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Molecular biology and structure-function of lignin-degrading heme peroxidases. Enzyme Microb. Technol. 30, 425-444
Abstract
Three peroxidases involved in lignin degradation are produced by white-rot fungi. Lignin peroxidase (LiP) is characterized by oxidation of high redox-potential aromatic compounds (including veratryl alcohol) whereas manganese peroxidase (MnP) requires Mn2+ to complete the catalytic cycle and forms Mn3+ chelates acting as diffusing oxidizers. Pleurotus and Bjerkandera versatile peroxidase (VP) is able to oxidize Mn2+ as well as non-phenolic aromatic compounds, phenols and dyes. Phanerochaete chrysosporium has two gene families including ten LiP-type and three MnP-type genes coding different isoenzymes expressed during secondary metabolism. Two VP genes have been recently cloned from Pleurotus eryngii. Phanerochaete chrysosporium MnP and P. eryngii VP are induced by H2O2, being Mn2+ involved in regulation of their transcript levels. At least eighteen more ligninolytic peroxidase genes have been cloned from other white-rot fungi. Protein sequence comparison reveals that typical MnP from P. chrysosporium and two other fungi (showing a longer C-terminal tail) are separated from other ligninolytic peroxidases, which form two main groups including P. chrysosporium LiP and Pleurotus peroxidases respectively.
... White-rot fungi secrete many kinds of MnPs and LiPs during the vegetative mycelial growth stage (Gold and Alic 1993). Generally, MnPs and LiPs occur as a series of isozymes encoded by a family of genes; many MnP and LiP isozymes have been purifi ed, and their encoding genes have been cloned from various white-rot basidiomycetes (Martínez 2002). Basidiomycete MnPs and LiPs are heme-containing class II fungal peroxidases (Welinder 1992). ...
... This result suggested that lemnp1 is a singlecopy gene. Amino acid residues known to be involved in peroxidase function, Arg72, His76, and His200 (Martínez 2002), were conserved in LeMnP1. The residues that constitute the Mnbinding sites of the Phanerochaete chrysosporium Burds MnP isozymes (Alic et al. 1997) and the Agaricus bisporus (Lange.) ...
... Kamitsuji et al. (2004) have reported that the production of MnP isozymes by P. ostreatus was controlled by culture conditions (nitrogen concentration) at the transcriptional level. In P. chrysosporium, the expression of the MnP gene can be regulated by Mn, nitrogen concentration, and heat shock (Martínez 2002). Considering that L. edodes was previously reported to produce only one MnP in oak wood culture (Forrester et al. 1990), LeMnP1 may be an isozyme of L. edodes repressed by the environmental conditions employed in the previous study. ...
A genomic DNA sequence and cDNA encoding a putative manganese peroxidase were isolated from the white-rot basidiomycete Lentinula edodes. The gene, called lemnp1, consists of a 1985-bp open reading frame interrupted by 16 introns and was flanked by an upstream region having putative CAAT, TATA, and heat shock elements and by a downstream region having polyadenylation signals. The lemnp1 gene encodes a protein of 364 amino acids that shows high sequence homology to manganese peroxidases of other basidiomycetes. The deduced N-terminal amino acid sequence is different from the L. edodes manganese peroxidase reported previously.
... and in several basidiomycete fungi have been cloned and characterized. Fungal MnPs and LiPs are members of the class II peroxidase family (Welinder 1992), which is further divided into four classes based on their amino acid sequence similarity (Martínez 2002). MnPs belong to two separate classes: I and II (Martínez 2002). ...
... Fungal MnPs and LiPs are members of the class II peroxidase family (Welinder 1992), which is further divided into four classes based on their amino acid sequence similarity (Martínez 2002). MnPs belong to two separate classes: I and II (Martínez 2002). Recently, genes of a third type of peroxidase family, the versatile peroxidases (VPs), were cloned from Pleurotus eryngii (DC.) ...
... VPs are enzymatically similar to both MnPs and LiPs in that they can oxidize not only Mn 2+ but also aromatic compounds or dyes in manganeseindependent reactions. The regulation of MnP genes has been well investigated in P. chrysosporium and has been reviewed by Gold and Alic (1993) and Martínez (2002). MnP production in fungi is regulated at the transcriptional level by nutrient nitrogen sources (Pribnow et al. 1989;Li et al. 1994). ...
Manganese peroxidase (MnP), which is one of the lignin-degrading enzymes from white-rot fungi, possesses oxidative activity against phenolic compounds, making it a useful enzyme for bioremediation. A novel MnP-encoding gene (lemnp2) was isolated from Lentinula edodes. The deduced amino acid sequence showed approximately 48.8% homology to LeMnP1. The cDNA clone was approximately 1.4 kbp whereas the genomic sequence was 1.9 kbp, and comparison of the two indicated that lemnp2 contains 13 introns. The upstream region of lemnp2 contains putative CAAT, TATA, and metal response elements. Additionally, LeMnP2 contains conserved motifs that are observed in fungal MnPs, including 10 cysteines, a Mn-binding site, and Ca²⁺-binding sites. The lemnp2 transcript was identified in mycelium cultivated on sawdust medium, and the protein was secreted into the medium. MnP activity was purified from the sawdust medium as one peak during purification. Western blot analysis confirmed that LeMnP2, but not LeMnP1, was secreted into the sawdust medium. These results collectively demonstrate that LeMnP2 is the major MnP secreted into sawdust medium.
... All fungal ligninolytic peroxidases are extracellular enzymes that share a similar topology and folding formed predominantly by alpha helices in two domains among which the heme group is tightly embedded. In all cases, the protein structure is stabilized by the presence of four disulfide bridges (five in the case of MnP) and by two structural Ca 2+ ions [227]. ...
... Therefore, organic chelators, such as malonate and oxalate, are required for allowing the dissociation of Mn(III) from the enzyme and for the stabilization of the diffusing oxidizers. The existence of such low molecular weight redox mediators is fundamental for the initial degradation of lignin since the compact molecular structure of the biopolymer prevents direct enzyme-lignin contact [227]. Therefore, Mn(III) is responsible for the direct oxidation of the minor phenolic substructures of lignin but also for the indirect oxidation of the non-phenolic lignin units through the generation of lipid peroxyl radicals. ...
... Elongation of the Fe-NHis bond is a consequence of the strong H-bond interaction between the axial histidine and the contiguous aspartate residue (His176-Asp238 in pristine LiP) as well as between the serine bound to proximal histidine and another aspartate belonging to a different protein helix (His177-Asp201 in pristine LiP). This H-bond causes the movement of the proximal histidine residue away from the heme and weakens the Fe-NHis bond, thereby increasing the heme iron electron deficiency [227,238,239,245]. ...
Molecular modeling techniques have become indispensable in many fields of molecular sciences in which the details related to mechanisms and reactivity need to be studied at an atomistic level. This review article provides a collection of computational modeling works on a topic of enormous interest and urgent relevance: the properties of metalloenzymes involved in the degradation and valorization of natural biopolymers and synthetic plastics on the basis of both circular biofuel production and bioremediation strategies. In particular, we will focus on lytic polysaccharide monooxygenase, laccases, and various heme peroxidases involved in the processing of polysaccharides, lignins, rubbers, and some synthetic polymers. Special attention will be dedicated to the interaction between these enzymes and their substrate studied at different levels of theory, starting from classical molecular docking and molecular dynamics techniques up to techniques based on quantum chemistry.
... On the other hand, it corresponded to the smallest extent (40%) to the N-terminal sequence of LiP BA45 B. adusta CX-9 (Bouacem et al., 2018). In general, the N-terminal sequences of peroxidases are characterized by the presence of cysteine residues, which are involved in the formation of the disulfide bond (Martínez, 2002). Studies conducted by other authors show that some fungal peroxidases produced by, Pleurotus eryngii, Thanatephorus cucumeris Dec1, and Bjerkandera adusta DSM strains involved in the breakdown of aromatic compounds lack cysteine in the initial N-terminal sequence (Martinez et al., 1996;Heinfling et al., 1998;Sugano et al., 2006) or an unidentified amino acid (unknown residue) is present at this position in the sequence (Heinfling et al., 1998). ...
... Like other fungal peroxidases, VP/Ba was characterized by the highest activity in the presence of 0.1 mM Al 3+ , Cu 2+ , Ca 2+ , and Mn 2+ ions (126%-176%), while Ag 2+ , Hg 2+ , and Ni + significantly decreased its activity. Metal ions, especially Ca 2+ , Cu 2+ and Mn 2+ are necessary for fungi since they participate as cofactors of peroxidases (Martínez 2002;Kostadinova et al., 2018;Robinson et al., 2021). Previously study indicated that Cu 2+ and Mn 2+ were observed to increase the MnP peroxidase activity up 1 mM. ...
... Peroxidases activity is strongly dependent on the presence of Ca 2+ . Some fungal peroxidases have a calmodulin binding domain and Ca 2+ may switch a peroxidase between different modes of action (Martínez 2002;Plieth and Vollbehr 2012). Xue and Tao (2008) indicated an increase of ligninolytic peroxidase activity in the presence of lower Al 3+ concentrations (10-50 μM). ...
The aim of the study was to purify and characterize a peroxidase synthesized by the B. adusta strain CCBAS 930. Moreover, optimal pH and temperature, substrate specificity (2,2′-azino-bis(3-ethylben-zothiazoline-6-sulfonate (ABTS), veratryl alcohol (VA), guaiacol (GA), o-dianisidine, 2,6-dimetoxyphenol (2,6-DMP), 2-methoxy-1,4-benzohydroquinone (MBQH2), 2,6-Dimethoxy-1,4-benzohydroquinone (DBQH2)) and the influence of different ions (Al³⁺, Mn²⁺, Mg²⁺, Ca²⁺, Cu²⁺, Fe²⁺, Co²⁺, Ag²⁺, Cd²⁺, Hg²⁺, and K⁺ and organic solvents (ethanol, methanol, DMSO, ethyl acetate, n-hexane, diethyl ether, and dichloromethane) on VP/Ba activity were determined. The purified VP/Ba had an RZ value (A407/A280) of 1.85, 45 kDa and an N-terminal sequence VTXPGKVNVVENSA. The catalytic activity of VP/Ba measured by the oxidation of 2,6-DMP with/without Mn²⁺ was noted at optimum temperature values of 50 °C and 55 °C, respectively. After 24h incubation in different pH conditions (pH = 3.0–7.0), residual VP/Ba activity ranging from 30 to 50% with the optimum at pH = 5.5 (50%) was observed. All the seven substrates tested were oxidized by purified VP/Ba, but the oxidation ratio of 2,6-DMP, VA, GA, and o-dianisidine in the presence of Mn²⁺ was higher. The highest activity of VP/Ba was detected in the presence of 0.1 mM Al³⁺, Cu²⁺, Ca²⁺, and Mn²⁺ ions (126%–176%), while Ag²⁺, Hg²⁺, Ni⁺, and K⁺ at all the concentrations tested (5, 1, and 0.1 mM) significantly decreased its activity. Organic solvents e.g. ethanol, methanol, DMSO, ethyl acetate, n-hexane, diethyl ether, and dichloromethane, partially decreased the activities of VP/Ba measured without and with Mn²⁺, with residual activities ranging from 10.33% to 69.16% and from 50% to 68.75%, respectively.
... They are globular proteins formed by 11-12 predominantly helices in two domains, delimiting a central cavity harboring the heme (up to four short sheets are also shown). In order to stabilize protein structure, they have eight cysteine residues forming four conserved disulfide bridges, exception for MnP that has a fifth bridge in its C-terminal region and two Ca 2þ binding sites (Choinowski et al., 1999;Martinez, 2002). ...
... not only is specific for Mn (II) as MnP but also oxidizes phenolic and non-phenolic substrates that are typical for LiP, including veratryl alcohol and lignin model compounds, in the absence of manganese ions. The catalytic versatility of VP is due to its molecular architecture, which has binding sites for Mn 2þ , such as in MnP, and the presence of catalytic Tryptophan residue like that found in LiP (Wong, 2009); however, the differences in catalytic properties and structural characteristics justify the description of VP as another peroxidase family in Class II (Martinez, 2002). ...
... After manual curating, three proteins were excluded based on their size: sequences 14 and 32 were considered too short (190 and 196 residues, respectively) while the sequence 42 was very large (688 residues) and seems to be truncated in the corresponding typical POD region. Considering that POD size varies between 345 to 427 residues (Janusz et al., 2013;Martinez, 2002;Wong, 2009), these sequences may have been wrongly annotated. Additionally, sequence 12 was removed because of its missed residues. ...
Functional annotation of Trametes villosa genome was performed to search Class II peroxidase proteins in this white-rot fungus, which can be valuable for several biotechnological processes. After sequence identification and manual curation, five proteins were selected to build 3 D models by comparative modeling. Analysis of sequential and structural sequences from selected targets revealed the presence of two putative Lignin Peroxidase and three putative Manganese Peroxidase on this fungal genome. All 3 D models had a similar folding pattern from selected 3 D structure templates. After minimization and validation steps, the best 3 D models were subjected to docking studies and molecular dynamics to identify structural requirements and the interactions required for molecular recognition. Two reliable 3 D models of Class II peroxidases, with typical catalytic site and architecture, and its protein sequences are indicated to recombinant production in biotechnological applications, such as bioenergy.
Communicated by Ramaswamy H. Sarma
... Therefore, this possibility can be excluded. 4. Compound I and Compound II both oxidize Mn 2+ exclusively, but in the latter case some of the resulting Mn 3+ oxidizes aldehydes before it can be released from the glutamate and aspartate residues that constitute the MnP binding site for Mn ions (4). Physiological oxalate competes with this process, because it removes some of the Mn 3+ to give a chelate that is thermodynamically more stable, less electropositive, and consequently less oxidizing. ...
... Physiological oxalate competes with this process, because it removes some of the Mn 3+ to give a chelate that is thermodynamically more stable, less electropositive, and consequently less oxidizing. The tendency of Mn 3+ produced by Compound II of MnP to remain associated with the enzyme in the absence of a chelator such as oxalate is well documented (4,8,41). Although it has not been shown that this enzyme-coordinated Mn 3+ can oxidize substrates, hypothesis 4 is the only one of the four that appears consistent with our data and with the literature. ...
The ability of some white rot basidiomycetes to remove lignin selectively from wood indicates that low molecular weight oxidants have a role in ligninolysis. These oxidants are likely free radicals generated by fungal peroxidases from compounds in the biodegrading wood. Past work supports a role for manganese peroxidases (MnPs) in the production of ligninolytic oxidants from fungal membrane lipids. However, the fatty acid alkylperoxyl radicals initially formed during this process are not reactive enough to attack the major structures in lignin. Here, we evaluate the hypothesis that the peroxidation of fatty aldehydes might provide a source of more reactive acylperoxyl radicals. We found that Gelatoporia subvermispora produced trans-2-nonenal, trans-2-octenal, and n-hexanal (a likely metabolite of trans-2,4-decadienal) during the incipient decay of aspen wood. Fungal fatty aldehydes supported the in vitro oxidation by MnPs of a nonphenolic lignin model dimer, and also of the monomeric model veratryl alcohol. Experiments with the latter compound showed that the reactions were partially inhibited by oxalate, the chelator that white rot fungi employ to detach Mn³⁺ from the MnP active site, but nevertheless proceeded at its physiological concentration of 1 mM. The addition of catalase was inhibitory, which suggests that the standard MnP catalytic cycle is involved in the oxidation of aldehydes. MnP oxidized trans-2-nonenal quantitatively to trans-2-nonenoic acid with the consumption of one O2 equivalent. The data suggest that when Mn³⁺ remains associated with MnP, it can oxidize aldehydes to their acyl radicals, and the latter subsequently add O2 to become ligninolytic acylperoxyl radicals.
IMPORTANCE
The biodegradation of lignin by white rot fungi is essential for the natural recycling of plant biomass and has useful applications in lignocellulose bioprocessing. Although fungal peroxidases have a key role in ligninolysis, past work indicates that biodegradation is initiated by smaller, as yet unidentified oxidants that can infiltrate the substrate. Here, we present evidence that the peroxidase-catalyzed oxidation of naturally occurring fungal aldehydes may provide a source of ligninolytic free radical oxidants.
... The crystal structure of ApeLiP maintains the heme cavity architecture found in other ligninolytic peroxidases [62]. The cofactor is buried in the structure and sandwiched by proximal (F) and distal (B) helices, which contain conserved residues involved in the heterolytic cleavage of H 2 O 2 to form CI (at the distal side) and in the modulation of the redox-potential of the enzyme (with a proximal histidine acting as fifth ligand of the heme iron). ...
... As reported for plant and fungal generic peroxidases and for P. eryngii VP, phenolic and other low redox-potential substrates can be oxidized directly by the heme through the channel that gives access to H 2 O 2 [41,55]. However, the narrowness of this channel in most LiPs impedes these aromatic substrates to directly interact with the cofactor [62]. The size and shape similarities between the ApeLiP and VP heme channels may explain the kinetic identification of a low-efficiency site for ABTS oxidation in the former enzyme, which remained when Trp166 was removed. ...
Lignin biodegradation has been extensively studied in white-rot fungi, which largely belong to order Polyporales. Among the enzymes that wood-rotting polypores secrete, lignin peroxidases (LiPs) have been labeled as the most efficient. Here, we characterize a similar enzyme (ApeLiP) from a fungus of the order Agaricales (with ~13,000 described species), the soil-inhabiting mushroom Agrocybe pediades. X-ray crystallography revealed that ApeLiP is structurally related to Polyporales LiPs, with a conserved heme-pocket and a solvent-exposed tryptophan. Its biochemical characterization shows that ApeLiP can oxidize both phenolic and non-phenolic lignin model-compounds, as well as different dyes. Moreover, using stopped-flow rapid spectrophotometry and 2D-NMR, we demonstrate that ApeLiP can also act on real lignin. Characterization of a variant lacking the above tryptophan residue shows that this is the oxidation site for lignin and other high redox-potential substrates, and also plays a role in phenolic substrate oxidation. The reduction potentials of the catalytic-cycle intermediates were estimated by stopped-flow in equilibrium reactions, showing similar activation by H2O2, but a lower potential for the rate-limiting step (compound-II reduction) compared to other LiPs. Unexpectedly, ApeLiP was stable from acidic to basic pH, a relevant feature for application considering its different optima for oxidation of phenolic and nonphenolic compounds.
... Figure 8B shows a detail of the IrlacDyP heme environment, which resembles that found in other DyPs. An aspartic (Asp172) and an arginine (Arg335) located over the heme plane (distal side) are expected to participate in the heterolytic cleavage of H 2 O 2 and enzyme activation forming the compound I, as distal Asp168 and Arg332 have been described to do in AauDyP, or as distal arginine and histidine do in other classical fungal peroxidases [54]. At the other side of the heme plane (proximal side), His312 acts as fifth coordination position of the heme iron, being conserved in all basidiomycete DyPs and classical fungal peroxidases. ...
... At the other side of the heme plane (proximal side), His312 acts as fifth coordination position of the heme iron, being conserved in all basidiomycete DyPs and classical fungal peroxidases. Near the proximal histidine and aspartic (Asp397) residues, a phenylalanine occupies the position of Val253 in AauDyP or tryptophan in cytochrome C peroxidase [54]. In H 2 O 2 -activated cytochrome C peroxidase, a catalytic protein radical is located in this tryptophan residue [55,56], but no radicals have been described at this position in other peroxidases. ...
A dye-decolorizing peroxidase (DyP) from Irpex lacteus was cloned and heterologously expressed as inclusion bodies in Escherichia coli. The protein was purified in one chromatographic step after its in vitro activation. It was active on ABTS, 2,6-dimethoxyphenol (DMP), and anthraquinoid and azo dyes as reported for other fungal DyPs, but it was also able to oxidize Mn2+ (as manganese peroxidases and versatile peroxidases) and veratryl alcohol (VA) (as lignin peroxidases and versatile peroxidases). This corroborated that I. lacteus DyPs are the only enzymes able to oxidize high redox potential dyes, VA and Mn+2. Phylogenetic analysis grouped this enzyme with other type D-DyPs from basidiomycetes. In addition to its interest for dye decolorization, the results of the transformation of softwood and hardwood lignosulfonates suggest a putative biological role of this enzyme in the degradation of phenolic lignin.
... LiP and MnP have three reactions of the catalytic cycle which are as follows a) Hydrogen peroxide oxidizes the enzyme to produce compound I (modified enzyme) and water, (b) The compound I (modified enzyme) catalyzes the production of compound II (second modified form of an enzyme), formed by the electron transfer from the reduced substrate along with a generation of free radical, (c) The compound II reacts with the molecule of a reduced substrate to produce another free radical and water. As the reaction continues the enzymes reduces to its native form, (Piontek et al. 1993;Sundaramoorthy et al. 1994;Choinowski et al. 1999;Martínez 2002;Dias et al. 2007;Piontek et al. 1993) and are represented in Fig. 11.2. ...
... The compound II then oxidizes Mn 2+ to Mn 3+ which is responsible to oxidize aromatic compounds. The converted Mn 3+ are than stabilized by organic acids which react nonspecifically with organic molecules thereby removing an electron and a proton from the substrates (Hofrichter 2002), The attack of the Mn 3+ which is a small size compound having high redox potential diffuses easily in the lignified cell wall thereby attacking inside the plant cell wall facilitating the penetration as well as the action of other enzymes (Martínez 2002;Hammel and Cullen 2008). ...
The enzymes and its utility have increased tremendously over the past decade, as the focus presently is diverting toward the development of technologies that are cyclic in nature. This idea depends on the fact that both the substrate and the end product should be biodegradable and should fit well with the idea of it being recycled and reused. The enzymes are biological molecules when used commercially can solve many issues e.g., agro-residues waste disposal, replacement of synthetic processes to natural more environment reliable processes. The effective utilization of agro-residues in biorefinery has been gaining attention but its application has been restricted due to higher lignin content and expensive chemical treatment. The biological delignification involving xylanase, cellulose, and ligninolytic enzymes is an effective method, cheap and carbon neutral as well. These enzymes have wide utility and with the advancement of techniques i.e., protein engineering has enabled the synthesis of enzymes that are industrially feasible, higher production yield and can tolerate harsh conditions. This has widened the application to the areas which were previously not known and were either not possible due to the restrictions. This chapter focuses on different enzymes, the method involved in the production, and its application in the bio-based economy.
... Extracellularly, under the assistance of laccases and peroxidases, monolignols are activated, typically producing radicals at phenol OH groups, and then polymerize oxidatively. Of note, the very same enzyme classes are also those capable of lignin degradation [134,135], always through oxidative mechanisms. ...
At a time when environmental considerations are increasingly pushing for the application of circular economy concepts in materials science, lignin stands out as an under-used but promising and environmentally benign building block. This review focuses (A) on understanding what we mean with lignin, i.e., where it can be found and how it is produced in plants, devoting particular attention to the identity of lignols (including ferulates that are instrumental for integrating lignin with cell wall polysaccharides) and to the details of their coupling reactions and (B) on providing an overview how lignin can actually be employed as a component of materials in healthcare and energy applications, finally paying specific attention to the use of lignin in the development of organic shape-memory materials.
... Cations are also vital to the lignocellulose-metabolizing mechanisms used by these fungi, either as reactants or as part of enzyme systems. The peroxidases used by white rot fungi to degrade ligninlignin peroxidases, manganese peroxidases, and versatile peroxidaseshave both calcium and iron in their structures, with manganese peroxidase and versatile peroxidase additionally utilizing manganese as a reactant (Martínez, 2002;Ruiz-Dueñas et al., 2013). The ROS based mechanism used by brown rot fungi relies on Fenton chemistry to generate hydroxyl radicals. ...
Fungi are the primary decomposers of wood across the globe. They redistribute the vast pool of wood carbon to other parts of the carbon cycle using mechanisms that also have intriguing potential in bioconversion and biotechnology applications. To make a living in the relatively sparse microenvironment of wood, these filamentous fungi must import many of their needs, including cations. Understanding the timing of cation import can help us validate the functions of cations in wood decay and create a clearer understanding of these complex wood degradation mechanisms. In this study, we resolved cation timing dynamics across space for two fungi (brown rot fungus Rhodonia placenta and white rot fungus Pleurotus ostreatus) using a wood wafer system. We found some expected patterns of the cation dynamics for both fungi, and a clear role for iron at the early stages of brown rot decay. On the other hand, the lack of an increase in manganese during initial white rot decay was surprising. Unexpectedly, we also saw a spike in copper during early brown rot decay that demands more investigation as a potential player in the brown rot mechanism.
... In addition, different types of enzymesencoding genes that involved in lignin degradation have been characterized (Janusz et al. 2013). To date, cDNA cloning of lip, mnp, and vp has been conducted in several species, including Polyporales, Agaricales, and Corticiales Basidiomycetes (Camarero et al. 1999;Ruiz-Dueñas et al. 1999Martinez 2002;Moreira et al. 2005;Mohorčič et al. 2009;Janusz et al. 2013;Schüttmann et al. 2014). ...
Inonotus obliquus is a pathogenic fungus found in living trees and has been widely used as a traditional medicine for cancer therapy. Although lignocellulose-degrading enzymes are involved in the early stages of host infection, the parasitic life cycle of this fungus has not been fully understood. In this study, we aimed to investigate the activities of laccase (Lac), manganese peroxidase (MnP), and lignin peroxidase (LiP) from I. obliquus cultivated in Kirk’s medium. The fungus was subjected to genome sequencing, and genes related to wood degradation were identified. The draft genome sequence of this fungus comprised 21,203 predicted protein-coding genes, of which 134 were estimated to be related to wood degradation. Among these, 47 genes associated with lignin degradation were found to have the highest number of mnp genes. Furthermore, we cloned the cDNA encoding a putative MnP, referred to as IoMnP1, and characterized its molecular structure. The results show that IoMnP1 has catalytic properties analogous to MnP. Phylogenetic analysis also confirmed that IoMnP1 was closely related to the MnPs from Pyrrhoderma noxium, Fomitiporia mediterranea, and Sanghuangporus baumii, which belong to the same family of Hymenochaetaceae. From the above results, we suggest that IoMnP1 is a member of MnPs.
... & Ryvarden, degraded polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) (Bumpus et al. 1985;Valli et al. 1992). The most general lignin-degrading enzymes are two closely related hemecontaining peroxidases (Martínez 2002;Morgenstern et al. 2008), manganese peroxidase (MnP; EC 1.11.1.13.) and lignin peroxidase (LiP; EC 1.11.1.14.), and a multi-coppercontaining phenoloxidase laccase (Lac; EC 1.10.3.2.). Studies on Phanerochaete chrysosporium developed a model of 2,7-diclorodibenzo-p-dioxin (2,7-DCDD) degradation, which was catalyzed by 1-electron oxidations of LiP (Bumpus et al. 1985;Valli et al. 1992;Joshi and Gold 1994). ...
A degradation experiment on dibenzo-p-dioxin (DD) and 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD) was carried out using basidiomycetous fungi belonging to the genera Coprinus, Coprinellus, and Coprinopsis. Some species showed a high rate of decrease in DD for the 2-week test period. Among them, Coprinellus disseminatus showed the highest ability to decrease the DD level, close to 100% by the end of 2 weeks. Further examination showed that Coprinellus disseminatus and Coprinellus micaceus, belonging to the genus Coprinellus, were able to metabolize 2,7-DCDD to a monohydroxylated compound, probably mediated by the P450 system. The metabolism of chlorinated DD by fungi capable of living in soil conditions is reported here for the first time.
... The structure of LiP was constructed based on the crystal structure of the native LiP from P. chrysosporium (PDB entry: 1B80, which was obtained at pH = 3.5 and with a resolution of 1.73 Å). 35 The P. chrysosporium LiP has been extensively studied by experiments, which has well-characterized crystal structures and biochemical data, [6][7][8][9][10][11][12]36,37 providing an excellent example for comparison with theoretical studies. Only chain A was retained in our study. ...
pH buffer plays versatile roles in both biology and chemistry. In this study, we unravel the critical role of pH buffer in accelerating degradation of the lignin substrate in lignin peroxidase (LiP) using QM/MM MD simulations and the nonadiabatic electron transfer (ET) and proton-coupled electron transfer (PCET) theories. As a key enzyme involved in lignin degradation, LiP accomplishes the oxidation of lignin via two consecutive ET reactions and the subsequent C-C cleavage of the lignin cation radical. The first one involves ET from Trp171 to the active species of Compound I, while the second one involves ET from the lignin substrate to the Trp171 radical. Differing from the common view that pH = 3 may enhance the oxidizing power of Cpd I via protonation of the protein environment, our study shows that the intrinsic electric fields have minor effects on the first ET step. Instead, our study shows that the pH buffer of tartaric acid plays key roles during the second ET step. Our study shows that the pH buffer of tartaric acid can form a strong H-bond with Glu250, which can prevent the proton transfer from the Trp171-H•+ cation radical to Glu250, thereby stabilizing the Trp171-H•+ cation radical for the lignin oxidation. In addition, the pH buffer of tartaric acid can enhance the oxidizing power of the Trp171-H•+ cation radical via both the protonation of the proximal Asp264 and the second-sphere H-bond with Glu250. Such synergistic effects of pH buffer facilitate the thermodynamics of the second ET step and reduce the overall barrier of lignin degradation by ∼4.3 kcal/mol, which corresponds to a rate acceleration of 103-fold that agrees with experiments. These findings not only expand our understanding on pH-dependent redox reactions in both biology and chemistry but also provide valuable insights into tryptophan-mediated biological ET reactions.
... It can oxidize low as well as high redox potential substrates [39]. It is fascinating to know that the VP enzyme shows dissimilar optimal pH for the oxidation of aromatic compounds with pH 3 or Mn2 + with pH 5 (Fig. 9d) [40,41]. Fig. 9 shows the catalytic activity of Lignin modifying enzymes. ...
Endocrine disruptors, also known as EDCs are omnipresent, as their presence can be easily observed in our day-to-day life. Endless exposure to EDCs can cause adverse health effects in organisms such as type 2 diabetes, cardiovascular diseases, obesity, growth inhibition, morphological deformities, behavioral changes, and certain types of cancer. The objective of this review is to deliver an overview of the environmental aspects of endocrine-disrupting chemicals and their effects on animals and human health. Based on their origin, EDCs are divided into two types: (a) Synthetic or man-made such as bisphenol A (BPA), triclosan (TCS), and other chemicals, and (b) natural hormones namely estrone E1, 17β-estradiol (E2), and estriol (E3); both are prone to human and animal health. Emphasis is given to the treatment of EDCs by eliminating them from the wastewater through advanced oxidation processes. However, these processes are generally expensive with limited specificity and side effects. An alternate approach is used for the post-treatment of EDCs by high redox potential enzyme peroxidases which are produced only by specific strains of ligninolytic fungi, helping in minimizing endocrine-disrupting compounds.
... Steroids are ubiquitously available bioactive compounds containing the tetracyclic ring system belonging to terpenes. >250 sterols and related compounds have been reported in lower eukaryotes, including yeast, fungi, insects, plants, and invertebrates [1][2][3]. Sterols and their derivatives are important components responsible for the structure of cell membranes. They are also precursors for the production of steroids hormones, bile, and vitamins [4,5]. ...
Glucosyltransferases catalyze the glucosidic bond formation by transferring a glucose molecule from an activated sugar donor to an acceptor substrate. Glucocorticoids (GCs) are adrenal-derived steroid hormones most widely used for anti-inflammatory treatments. In this study, we biotransformed two selected GCs, cortisone and prednisone, into their O-glucoside derivatives using a versatile UDP-glycosyltransferase UGT-1. Complete structural assignment of glucosylated products revealed that the bioconversion by regio-selective glucosylation of cortisone and prednisone produced cortisone 21-glucoside and prednisone 21-glucoside, respectively. We also combined molecular dynamics (MD) simulation to study the binding feature and mechanism of glucosylation. MD simulation studies showed the formation of a stable complex between protein, glucose donor, and substrate, stabilized by hydrogen bonds. Overall, we were able to provide explanations for the experimentally observed selectivity for glucosylation by integrating experimental and computational techniques.
... This agrees with the visual colonization of the microcosms that was more vigorous for A. bisporus and P. eryngii than for the other fungi. MnP has a high redox potential and is characterized by oxidizing phenolic structures [54]. It was observed that all the bioaugmented treatments showed a relatively higher value than the control, highlighting again P. eryngii and A. bisporus. ...
Bioremediation techniques are being developed as substitutes for physical–chemical methodologies that are expensive and not sustainable. For example, using the agricultural waste spent mushroom substrate (SMS) which contains valuable microbiota for soil bioremediation. In this work, SMSs of four cultivated fungal species, Pleurotus eryngii, Lentinula edodes, Pleurotus ostreatus, and Agaricus bisporus were evaluated for the bioremediation of soils contaminated by petroleum hydrocarbons (TPHs). The bioremediation test was carried out by mixing the four different SMSs with the TPH-contaminated soil in comparison with an unamended soil control to assess its natural attenuation. To determine the most efficient bioremediation strategy, hydrolase, dehydrogenase, and ligninolytic activities, ergosterol content, and percentage of TPHs degradation (total and by chains) were determined at the end of the assay at 40 days. The application of SMS significantly improved the degradation of TPHs with respect to the control. The most effective spent mushroom substrate to degrade TPHs was A. bisporus, followed by L. edodes and P. ostreatus. Similar results were obtained for the removal of aliphatic and aromatic hydrocarbons. The results showed the effectiveness of SMS to remove aliphatic and aromatic hydrocarbons from C10 to C35. This work demonstrates an alternative to valorizing an abundant agricultural waste as SMS to bioremediate contaminated soils.
... La manganeso peroxidasa contiene un grupo hemo (hierro protoporfirina IX) alojado entre dos dominios con abundancia de hélices α (Martínez 2002) (Figura 1.3). ...
Armillaria es un género de hongos de la pudrición blanca y constituye uno de los
patógenos más ampliamente estudiados por las pérdidas que ocasiona en la actividad forestal. En los últimos años se ha comenzado a utilizar la batería enzimática ligninolítica de este organismo en actividades de biorremediación que involucran la producción de enzimas extracelulares y la transformación de colorantes. En Argentina este patógeno está asociado a bosque nativo e implantado de Patagonia y recientemente han sido dilucidadas su taxonomía y filogenia estableciéndose cuatro linajes correspondientes a cuatro especies. El objetivo de este trabajo fue caracterizar la enzimología ligninolítica de especies de Armillaria en Patagonia y su capacidad de biodegradar compuestos contaminantes. Se cuantificaron las actividades lacasa y manganeso peroxidasa basales de 17
aislamientos y se evaluó su inducción por aditivos xenobióticos (guayacol y cobre) en el cultivo en 6 aislamientos representativos de las cuatro especies. Se determinaron los perfiles electroforéticos de isoenzimas de lacasa de 12 aislamientos y de tres aislamientos cultivados con los distintos xenobióticos. Se evaluó la capacidad de decoloración del colorante trifenilmetano verde de malaquita.
La actividad lacasa, su perfil de isoenzimas, la susceptibilidad de ser inducida y la
capacidad degradadora del colorante sugirieron ser caracteres quimiotaxonómicos útiles para Armillaria en Patagonia, no así la actividad manganeso peroxidasa que no resultó de interés a estos fines y registró actividades muy bajas o indetectables en concordancia con otras especies de la pudrición blanca citadas en bibliografía.
Todas las especies demostraron ser efectivas en degradar el colorante verde de malaquita pero A. montagnei y A. novae-zelandiae exhibieron las mayores capacidades degradadoras, asociadas a las mayores actividades lacasa. En A. umbrinobrunnea y A. sparrei la degradación no estuvo asociada a actividades lacasa significativas, por lo que otras ligninasas u otros procesos oxidativos podrían ser los responsables de la transformación del contaminante. A. novae-zelandiae presentó la mayor tendencia a inducción de la enzima lacasa, resultando condicionante la presencia de aditivos xenobióticos para el registro de actividades enzimáticas elevadas en condiciones de cultivo; la inducción evidenció ser diferencial para algunas isoformas de lacasa en esta especie. El perfil electroforético reveló tres isoenzimas de lacasa, dos de ellas nuevas para el género, y la heterogeneidad interespecífica suficiente para resultar útil en caracterizar las especies patagónicas; ambos elementos resultan novedosos en el marco actual de conocimiento del género.
... Versatile peroxidases are attractive wild-type ligninolytic enzymes having characteristic bifunctional oxidative capability and a broad spectrum substrate preference (Camarero et al. 1999). The genomic studies have revealed that VPs share the catalytic properties of both MnP and LiP and are capable of oxidizing Mn 2+ to Mn 3+ , as well as degradation of high redox potential non-phenolic lignin substrates like MnP and LiP, respectively (Martı nez 2002;Pérez-Boada et al. 2005). Unlike LiP and MnP, VPs are also competent in catalyzing the oxidation of complex lignin structures such as hydroquinone and substituted phenol units without any mediator ). ...
Lignin is the most abundant polyphenolic aromatic biopolymer on Earth, which is extremely recalcitrant toward biodegradation, owing to its heterogeneous structure and biochemical composition. Extensive research efforts have been made to understand the polymeric structure of lignin in a better way and develop a simple, cost-competitive, and eco-friendly method for its degradation. Over the past few years, wood-rotting fungi, especially white-rot fungi have emerged as a crucial group of microorganism capable of mineralizing lignin biopolymers more efficiently. Such fungi have evolved to produce a unique set of extracellular oxidative enzymes in different combinations. Further, they also produce enzymes in multiple isoforms and isozymes that catalyze ligninolysis using radical mediated oxidative reactions. The major ligninolytic enzymes include laccase, manganese peroxidase, lignin peroxidase, and versatile peroxidase. The ligninolytic activities of these enzymes can be enhanced by various natural and/or chemical redox mediators as well as some other auxiliary enzymes (aryl-alcohol oxidase, glyoxal oxidase, quinone reductases, aryl-alcohol dehydrogenases, and feruloyl esterase) to facilitate lignin degradation process. These enzymes have attracted attention of several researchers due to their broad substrate specificity, which make them readily available for numerous biotechnological and industrial applications including paper and pulp industry, food-feed and beverage industry, biofuel industry, bioremediation of hazardous pollutants, and degradation of toxic textile dye effluents. In this chapter, we appraise different ligninolytic fungi from Indian subcontinent and the research findings by native microbiologists and biotechnologists on the fungal enzymatic systems. Finally, the biotechnological and industrial applications of ligninolytic fungi and their enzyme arsenals are also discussed.KeywordsLigninLigninolytic fungiLigninolytic enzymesLaccaseLignin peroxidaseManganese peroxidaseLignin degradation
... Then the positive charge peroxidase (Fe 4+ -R + •) oxidises the substrate molecule, this forms a substrate radical and an intermediate (Fe 4+ ). In a second step the intermediate is further reduced by a second substrate molecule, regenerating the peroxidase and producing another free radical (Martıńez, 2002). The catalytic cycle is further illustrated in the Figure 2-4. ...
Dans ce travail de recherche, des monolithes en silice présentant une très grande porosité (83 %), une double distribution de taille de pores (des macropores de diamètre 20 μm et des méseopores de diamètre 20 nm) et une très grande surface spécifique (370 m2 g-1) ont été utilisés comme supports pour immobiliser une laccase de Trametes versicolor par greffage covalent avec du glutaraldéhyde. Les monolithes enzymatiques ont été utilisés pour dégrader la tétracycline (TC) en solution aqueuse dans un réacteur de configuration tubulaire type "Flow Through Reactor" avec recyclage. Au cours des 5 premières heures de réaction à pH 7 ; 40 à 50 % de la TC a été dégradée, puis un seuil a été atteint. Une des hypothèses pouvant expliquer ce comportement est un éventuel manque de co-substrat (oxygène) à proximité des sites catalytiques. Des monolithes enzymatiques ont été utilisés pendant 75 h de fonctionnement séquentiel sans perte d'activité. L'efficacité de la dégradation de la TC a pu être simulée à travers un modèle mathématique construit en couplant la cinétique de la réaction (Michaelis-Menten) avec un bilan matière en régime dynamique. Les résultats de la simulation ont révélé que le procédé global est contrôlé par la cinétique enzymatique mais que la taille des monolithes pouvait être adaptée pour dégrader 100 % de la CT en un seul passage à travers un monolithe.
... The production of microbial extremophilic MnP enzymes is listed in Table 29.1. The acidic glycoproteincontaining MnP enzymes have a molecular weight between 38 and 62.5 kDa, with a high isoelectric point (pI 4.5) (Martınnez, 2002). The molecular structures of MnP enzymes are similar (43%) to LiP. ...
Extremophiles are specialized groups of microorganisms to survive in a stress environment. The ligninolytic enzymes are capable of functioning at a broad range of environmental parameters due to their specialized protein structure containing the salt bridge between disulfide bond and metallic ions. Laccase, lignin peroxidase (LiP), and manganese peroxidase (MnP) are well-known ligninolytic enzymes that contain copper, calcium, sodium, and magnesium ions interlinked with ligands and hydrogen bonds, which provides the stability for its functions. The broad range substrate degradation by laccase, LiP, and MnP has been reported in Bacillus sp., Streptomyces psammoticus, Paenibacillus sp., Serratia liquefaciens, and Klebsiella pneumonia which are well reported for biodegradation of various industrial waste in stress conditions. The aromatic ring cleavage along with Cα-Cβ breakdown of lignin by LiP from lignocellulosic waste degradation is well known. Simultaneously, the role of laccase and MnP is well reported for depolymerization and detoxification of phenolic and nonphenolic polymers containing dyes, pesticides, endocrine disruptor, and polycyclic aromatic hydrocarbons from various industrial waste. This provides a broad perspective of their biotechnological applications.
... The optimal pH for Mn (II) oxidation and for aromatic compounds oxidation was found to be pH 5 and pH 3, respectively. These optimum pH values were close to the corresponding pH values observed for MnPs and LiPs, respectively [53,139]. VPs undergo the same catalytic cycle as other peroxidases, which uses the two intermediary compounds I and II, but are much more complex due to the array of substrates they can consume [120]. VPs constitutes an important enzyme component for unravelling architecture of lignin. ...
Lignin is the 3rd most abundant biopolymer surpassed by cellulose and hemicellulose and is the most abundant aromatics resource available on earth for utilization by mankind. It was considered undesirable historically which was usually burned as inefficient fuel. Lignin’s 3D recalcitrant nature caused hinderance to feasible biorefinery of holocellulosic fraction of biomass; however, with the rise of lignin biorefinery the concept has changed completely. Now modern biorefinery of biomass insists on making complete value of all the streams including lignin by valorising into variety of phenolics, biopolymers and other high value-added chemicals. Biological depolymerisation of lignin via enzymes is environmentally benign and preferred approach by virtue of low chemical requirement and disposal and energy demand; however, economic challenges are ahead. Robust enzymes are available in nature which can either modify or depolymerise lignin to add further value. Lignin modifying as well as lignin degrading auxiliary enzymes are instrumental and pave the way to a green process for lignin valorisation. This review article is focussed on various lignin degrading as well as lignin modifying enzymes produced by microorganisms especially fungi for degradation or modification of lignin, and its mechanisms, along with the strength and challenges for sustainable bio-based economy development.
... In addition, different types of genes involved in lignin degradation have also been characterized 24 . Molecular characterizations of MnP and LiP have also been conducted in many species using cDNA clone sequences, including Polyporales, Agaricales, and Corticiales basidiomycetes [24][25][26] . By contrast, complementary DNA (cDNA) from VP genes has been cloned and characterized in only a few species, such as P. eryngii 15,16 , P. sapidus 27 , Bjerkandera strain 28 , and B. adusta 29 . ...
Inonotus obliquus is pathogenic fungus on living trees and has been widely used as a traditional medicine for cancer therapy. Although it has been reported that lignocellulose-degrading enzymes are involved in the early stage of host infection, the parasitic life cycle of this fungus has not been fully clarified. In the present study, we investigated the activities of lignin-degrading enzymes from I. obliquus and analyzed the degradation products. Our results revealed that I. obliquus is a pathogenic canker-rot fungus that does not produce lignin peroxidase (LiP), yet is capable of degrading the non-phenolic unit of lignin. The draft genome sequence of this fungus consisted of 21,203 predicted protein coding genes, of which 136 genes were estimated to be related to wood degradation. Furthermore, we found genes encoding putative versatile peroxidase (VP) and dye-decolorizing peroxidase (DyP), which are considered to be involved in the degradation of the non-phenolic unit of lignin. Thus, we cloned the cDNA encoding putative VP, referred as IO-Px, and characterized its molecular structure. From the above results, we suggested that: 1) IO-Px is a new member of MnP, and 2) the ability of I. obliquus to degrade non-phenolic lignin unit might arise from DyP properties.
... Ferreira-Leitao et al. (2007) reported the role of plant Horseradish peroxidase and LiP from Penicillium chrysosporium in the oxidation of Methylene Blue (Basic Blue 9) and Azure B dyes. Recently Versatile Peroxidase (VP) has been purified and described as a new family of ligninolytic peroxidases, along with LiP and MnP obtained from P. chrysosporium (Martinez, 2002). Interestingly, these enzymes have shown the activity of both LiP and MnP and they have potential to oxidize Mn 2+ to Mn 3+ at around pH 5.0 while aromatic compounds at around pH 3.0, despite the presence of Mn 2+ (Heinfling et al., 1998;Ruiz-Duenas et al., 2001). ...
Synthetic dyes are widely used in textile, paper, food, cosmetics and pharmaceutical industries with the textile industry as the
largest consumer. Among all the available synthetic dyes, azo dyes are the largest group of dyes used in textile industry.
Textile dyeing and finishing processes generate a large amount of dye containing wastewater which is one of the main
sources of water pollution problems worldwide. Several physico-chemical methods have been applied to the treatment of
textile wastewater but these methods have many limitations due to high cost, low efficiency and secondary pollution
problems. As an alternative to physico-chemical methods, biological methods comprise bacteria, fungi, yeast, algae and
plants and their enzymes which received increasing interest due to their cost effectiveness and eco-friendly nature.
Decolorization of azo dyes by biological processes may take place either by biosorption or biodegradation. A variety of
reductive and oxidative enzymes may also be involved in the degradation of dyes. This review provides an overview of
decolorization and degradation of azo dyes by biological processes and establishes the fact that these microbial and plant
cells are significantly effective biological weapon against the toxic azo dyes.
... These TLS are also very much involved in high-redox catalytic oxidation of complex aromatic hydrocarbons (Additional file 1: Table S2). Peroxidases (LiP, MnP, and VP) generally have a conserved primary, secondary and tertiary architectures including Ca ion binding sites, heme-pocket residues, and four disulfide bridges [64]. ...
Background: Many fungi grow as saprobic organisms and obtain nutrients from a wide range of dead organic materials. Among saprobes, fungal species that grow on wood or in polluted environments have evolved prolific mechanisms for the production of degrading compounds, such as ligninolytic enzymes. These enzymes include arrays of intense redox-potential oxidoreductase, such as laccase, catalase, and peroxidases. The ability to produce ligninolytic enzymes makes a variety of fungal species suitable for application in many industries, including the production of biofuels and antibiotics, bioremediation, and biomedical application as biosensors. However, fungal ligninolytic enzymes are produced naturally in small quantities that may not meet the industrial or market demands. Over the last decade, combined synthetic biology and computational designs have yielded significant results in enhancing the synthesis of natural compounds in fungi.
Main body of the abstract: In this review, we gave insights into different protein engineering methods, including rational, semi-rational, and directed evolution approaches that have been employed to enhance the production of some important ligninolytic enzymes in fungi. We described the role of metabolic pathway engineering to optimize the synthesis of chemical compounds of interest in various fields. We highlighted synthetic biology novel techniques for biosynthetic gene cluster (BGC) activation in fungo and heterologous reconstruction of BGC in microbial cells. We also discussed in detail some recombinant ligninolytic enzymes that have been successfully enhanced and expressed in different heterologous hosts. Finally, we described recent advance in CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR associated) protein systems as the most promising biotechnology for large-scale production of ligninolytic enzymes.
Short conclusion: Aggregation, expression, and regulation of ligninolytic enzymes in fungi require very complex procedures with many interfering factors. Synthetic and computational biology strategies, as explained in this review, are powerful tools that can be combined to solve these puzzles. These integrated strategies can lead to the production of enzymes with special abilities, such as wide substrate specifications, thermo-stability, tolerance to long time storage, and stability in different substrate conditions, such as pH and nutrients.
... It can perform the oxidation of phenolic compounds though long-range electron transfer system. Versatile peroxidase combines typical properties of manganese and lignin peroxidase, coupled with both substrate ring (Camarero et al., 2001;Ruiz Due Nas et al., 2009) This enzyme has high affinity for manganese and dye but also have affinity to oxidize 2,6-dimethoxy phenol (DMP) and veratryl alcohol (VA) in a manganese independent reaction (Martinez, 2002). The all peroxidase present in fungus help in the lignin degradation collectively called general peroxidases. ...
Seven different strains of Flammulina velutipes collected from the North East and North West Himalayas were evaluated for total protein and ligninolytic enzymes on wheat straw and malt extract liquid broth medium respectively. Nutritional analyses of 6 strains were also done followed with yield evaluation. Maximum mycelia protein was observed in strain DMRX-166 on the 20th day in malt extract medium and DMRX-768 and DMRX-769 in wheat straw medium. Minimum laccase production was observed in strain (DMRO-253). Laccase appearance starts on the 5 th day of incubation and continuously increased upto 20 th day. Highest peroxidase activity was observed in DMRX-897 in wheat straw medium on 15 th day followed by DMRO-253. In general, more peroxidase activity observed in wheat straw media as compared to malt extracts media.On the 15th day strain no. DMRX-1618 produced maximum ligninase activity (175 U/ml) followed by DMRX-1446 on malt extract medium. Wheat straw broth medium favored early lignin peroxidase activity on the 10th day in 3 strains while 4 strains produce more lignin peroxidase on the 20th day. Least lignin peroxidase activity was recorded in DMRX-897 in the malt extract medium. Manganese peroxidase was observed in DMRO-253 in malt extract medium. Nutritional analysis showed highest protein content in DMRX-1446 while highest vitamin C and D 2 were found in DMRX-767. The current study elucidated enzyme and nutritional relationship with time will help for developing efficient cultivation practices in Flammulina in near future.
... and laccase (Lac; EC 1.10.3.2) [2,3]. MnP oxidizes Mn 2+ to Mn 3+ and nonphenolic aromatic compounds with high oxidation-reduction potentials such as lignin [4]. Lac, which is catalyzed by the redox ability of copper ions, can also oxidize nonphenolic substrates with high oxidation-reduction potentials, concomitantly with the reduction of oxygen to water [5][6][7]. ...
Ganoderma produces lignolytic enzymes that can degrade the lignin component of plant cell walls, causing basal stem rot to oil palms. Nitrogen sources may affect plant tolerance to root pathogens while hydrogen peroxide (H2O2), salicylic acid (SA) and jasmonic acid (JA) play important roles in plant defense against pathogens. In this study, we examined the expression of genes encoding manganese peroxidase (MnP) and laccase (Lac) in Ganoderma boninense treated with different nitrogen sources (ammonium nitrate, ammonium sulphate, sodium nitrate and potassium nitrate), JA, SA and H2O2. Transcripts encoding MnP and Lac were cloned from G. boninense. Of the three GbMnP genes, GbMnP_U6011 was up-regulated by all nitrogen sources examined and H2O2 but was down-regulated by JA. The expression of GbMnP_U87 was only up-regulated by JA while GbMnP_35959 was up-regulated by ammonium nitrate but suppressed by sodium nitrate and down-regulated by H2O2. Among the three GbLac genes examined, GbLac_U90667 was up-regulated by ammonium nitrate, JA, SA and H2O2; GbLac_U36023 was up-regulated by JA and H2O2 while GbLac_U30636 was up-regulated by SA but suppressed by ammonium sulphate, sodium nitrate, JA and H2O2. Differential expression of these genes may be required by their different functional roles in G. boninense.
Industrial development has enhanced the release into the environment of large quantities of chemical compounds with high toxicity and limited prospects of degradation. The pollution of soil and water with xenobiotic chemicals has become a major ecological issue; therefore, innovative treatment technologies need to be explored. Fungal bioremediation is a promising technology exploiting their metabolic potential to remove or lower the concentrations of xenobiotics. In particular, white rot fungi (WRF) are unique microorganisms that show high capacities to degrade a wide range of toxic xenobiotic compounds such as synthetic dyes, chlorophenols, polychlorinated biphenyls, organophosphate pesticides, explosives and polycyclic aromatic hydrocarbons (PAHs). In this review, we address the main classes of enzymes involved in the fungal degradation of organic pollutants, the main mechanisms used by fungi to degrade these chemicals and the suitability of fungal biomass or extracellular enzymes for bioremediation. We also exemplify the role of several fungi in degrading pollutants such as synthetic dyes, PAHs and emerging pollutants such as pharmaceuticals and perfluoroalkyl/polyfluoroalkyl substances (PFASs). Finally, we discuss the existing current limitations of using WRF for the bioremediation of polluted environments and future strategies to improve biodegradation processes.
The lingocellulosic wastes are produced by industries, forestry, agriculture and municipalities. The accumulation of these wastes result in several environmental problems, health issues and safety hazards. These lingocellulosic wastes are economically attractive materials for cellulosic bioethanol production because of the large amount of potential sugar for fermentation and bioenergy production. However, the conversion of lignocellulosic biomass is challenged by its recalcitrant structure. For efficient conversion to bioethanol, it is important to study the composition of the raw lingocellulosic residues and devise appropriate delignification and saccharification strategies. The discovery of lignin degrading enzymes (lignin peroxidases, manganese peroxidases, versatile peroxidases and laccases) in cultures of the white rot fungi marked the beginning of the development of enzymatic systems for applied biomass delignification. Degradation of resulting cellulosic biomass is performed by a mixture of hydrolytic enzymes collectively known as glycosyl hydrolases (cellulases and hemicellulases), which act in a synergistic manner in biomass-degrading microorganisms. In comparison with conventional physico-chemical processes, enzymatic delignification and saccharification treatments of lignocellulosic materials are advantageous due to their specificities, low energy requirement, mild operational conditions, absence of substrate loss due to chemical modifications, and no byproduct formation. This review examines what is currently known regarding recent enzymatic technologies for delignification and saccharification of lignocellulosic materials that are used in production of second generation bioethanol.
An increasing attention to biomining technologies has emerged in the mining industry as a result of stricter environmental regulations and economic concerns to process ores with more complex mineralogy having high sulfur and carbon content. The application of bacteria and archaea in biomining has been broadly investigated. Nevertheless, to overcome the present challenges in biomining, there is a potential opportunity for fungi to be employed for the treatment of double/refractory sulfidic ores. This review, for the first time, provides a detailed investigation of the reported fungi used as biological engines to alter numerous sulfur and carbon matrices, including low and high-rank coals, organic and inorganic sulfur, as well as sulfide and carbon minerals. This study illustrates the potential applicability of fungi in biomining technologies, and summarizes microorganisms that have been successfully used with different sulfur and carbon sources. The fundamentals of fungi and their applications have been discussed in detail. Future directions that require further research to foster biomining technologies assisted by fungi have also been provided.
The extensive utilisation of microorganisms namely fungi and bacteria for treating organic wastes has been
attributed to their efficiency in eliminating pathogen and accelerating the degradation process. Their uses have
been found considerably efficient for enhancing waste treatment. Among many methods employed,
composting mediated by indigenous microbial communities has gained significant popularity in treating
organic waste.
Recently, the desire for a safe and effective method for skin whitening has been growing in the cosmetics industry. Commonly used tyrosinase-inhibiting chemical reagents exhibit side effects. Thus, recent studies have focused on performing melanin decolorization with enzymes as an alternative due to the low toxicity of enzymes and their ability to decolorize melanin selectively. Herein, 10 different isozymes were expressed as recombinant lignin peroxidases (LiPs) from Phanerochaete chrysosporium (PcLiPs), and PcLiP isozyme 4 (PcLiP04) was selected due to its high stability and activity at pH 5.5 and 37 °C, which is close to human skin conditions. In vitro melanin decolorization results indicated that PcLiP04 exhibited at least 2.9-fold higher efficiency than that of well-known lignin peroxidase (PcLiP01) in a typical human skin-mimicking environment. The interaction force between melanin films measured by a surface forces apparatus (SFA) revealed that the decolorization of melanin by PcLiP04 harbors a disrupted structure, possibly interrupting π-π stacking and/or hydrogen bonds. In addition, a 3D reconstructed human pigmented epidermis skin model showed a decrease in melanin area to 59.8% using PcLiP04, which suggests that PcLiP04 exhibits a strong potential for skin whitening.
Lignin present in the inner core of natural lignocellulosic fibers is preferably removed for use in the textile industry as it hinders fiber spinnability. This study focuses on integrating the bioprocessing of agro residues and a natural fiber source (coir) for the effective production of lignin‐modifying enzymes while simultaneously softening the natural fibers. The lignin content in the raw fibers (42.17%) could be reduced by 14–31% by treating them with Phanerochaete chrysosporium for 30 days in the presence of agro waste, such as paddy straw, groundnut husk, corn husk, coir pith, sugarcane bagasse, saw dust and coconut leaf. Among the lignocellulosic wastes tested, P. chrysporium‐fermented groundnut husk with coir produced the maximum yield of lignin‐modifying enzymes (lignin peroxidase, 2.05 U mg⁻¹; manganese peroxidase, 7.50 U mg⁻¹; laccase, 0.43 U mL⁻¹), while the tensile strength of the fiber was decreased by 9.9% compared with the raw fiber (110.14 MPa) on 30 days of incubation. Similarly, SB dust demonstrated good lignin‐modifying enzyme activity (lignin peroxidase, −1.57 U mg⁻¹; manganese peroxidase, 8.14 U mg⁻¹; and laccase, 0.35 U mg⁻¹) as well as the tensile strength of the coir also being increased. A reduction in flexural rigidity owing to delignification was observed and hence a minimum 16% increase in softness was achieved in the integrated process. Our study presents an efficient strategy that exploits low‐cost agro residues for enzyme production and generates bio‐softened coir of acceptable quality in the export and domestic markets. © 2023 Society of Industrial Chemistry and John Wiley & Sons Ltd.
The aim of the study was to study the effect of basidiomycetes on the aromatic and carbohydrate components of plant materials under the influence of the mycelium of the Pleurotus ostreatus fungus. To achieve this goal, we carried out a component analysis of the initial plant raw materials and raw materials after exposure to the mycelium of the basidiomycete. The moisture content in the raw material was determined, and the analysis of the content of: lignin, cellulose, easily and difficultly hydrolysable polysaccharides was carried out. The quantitative analysis of the structural components of plant raw materials was carried out according to the methods generally accepted in the chemistry of plant raw materials, in terms of absolutely dry raw materials. Chemical analysis of substrates showed that polysaccharides undergo bioconversion in the first place; difficultly hydrolyzable polysaccharides underwent biodegradation to a greater extent. It can be concluded that lignin does not completely destroy lignin on annual plants, as a result of which the growth and productivity of mushrooms increases.
Lignin is the most abundant source of renewable aromatic biopolymers and its valorization presents significant value for biorefinery sustainability, which promotes the utilization of renewable resources. However, it is challenging to fully convert the structurally complex, heterogeneous, and recalcitrant lignin into high-value products. The in-depth research on the lignin degradation mechanism, microbial metabolic pathways, and rational design of new systems using synthetic biology have significantly accelerated the development of lignin valorization. This review summarizes the key enzymes involved in lignin depolymerization, the mechanisms of microbial lignin conversion, and the lignin valorization application with integrated systems and synthetic biology. Current challenges and future strategies to further study lignin biodegradation and the trends of lignin valorization are also discussed.
The heightened increase in pollutants in the environment including soil due to the addition of different toxic chemical compounds resulting from geogenic and various anthropogenic activities is a worldwide concern. There have been a number of evidences regarding involvement of bacterial enzymes in soil bioremediation of these toxic pollutants but little work has been accomplished regarding potential fungal enzymes. In this context, development of bioremediation techniques with filamentous fungi and their oxidative enzymatic activities could be of great potent to reduce toxicity of soil pollutants namely polyaromatic hydrocarbons (PAHs), halogenated compounds, polyphenols, heavy metals, etc. Although, bioremediation through enzymatic activities of fungi is a cost-effective and eco-friendly technology, and many studies have already been performed using fungal cultures, but its study has been restricted. Therefore, more work needs to be done in this field for a commercial breakthrough regarding the use of fungal enzymes in soil bioremediation. A variety of lignolytic and filamentous fungi have been studied to perform the function employing their capability to transform or degrade specific contaminants using their enzymatic activities.KeywordsFungal enzymesHeavy metalsPAHLignolytic fungiFilamentous fungi
Abstract
Ganoderma spp. are wood decay fungi forming a white rot of wood by secretion of various extracellular ligninolytic enzymes. In this study, growth and ligninolytic activity of two Ganoderma species on sawdust of European ash ( Fraxinus excelsior ) wood were monitored over an eight week incubation period. One of these species, G. mbrekobenum is a recently-discovered species found growing on Citrus limon trees in Egypt, whilst the second G. lucidum is one of the best studied species in the genus and both a mononkaryon (1N) and a dikaryon (2N) strain of the species were studied here. Growth on sawdust was monitored using the fungal specific sterol (ergosterol) and the activities of three ligninolytic enzymes (Manganese independent peroxidase [MiP], manganese dependant peroxidase [MnP] and laccase). Chemical changes in ash wood composition were monitored using TMAH-thermochemolysis (Pyrolysis–gas chromatography–mass spectrometry, in the presence of tetramethylammonium hydroxide).
Keywords: Wood decay- white rot fungi- ligninolytic enzymes- tree pathogens- pyrolysis-TMAH-thermochemolysis.
Dye is one of the integral parts of human civilization. It has been used in day-to-day life from prehistoric periods. There are more than ten thousand different types of dyes present in the market which are used in industries related to food, textile, paint, cosmetics, paper and pharmaceuticals. Most of the recent dyes are synthetic in nature and have xenobiotic, toxic, mutagenic and cancer-causing properties. There are various classes of dyes based on their chemical structure or based on their mode of action, some common classes of dyes which are used in industries are azo dyes, vat dyes, acidic dyes, basic dyes, reactive dyes, disperse dyes and others Theses dyes after being used in the various process are discharged to various water resources by various industries without proper treatment or by partial treatment, which leads to water pollution and affects the aquatic ecosystem and human health. Several conventional physicochemical methods based on principles of coagulation, membrane filtration, oxidation, reverse osmosis and others have been used but these methods are associated with several drawbacks related to cost, complexity, end product and efficiency. These limitations can be overcome by using biological methods in which various microflora having suitable properties are used. Methods using microorganisms are commercially viable, have a low initial investment, simple and ecologically suitable. Degradation of dyes by microflora can be achieved by either biosorption or enzymatic action. There are several oxidizing and reducing enzymes produced by microflora are used in dye decolourization with effective result. This chapter focuses on various biological methods for dye decolourization, advantages of using the biological method over conventional methods and the future in the field of dye removing by microflora.KeywordsDyesBioremediationMicrofloraBiosorptionDecolourization
Mud volcanoes (MVs) are visible signs of oil and gas reserves present deep beneath land and sea. The Marac MV in Trinidad is the only MV associated with natural hydrocarbon seeps. Petrogenic polyaromatic hydrocarbons (PAHs) in its sediments must undergo biogeochemical cycles of detoxification as they can enter the water table and aquifers threatening ecosystems and biota. Recurrent hydrocarbon seep activity of MVs consolidates the growth of hydrocarbonoclastic fungal communities. Fungi possess advantageous metabolic and ecophysiological features for remediation but are underexplored compared to bacteria. Additionally, indigenous fungi are more efficient at PAH detoxification than commercial/foreign counterparts and remediation strategies remain site-specific. Few studies have focused on hydrocarbonoclastic fungal incidence and potential in MVs, an aspect that has not been explored in Trinidad. This study determined the unique biodiversity of culturable fungi from the Marac MV capable of metabolizing PAHs in vitro and investigated their extracellular peroxidase activity to utilize different substrates ergo their extracellular oxidoreductase activity (> 50% of the strains decolourized of methylene blue dye). Dothideomycetes and Eurotiomycetes (89% combined incidence) were predominantly isolated. ITS rDNA sequence cluster analysis confirmed strain identities. 18 indigenous hydrocarbonoclastic strains not previously reported in the literature and some of which were biosurfactant-producing, were identified. Intra-strain variability was apparent for PAH utilization, oil-tolerance and hydroxylase substrate specificity. Comparatively high levels of extracellular protein were detected for strains that demonstrated low substrate specificity. Halotolerant strains were also recovered which indicated marine-mixed substrata of the MV as a result of deep sea conduits. This work highlighted novel MV fungal strains as potential bioremediators and biocatalysts with a broad industrial applications.
Introduction:
Silver fir (Abies alba Mill.) is one of the most valuable conifer wood species in Europe. Among the main opportunistic pathogens that cause root and butt rot on silver fir are Armillaria ostoyae and Heterobasidion abietinum. Due to the different enzymatic pools of these wood-decay fungi, different strategies in metabolizing the phenols were available.
Objective:
This work explores the changes in phenolic compounds during silver fir wood degradation.
Methodology:
Phenols were analyzed before and after fungus inoculation in silver fir macerated wood after 2, 4 and 6 months. All samples were analyzed using high-performance liquid chromatography coupled to a hybrid quadrupole-orbitrap mass spectrometer.
Results:
Thirteen compounds, including simple phenols, alkylphenyl alcohols, hydroxybenzoketones, hydroxycinnamaldehydes, hydroxybenzaldehydes, hydroxyphenylacetic acids, hydroxycinnamic acids, hydroxybenzoic acids and hydroxycoumarins, were detected. Pyrocatechol, coniferyl alcohol, acetovanillone, vanillin, benzoic acid, 4-hydroxybenzoic acid and vanillic acid contents decreased during the degradation process. Methyl vanillate, ferulic acid and p-coumaric were initially produced and then degraded. Scopoletin was accumulated. Pyrocatechol, acetovanillone and methyl vanillate were found for the first time in both degrading and non-degrading wood of silver fir.
Conclusions:
Despite differences in the enzymatic pool, both fungi caused a significant decrease in the amounts of phenolic compounds with the accumulation of the only scopoletin. Principal component analysis revealed an initial differentiation between the degradation activity of the two fungal species during degradation, but similar phenolic contents at the end of wood degradation.
Lignocellulosic biomass has been admitted as sustainable, renewable, and carbon-neutral energy sources which can be the potential alternative to the fossil fuel. Although the biomass mainly consists of cellulose, hemicellulose, and lignin, each of them does not exist separately. These components are cross-linked to form lignin-carbohydrate complexes (LCCs) which should be considered as the basic structure in cell wall. The existence of LCC is being realized to directly restrict the fractionation and cause the recalcitrance in biomass utilization. Still, there are controversial debates regarding the properties, structure, and composition of LCCs and its effect on biorefinery, which can be attributed to the lack of attention on LCCs which makes progress more sluggish in this field. Discussion in this review critically emphasizes on the fractionation of LCCs, presence and susceptibility of lignin-carbohydrate bonds, and direct/indirect analytical techniques to evoke the attention of researchers toward this, when the world is surrounded by energy and environment issues and realization of biorefinery needs to be addressed.
To date, much attention has been paid on developing new strategies for the valorization of abundant, inexpensive, and renewable lignocellulosic biomass into liquid biofuels and chemicals. Lipids are one kind of value-added energy-rich compounds, which can produce by oleaginous microorganisms using biomass and/or biomass-hydrolysates. Recently, the conversion of lignocellulosic biomass into microbial lipid has received significant attention replacing fossil fuels. However, biomass is highly recalcitrant due to its complex structure with cellulose, hemicellulose, and lignin. Pretreatment of biomass is a critical process in the conversion due to the nature and structure of the biomass cell wall that is complex. Although green technologies for microbial production are advancing, the productivity and yield from these techniques are low. Over the past years, various biomass pretreatment techniques have been developed to disrupt the plant cell-wall structure of lignocellulosic biomass, facilitate subsequent enzymatic hydrolysis and microbial lipid fermentation, and successfully employed to improve biomass-to-lipid technology. In this chapter, the progress of pretreatment for enhancing the enzymatic digestion of lignocellulosic material is introduced. In addition, microbial lipid production from lignocellulosic biomass pretreated by effective pretreatment is discussed.
Using lignin to produce high-value-added aromatic fine chemicals and high-grade biofuels such as aromatics, cycloalkanes, and alkanes can reduce the dependence on fossil resources and highly improve the competitiveness of biorefining industry. The biological valorization of lignin includes the biological depolymerization and bioconversion of lignin. With the development of bioprospecting and systems biology technology, more and more lignin-degrading microorganisms have been discovered and separated from the natural habitat of lignin decomposition. The physiological and biochemical characteristics of microorganisms and the molecular- and systematic-level degradation mechanism on lignin and lignin-derived aromatic compounds have also been deeply recognized. All of these have laid a theoretical foundation for precisely controlling the depolymerization and metabolism of lignin and establishing the biological processing pathway of lignin. This chapter will introduce the research progress of lignin valorization from the aspects of lignin-degrading microorganisms and enzymes, lignin degradation metabolic pathways, and the application of biosynthesis in lignin conversion.
The development of a lignin peroxidase (LiP) that is thermostable even under acidic pH conditions is a main issue for efficient enzymatic lignin degradation due to reduced repolymerization of free phenolic products at acidic pH (< 3). Native LiP under mild conditions (half-life (t 1/2) of 8.2 days at pH 6) exhibits a marked decline in ther-mostability under acidic conditions (t 1/2 of only 14 min at pH 2.5). Thus, improving the thermostability of LiP in acidic environments is required for effective lignin depolymerization in practical applications. Here, we show the improved thermostability of a synthetic LiPH8 variant (S49C/A67C/H239E, PDB: 6ISS) capable of strengthening the helix-loop interactions under acidic conditions. This variant retained excellent thermostability at pH 2.5 with a 10-fold increase in t 1/2 (2.52 h at 25 • C) compared with that of the native enzyme. X-ray crystallography analysis showed that the recombinant LiPH8 variant is the only unique lignin peroxidase containing five disulfide bridges, and the helix-loop interactions of the synthetic disulfide bridge and ionic salt bridge in its structure are responsible for stabilizing the Ca 2+-binding region and heme environment, resulting in an increase in overall structural resistance against acidic conditions. Our work will allow the design of biocatalysts for ligninolytic enzyme engineering and for efficient biocatalytic degradation of plant biomass in lignocellulose biorefineries.
Many of the extracellular lignin-degrading peroxidases from the wood-degrading fungus Phanerochaete chrysosporium are phosphorylated. Immunoprecipitation of the extracellular fluid of cultures grown with H2K³²PO4 with a polyclonal antibody raised against one of the lignin peroxidase isozymes, H8 (pI 3.5), revealed the incorporation of H2K³²PO4 into lignin peroxidases. Analyses of the purified isozymes from labeled cultures by isoelectric focusing showed that, in addition to isozyme H8, lignin peroxidase isozymes H2 (pI 4.4), H6 (pI 3.7), and H10 (pI 3.3) are also phosphorylated. These analyses also showed that lignin peroxidase isozyme H1 (pI 4.7) and manganese-dependent peroxidase isozymes H3 (pI 4.9) and H4 (pI 4.5) are not phosphorylated. Phosphate quantitation indicated the presence of one molecule of phosphate/molecule of enzyme for all of the phosphorylated isozymes. To locate the site of phosphorylation, one-dimensional phosphoamino acid analysis was performed with hydrolyzed ³²P-protein. However, phosphotyrosine, phosphoserine, and phosphothreonine could not be identified. Coupled enzyme assays of acid hydrolysate indicated the presence of mannose 6-phosphate as the phosphorylated component on the lignin peroxidase isozymes. Digestion of the isozymes with N-glycanase released the phosphate component, indicating that the mannose 6-phosphate is contained on an asparagine-linked oligosaccharide.
A cDNA clone encoding a manganese-dependent peroxidase from the filamentous fungus Phanerochaete chrysosporium was isolated and characterized. The clone, λMP-1, was isolated by screening a λgt11 expression library with polyclonal antibodies raised against a purified manganese-dependent peroxidase (isozyme H4, pI 4.5). The λMP-1 cDNA sequence predicts a mature protein containing 358 amino acids with a molecular weight of 37,711 preceded by a leader peptide of 24 amino acid residues. The N-terminal amino acid sequence of a purified manganese-dependent peroxidase (H4) corresponds to the sequence deduced from the cDNA. Some homology (58% in nucleotide sequence and 65% in amino acid sequence) is observed between the manganese-dependent peroxidase and lignin peroxidase isozyme H8. The highest degree of similarity is observed near the enzyme active site. Residues essential for peroxidase activity, the distal and proximal histidines, can be identified in the amino acid sequence. Near these residues, homology is also observed with several other peroxidases. Northern blot analysis of poly(A)⁺ RNA from nitrogen-limited P. chrysosporium cultures indicates that the level of messenger RNA correlates with expression of the enzyme and its activity. This is consistent with the regulation of the enzyme being at the level of transcription.
The crystal structure of the major lignin peroxidase isozyme from Phanerocheate chrysosporium has been refined to an R = 0.15 for data between 8 A and 2.03 A. The refined model consists of 2 lignin peroxidase molecules in the asymmetric unit, 2 calcium ions per monomer, 1 glucosamine per monomer N-linked to Asn-257, and 476 water molecules per asymmetric unit. The model exhibits excellent geometry with a root mean square deviation from ideality in bond distances and angles of 0.014 A and 2.9 degrees, respectively. Molecule 1 consists of all 343 residues, while molecule 2 consists of residues 1-341. The overall root mean square deviation in backbone atoms between the 2 molecules in the asymmetric unit is 0.36 A. The refinement at 2.0 A confirms our conclusions based on the partially refined 2.6-A structure (Edwards, S. L., Raag, R., Wariishi, H., Gold, M. H., and Poulos, T. L. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 750-754). The overall fold of lignin peroxidase closely resembles that of cytochrome c peroxidase. A superimposition of alpha-carbons gives a root mean square deviation of 2.65 A between the two peroxidases and 1.66 A for the helices. The active sites also are similar since both contain a proximal histidine heme ligand hydrogen-bonded to a buried aspartate residue and both contain histidine and arginine residues in the distal peroxide binding pocket. The most obvious difference in the active site is that whereas cytochrome c peroxidase has tryptophan residues located in the proximal and distal heme pockets, lignin peroxidase has phenylalanines. There are four other especially noteworthy differences in the two structures. First, although the heme in cytochrome c peroxidase is recessed about 10 A from the molecular surface, the heme pocket is open to solvent. The analogous opening in lignin peroxidase is smaller which can explain in part the differences in reactivity of the two hemes. This same opening may provide the site for binding small aromatic substrates. Second, lignin peroxidase has a carboxylate-carboxylate hydrogen bond important for heme binding that is not present in cytochrome c peroxidase. Third, lignin peroxidase contains 2 structural calcium ions while cytochrome c peroxidase contains no calcium. The calciums in lignin peroxidase coordinate to residues near the C-terminal ends of the distal and proximal helices and hence are probably important for maintaining the integrity of the active site. Fourth, the extra 49 residues in lignin peroxidase not present in cytochrome c peroxidase constitutes the C-terminal end of the molecule with the C terminus situated at the “front” end of the molecule between the two heme propionates.
Manganese oxidation by manganese peroxidase (MnP) was investigated. Stoichiometric, kinetic, and MnII binding studies demonstrated that MnP has a single manganese binding site near the heme, and two MnIII equivalents are formed at the expense of one H2O2 equivalent. Since each catalytic cycle step is irreversible, the data fit a peroxidase ping-pong mechanism rather than an ordered bi-bi ping-pong mechanism. MnIII-organic acid complexes oxidize terminal phenolic substrates in a second-order reaction. MnIII-lactate and -tartrate also react slowly with H2O2, with third-order kinetics. The latter slow reaction does not interfere with the rapid MnP oxidation of phenols. Oxalate and malonate are the only organic acid chelators secreted by the fungus in significant amounts. No relationship between stimulation of enzyme activity and chelator size was found, suggesting that the substrate is free MnII rather than a MnII-chelator complex. The enzyme competes with chelators for free MnII. Optimal chelators, such as malonate, facilitate MnIII dissociation from the enzyme, stabilize MnIII in aqueous solution, and have a relatively low MnII binding constant.
A versatile ligninolytic peroxidase has been cloned from Pleurotus eryngii and its allelic variant MnPL2 expressed in Aspergillus nidulans, with properties similar to those of the mature enzyme from P. eryngii. These include the ability to oxidize Mn2+ and aromatic substrates, confirming that this is a new peroxidase type sharing catalytic properties of lignin peroxidase and manganese peroxidase.
Lignin peroxidase (LiP) and manganese peroxidase (MnP) have been investigated in Phanero-chaete chrysosporium. A third ligninolytic peroxidase has been described in Pleurotus and Bjerkandera. Two of these versatile peroxidases (VPs) have been cloned, sequenced and characterized. They have high affinity for Mn²⁺, hydro-quinones and dyes, and also oxidize veratryl alcohol, dimethoxybenzene and lignin dimers. The deduced sequences show higher identity with Ph. chrysosporium LiP than MnP, but the molecular models obtained include a Mn²⁺-binding site. Concerning aromatic substrate oxidation, Pl. eryngii VP shows a putative long-range electron transfer pathway from an exposed trytophan to haem. Mutagenesis and chemical modification of this tryptophan and the acidic residues forming the Mn²⁺-binding site confirmed their role in catalysis. The existence of several substrate oxidation sites is supported further by biochemical evidence. Residue conservation in other fungal peroxidases is discussed.
A cDNA coding for laccase was isolated from the ligninolytic fungus Trametes versicolor by RNA-PCR. The cDNA corresponds to the gene lcc1, which encodes a laccase isoenzyme of 498 amino-acid residues preceded by a 22-residue signal peptide. The lcc1 cDNA was cloned into the vector pHIL-D2 for expression in Pichia pastoris under the control of the AOX1 promoter. Transformants were found to secrete active recombinant enzyme after induction with methanol. The use of growth
medium buffered to pH 6.0 and control of pH during cultivation were found to be important, or even necessary, for obtaining
activity in liquid cultures. The effect of exchanging the native secretion signal for the Saccharomyces cerevisiae α-factor pre-pro secretion signal was studied by cloning the portion encoding the mature enzyme into the vector pPIC9. The
activity obtained for the construct encoding the native laccase signal sequence was found to be seven-fold higher than for
the construct encoding the α-factor secretion signal. Utilisation of the P. pastoris pep4 mutant strain SMD1168 was found to provide a two-fold higher level of activity compared with P. pastoris GS115.
Three new genes (Cs-mnp2A, Cs-mnp2B and Cs-mnp3) coding for manganese-dependent peroxidase (MnP) have been identified in the white-rot basidiomycete Ceriporiopsis subvermispora. The mature proteins contain 366 (MnP2A and MnP2B) and 364 (MnP3) amino acids, which are preceded by leader sequences of 21 and 24 amino acids, respectively. Cs-mnp2A and Cs-mnp2B appear to be alleles, since the corresponding protein sequences differ in only five residues. The upstream region of Cs-mnp2B contains a TATA box, AP-1 and AP-2 sites, as well as sites for transcription regulation by metals (two), cAMP (two) and xenobiotics (one). Some of these elements are also found in the regulatory region of Cs-MnP3. Transcription of Cs-mnp2A and Cs-mnp2B, but not that of Cs-mnp3, is activated by manganese.
A cDNA (MnP13-1) and the Cs-mnp1 gene encoding for an isoenzyme of manganese peroxidase (MnP) from C. subvermispora were isolated separately and sequenced. The cDNA, identified in a library constructed in the vector Lambda ZIPLOX, contains 1285 nucleotides, excluding the poly(A) tail, and has a 63% G+C content. The deduced protein sequence shows a high degree of identity with MnPs from other fungi. The mature protein contains 364 amino acids, which are preceded by a 24-amino-acid leader sequence. Consistent with the peroxidase mechanism of MnP, the proximal histidine, the distal histidine and the distal arginine are conserved, although the aromatic binding site (L/V/I–P–X–P) is less hydrophilic than those of other peroxidases. A gene coding for the same protein (Cs-mnp1) was isolated from a genomic library constructed in Lambda GEM-11 vector using the cDNA MnP13-1 as a probe. A subcloned SacI fragment of 2.5 kb contained the complete sequence of the Cs-mnp1 gene, including 162 bp and 770 bp of the upstream and downstream regions, respectively. The Cs-mnp1 gene possesses seven short intervening sequences. The intron splice junction sequences as well as the putative internal lariat formation sites adhere to the GT–AG and CTRAY rules, respectively. To examine the structure of the regulatory region of the Cs-mnp1 gene further, a fragment of 1.9 kb was amplified using inverse PCR. A putative TATAA element was identified 5′ of the translational start codon. Also, an inverted CCAAT element, SP-1 and AP-2 sites and several putative heat-shock and metal response elements were identified.
The white rot basidiomycete Phanerochaete chrysosporium completely degrades lignin and a variety of aromatic pollutants during the secondary metabolic phase of growth. Two families of secreted heme enzymes, lignin peroxidase (LiP) and manganese peroxidase (MnP), are major components of the extracellular lignin degradative system of this organism. MnP and LiP both are encoded by families of genes, and the lip genes appear to be clustered. The lip genes contain eight or nine short introns; the mnp genes contain six or seven short introns. The sequences surrounding active-site residues are conserved among LiP, MnP, cytochrome c peroxidase, and plant peroxidases. The eight LiP cysteine residues align with 8 of the 10 cysteines in MnP. LiPs are synthesized as preproenzymes with a 21-amino-acid signal sequence followed by a 6- or 7-amino-acid propeptide. MnPs have a 21- or 24-amino-acid signal sequence but apparently lack a propeptide. Both LiP and MnP are regulated at the mRNA level by nitrogen, and the various isozymes may be differentially regulated by carbon and nitrogen. MnP also is regulated at the level of gene transcription by Mn(II), the substrate for the enzyme, and by heat shock. The promoter regions of mnp genes contain multiple heat shock elements as well as sequences that are identical to the consensus metal regulatory elements found in mammalian metallothionein genes. DNA transformation systems have been developed for P. chrysosporium and are being used for studies on gene regulation and for gene replacement experiments.
The crystal structure of the major lignin peroxidase isozyme from Phanerocheate chrysosporium has been refined to an R = 0.15 for data between 8 angstrom and 2.03 angstrom. The refined model consists of 2 lignin peroxidase molecules in the asymmetric unit, 2 calcium ions per monomer, 1 glucosamine per monomer N-linked to Asn-257, and 476 water molecules per asymmetric unit. The model exhibits excellent geometry with a root mean square deviation from ideality in bond distances and angles of 0.014 angstrom and 2.9-degrees, respectively. Molecule 1 consists of all 343 residues, while molecule 2 consists of residues 1-341. The overall root mean square deviation in backbone atoms between the 2 molecules in the asymmetric unit is 0.36 angstrom. The refinement at 2.0 angstrom confirms our conclusions based on the partially refined 2.6-angstrom structure (Edwards, S. L., Raag, R., Wariishi, H., Gold, M. H., and Poulos, T. L. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 750-754). The overall fold of lignin peroxidase closely resembles that of cytochrome c peroxidase. A superimposition of alpha-carbons gives a root mean square deviation of 2.65 angstrom between the two peroxidases and 1.66 angstrom for the helices. The active sites also are similar since both contain a proximal histidine heme ligand hydrogen-bonded to a buried aspartate residue and both contain histidine and arginine residues in the distal peroxide binding pocket. The most obvious difference in the active site is that whereas cytochrome c peroxidase has tryptophan residues located in the proximal and distal heme pockets, lignin peroxidase has phenylalanines. There are four other especially noteworthy differences in the two structures. First, although the heme in cytochrome c peroxidase is recessed about 10 angstrom from the molecular surface, the heme pocket is open to solvent. The analogous opening in lignin peroxidase is smaller which can explain in part the differences in reactivity of the two hemes. This same opening may provide the site for binding small aromatic substrates. Second, lignin peroxidase has a carboxylate-carboxylate hydrogen bond important for heme binding that is not present in cytochrome c peroxidase. Third, lignin peroxidase contains 2 structural calcium ions while cytochrome c peroxidase contains no calcium. The calciums in lignin peroxidase coordinate to residues near the C-terminal ends of the distal and proximal helices and hence are probably important for maintaining the integrity of the active site. Fourth, the extra 49 residues in lignin peroxidase not present in cytochrome c peroxidase constitutes the C-terminal end of the molecule with the C terminus situated at the ''front'' end of the molecule between the two heme propionates.
Previously, we reported that Arg177 is involved in MnII binding at the MnII binding site of manganese peroxidase isozyme 1 (MnP1) of Phanerochaete chrysosporium by examining two mutants: R177A and R177K. We now report on additional mutants: R177D, R177E, R177N, and R177Q. These new mutant enzymes were produced by homologous expression in P. chrysosporium and were purified to homogeneity. The molecular mass and the UV/visible spectra of the ferric and oxidized intermediates of the mutant enzymes were similar to those of the wild-type enzyme, suggesting proper folding, heme insertion, and preservation of the heme environment. However, steady-state and transient-state kinetic analyses demonstrate significantly altered characteristics of MnII oxidation by these new mutant enzymes. Increased dissociation constants (Kd) and apparent Km values for MnII suggest that these mutations at Arg177 decrease binding of MnII to the enzyme. These lowered binding efficiencies, as observed with the R177A and R177K mutants, suggest that the salt-bridge between Arg177 and the MnII binding ligand Glu35 is disrupted in these new mutants. Decreased kcat values for MnII oxidation, decreased second-order rate constants for compound I reduction (k2app), and decreased first-order rate constants for compound II reduction (k3) indicate that these new mutations also decrease the electron-transfer rate. This decrease in rate constants for compounds I and II reduction was not observed in our previous study on the R177A and R177K mutations. The lower rate constants suggest that, even with high MnII concentrations, the MnII binding geometries may be altered in the MnII binding site of these new mutants. These new results, combined with the results from our previous study, clearly indicate a role for Arg177 in promoting efficient MnII binding and oxidation by MnP.
The results of the transient-state kinetic analyses suggest the inhibition by CdII of MnII binding to the enzyme intermediates, MnPI and MnPII. Because reduction of MnPI by MnII is a second-order process, information on binding and catalysis are combined in the rate constant and cannot be distinguished (Scheme 1). In contrast, the reduction of MnPII displays ‘saturation kinetics’, indicating that at high concentrations the formation of the productive ES complex consisting of MnII-bound MnPII (Scheme 2) can be observed. In this case, binding is so much faster than catalysis that the ES accumulates and the kinetics of binding and catalysis can be separated into an apparent binding constant, Kd, and apparent first-order catalytic rate constant, k3,app. The addition of CdII to the reaction (Scheme 2) most likely results in depletion of the functional ES complex via formation of a competing EI complex, resulting in an increase in the apparent Kd for MnII, as shown in the Fig. 4 inset. At CdII concentrations exceeding the apparent equilibrium Ki,MnPII for CdII (≈ 5 µm), the formation of EI may dominate. In this case, the rate of formation of ES probably becomes limiting; thus, ES forms at the same rate or more slowly than it is subsequently converted into product. Such an effect would presumably result in apparent second-order kinetics, where binding and catalysis are again combined into a single rate constant. The linear plots of kobs vs. [S] for reactions containing > 10 µm CdII support this probable transition (Fig. 4).
During screening of basidiomycetes for wheat straw delignification, considerable lignin degradation with a limited attack to cellulose was attained with Pleurotus eryngii. Straw solid-state fermentation (SSF) was optimized, and the enzymatic mechanisms for lignin degradation were investigated. No lignin peroxidase was detected under liquid or SSF conditions, but high laccase and aryl-alcohol oxidase levels were found. The latter enzyme has been fully characterized in PI. eryngii and it seems to be involved in a cyclic redox system for H202 generation from aromatic compounds. Results obtained using homoveratric acid suggest that Pleurotus laccase could be involved in degradation of phenolic and non-phenolic lignin moieties. Histological and ultrastructural studies provided some general morphological characteristics of the fungal attack on wheat straw. Whereas a simultaneous degradation pattern was observed in straw treated with Phanerochaete chrysosporium, PI. eryngii caused partial degradation of middle lamella and separation of individual sclerenchymatic fibers. When these straw samples were subjected to refining tests, energy saving after biological treatment was the highest in the case of straw treated with PI. eryngii, which also produced the lowest substrate loss. From these results, a correlation between preferential removal of lignin, separation of sclerenchymatic fibers and pulping properties was provided during fungal treatment of wheat straw.
The mechanism of oxidation of veratryl alcohol and β-0–4 dimeric lignin models is reviewed. Veratryl alcohol radicals are intermediates in both oxidation pathways. The possible role of the veratryl alcohol radical cation as a mediator is discussed. The lignin peroxidase (LIP) redox cycle is analyzed in terms of the Marcus theory of electron transfer. Reduction of both LiP-Compound I (LiP-I) and LiP-Compound II (LiP-II) by veratryl alcohol occurs in the endergonic region of the driving force. The reduction of LiP-II has a higher reorganization energy due to the change in spin state and the accompanying conformational change in the protein. It is suggested that a reversible nucleophilic addition of a carbohydrate residue located at the entrance of the active site channel plays a key role in the LiP redox cycle. Moreover. (polymeric) hydroxysubstituted benzyl radicals may reduce LiP-II via long-range electron transfer.
Lignin peroxidase (LiP), manganese peroxidase (MnP) from Phanerochaete chrysosporium and laccase from Pleurotus eryngii were separately used to degrade alkali wheat straw lignin (AL). In order to characterize the catalytic action of the different enzymes, the chemical structure and the hydrodynamic properties of treated lignin were analyzed by thioacidolysis-gas chromatography and molecular size exclusion chromatography. The results confirmed that only LiP was able to degrade guaiacyl (G) and syringyl (S) structures in non-phenolic methylated lignins. However, Provided that some phenolic terminal structures are present, MnP and laccase were able to degrade the non-phenolic part of the polymer linked by beta-O-4 alkyl aryl ether bonds. This suggested that the oxidative reactions catalyzed in alkali straw lignin could progress through bond cleavages generating phenoxy radicals. The molecular size distribution of both thioacidolysis products and the oxidized polymer showed that AL underwent condensation side-reactions regardless of the enzymic treatment, but only UP oxidation led to the increase in the hydrodynamic volume of the recovered lignin. This indicated that modification by enzymes of bonding patterns in lignin is not always associated with alterations in the spatial network of the polymer.
The purpose of this study was to examine changes in cell wall void (pore) volume and pore size distribution in sweetgum wood during decay by a white-rot fungus, Phanerochaete chrysosporium Burds. Results of the study may provide quantitative answers to questions regarding the accessibility of degradative proteins to their respective substrates within the cell wall. Sweetgum (Liquidambar styraeiflua L.) wood blocks were decayed by Phanerochaete chrysosporium Burds. in soil-block cultures. Lignin, cellulose and hemicellulose were removed at approximately equal rates with progression of decay. Decay was terminated at various weight losses, and the pore volumes available to probes of various molecular weight and diameter were determined by the solute exclusion technique. The cell wall void volume in sound sweetgum wood was 0.35 ml . g-1 and the maximum pore diameter, 2 nm (20 angstrom). In white-rot decayed wood, cell wall void volume increased to 0.6 ml . g-1 at 40% weight loss, and maximum pore diameter increased to more than 5 nm (50 angstrom). Most of the cell wall void volume increase resulted from the creation of pores of 2 to 5 nm (20 to 50 angstrom) diameter. Assuming a model in which the cell wall is built of microfibrils laterally associated to form lamellae, we conclude that ligninolytic enzymes are expected to penetrate only a small fraction of new cell wall void volume, even after extensive decay, whereas small enzymes of 2 to 3 nm (20 to 30 angstrom) may gain access to considerable new cell void volume.
The activity of aryl-alcohol oxidase was detected in the mycelial extracts of a lignin-degrading basidiomycete, Phanerochaete chrysosporium. The induction of production of the enzyme by aryl-alcohols was suggested. The enzyme was purified to homogeneity. The molecular weight was estimated to be about 78,000. The prosthetic group was found to be FAD. Several aryl alcohols can serve as substrates but aliphatic alcohols are inert.
White-rot fungi produce extracellular lignin-modifying enzymes, the best characterized of which are laccase (EC 1.10.3.2), lignin peroxidases (EC 1.11.1.7) and manganese peroxidases (EC 1.11.1.7). Lignin biodegradation studies have been carried out mostly using the white-rot fungus Phanerochaete chrysosporium which produces multiple isoenzymes of lignin peroxidase and manganese peroxidase but does not produce laccase. Many other white-rot fungi produce laccase in addition to lignin and manganese peroxidases and in varying combinations. Based on the enzyme production patterns of an array of white-rot fungi, three categories of fungi are suggested: (i) lignin-manganese peroxidase group (e.g.P. chrysosporium and Phlebia radiata), (ii) manganese peroxidase-laccase group (e.g. Dichomitus squalens and Rigidoporus lignosus), and (iii) lignin peroxidase-laccase group (e.g. Phlebia ochraceofulva and Junghuhnia separabilima). The most efficient lignin degraders, estimated by 14CO2 evolution from 14C-[Ring]-labelled synthetic lignin (DHP), belong to the first group, whereas many of the most selective lignin-degrading fungi belong to the second, although only moderate to good [14C]DHP mineralization is obtained using fungi from this group. The lignin peroxidase-laccase fungi only poorly degrade [14C]DHP.
New peroxidase structures have significantly increased our understanding of the evolutionary and functional relationships within the plant peroxidase superfamily. Three distantly related structural classes have emerged: mitochondrial yeast cytochrome c peroxidase, chloroplast and cytosol ascorbate peroxidases, and gene duplicated bacterial peroxidase (class I); secretory fungal peroxidases (class II); and, classical, secretory plant peroxidases (class III).
Lignin peroxidases, although believed to catalyze the first step of fungal ligninolysis in vivo, have never been demonstrated to depolymerize unmodified lignin in vitro. It is shown here that crude Phanerochaete chrysosporium lignin peroxidase, in the presence of H2O2 and veratryl alcohol, will catalyze the partial fragmentation of a 14Cβ-labeled synthetic hardwood lignin in Na acetate (pH 4.5)/N,N-dimethylformamide, 9:1. Gel permeation chromatography of the treated lignin demonstrated that fragments with molecular weights as low as ca. 170 were products of this reaction. Determinations of total 14C showed that 25–30% of the radiolabel originally present was converted to volatile products during enzymatic oxidation. No evidence for depolymerization was found in control reactions from which lignin peroxidase or H2O2 had been omitted, and little change in the lignin was discernible in reactions that lacked veratryl alcohol.
A gene encoding a manganese-dependent peroxidase (MnP) of the oriental mushroom Elfvingia applanata has been cloned into ?gt10 and characterized. This gene, Ea.mnp1, consists of a 1, 095 bp open reading frame coding for 364 amino acid residues. Northern blot analysis revealed that transcription of the Ea.mnp1 gene was elevated by an increase in Mn2+ to 200 mM or 0.1 g 2, 5-xylidine l-1. The Ea.mnp1 mRNA transcript levels changed in parallel with the observed changes in activity of MnP in E. applanata.
We have used NMR spectroscopy to study the cyanide derivative of lignin peroxidase with the goal of characterizing the electronic structure of this derivative of peroxidases. By using varions homonuclear and heteronuclear 2D NMR techniques and by making use of the available X-ray structure of the cyanide-free protein, it has been possible to extend the assignment of the heme substituents and of the protons of some active-site residues. An estimate of the anisotropy and direction of the magnetic susceptibility anisotropy tensor has been obtained from the pseudocontact shifts of protons of residues not directly bound to the heme iron ion. Finally, a factoring of the hyperfine shifts of the heme and proximal histidine protons, as well as of the ¹³C heme methyls and ¹âµN of the cyanide moiety, is obtained. The contact shift pattern of the heme protons is related to the orientation of the histidine plane. Very large upfield contact shifts are experienced by the aromatic protons of the proximal histidine. The axial magnetic anisotropy is smaller than in metmyoglobin-CN and slightly larger than in horseradish peroxidase-CN. This may reflect the order of the donor strength of the proximal histidine. The z axis of the magnetic susceptibility tensor is found essentially perpendicular to the heme plane. 96 refs., 7 figs., 3 tabs.
The extracellular peroxidase isozymes secreted by the white rot fungusPhanerochaete chrysosporiumhave been classified as manganese peroxidases (isozymes H3, H4, H5, and H9) and lignin peroxidases (isozymes H1, H2, H6, H7, H8, and H10). Recently we reported that lignin peroxidase isozyme H2 can also oxidize Mn2+(Khindariaet al.,1995,Biochemistry34, 7773–7779). This lignin peroxidase isozyme oxidized Mn2+with both of the enzyme intermediates, compound I and compound II, at the same rates as manganese peroxidase isozyme H4. The results of single-turnover kinetic studies have now demonstrated that compound I of the other lignin peroxidase isozymes (H1, H6, H7, H8, and H10) also readily oxidized Mn2+, but that the rate of Mn2+oxidation by compound II was extremely slow. Compound III rapidly formed in the presence of Mn2+, oxalate, and H2O2. However, upon the addition of veratryl alcohol, the results indicate that veratryl alcohol served to reduce compound II. Under such conditions, compound III did not accumulate, and a steady-state rate of Mn2+oxidation was observed. The rate of Mn2+oxidation was the same as for the reduction of compound II by veratryl alcohol. The dependence of the rate of Mn2+oxidation on the concentration of veratryl alcohol was consistent with a mechanism in which Mn2+is oxidized by compound I and veratryl alcohol is oxidized by compound II. Therefore, under physiologically relevant conditions, in which both veratryl alcohol and Mn2+are present, all lignin peroxidase isozymes would be capable of oxidizing Mn2+to Mn3+which can serve as a diffusible oxidant.
A peroxidase was purified 98.3-fold from the culture filtrate of Pleurotus ostreatus with an overall yield of 12.4%. The molecular mass determined by gel filtration was found to be approx. 140 kDa. SDS-PAGE revealed that the enzyme consists of two identical subunits with a molecular mass of approx. 72 kDa. The pI value of this enzyme is approx. 4.3. The enzyme contains 41% carbohydrate by weight, and aspartic acid and asparagine (16.8%), and glutamic acid and glutamine (12.0%). The enzyme has the highest affinity toward sinapic acid and affinity towards various phenolic compounds containing methoxyl and p-hydroxyl groups, directly attached to the benzene ring. However, the enzyme does not react with veratryl alcohol and shows no affinity for nonphenolic compounds. The optimal reaction pH and temperature are 4.0 and 40°C, respectively. The catalytic mechanism of the enzymic reaction is of the Ping-Pong type. The activity of the enzyme is competitively inhibited by high concentrations of H202 and its Ki value is 1.70 mM against H2O2. This enzyme contains approx, 1 mol of heme per mol of one subunit of the enzyme. The pyridine hemochrome spectrum of the enzyme indicates that the heme of P. ostreatus peroxidase is iron protoporphyrin IX. The EPR spectrum of the native peroxidase shows the presence of a high-spin ferric complex with g values at 6.102, 5.643 and 1.991
We report cloning and sequencing of gene ps1 encoding a versatile peroxidase combining catalytic properties of lignin peroxidase (LiP) and manganese peroxidase (MnP) isolated from lignocellulose cultures of the white-rot fungus Pleurotus eryngii. The gene contains 15 putative introns, and the deduced amino acid sequence consists of a 339-residue mature protein with a 31-residue signal peptide. Several putative response elements were identified in the promoter region. Amino acid residues involved in oxidation of Mn2+ and aromatic substrates by direct electron transfer to heme and long-range electron transfer from superficial residues as predicted by analogy with Phanerochaete chrysosporium MnP and LiP, respectively. A dendrogram is presented illustrating sequence relationships between 29 fungal peroxidases.
The crystal structures of ascorbate peroxidase (APX) and cytochrome c peroxidase (CCP) show that the active site structures are nearly identical. Both enzymes contain a His-Asp-Trp catalytic
triad in the proximal pocket. The proximal Asp residue hydrogen bonds with both the His proximal heme ligand and the indole
ring nitrogen of the proximal Trp. The Trp is stacked parallel to and in contact with the proximal His ligand. This Trp is
known to be the site of free radical formation in CCP compound I and also is essential for activity. However, APX forms a
porphyrin radical and not a Trp-centered radical, even though the His-Asp-Trp triad structure is the same in both peroxidases.
We found that conversion of the proximal Trp to Phe has no effect on APX enzyme activity and that the mutant crystal structure
shows that changes in the structure are confined to the site of mutation. This indicates that the paths of electron transfer
in CCP and APX are distinctly different. The Trp-to-Phe mutant does alter the stability of the APX compound I porphyrin radical,
by a factor of two. Electrostatic calculations and modeling studies show that a potassium cation located about 8 Å from the
proximal Trp in APX, but absent in CCP, makes a significant contribution to the stability of a cation Trp radical. This underscores
the importance of long-range electrostatic effects in enzyme catalyzed reactions.
A ligninase gene has been cloned from a Phanerochaete chrysosporium genomic DNA library. Nucleotide sequencing of the gene has revealed that the ligninase structural gene contains 1116 bp of the protein-encoding sequence, of which 84 bp encode the signal peptide. The protein-encoding sequence is interrupted by eight introns which conform to the universal G-T/A-G splicing rule observed for the 3 and 5 intron boundaries. The putative eukaryotic regulatory sequences, i.e. CAAT and TATA box-like sequences, are present in the 5 flanking region.
We have isolated the cDNA and genomic sequences encoding the major isozyme of manganese peroxidase, MnP3, from the white rot
basidiomycetePleurotus ostreatus strain IS1. The genemnp3 is interrupted by 10 introns and encodes a mature protein of 357 amino acid residues with a 26-amino-acid signal peptide.
The amino acid residues known to be involved in peroxidase function and those that form the Mn-binding site in thePanerochaete chrysosporium MnP isozyme are conserved in MnP3. Comparison of the deduced primary structure of MnP3 with those of other peroxidases from
various white rot fungi suggested that MnPs fromP. ostreatus andTrametes versicolor belong to a subgroup that is more similar to the lignin peroxidases than MnPs fromP. chrysosporium orCeriporiopsis subvermispora.
A survey for enzymes involved in lignin degradation was carried out from 90 strains of 68 species of different groups of basidiomycetes. Laccase activity was found in 50% of the fungi tested, whereas aryl-alcohol oxidase (AAO) and Mn-dependent peroxidase (MnP) were detected in 40% and 29% of species, respectively. Laccase activity of more than 200 U l−1 was obtained in cultures of Trametes versicolor, Phellinus torulosus, Cerrena unicolor and Pleurotus eryngii, whereas the AAO and MnP levels were comparatively lower, and lignin peroxidase was not detected in the different fungi tested. Previously known AAO-producing fungi, P. eryngii and Bjerkandera adusta, showed the highest AAO activities, and the enzyme was detected for the first time in fungi from very different taxonomic groups, including the gasteromycete Cyathus olla. In contrast to laccase and AAO, MnP seemed to be restricted to species of Aphyllophorales, with the exception of the tremellaceous fungus Exidia glandulosa. It is interesting to note that MnP was found in most Phellinus species, P. ribis, P. trivialis and P. torulosus producing the highest activities among the fungi studied. Simultaneous production of MnP and H2O2-producing AAO was found in several polyporaceous fungi, but the latter enzyme was absent from the above Phellinus species.
Soybean peroxidase (SBP), an acidic peroxidase isolated from the hulls of the bean, catalyzes the efficient oxidation of veratryl alcohol to veratraldehyde in the presence of H2O2. The reaction is optimal at pH 2.4 in the presence of 0.2 m CaCl2. Soybean peroxidase is highly thermostable at pH 2.4, with half-lives of 210 and 2.5 h at 30 and 50°C, respectively. This compares favorably to the thermostability of lignin peroxidase (LiP) from Phanerochaete chrysosporium under equally acidic conditions. In fact, SBP is at least 150-fold more stable than LiP at 30°C and the latter is completely labile at 50°C. Soybean peroxidase follows a ping-pong, bi-bi catalytic reaction mechanism with a (veratryl alcohol) of2.47 · 102m−1s−1, ca. 1500-fold lower than a similar value for lignin peroxidase. This lower value of catalytic efficiency is due both to a higher Km (veratryl alcohol) and lower kcat for SBP as compared to LiP. Oxidation of methoxybenzenes suggests that the approximate oxidation potential of SBP is 1.42 V, yet this is high enough to effect the oxidation (and eventual β-ether cleavage) of 1-(3,4-dimethoxyphenyl)-2-(phenoxy)propan-1,3-diol, a lignin model dimer. In addition to SBP, horseradish peroxidase (HRP) is also capable of oxidizing veratryl alcohol as well as methoxybenzenes (the latter up to an oxidation potential of 1.34 V). Horseradish peroxidase, however, is extremely labile at pH 2.4 and is inactivated within minutes under such acidic conditions. The oxidization bySBP, and to a lesser extent by HRP, of veratryl alcohol directly in the presence of H2O2 is the first reported case of plant peroxidases catalyzing the efficient oxidation of high oxidation potential nonphenolics and demonstrates that SBP may be an effective alternative to lignin peroxidase.
Two H2O2-dependent oxidases found in the extracellular medium of the white rot fungus Phanerochaete chrysosporium were separated by chromatography on blue agarose. The first enzyme fraction to elute from the column generated ethylene from 2-keto-4-thiomethylbutyric acid (KTBA) in the presence of veratryl alcohol, and catalyzed the α,β cleavage of the diarylpropane 1-(3,4-diethoxyphenyl)1,3-dihdyroxy-(4-methoxyphenyl)propane (I). During the diarylpropane cleavage, 18O from 18O2 was incorporated specifically into the α-position of the product 1-(4-methoxyphenyl)1,2-dihdyroxyethane (III), suggesting that this enzyme is an H2O2-dependent oxygenase. The second enzyme which binds to blue agarose is an Mn2+-dependent, lactate-activated peroxidase. The enzyme catalyzed the oxidation of phenol red, o-dianisidine, Poly R, and a variety of other dyes. It was also capable of decarboxylating vanillic acid.
Heterologous expression of Pleurotus eryngii versatile peroxidase (VP) in Escherichia coli was investigated. The cDNA encoding the mature sequence of the allelic variant VPL2 was cloned into the expression vector pFLAG1 and expressed in E. coli W3110. After induction with isopropyl-β-D-thiogalactopyranoside (IPTG), the recombinant polypeptide (VPL2∗) was found to be the major protein located in inclusion bodies. In vitro folding of VPL2∗ was initially performed under conditions previously determined for folding of recombinant lignin peroxidase from Phanerochaete chrysosporium (LiPH8∗) but a very low yield of active enzyme was obtained (<0.1% of the total protein in the folding reaction). The influence of different parameters in VPL2∗ folding was investigated and the results compared with those obtained for other peroxidases. Up to 7% folding yield was achieved with VPL2∗ using optimised conditions which included: 0.15 M urea, 5 mM Ca2+, 20 μM haemin, a 4:1 oxidised-glutathione/reduced-glutathione ratio and 0.1 mg/ml protein concentration at pH 9.5, a yield twice as high as previously obtained for other peroxidases from Classes II or III. The enzyme presented spectral and kinetic properties identical to those of the fungally derived protein. It was fully functional in both Mn-mediated and Mn-independent peroxidase assays.
Coexpression of two classes of folding accessory proteins, molecular chaperones and foldases, can be expected to improve the productivity of soluble and active recombinant proteins. In this study, horseradish peroxidase (HRP), which has four disulfide bonds, was selected as a model enzyme and overexpressed in Escherichia coli. The effects of coexpression of a series of folding accessory proteins (DnaK, DnaJ, GrpE, GroEL/ES, trigger factor (TF), DsbA, DsbB, DsbC, DsbD, and thioredoxin (Trx)) on the productivity of active HRP in E. coli were examined. Active HRP was produced by very mild induction with 1 μM isopropyl-β-d-thiogalactopyranoside (IPTG) at 37°C, whereas the amount of active HRP produced by the induction with 1 mM IPTG was negligibly small. Active HRP production was increased significantly by coexpression of DsbA-DsbB (DsbAB) or DsbC-DsbD (DsbCD), while coexpression of molecular chaperones did not improve active HRP production. The growth of E. coli cells was inhibited significantly by the induction with 1 mM IPTG in a HRP single expression system. In contrast, when HRP was coexpressed with DsbCD, the growth inhibition of E. coli was not observed. Therefore, coexpression of Dsb proteins improves both the cell growth and the productivity of HRP.
A novel decolorizing peroxidase gene (dyp) was cloned from a cDNA library of a newly isolated strain of fungus Geotrichum candidum Dec 1. The open reading frame of 1494 nucleotides which corresponds to dyp predicts a primary translation product of 498 amino acids, Mr 53,306. The deduced amino acid sequence of DyP does not contain the typical conserved motif which is characteristic of heme-containing peroxidases in the plant peroxidase superfamily. Comparison of the deduced amino acid sequence of DyP with that of a peroxidase from Polyporaceae sp. suggests that these proteins share highly homologous regions.
The kinetics of oxidation of phenolic compounds by ligninase was investigated in the presence and absence of dimethoxylated compounds (veratryl alcohol and 3,4-dimethoxyphenyl acetic acid). In all cases, the phenolic compounds were found to be preferentially oxidised compared to the dimethoxylated compounds. Veratryl alcohol but not 3,4-dimethoxyphenyl acetic acid enhanced their oxidation, but only when present in at least 200-fold molar excess compared with the phenolic compounds. Ligninase was inactivated in the course of oxidation of the phenolic compounds. Inactivation was associated with the accumulation of compound III, formed by reaction of compound II with H2O2. Inactivation was reversed with additions of more enzyme but not with additions of veratryl alcohol. Evidence of inactivation was also obtained during the course of veratryl alcohol oxidation, but the extent was much less, supporting the concept of a substrate-modified compound II intermediate able to promote reaction with reductant over reaction with H2O2. A model to describe the mechanism by which ligninase catalyses net depolymerisation of lignin as opposed to further polymerisation is presented. It involves spatial separation between ligninase, lignin and phenolic lignin breakdown products and invokes the concepts of radical cations as mediators between enzyme and lignin as well as of radical cation charge transfer reactions through the structure of lignin.
To study the mechanism of regulation and structure/function relationship of the Pleurotus ostreatus manganese (11) peroxidase (MnP), we amplified the full-length genomic and complementary DNAs for the major isozyme of the MnP mainly by the cassette-primer PCR technique and then sequenced them. The cDNA contained an open reading frame of 1083 by encoding for a polypeptide of 361 amino-acid residues, including the suggested signal peptide of 29 amino-acid residues with a prepro structure. The predicted amino-acid sequence of the protein shared several common characteristics with those of fungal lignin and manganese (11) peroxidases. We could find a suggested metal response element and two heat-shock element-like sequences in the 5′-flanking region of the structural gene. The structural gene contained 15 introns, many of which lie identical to those in lignin peroxidase genes rather than to those in the known MnP genes.