c) Coal depolymerization and degradation of other xenobiotics: The ligninolytic enzyme system of microbes has been used in the degradation of several xenobiotics. These includes chlorophenols, polycyclic aromatic hydrocarbons (PAHs), organophosphorus, and phenols. Furthermore, these compounds which are released from different anthropogenic sources are categorized as major environmental pollutants. Some of these compounds are active components of pesticides, disinfectants, herbicides, explosives, dyes and others which are found in daily industrial application. These xenobiotics, accumulates in the soil, ground water, and air contaminating the environment and results in public health issues. Therefore, effective removal of these environmental pollutants has become very importance when considering the risk it poses to human health. Extracellular peroxidases, from ligninolytic microbes, have been reported to play a significant role in the degradation of xenobiotic compounds. Lignin peroxidase from both fungi and bacteria, has been reported to mineralize different types of recalcitrant aromatic compounds including three-and four-ring polycyclic aromatic hydrocarbons, polychlorinated biphenyl, chlorophenols, and synthetic dyes indicating the potential it has in the treatment of these pollutants.5, 8
d) Melanin oxidation-novel cosmetic lightning agents: Although one of the biological functions of melanin in human may be to protect the underlying tissues from harmful ultraviolet radiation, many hyperpigmented women in Africa and other black nations desire a light face and skin. However, skin-lightening by inhibition of melanin synthesis is slow in achieving the desired results. Hence, there is the need to explore alternative agents with the potential to directly decolorize melanin pigment through oxidation as a means of skin-lightening agents. The structural characteristics of melanin is similar to that of lignin where the polymers are made up of indole and phenolic subunits. The ability of ligninolytic enzymes to oxidize a wide range of structurally different substrates makes them suitable candidates for the oxidation of melanin which is structurally similar to lignin. Therefore, ligninolytic enzymes with melanolytic ability have the potential for application in the cosmetics industry.5
Lignin peroxidase structure
Lignin peroxidase folds to form a globular shape. It is segregated into proximal and distal domains by the heme which is in the protein but is made accessible through two small channels.
The lignin peroxidase folding motif contains;
¬ Eight major α-helices
¬ Eight minor helices
¬ Three short antiparallel β sheets
Overall catalytic cycle of lignin peroxidase is similar to that of a typical heme-peroxidase. The molecular weight range of lignin peroxidase has been documented as 38 kDa to 43 kDa, isoelectric point ranging from of 3.3 to 4.7, and a very low pH optimum of 3.0 with veratryl alcohol as the substrate. The low pH optimum of lignin peroxidase distinguishes it from other peroxidases.5
Crystallographic studies of cytochrome c peroxidase (CcP) which is a class I intracellular fungal peroxidase (e.g. in yeast) and lignin peroxidase which is a class II extracellular fungal peroxidase revealed some structural differences:5, 9
¬ Lignin peroxidase possesses four disulfide bonds while cytochrome c peroxidase has none.
¬ Lignin peroxidase is larger in size and contains about 343 amino acid residues while cytochrome c peroxidase is made up of 294 residues.
¬ Lignin peroxidase has three tryptophans and eight methionines as oxidizable amino acids whereas cytochrome c peroxidase has seven tryptophans, fourteen tyrosine residues, five methionines, and one cysteine as oxidizable amino acids. Tyrosine is absent in LiP and it also does not have free cysteine.
¬ Lignin peroxidase has phenylalanines at the contact point between the distal and proximal heme surfaces whereas cytochrome c peroxidase has tryptophans at the contact point between the distal and proximal heme surfaces.
¬ Lignin peroxidase has Asp-183 hydrogen bonded to heme propionate whereas Asn-184 is hydrogen bonded to heme propionate in cytochrome c peroxidase. This has been suggested to partly account for the low pH optimum of lignin peroxidase as the disruption of the aspartic acid-propionate hydrogen bond would be expected to result in the destabilization of the heme pocket.
¬ In lignin peroxidase the bond between the heme iron and the Nɛ2 atom of the proximal histidine residue is longer than that in cytochrome c peroxidase. The weaker iron-nitrogen bond observed in lignin peroxidase makes the heme more electron deficient, thereby destabilizing the high oxidation states. This results in the higher redox potential of lignin peroxidase when compared to cytochrome c peroxidase.
Lignin peroxidase activity
Lignin peroxidase, which is also referred to as diaryl propane oxygenase, is a heme-containing enzyme that catalyzes hydrogen peroxide-dependent oxidative degradation of lignin (Figure 2).
Figure 2: Oxidative cleavage of β-1 linkage in lignin structure by lignin peroxidase5
The heme cofactor in the lignin peroxidase is nonplanar which is also true for other enzymes in class-II peroxidases. Lignin peroxidase was first discovered in extracellular medium of white-rot fungus P. chrysosporium. Furthermore, various isozymes have been identified in the following organisms: P. chrysosporium, Tramates versicolor, Phlebia radiata, and Phanerochaete sordida.5
Several kinds of isoenzymes associated with lignin peroxidase has been identified; e.g. six isozymes of lignin peroxidase designated as H1, H2, H6, H7, H8, and H10 which is found in the extracellular fluid of cultures of P. chrysosporium BKM-F-1767. Studies to these isoenzymes have shown that the purified isozymes had different isoelectric point, sugar content, substrate specificities, and stability. The N-terminal sequences of their amino acids were also found to be different, suggesting that they were encoded by different genes. Gene sequencing of a lignocellulose-degrading fungus; P. chrysosporium strain RP78, revealed about 10 lip genes, confirming the existence of isozymes of lignin peroxidase. Furthermore, it has been found that P. chrysosporium genome harbors 10 lip genes designated lip A-J and they, respectively, encode different isoforms of lignin peroxidase.5
Apart from the white-rot and brown-rot fungi which produces lignin peroxidase, it has been found out that some bacteria have ligninolytic abilities with the potential of producing ligninases. This group of bacteria include actinomycetes, α-proteobacteria, and γ-proteobacteria. But compared to lignin peroxidases found in fungi, bacterial lignin peroxidase has been less studied.5
Lignin peroxidase catalytic reaction
Proposed mechanism for mode of action
¬ Direct oxidation pathway: The catalytic cycle of lignin peroxidase involves three steps (Figure 3).5, 7, 8, 10
Figure 3: Lignin peroxidase (LiP)-catalyzed oxidation of nonphenolic β-O-4 lignin model compound7
⎫ Step 1: Oxidation of the resting ferric enzyme [Fe (III)] by hydrogen peroxide as an electron acceptor resulting in the formation of compound I oxo-ferryl intermediate (LiP-I).
⎫ Step 2: The oxo-ferryl intermediate (deficient of two electrons) is reduced by a molecule of substrate such as non-phenolic aromatic substrate (e.g. Nonphenolic beta-O-4 lignin model compound) which donates one electron to compound I (LiP-1) to form the second intermediate, compound II (LiP-II) which is deficient of one electron.
⎫ Step 3: Subsequent donation of a second electron to compound II by the reduced substrate thereby returning lignin peroxidase to the resting ferric oxidation state which indicates the completion of the oxidation cycle. This result in the oxidation of halogenated phenolic compounds, polycyclic aromatic compounds and other aromatic compounds followed by a series of nonenzymatic reactions (Figure 3).
¬ Indirect oxidation pathway: In many cases, chemicals are not directly accessible to heme of lignin peroxidase and thus direct oxidation does not occur. In such cases involvement of redox mediator plays an important role. Veratryl alcohol (VA) is an excellent substrate for lignin peroxidase. Veratryl alcohol serves as an electron mediator to facilitate oxidation of pollutants. There are two proposed mechanisms how veratryl alcohol helps in the indirect oxidation pathway.8, 5
⎫ Mechanism 1: Veratryl alcohol is oxidized by lignin peroxidase to veratryl alcohol cation radical (VA•+) which is a strong oxidant responsible for indirect oxidation of lignin and pollutants. E.g. EDTA was found to be indirectly decarboxylated by lignin peroxidase. The apparent inhibition of veratryl alcohol oxidase activity of lignin peroxidase by EDTA is suggestive of the reduction of VA•+ back to VA during oxidation of EDTA (Figure 4).8
⎫ Mechanism 2: The lignin peroxidase (oxidized) gains an electron from veratryl alcohol returns to its native reduced state, and veratryl aldehyde is formed. Veratryl aldehyde then again gets reduced back to veratryl alcohol by gaining an electron from substrate.7
Figure 4: Lignin peroxidase catalyzed indirect oxidation8
Chemicals that have been found to be indirectly oxidized by lignin peroxidase include herbicide aminotriazol, pentachlorophenol, phenol, etc.
¬ Reduction pathway:8 In the presence of veratryl alcohol, lignin peroxidase is able to catalyze reduction of various chemicals. Veratryl alcohol is oxidized by lignin peroxidase to veratryl alcohol cation radical (VA•+) which oxidizes carboxylic acids to respective acid derived anion radicals, which in turn serve as reductant. Such radicals are able to reduce cytochrome c, nitro blue tetrazolium, ferric ion and molecular oxygen. They are also involved in the reduction of carbon tetrachloride to the trichloromethyl radical which is neither a substrate for enzyme nor a good reductant.
Veratryl alcohol cation radical oxidizes EDTA as well as oxalate to their corresponding anion radicals. In the absence of another electron acceptor, these carboxylate anion radicals reduce molecular oxygen to O2•-, which will reduce ferric iron to ferrous iron. Hydrogen peroxide then readily reacts with chelated ferrous iron to produce hydroxy radical (•OH). Hydroxy radicals have incredible oxidizing ability and makeup a potential non-enzymaticbiological system known as Fenton reagent. Fenton’s reaction has been widely used for degradation of xenobiotic compounds including PCBs, herbicides and dyes (Figure 5).
Figure 5: Lignin peroxidase catalyzed reduction and generation of radicals8
Factors effecting lignin peroxidase activity, stability, and substrate specificity
The catalytic activity of lignin peroxide to oxidize lignin and other high redox potential compounds has been attributed to its exposed tryptophan residue (Trp171). This tryptophan residue (Trp171) forms a tryptophanyl radical on the surface of the enzyme through long-range electron transfer (LRET) to the heme. Furthermore, variation in the tryptophan environment (e.g. pH) has been identified as a factor capable of modulating the enzyme activity, stability, and substrate specificity. This variation in the tryptophan environment which results in enzyme activity, is hypothesized to accounts for the variation in the catalysis of versatile peroxidase and lignin peroxidase as lignin peroxidase is able to oxidize veratryl alcohol more effectively than versatile peroxidase. This ability of lignin peroxidase is attributed to the acidic environment of Trp171 in P. chrysosporium lignin peroxidase as it facilitates the stabilization of veratryl alcohol cation radical.5
Chemical tools used to probe enzyme activity
Using absorbance to monitor lignin peroxidase activity
Lignin peroxidase activity can be monitored at 310 nm by the oxidation of 2.0 mM veratryl alcohol to veratraldehyde (ɛ310 = 9.3 × 103 M−1 cm−1) in 50 mM sodium tartrate buffer at pH 2.5 or 4.5 in the presence of 0.1 mM hydrogen peroxide. The drawback of this method is phenolic and other aromatic compounds with high absorbance at 310 nm may interfere with this assay. Furthermore, nonperoxidizing veratryl alcohol oxidases from some white-rot fungi (such as Pleurotus sajorcaju) also catalyze the same reaction.11
Alternative to this method is to use Azure B which has higher specificity than veratryl alcohol with respect to lignin peroxidase activity since the dye is not oxidized by P.sajor-caju veratryl alcohol oxidase. Lignin peroxidase catalyzed oxidation of 32 mM Azure B (ɛ651 = 4.88 × 104 M−1 cm−1) may be monitored by the reduction in absorbance at 651 nm in 50 mM sodium tartrate buffer at pH 2.5 or 4.5 containing 0.1 mM hydrogen peroxide. The presence of lignin or other aromatic compounds usually does not interfere with this assay.11
Immobilization as a tool to stabilize lignin peroxidase
Immobilised enzymes are enzymes that are physically confined or localized, with retention of its catalytic activity, so that they can be used repeatedly and continuously. The immobilization of enzymes reduces some disadvantages of the use of enzymes, mainly the cost factor because immobilised enzymes can be easily reused. Lowering the costs of enzymatic conversions will help to produce biobased chemicals and transportation fuels. Enzyme immobilization techniques can be classified as follows:12
1. Binding to a solid support: by adsorption (by hydrogen bonds or hydrophobic interaction), by ionic binding (e.g. to ion exchange resins), by covalent binding (e.g. to epoxy groups)
2. Cross-linking (e.g. by glutaraldehyde)
3. Entrapment: in gels (e.g. calcium alginate), in membrane reactors (e.g. hollow fibre reactors), in reversed micelles, microemulsions, etc.
The standard assay for lignin peroxidase is immobilised lignin peroxidase assay where oxidation of the lignin building block veratryl alcohol (VA) to veratraldehyde occurs. The first report on the immobilisation of lignin peroxidase from Phanerochaete chrysosporium, the enzyme was immobilised by covalent coupling of native enzyme to glutaraldehyde-activated glass beads and to epoxy-activated acrylic beads, and by coupling of NaIO4- oxidized lignin peroxidase to hydrazide-derivatized agarose. The glutaraldehyde-glass method gave the highest immobilisation yield, but the activity was low due to diffusion limitation. Immobilisation protected the enzyme against inactivation by hydrogen peroxide.12
In another method carboxylate groups of the enzyme were coupled to the amino beads. In the second method the amino groups of the beads were reacted with succinic acid anhydride, yielding carboxylate-functionalized glass beads. The third method involved reacting the aldehyde groups derived from periodate oxidation of the sugars of this glycoprotein to the amino beads. For all three enzyme preparations, Vmax for veratryl alcohol conversion was lower than for free enzyme. The KM for hydrogen peroxide was decreased for all three enzyme preparations.12
Another study on the covalent immobilisation of lignin peroxidase from Phanerochaete chrysosporium involved three similar methods: coupling to CNBr-activated Sepharose, to N-hydroxysuccinimide-activated Affigel 15, and to the amine groups of Affigel 102 using EDC. The CNBr-Sepharose method gave best results, although the activity towards veratryl alcohol dropped to only 35%. Effect on pH profile and vulnerability towards organic solvents was not observed, and there was a small increase in temperature stability. The stability towards low pH, was strongly increased. This is important because the activity of the enzyme is highest at low pH.12
When lignin peroxidase from P. chrysosporium is entrapped in xerogel the immobilised enzyme is more active than the soluble enzyme, when considering the conversion of veratryl alcohol. Entrapping the enzyme makes it much more stable at temperatures above 40 0C and above pH 4. When lignin peroxidase from P. chrysosporium is entrapped between polyelectrolytes using the LbL technique, enzyme activity measured by veratryl alcohol oxidation, was decreased tenfold by the immobilisation, but stability was increased.12
Immobilizing lignin peroxidase and glucose oxidase to nanoporous gold can also be carried out. The second enzyme produces hydrogen peroxide upon oxidizing glucose, which forms in a slow and continuous way. As a result, inactivation of lignin peroxidase by hydrogen peroxide was minimized. The enzyme is only physisorbed to the support yet is very strongly bound because the enzyme does not detach in citrate buffer. Immobilisation strongly increased lignin peroxidase stability at 45 0C as well as stability under storage and turnover conditions. The enzyme has 65% of its original activity after seven reuses. Furthermore, any loss of activity upon immobilisation was not found.12
Protein engineering for improved lignin peroxidase function
Different obstacles prevent lignin peroxidases from being used in industrial and environmental applications including low stability towards their natural oxidizing-substrate hydrogen peroxide. This process has been described as a suicide inactivation related to the formation of Compound III, a peroxidase intermediate that is not part of the catalytic cycle which leads to enzyme inactivation. Both Compound I and Compound II which are intermediates of the catalytic cycle are very reactive intermediates that, in the absence of a normal reducing substrate and in the presence of high H2O2 concentration, can be converted to Compound III, a superoxide anion containing Fe3+ species. Once formed, Compound III can follow different decomposition pathways under excess of H2O2 generating reactive oxygen species able to oxidize the porphyrin moiety or amino acid side chains (such as those of methionines) leading to enzyme inactivation. Protein engineering can be used to overcome this where the lignin peroxides is more stable to hydrogen peroxide.13
In one of the studies, versatile peroxidase was taken as a model ligninolytic peroxidase, to improvise different strategies to improve hydrogen peroxide stability. Oxidation of the methionine residues was produced during enzyme inactivation by hydrogen peroxide excess. In one strategy substitution of these methionine residues, located near the heme cofactor and the catalytic tryptophan, rendered a variant (variant I) with a 7.8-fold decreased oxidative inactivation rate.13
A second strategy consisted in mutating two residues (Thr45 and Ile103) near the catalytic distal histidine with the aim of modifying the reactivity of the enzyme with hydrogen peroxide. This variant (Variant II) showed a 2.9-fold slower reaction rate with hydrogen peroxide and 2.8-fold enhanced oxidative stability.
Finally, both strategies were combined to give another variant (Variant III), whose stability in the presence of hydrogen peroxide was improved 11.7-fold. This variant showed an increased half-life, over 30 minutes compared with 3.4 minutes of the native enzyme, under an excess of 2000 equivalents of hydrogen peroxide. The stability improvement achieved was related with slower formation, subsequent stabilization and slower bleaching of the enzyme Compound III, which is a peroxidase intermediate that is not part of the catalytic cycle and leads to the inactivation of the enzyme.13
The major drawback of these methods to come up with variants is the difficulty and accuracy of selectively modify amino acid moieties. A better approach would be to change the heme moiety in lignin peroxidase with another suitable metal which could potentially enhance reactivity and stability of lignin peroxidase.
Conclusion
Lignin peroxidase has several potential applications in which some provides solutions for major environmental concerns with regards to pollutants. But for its commercial application there is much work to be done to enhance the activity and stability of lignin peroxidase where there is much effort to overcome these drawbacks using immobilization and protein engineering.
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