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Biology of Pleurotus eryngii and role in biotechnological processes: a reviewMirjana Staji a; Jelena Vukojevi a; Sonja Duleti-Lauševi a

a Institute of Botany, Faculty of Biology, University of Belgrade, Belgrade, Serbia

Online Publication Date: 01 March 2009

To cite this Article Staji, Mirjana, Vukojevi, Jelena and Duleti-Lauševi, Sonja(2009)'Biology of Pleurotus eryngii and role inbiotechnological processes: a review',Critical Reviews in Biotechnology,29:1,55 — 66

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Critical Reviews in Biotechnology, 2009; 29(1): 55–66

R e v i e w A R t i c l e

Biology of Pleurotus eryngii and role in biotechnological processes: a review

Mirjana Stajic, Jelena Vukojevic and Sonja Duletic-Lauševic

Institute of Botany, Faculty of Biology, University of Belgrade, Belgrade, Serbia

Address for Correspondence: Mirjana Stajic, stajicm@bio.bg.ac.rs

(Accepted 11 November 2008)

Introduction

Pleurotus eryngii (DC.:Fr.) Quél. is an edible and medicinal species widespread in the Mediterranean, central Europe, central Asia, and north Africa. It is one of two species of the genus Pleurotus, which is a weak parasite, and can live on the root or stem base of living plants of the family Apiaceae (Zervakis et al., 2001a; Lewinsohn et al., 2002). Wide geo-graphical distribution has resulted in significant morpho-logical, biochemical and genetic variations in the P. eryngii taxon. Classification and relationships within it are therefore still debatable and the topic of many studies. P. eryngii is also used in different medicinal, pharmaceutical, and biotech-nological studies (Couto and Herrera, 2006; Gregori et al., 2007). It has the ability to produce various biologically active compounds and possesses a well-developed ligninolytic enzyme system that participates in the degradation of lignin and different aromatic compounds (Cohen et al., 2002). Additionally, due to a remarkable flavor and high nutritional value, the commercial production of P. eryngii has, in the last few decades, increased rapidly worldwide (Manzi et al., 1999).

Genetic polymorphism within Pleurotus eryngii

P. eryngii is now described as a ‘complex’ due to the sig-nificant variations in morphology (Lewinsohn et al., 2002), isoenzymes (Zervakis et al., 1994), and genetic character-istics (Lewinsohn et al., 2001), which are the result of geo-graphical and/or ecological differences in the environment (Zervakis et al., 2001a). Early classification of the aforemen-tioned complex was based only on morphological similari-ties and differences of cultures and basidiomata. Although in recent studies, molecular/genetic approaches were also used in attempts to resolve classification issues in P. eryngii, numerous ambiguities still exist. According to the results of Zervakis and Balis (1996), based on mating com-patibility, the P. eryngii complex is comprised of three vari-eties: var. eryngii, var. ferulae, and var. nebrodensis. These varieties showed limited intertaxa compatibility under laboratory conditions, and gene flow between these taxa was reduced even further in nature by host specificity (De Gioia et al., 2005). However, Venturella (2000), according to the host specialization, raised var. nebrodensis to species

ISSN 0738-8551 print/ISSN 1549-7801 online © 2009 Informa UK LtdDOI: 10.1080/07388550802688821

AbstractPleurotus eryngii is considered a complex species owing to a perplexed structure within species and a wide geo-graphical distribution. Due to its remarkable flavor, high nutritional value, and numerous medicinal features, P. eryngii is commercially cultivated on various raw plant materials. Its efficacy in using nutrients from lignocellulose residues is based on possession of a potent ligninolytic enzyme system, constituted of laccase, Mn-oxidizing peroxidases, and aryl-alcohol oxidase, which successfully degrade different aromatic compounds. Similarly, due to the ability of these enzymes, P. eryngii plays a very important role in many biotechnological processes, such as food production (edible basidiomata), biotransformation of raw plant materials to feed, biopulping and biob-leaching of paper pulp, as well as bioremediation of soil and industrial waters.

Keywords: Biodegradation; cultivation; ligninolytic enzyme system; medicinal features; nutritional value; taxonomy

http://www.informapharmascience.com/bty

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56 M. Stajic et al.

level (P. nebrodensis), while Zervakis et al. (2001a), accord-ing to the genetic and phenotypic distances, separated this species into two varieties, viz. var. nebrodensis and var. elaeoselini, and two debatable species (P. hadamardii and P. fossulatus). Lewinsohn et al. (2002) studied the P. eryngii population growing in close association in Israel with Ferula tingitana, and described a new variety, var. tingitanus, based on significant differences in spore size, mycelial and basidiomata characteristics, host plant, and ecology, compared to var. ferulae, although no common incompatibility alleles existed between them. Although, De Gioia et al. (2005) generally accepted the classifica-tion of Zervakis and Balis (1996), they placed vars. ferule and eryngii at a subspecies level according to the genetic distance between them determined by M13 and RAPD markers. Contrary to those results, Urbanelli et al. (2007) showed that P. eryngii and P. ferulae are two distinct spe-cies and that P. nebrodensis is a variety of P. eryngii based on allozymes and PCR fingerprinting, as well as AFLP and RFLP analyses of laccase and Mn-dependent peroxidase genes. However, Zhang et al. (2006), on the basis of results obtained by mating tests and analysis of ITS sequence, separated P. eryngii var. ferulae and P. nebrodensis, which had obvious differences in ribosomal DNA, although they live under the same climatic conditions and have similar basidiomata morphology. Ro et al. (2007a), by analysis of the ITS sequence and RAPD fingerprinting, arranged all analyzed P. eryngii strains into five groups and emphasized that all of them were clearly distinguished from the P. feru-lae strain, which was used as the control.

It can be concluded that the classification of the P. eryngii complex is based on the classification of Zervakis and Balis (1996), and that only the taxonomical level of vars. ferule and nebrodensis varies according to the opinions of various authors.

Nutritional value of basidiomata

Pleurotus eryngii basidiomata have a high nutritional value. According to Manzi et al. (1999; 2004), they are especially rich in carbohydrates (9.6% of fresh weight), and a signifi-cant amount corresponds to dietary fibers (4.64% of fresh weight; 4.11% is insoluble and 0.53% is soluble dietary

fibers), chitin (0.50% of fresh weight), and polysaccharides (0.41% of fresh weight). The total nitrogen content is around 5.30%, and protein content is between 1.88% and 2.65%. The most abundant amino acids are aspartic acid, glutamic acid, and arginine.

Significant concentrations of vitamins (C, A, B2, B

1, D, and

niacin) and minerals (especially K, Mg, Na, and Ca), very low amounts of lipid (0.8% of fresh weight), and high moisture (between 86.6% and 91.7%) are present in the basidiomata (Manzi et al., 1999). However, these authors obtained those values by cultivation of three P. eryngii strains on a wheat straw–sugar beet compost, and it is known that the chemical and nutritional characteristics of basidiomata depend on the strain and the developmental stage, as well as the substrate type (Bernas et al., 2006).

P. eryngii has the ability to absorb some microelements from the medium and incorporate them into organic compounds (Baeza et al., 2000; 2002). Stajic et al. (2006a) showed that mycelia of various strains of different origins absorbed selenium from a liquid synthetic medium which was enriched with different inorganic selenium sources (Na

2SeO

3, Na

2SeO

4, or SeO

2) at different concentrations.

Likewise, among 32 tested wild mushroom species, P. eryngii strains accumulated the highest levels of lead (Pb) in basidi-omata (Dogan et al., 2006). The above reports may suggest that P. eryngii could be an excellent dietary source of some useful microelements or an indicator of environmental pol-lution by heavy metals.

Besides the high nutritional value of basidiomata, many commercial P. eryngii strains produce significant concentra-tions of hydrogen cyanide (HCN), which interrupts electron transfer and ATP production, and causes cell death (Stijve and de Meijer, 1999). However, Chou et al. (2006) empha-sized that the HCN content in the majority of tested basidi-omata was insignificant (< 1.0 mg/kg) and that this amount could be decreased with cooking.

Medicinal features

Pleurotus eryngii is reported to synthesize various biologi-cally active compounds (Table 1). Polysaccharides, among others, are the main biologically active compounds in P. eryngii, which are classified pharmacologically as biological

Table 1. The medicinal effects of various biological active compounds produced by Pleurotus eryngii.

Biologically active compounds

Medicinal effects

Immuno modulating Anti tumor

Anti hyper cholesterolic

Anti bacterial Anti viral Anti fungal Anti oxidant

Anti osteo porotic

-1,3-glucans × ×

Lovastatin ×

Pleureryn ×

Eryngin ×

Ribonuclease × ×

17-Estradiol ×

Eryngeolysin × ×

Ergothioneine ×

Protein xb68AB ×

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Pleurotus eryngii biology and its role in biotechnology 57

response modifiers, due to the enhancement of the host immune system as a response to various diseases.

Besides polysaccharides, numerous other biologically active compounds were isolated from P. eryngii basidiomata. Lovastatin has been found to be an effective natural com-pound that can decrease cholesterol level in blood (Wasser and Weis, 1999). Pleureryn, a compound with a molecular mass of 11.5 kDa and protease activity, showed inhibitory effects on translation in a rabbit reticulocyte lysate system, as well as on HIV-1 reverse transcriptase (Wang and Ng, 2001). The same authors, during further studies, isolated eryngin, an antifungal peptide of molecular mass of 10 kDa (Wang and Ng, 2004). Thermostable ribonuclease, with polyhomoribonucleotide specificity and a molecular mass of 16 kDa, ensures antiviral, immunomodulating, and anti-neoplastic activities (Ng and Wang, 2004). Laccase isolated from basidiomata also has an inhibitory activity toward HIV-1 reverse transcriptase (Wang and Ng, 2006).

Kim et al. (2006) showed that an extract of unidentified content from P. eryngii basidiomata played an important role in bone metabolism. This extract caused enhancement of the osteoblasts’ alkaline phosphatase and luciferase activity, the expression level of the Runx2 gene, the secre-tion of osteoprotegerin, and the decrease of resorption areas. Similarly, Shimizu et al. (2006) demonstrated that an ethanol extract of P. eryngii basidiomata prevented ovariec-tomy-induced bone loss without any substantial effect on the uterus. These authors also isolated 17-estradiol from the basidiomata (a nonsteroidal compound with the strong-est estrogen-like activity among many naturally occurring compounds). Although estrogen replacement therapy plays an important role in the functions of the skeletal, central nervous, and cardiovascular system, as well as in the bone maintenance in postmenopausal women, it increases the risk of breast and endometrial cancers with long-term usage (Persson et al., 1999). However, besides nonsteroidal com-pounds, P. eryngii may also produce some sterols (Yaoita et al., 2002).

From basidiomata, Ngai and Ng (2006) isolated eryn-geolysin, a monomeric 17 kDa hemolysin, which exhibits cytotoxicity toward leukemia cells and antibacterial activity against Bacillus spp., but no antifungal effects. P. eryngii anti-oxidant capacity is based on the production of ergothioneine (Dubost et al., 2007), while protein xb68Ab is responsible for its antiviral effect (Fu et al., 2003).

The studies reported involving different methods and compounds derived from P. eryngii have led to the general belief that this fungus has anti-hypertensive, antioxidant, anti-hypercholesterolic, anti-hyperglycemic, immunomod-ulating, antitumor, antibacterial, antiviral, antifungal, anti-inflammatory, and anti-osteoporotic effects (Wasser and Weis, 1999). Although species of the genus Pleurotus synthe-size different types of lectins, P. eryngii does not have that ability (Berne et al., 2002). But in addition to its numerous medicinal properties, P. eryngii spores may cause hypersen-sitive pneumonitis based either on the activation of type II cell pneumocytes or peripheral airway epithelium, or an

increase in the permeability of lung structures (Saikai et al., 2002).

Cultivation

Pleurotus eryngii, commonly called king oyster mushroom, has high nutritional and medicinal values, and as such has become a highly valued species among consumers in Europe, Asia, and north America. Commercial cultivation began in Italy in the mid-1970s and nowadays it is cultivated in over a dozen countries worldwide, with the trend being one of increasing production. For example, in China pro-duction increased from 7300 t in 2001 to 114,000 t in 2003 (Chang, 2005).

This species is cultivated successfully on various raw plant materials such as sawdust, sugarcane bagasse, cottonseed hulls, chopped rice straw, rice husks, etc with or without various supplements. The type and amount of nutrients and cultivation conditions, as well as the cultivated strain, have a significant influence on productivity. Thus Peng et al. (2000) showed differences in the nutrient requirements between two P. eryngii strains (one originating from Czechoslovakia and another from Holland), which needed 48% and 37% of rice bran, respectively, to reach the maximum yield level. Zervakis et al. (2001b) reported that the highest mycelium extension rates were during the cultivation on cotton gin-trash, peanut shells, and poplar sawdust, at a temperature of 25°C. In recent years in Japan, numerous studies have been conducted with the aim of finding the optimum conditions for the use of rice husks (one of the most important waste products) for P. eryngii cultivation. The results of Hanai et al. (2005) showed that nontreated and methanol-soaked rice husks, at a concentration of 20 mg mL−1 and 160 mg mL−1, respectively, stimulate mycelial growth, while at the higher concentration they inhibit it. The authors explained that this was caused by the composition of the rice husks, which contain both stimulatory and inhibitory substances, such as phytoalexins, momilactone A, silicic acid, etc. On the other hand, although methanol-soaked rice husks promote myc-elial growth, the biggest problem in using them is the cost of methanol treatment. A mixture of sugarcane bagasse and rice bran was also shown as being a good substrate for P. eryngii cultivation and productivity; the average yield was 74.3 g per culture bottle, at the first flush (Okano et al., 2007). According to the results of Rodriguez Estrada and Royse (2007), the yield as well as the N, P, Mg, Fe, B, and Zn compo-sition of fruiting bodies, were significantly dependent on the amount of soybean added to the basal cottonseed hulls/red oak sawdust/corn distiller’s waste substrate. These authors also showed that the addition of some microelements to the basal substrates stimulated mycelial growth, yield, and sig-nificantly influenced N, P, Fe, B, and Zn content in the fruit-ing bodies. Thus the presence of Mn at a concentration of 50 µg/g led to the highest yield, and the positive effect of that microelement is explained by its participation in transcrip-tion and activation of Mn-oxidizing peroxidases that are responsible for lignin mineralization. Similarly, Rodriguez

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58 M. Stajic et al.

Estrada and Royse (2007) reported that substrate enrich-ment with 150 µg/g or 250 µg/g of Cu are associated with the activation of various enzymes.

Various pathogens are reported to cause substantial crop failure during commercial cultivation of P. eryngii, among which are Pseudomonas tolaasii and P. eryngii Spherical Virus (PeSV). Brown blotch disease, caused by P. tolaasii, leads to about 25–35% of crop loss per year, and the critical factors for its appearance are the density of the bacterial population and the organism’s ability to spread through the substrate (Russo et al., 2003). The pathogenicity of P. tolaasii is based on its ability to produce lipodepsipeptide tolaasin (Soler-Rivas et al., 1999). Murata and Magae (1996) showed that chemical composition of the mycelium and basidi-omata affects activation and strength of the P. eryngii signals that activate toxin production in P. tolaasii. Thus, an increase in the N content in the substrate, as well as substrate enrich-ment with Cu, is associated with disease severity (Rodriguez Estrada and Royse, 2007). A serious problem in disease pre-vention is the absence of a resistant commercial P. eryngii strain.

The infection by PeSV causes a few significant phenotypic effects such as short and stout stems, flattened caps with irregular shapes, as well as the abnormally retarded growth of mycelia, obtained from the infected basidiomata, on a solid medium (Ro et al., 2007b). The authors isolated this mycovirus from diseased basidiomata, and characterized and named it. The virus is spherical and is not related to any known mycovirus. However, effective control of the disease is the use of virus-free spawn in cultivation.

The ligninolytic enzyme system of Pleurotus eryngii

Pleurotus eryngii produces laccases, Mn-oxidizing per-oxidases, and aryl-alcohol oxidase and belongs to the group generally referred to as white-rot fungi (Muñoz et al., 1997; Camarero et al., 1999; Martinez, 2002; Pérez-Boada et al., 2005).

LaccasesCatalytic propertiesLaccases (Lac) are extracellular glycoproteins which catalyze the one-electron oxidation of numerous organic and inor-ganic phenolic and non-phenolic substrates, as well as Mn2+, in the presence of mediators. During that process, molecular oxygen, which is used as the electron acceptor, is reduced to water and Cu2+ to Cu+ (Thurston, 1994; Muñoz et al., 1997).

Muñoz et al. (1997) showed that Lac has wide substrate specificity, but that aromatic amines and phenols, espe-cially those with electron-donor groups (methoxy, methyl, amino, and hydroxy) in the ortho and para positions, are the best substrates. This is important since the natural habitats of white-rot fungi are rich in methoxy-substituted monophenolic compounds and hydroquinones, which are metabolized during lignin degradation by fungal lignino-lytic enzymes (Schoemaker et al., 1989). The Lac of P. eryngii

oxidize hydroquinones to quinones and superoxide anion radical (O

2–). This radical is converted to H

2O

2 in two ways:

(1) in the presence of superoxide dismutase and with the production of oxygen and (2) by the oxidation of Mn2+ to Mn3+ (Figure 1). The superoxide dismutase and Mn2+ ion do not exert any effect on Lac activities (Muñoz et al., 1997).

Since Lacs have lower redox potentials than non-phenolic compounds, they can only oxidize them in the presence of small molecules, i.e. redox mediators. Pleurotus sp. may synthesize from glucose several phenolic compounds which could be involved in the degradation of non-phenolic struc-tures (Gutiérrez et al., 1994), or they may use some chemi-cals as mediators (Couto and Herrera, 2006).

Isoenzymes: purification and characterizationTwo Lac isoenzymes, Lac I and Lac II, were isolated from P. eryngii mycelia cultures (Muñoz et al., 1997; Stajic et al., 2006b). Muñoz et al. (1997) reported that they differ in molecular mass (65 kDa and 61 kDa, respectively), the number of N-terminal amino acid residues, optimal pH (4.0 for Lac I and 3.5 for Lac II) and temperature (65°C for Lac I and 55°C for Lac II), as well as in substrate specificity (Lac I more efficiently oxidized phenols and Lac II amines).

However, only one laccase was isolated from P. eryngii basidiomata (Wang and Ng, 2006). In contrast to mycelial Lac, which are monomeric, basidiomata Lac is dimeric with a molecular mass of 34 kDa, an optimal temperature of 70°C and pH values of 3–5, and does not exhibit any similarity in the N-terminal sequences to mycelial Lac.

Molecular structureThe fungal Lacs are evolutionarily very ancient enzymes presenting with a single monophyletic branch. The crystal Lac structure was studied in only a few fungal species, i.e. Coprinus cinereus (Ducros et al., 2001), Trametes versicolor (Piontek et al., 2002), Melanocarpus albomyces (Hakulinen et al., 2002), and Lentinus tigrinus (Ferraroni et al., 2007).

Figure 1. Mechanisms of aromatic compounds’ degradation by versa-tile peroxidase (VP), laccase (Lac), and aryl-alcohol oxidase (AAO) of Pleurotus eryngii.

H2O2

H2O2

H2O2O2O2

H2O2

VP

Lac AutoxidationQuinone Superoxide anion

radicalSemiquinoneradical

Superoxidedismutase

Hydroquinone

Oxidationp-anysilalcohol

QH2 Q*− Q + O2*−

*OH

O2

O2

H− Mn2+ + H−

Mn3+ O2

AAOp-anysaldehyde p-anisic acid

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Pleurotus eryngii biology and its role in biotechnology 59

Crystallographic and phylogenetic studies showed that Lac copper-binding domains, its active site and its environment are highly conserved even when the rest of the molecule shows wide variability (Alcalde, 2007). Muñoz et al. (1997) reported that Lacs of P. eryngii are monomeric and dimeric globular glycoproteins containing Cu atoms, but did not study and present their crystal structure. In relation to the fact that Lacs are highly conserved molecules, their molecu-lar structure is presented in the sample of L. tigrinus Lac dis-cussed by Ferraroni et al. (2007). The Lac molecule is organ-ized into three sequentially arranged domains. The presence of six N-glycosylation sites indicates molecular glycosylation. The structure is stabilized by two sulphur bridges.

Lac contain four Cu atoms per monomer and no other co-factor (Alcalde, 2007). These Cu atoms are bound to three redox sites designated T1, T2, and T3. Cu1 (at site T1), known as ‘blue’ Cu, gives a greenish-blue colour due to its covalent bonds with two histidine N atoms and a cysteine S atom. It is involved in the oxidation of the reducing substrate. Cu2 (at site T2), known as ‘normal’ or ‘non-blue’, behaves as a mono-nuclear site, while the spin-coupled Cu3–Cu4 pair (at site T3) forms a binuclear site where they are coupled through an hydroxyl bridge. Together the T2 and T3 Cu form a trinuclear cluster, where reduction of O

2 and the release of water take

place. Two channels, one broad and one narrow, facilitate the access of solvent molecules to the trinuclear site. The narrow channel leads to the T2 Cu and the broad channel to Cu3.

The substrate binding site is a relatively large superficial zone near the T1 Cu, located in domain 3 (Ferraroni et al., 2007). His457, the T1 Cu ligand, and several surrounding amino acids are potentially involved in the interactions with different substrates which are oxidized at that site. The vari-ety of this region explains the large substrate specificity.

The effect of cultivation conditions on production and properties of laccasesLaccase production depends on the cultivation conditions: submerged or solid-state fermentation, carbon and nitrogen sources and concentrations, presence of inducers and other small molecules, as well as the presence of different micro-elements and their concentrations (Galhaup and Haltrich, 2001; Stajic et al., 2004; 2006b; 2006c). Likewise, significant P. eryngii intraspecies diversity in the production of these enzymes has been reported (Stajic et al., 2004).

According to the results of Muñoz et al. (1997), straw alkali lignin was the best substrate for Lac production. Stajic et al. (2006b) showed that the analyzed P. eryngii strain had the highest level of Lac activity (999.5 U/L) during cultiva-tion under conditions of submerged fermentation of dry ground tangerine fruit peels. Significant production was noted under the same conditions in the presence of xylan and D-gluconic acid sodium salt (134.4 U/L and 121.5 U/L, respectively), which could be explained by the results of Martínez et al. (1994b), who showed that xylan degradation was parallel to lignin degradation.

Numerous studies have shown that the optimal nitro-gen source for Lac production is (NH

4)

2SO

4, at a nitrogen

concentration of 20 mM (Muños et al., 1997; Hammel, 1997; Stajic et al., 2006b).

One of the effective methods for regulating and increas-ing Lac production is the addition of the appropriate induc-ers to the medium. Lignin preparation, xylidine, p-anisidine, aliphatic alcohols, aqueous plant extracts, aromatic com-pounds such as acids, alcohols, and aldehydes, as well as some metal ions, are the most efficient inducers (Galhaup and Haltrich, 2001).

Heavy metals, which are present in the environment either naturally (Cu) or as a result of human activities (Cd, Hg, Pb), are an important group of modulators of Lac activ-ity. Cu2+ participates in the regulation of Lac gene transcrip-tion (Palmeiri et al., 2000; Galhaup and Haltrich, 2001) and has positive effects on the enzyme activity and stability (Baldrian and Gabriel, 2002). Its positive effect on Lac syn-thesis is reflected by the fact that Cu2+ at the concentration of 1 mM decreases the activity of the extracellular protease, which is responsible for Lac degradation (Palmeiri et al., 2001). Contrary to the results of Muñoz et al. (1997), who showed that CuSO

4 at a concentration of 5 mM did not affect

Lac activity, Stajic et al. (2006c) noted the highest level of Lac activity at a Cu2+ concentration of 1 mM and a significant decrease at higher concentrations.

Some other microelements and macroelements also influence Lac production in P. eryngii. Muñoz et al. (1997) reported that CaCl

2, ZnCl

2, and KCl (at concentrations of

1.0 mM and 2.5 mM), as well as FeCl3 (at concentrations of

0.25 mM and 0.5 mM) inhibited Lac production, and total inhibition was noted in the presence of 0.05 mM NaN

3. Stajic

et al. (2006c) studied the effect of different concentrations of Mn2+, Fe2+, Zn2+, and Se (added in the forms of MnSO

4·H

2O,

FeSO4·7H

2O, ZnSO

4·7H

2O, Na

2SeO

3, Na

2SeO

4 and SeO

2)

on Lac production during submerged fermentation of dry ground tangerine fruit peels. Their results indicated that the highest level of Lac activity was at an Mn2+ concentration of 5 mM; Lac activity significantly increased with the addition of 1 mM of Zn2+ and Fe2+ to the medium, and the presence of 1 mM of Se decreased Lac activity.

Mn-oxidizing peroxidases

Catalytic propertiesThe Mn-oxidizing peroxidase group is composed of two enzymes: (1) Mn-dependent peroxidase (MnP) that oxi-dizes Mn2+ to Mn3+, which then directly oxidizes lignin and other phenolic aromatic compounds, and (2) versatile peroxidase (VP) that oxidizes phenolic and non-phenolic aromatic compounds, as well as Mn2+ to Mn3+ (Martinez, 2002). VP was reported for the first time in P. eryngii. Ruiz-Dueñas et al. (2001) noted the highest VP activity on Mn2+, moderate on different aromatic substrates, and the lowest on guaiacol and other dyes. This enzyme can oxidize hyd-roquinones, in the absence of Mn2+ and in the presence of H

2O

2, to semiquinone radicals, which are autoxidized

to an O2

*–, which spontaneously dismutated to H2O

2 and

O2 (Figure 1). However, glyoxylic and oxalic acids can

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60 M. Stajic et al.

be oxidized by VP only in the presence of Mn2+ (GÓmez-Toribio et al., 2001).

Although conversion of hydroquinones into semiquinone radicals under natural conditions can be catalyzed by Lac and aryl-alcohol oxidase, their oxidation by Mn2+ is relevant dur-ing the initial stages of wood biodegradation. As ligninolytic enzymes are too large to penetrate into non-modified wood cell walls, Mn3+ and the hydroxyl radical (–OH), derived from H

2O

2 during the Fenton reaction, present oxidizing agents

involved in the initial attack on lignocellulose by white-rot fungi (GÓmez-Toribio et al., 2001). Some organic and inor-ganic compounds (organic acids, NADH, hydroquinones, and thiols) can support peroxidase activity in the absence of H

2O

2 by self-oxidation catalyzed by free metal ions, and

by the production of a minor amount of H2O

2 (Li and Trush,

1993).

Isoenzymes: purification and characterizationMn-oxidizing peroxidases are produced in several isoen-zymes and encoded by multiple genes (Martinez, 2002). Two isoenzymes with a molecular mass of 42–45 kDa, a pI from 3 to 4, an optimal pH of 4 (in the presence of Mn2+) and 3 (in the absence of Mn2+), and high Mn-independent activity against 2,6-dimethoxyphenol and veratryl alcohol, were purified from a glucose-peptone-submerged culture of P. eryngii, and it was suggested that they are encoded by two genes (Martinez et al., 1996). Camarero et al. (1999) and Ruiz-Dueñas et al. (1999a) also purified two peroxidases, PL1 and PL2, under the same conditions, encoded by two alleles of the mnpl gene. Smith et al. (1990) obtained an inactive recombinant of PL2 by its gene expression in Escherichia coli. An active form of this isoenzyme, with the same properties as those of the fungal enzyme, was obtained by gene expres-sion in Aspergillus nidulans (Ruiz-Dueñas et al., 1999b) and E. coli (Pérez-Boada et al., 2002), but its yield was very low.

Camarero et al. (1999) purified three Mn2+-oxidizing peroxidases (PS1, PS2, and PS3) from a solid-state culture of P. eryngii grown on wheat straw. All of them were able to oxidize Mn2+ to Mn3+, while PS1 and PS2 also oxidized sub-stituted phenols, hydroquinones, veratryl alcohol, and some dyes in the absence of Mn2+. However, Caramelo et al. (1999) purified two peroxidases, PS1 and PS3, under the same cul-tivation conditions, and emphasized that PS2 is a tail of PS1. According to molecular masses, pI, and ability to oxidize some aromatic compounds, peroxidases from solid-state cultures were similar to those from submerged cultures, while significant differences were noted in their N-terminal sequences (Camarero et al., 1999; Ruiz-Dueñas et al., 2001). Camarero et al. (2000) also showed, by analyzing genomic or cDNA sequences of 29 fungal peroxidases, that the amino acid sequence of PS1 peroxidase had a high similarity to the sequences of PL (74% identity).

Molecular structureThe Mn-oxidizing peroxidases are globular proteins formed by 11–12 predominantly -helices in two domains delimit-ing a central cavity (Ruiz-Dueñas et al., 1999b; Camarero

et al., 2000; Martinez, 2002). Four disulfide bridges (MnP has the fifth bridge in the C-terminal region), composed of eight cysteine residues, and two Ca2+ binding sites, have an impor-tant role in the stabilization of the protein structure. Heme cofactors are located at an internal cavity (heme pocket) and connected to the protein surface by two small access chan-nels. One of them is the site where Mn2+ is oxidized to Mn3+, and the second channel is used by H

2O

2 to reach the heme

and react with Fe3+ forming the two-electron-activated form of the enzyme. Several amino-acids residues at both sides of the heme pocket are involved in substrate oxidation.

Due to the fact that the heme channel is narrow and that direct contact between the large lignin polymer and heme is prevented, low-molecular-mass compounds are involved in the process of lignin degradation. However, in some cases, small compounds may also seem too large to approach the heme through the channel, and oxidation of redox media-tors, aromatic substrates, and polymeric lignin could be explained by long-range electron transfer from the enzyme surface to the heme cofactor (Martinez, 2002). According to Pérez-Boada et al. (2005), there are three possibilities for long-range electron transfer for the oxidation of high-redox-potential aromatic compounds.

Effect of cultivation conditions on the production and properties of Mn-oxidizing peroxidasesProduction and properties of Mn-oxidizing peroxidases in P. eryngii also depend on the cultivation conditions (Martinez et al., 1996; Ruiz-Dueñas et al., 1999a; Stajic et al., 2006b; 2006c). Martínez et al. (1996) and Ruiz-Dueñas et al. (1999a) noted the highest levels of peroxidase activity in glucose-peptone medium, while no activity was detected in the medium containing malt extract and corn-steep liquor, respectively. These authors showed that yeast extract added to the glucose-peptone medium improved peroxidase pro-duction, but when it was used as a unique nitrogen source, peroxidase production levels were very low. The presence of peptone caused transcription of the mnpl gene. Stajic et al. (2006b) noted that the presence of peptone or (NH

4)

2SO

4 as

a nitrogen source elicited the highest levels of Mn-oxidizing peroxidases activities in P. eryngii, during the submerged fermentation of dry ground tangerine fruit peels.

Mn2+ plays an important role in biological oxidation and in lignin biodegradation, both as an active mediator of Mn-oxidizing peroxidases and as a regulator of their pro-duction (Kerem and Hadar, 1993). Mn2+ concentrations did not significantly affect the production of the Mn-oxidizing peroxidases during solid-state fermentation of wheat straw. On the contrary, in a submerged culture containing glucose-peptone-yeast extract, the absence of Mn2+ resulted in the highest levels of peroxidases activities, while an Mn2+ con-centration of 5 µM induced a strong decrease (90%), and no activity was found at an Mn2+ concentration of 25 µM (Martinez et al., 1996). Ruiz-Dueñas et al. (1999a) also detected the highest levels of mnpl mRNA and peroxidase production in a glucose-peptone medium without Mn2+, and their absence at an Mn2+ concentration of 25 µM or higher.

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Pleurotus eryngii biology and its role in biotechnology 61

Contrary to previous results, Stajic et al. (2006c) reported the highest levels of Mn-oxidizing peroxidase activity during the submerged fermentation of dry ground tangerine fruit peels, in the presence of 5 mM Mn2+ in the medium. This was approximately twice as high as in the control medium. This can be explained by differences in the potential of the strains to produce the enzymes mentioned, as well as by the vari-ability in the substrate compositions.

Other microelements also affect the activity of Mn-oxidizing peroxidases. The addition of Cu2+ led to an increase of the peroxidase levels and peaks of activities were recorded at a Cu2+ concentration of 10 mM (Stajic et al., 2006c). Ruiz-Dueñas et al. (1999a) reported that the pres-ence of Fe3+, at a concentration of 500 µM, caused the strong induction of mnpl gene expression, and Stajic et al. (unpub-lished results) found that the presence of 1 mM of Zn2+, Fe2+, or Se (especially in the form of SeO

2) increased activities.

Aryl-alcohol oxidaseCatalytic propertiesThe aryl-alcohol oxidase (AAO) of P. eryngii is an extracel-lular flavoenzyme with a broad substrate specificity. It oxidizes various aromatic compounds, as well as aliphatic polyunsaturated alcohols, but the highest affinity is for p-an-isyl alcohol (Varela et al., 2001). According to the results of Gutiérrez et al. (1994), the enzyme oxidizes p-anisyl alcohol to p-anisaldehyde, which can be slowly oxidized to p-anisic acid (Figure 1). In these oxidation processes, AAO provides H

2O

2, which is needed for the activity of the Mn-oxidizing

peroxidases and the generation of the . OH radical that is involved in the initial attack on lignocellulose (Evans et al., 1991). Similarly, AAO has a synergistic action with Lac in the process of oxidation of hydroquinines. This enzyme directly reduces O

2 to H

2O

2, and prevents repolymerization of prod-

ucts released during the enzymatic degradation of lignin and other aromatic compounds (Guillén et al., 2000). Due to the important AAO role in the processes of biodegrada-tion of different aromatic compounds, enzyme activation was attempted. After corresponding cDNA expression in A. nidulans, which was not very successful because of low enzyme yield (Varela et al., 2001), Ruiz-Dueñas et al. (2006) activated the enzyme by gene expression in E. coli.

Isoenzymes: purification and characterizationThe aryl-alcohol oxidase of P. eryngii was purified and char-acterized for the first time by Guillén et al. (1990). It is pro-duced in only one isoform with a molecular mass of 70 kDa, an optimum pH of 6.0–6.5, a temperature of 45–50°C, a K

m

value of 1.2 mM, and a pI of 3.8 (Guillén et al., 1990; Varela et al., 2001).

Literature data on the effect of cultivation conditions on the production and properties of AAO are still unavailable.

Molecular structureAccording to Varela et al. (2000), AAO is a monomeric protein composed of 593 amino acids and 27 of them form a signal peptide. This enzyme contains 13 putative -helices and two

major -sheets, each of them formed by six -strands. Since AAO has very broad substrate specificity, its substrate bind-ing site is large and readily accessible, and in the vicinity of the flavin ring. Residues potentially involved in catalysis and substrate binding include two histidines and several aro-matic residues of which phenylalanine makes 85% (Varela et al., 2000).

BiodegradationRecently, there has been a growing interest in the study of the lignin-modifying enzymes of the white-rot fungi with the aim of finding better aromatic compound-degrading systems, which could be applied in various biotechnologi-cal processes, such as food production, biotransformation of raw plant materials to feed and fuel, as well as in biopulping, the biobleaching of paper pulp, and the bioremediation of soil and industrial waters polluted with toxic chemicals and dyes (Reddy, 1995; Cerniglia, 1997; Ooi, 2000).

Food industryCouto and Herrera (2006) reported that Lac can be applied in the food and beverage industry, for the elimination of undesirable phenols which are responsible for the brown-ing and turbidity development in clear juice, beer, and wine, and for the determination of ascorbic acid and sugar beet pectin gelation. Since P. eryngii is a good Lac producer, it has the potential for usage in this industrial branch (Figure 2).

Conversion of agricultural residues into feedsThe production of agricultural plants is considerable world-wide (123 × 106 t/year), and approximately half of this amount is not used for either food or feed, or for the production of textiles and paper (Villas-Bôas et al., 2002). Lignin is a major aromatic polymer in the plant cell wall, which is composed of oxygenated phenylpropanoid units. Chemical and physi-cal degradation is a very expensive and inefficient process, while biological degradation is more acceptable (Martinez et al., 2001). According to Hammel (1997), lignin degradation is an extracellular, oxidative, nonspecific, and slow process. White-rot fungi play an important part in lignin degradation, owing to their ability to produce the extracellular ligninolytic enzymes and to preferentially degrade both lignin and xylan, and also cellulose slightly (Martinez et al., 1994a). Camarero et al. (1994) noted a lignin loss of 47%, a xylan loss of 43%, and only a 14% cellulose loss after 80 days of P. eryngii culti-vation on wheat straw. According to Barrasa et al. (1998) and Martinez et al. (2001), Mn-oxidizing peroxidases and AAO play a main part in wheat straw degradation (Figure 2).

However, apart from the production of ligninolytic enzymes, the presence of wheat straw stimulates the secre-tion of extracellular polysaccharides by P. eryngii and the formation of lignin–polysaccharide complexes, where lignin composition is modified due to the different incorporation of lignin units into the polysaccharides (Barrasa et al., 1998). These authors showed that the polysaccharides, which form a thin layer around hyphae, are involved in: (1) mycelial adhesion to the straw cell wall during degradation, (2) AAO

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62 M. Stajic et al.

immobilization on the hyphal surfaces and further penetra-tion of the plant cell wall, and (3) immobilization of degra-dation products that are incorporated into polysaccharides. As a consequence, P. eryngii can be cultivated on different lignocellulose residues, producing both basidiomata and protein-rich cells that remain within the substrate after the fermentation process. Substrate protein content is therefore increased, its digestibility is improved, and it can be con-verted to feed (Villas-Bôas et al., 2002; Okano et al., 2006).

Paper industryThe ligninolytic enzyme system plays a very important role in the paper industry. Due to the fact that Lacs are more available and easier to manipulate than Mn-oxidizing per-oxidases, successful pulp delignification and bleaching can be achieved by using wild Lac and AAO (Couto and Herrera, 2006) (Figure 2).

Today, especially in industrialized countries, consump-tion of paper is increasing, and with the aim of protect-ing the autochthonous forests as well as the environment

from excess quantities of agricultural residues, some Asian countries have begun modification of raw plant materials to paper pulp using white-rot mushrooms (Martinez et al., 1994a). P. eryngii has been shown to be the most efficient species for the delignification of wheat straw among the numerous white-rot fungi tested. However, it should be emphasized that although obtaining non-wood paper pulps is easier and more acceptable (Hatfield et al., 1999), non-wood pulps are more difficult to bleach than wood pulps, due to their anatomical and chemical characteristics (Akin et al., 1996).

Soil and water bioremediationCurrently numerous xenobiotics pollute the soil and water ecosystems. Besides bacteria, some Basidiomycetes play an important role in the degradation of a large variety of aro-matic xenobiotics (Bogan et al., 1999). The same non-specific system, which is responsible for lignin degradation, partici-pates in these processes (Schoemaker et al., 1991). Pleurotus spp. have the ability to grow into the soil contaminated with

Figure 2. Participation of Pleurotus eryngii ligninolytic enzymes in various biotechnological processes.

AROMATICACID

MODIFIEDDIGESTIBLE

WASTES

FOOD ANDBEVERAGES

UNDESIRABLEPHENOLS

CRUDEFOOD AND

BEVERAGES

POLYCYCLICAROMATIC

HYDROCARBONS

PETROLEUM

NOT TOXICPRODUCTS

WOODIncomplete

combustion

Incom

plete

combu

stion

AROMATICALDEHYDES

AROMATICALCOHOLS

HO−

H2O2−

H2O2

H2O2

H2O2

O2

HO−

H2O2

H2O2

H2O2

LAC

LAC

oxidatio

n

Delig

nific

atio

nBl

each

ing

AAO

AAO

WOOD ORAGRICULTURAL

WASTE

INDUSTRIALDYES

AGRICULTURALWASTES

Pleurotus eryngii

PAPER PULP

VP

VP

O2

O2

O2

O2

AA

O

H2O2

O2

H2O2

O2

Lac

Lac

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Pleurotus eryngii biology and its role in biotechnology 63

different hazardous wastes, such as polycyclic aromatic hydrocarbons (PAHs), industrial dyes, and other pollutants and to secrete the ligninolytic enzymes involved in bioreme-diation (Cohen et al., 2002). PAHs constitute abundant aro-matic pollutants with mutagenic and carcinogenic effects, which are formed by the incomplete combustion of wood and petroleum, and are bioconcentrated in trophic chains (Kotterman et al., 1998).

A commonly used bioremediation strategy is the addi-tion of lignocellulose substrate to contaminated soil, which is poor in organic matter, with the aim of promoting micro-bial growth. Morgan et al. (1993) reported that wheat straw is a good substrate for the promotion of soil colonization by microorganisms, because it improves the soil porosity and water-holding capacity, thus making microorganism growth and metabolism easier. Mushroom compost and spent mushroom compost have also been applied for the remediation of organopollutant-contaminated sites (Trejo-Hernandez et al., 2001). Lau et al. (2003) detected Lac and Mn-oxidizing peroxidases in spent mushroom compost, which degraded PAHs to products that were not toxic (Figure 2). When one considers that Pleurotus production has increased by over 500% in the past 10 years (Chiu et al., 2000), and that 5 kg of spent mushroom compost is produced during the production of 1 kg of basidiomata (Semple et al., 2001), the usage of spent compost in soil bioremediation is perfectly feasible.

Pleurotus spp. are also involved in the mineralization of different chlorophenols, which have been used extensively in pesticides, herbicides, and wood preservatives, and these have been the major toxic component in paper-pulp-bleach effluent for many years (Owens, 1991). Rodriguez et al. (2004) showed that P. eryngii successfully degraded both 2,4-dichlorophenol and benzo(a)pyrene by VP, Lac, and AAO. Martinez et al. (1994b) reported that the Lac of P. eryngii was involved in the degradation of homoveratric acid, the com-mon product of combustion of different woody plant spe-cies, and mineralization was not stimulated by the presence of H

2O

2.

Nowadays more than 100,000 commercial dyes are pro-duced in enormous amounts worldwide, about 7 × 105 t/year (Zollinger, 2002). These dyes are carcinogenic compounds and are resistant to fading due to their chemical structure. Since most currently existing processes for their removal from wastewaters are ineffective, and not economically acceptable, Kotterman et al. (1998) emphasized that Lac, Mn-oxidizing peroxidases, as well as the AAO of white-rot fungi, could play an important role in these processes (Figure 2). Cohen et al. (2002) showed that P. eryngii VP can oxidize six industrial azo and phthalocyanine dyes, and due to that ability, can be used for textile bleaching.

Conclusions

From the results obtained in the studies of P. eryngii, it can be concluded that there is great scope for its usage in a range of biotechnological processes. However, there is still a need

for considerable additional studies of this species for the fol-lowing reasons:

Scientists agree that • P. eryngii is a complex taxon and that several ambiguities in taxa classification and rela-tionships within the complex need to be resolved.The determination of the optimum substrate composi-•tion and cultivation conditions, as well as the finding of high-yield strains, is required.The interactions between • P. eryngii and its main patho-gens in farms need to be determined, and the develop-ment of efficient methodologies for infection preven-tion is needed.The better introduction of biologically active com-•pounds, with special attention to those which are applicable to the pharmaceutical industry, must be addressed.Obtaining active recombinants of ligninolytic enzyme •genes in significant amounts is required.

Acknowledgments

Declaration of interest: The authors report no conflicts of interest.

The authors offer special thanks to Prof. Dr. Solomon P. Wasser from the Institute of Evolution, University of Haifa, Israel, for the critical reading of the manuscript and for his helpful suggestions.

This study was carried out with the financial support of the Ministry of Science of the Government of Serbia, Project No. 143041.

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