White rot fungi are so named because they are able to preferentially degrade lignin in wood. When this occurs in nature substantial amounts of cellulose often remain giving the decayed wood a white appearance. Conversely when brown rot fungi attack wood residual lignin, which is brown in color, remains. Thus one of the consequences of biodegradation by white rot fungi is decolorization of naturally occurring substances. The enzymes responsible for the initial stages of biodegradation and decolorization are oxidative enzymes, primarily lignin peroxidases (ligninases), manganese peroxi-dases, and laccases (Kirk and Farrell 1987). Lignin peroxidases and manganese peroxidases have reaction mechanisms similar to those of other peroxidases (e.g., horseradish peroxidases and lactoperoxidase) (Dunford 1999). These peroxidases contain a heme prosthetic group and require hydrogen peroxide as an oxidizing cosubstrate. In the first step of the peroxidase reaction mechanism the enzyme undergoes a two-electron oxidation in which it is converted to compound I. Compound I is an activated form of the enzyme in which the heme iron exists as an oxyferryl species in the 4 + oxidation state. The other equivalent exists as a porphyrin cation radical or, in some cases, as another radical species. The oxygen atom in the oxyferryl species is donated by hydrogen peroxide. The other oxygen atom originally present in hydrogen peroxide is reduced to water. Compound I is a good oxidant and is able to mediate the one-electron oxidation of a variety of organic pollutants, including several azo dyes. During the one-electron oxidations mediated by compound I, this activated peroxidase intermediate is reduced to compound II, which is also an activated oxyferryl peroxidase intermediate. Unlike compound I, which has an oxidation state that is two electron equivalents greater than the ferric resting state, the oxidation state of compound II is one electron equivalent greater than the ferric resting stating. Thus another one-electron oxidation of an azo dye or other reducing substrate converts compound II back to the resting state thereby completing the reaction cycle.
Lignin peroxidases and manganese peroxidases are relatively nonspecific in that they are able to oxidize a variety of substrates. On the other hand, these enzymes also exhibit some specificity. This has been demonstrated nicely by Pasti-Grigsby et al. (1992) who showed that some azo dyes are preferentially oxidized by lignin peroxidases whereas others are preferentially oxidized by manganese peroxidases.
Veratryl alcohol is an endogenous substrate for lignin peroxidases produced by Phanerochaete chrysosporium. When this compound serves as substrate it undergoes two successive one-electron oxidations to form veratraldehyde. Of interest to this review is the fact that veratryl alcohol enhances oxidation of some substrates, including some azo dyes. It appears that some azo dyes (e.g., Biebrich Scarlet and Tartrazine) can undergo a one-electron oxidation by lignin peroxidase compound I forming the one-electron oxidation product of the dye and lignin peroxidase compound II (Paszyzynski and Crawford 1991). However, compound II does not appear to be able to mediate oxidation of the azo dye. This results in accumulation of lignin peroxidase compound II and the reaction ceases. When veratryl alcohol is added, compound II oxidizes it and returns to the ferric resting state of the enzyme which may then participate in another round of catalysis. By preventing accumulation of compound II veratryl alcohol enhances oxidation of the azo dye.
Wu et al. (1996) showed that nutrient carbon or nutrient nitrogen limited cultures of P. chrysosporium are able to decolorize Reactive Red 22. It was also reported that a crude preparation of lignin peroxidases mediated decolorization of Reactive Red 22 and another azo dye, Evan's Blue. Similar to the observations of Paszyzynski and Crawford (1991) it was found that the presence of veratryl alcohol enhanced decolorization of Reactive Red 22.
Manganese peroxidases are so named because they mediate the one-electron oxidation of Mn(II) to Mn(III) (Kuwahara et al. 1984; Glenn et al. 1986; Wariishi et al. 1988). In vitro, this reaction is often performed in the presence of a metal chelator such as lactate or tartrate, the purpose of which is to facilitate dissociation of Mn(III) from the enzyme in the form of a Mn(III)-lactate or Mn(III)-tartrate complex (Wariishi et al. 1992).
In vivo, oxalate or malonate likely performs this function. This is of interest because Mn(III)-complexes are relatively good oxidants. They are also relatively stable. It is thought that Mn(III)-complexes are stable enough to diffuse away from the active site and mediate oxidation of lignin and other materials that are inaccessible or otherwise not amenable to direct oxidation by laccases, lignin peroxidases, or manganese peroxidases. Recent studies have shown that the specificity of manganese peroxidases varies considerably between fungal species. For example, Heinfling et al. (1998a,b) showed that Reactive Orange 96 and Reactive Red 198 were not oxidized by manganese peroxidase P1 from P. chrysosporium and that oxidation of Reactive Blue 5 by this enzyme was negligible. This enzyme slowly oxidized Reactive Violet 5 and activity was increased by.approximately five-fold when 0.3 mM MnSO4 was present in the reaction mixture. In contrast, the presence of 0.3 mM MnSO4 did not enhance oxidation of the other azo dyes studied. The ability of manganese peroxidases from other white rot fungi to oxidize these azo dyes was also studied. Of importance is the observation that substantial rates of dye oxidation occurred in reactions that were independent of manganese. Indeed, Mn(II) was shown to function as a noncompetitive inhibitor of Reactive Black 5 oxidation by MnP1 from B. adusta. Manganese peroxidases from these fungi allow a strategy of direct oxidation (i.e., by the enzyme) or indirect oxidation (by Mn(III)-complexes) to be pursued. This may become important in bioreactor design and strategy development.
In addition to lignin peroxidases and manganese peroxidases, white rot fungi often produce laccases. These oxidases are also important in lignin degradation but unlike peroxidases, they do not contain heme. Laccases are more formally known as benzenediol:O2 oxidoreductases, EC 126.96.36.199). Instead of heme, laccases require active site Cu(II) ions for activity. During laccase-mediated reactions, diphe-nolic compounds undergo a four-electron oxidation. During this reaction, Cu(II) is reduced to Cu(I). During the next step in the reaction, Cu(I) reduces molecular oxygen (O2) to produce two molecules of water. During this reaction Cu(I) is oxidized back to Cu(II) thus completing the reaction cycle. Laccases can mediate other reactions. Wong and Yu (1999) showed that Trametes versicolor decolorized Acid Violet 7. Of interest was the observation that laccase did not directly oxidize this azo dye. However, in the presence of a low molecular weight compound substantial oxidation (i.e., decolorization) of this dye occurred. It was proposed that the low molecular weight compound was oxidized by laccase producing a reactive radical species, which then oxidized the dye molecule resulting in a colorless product.
Pointing and Vrijmoed (2000) have presented evidence implicating laccases produced by Pycnoporus sanguineus in the oxidation of the azo dyes Orange G and Amaranth and the triphenylmethane dyes Bromophenol Blue and Malachite Green. Evidence for this, however, was indirect as oxidation of these dyes was only correlated with laccase activity in cultures of this fungus. It should be noted, however, that P. sanguineus did not produce lignin peroxidase or manganese peroxidase.
Yesilada and Ozcan (1998) studied the ability of crude culture filtrates to decolorize Orange II. It was shown that filtrates from Coriolus versicolor but not P. chrysosporium, were able to mediate substantial decolorization of water contaminated with this dye. Decolorization was dependent on the age of the culture and was not dependent on the presence of hydrogen peroxide. Decolorization activity was inactivated by heat. Given the fact that C. versicolor produces laccase in abundance it is reasonable to suggest that this enzyme is responsible for the decolorization observed.
Production of veratryl alcohol radical and Mn(III) complexes may be of importance in lignin degradation as it has been suggested that these low molecular weight reactive species may be stable enough to diffuse into the 3-D lignin structure and mediate oxidations that are not accessible by direct enzymatic oxidation by lignin peroxidases, manganese peroxidases, or laccases (Barr and Aust 1994; Harvey et al. 1986; Glenn et al. 1986). Veratryl alcohol radical may be too unstable and short-lived to accomplish this. However, Mn(III) complexes do appear to be sufficiently stable for this purpose. Mn(III) complexes appear to be able to oxidize phenolic units in lignin. They do not appear to be able to oxidize nonphenolic units (Popp and Kirk 1992; Wariishi et al. 1992). However, when unsaturated fatty acids are added to reaction mixtures containing manganese peroxidase, Mn(II) complexes, and hydrogen peroxide, lipid peroxidation occurs (Kapich et al. 1999). Of interest is the observation that oxidants are produced, in such reaction mixtures, which are capable of oxidizing nonphenolic subunits in lignin as well as selected organic pollutants such as phenanthrene. Kapich et al. (1999) suggest that peroxyl radicals are produced that are responsible for the oxidations that occur.
Lignin peroxidases, manganese peroxidases, and laccases are by far the most studied oxidases found in white rot fungi. Because of their ability to degrade a wide range of organic compounds, it was reasonable to suspect that cytochrome P-450 monooxygenases may be responsible for some of the oxidations that have been observed. Indeed, some of the documented oxidations that are mediated by P. chrysosporium have not been attributed to lignin peroxidases, manganese peroxidases, or laccases. As a case in point, during DDT degradation, the parent compound undergoes hydroxylation, forming dicofol (Bumpus et al. 1985). To date, the enzyme responsible for this oxidation has not been identified and may well, indeed, be a cytochrome P-450 monooxygenase. This family of enzymes has not been extensively investigated in white rot fungi. Studies by Knapp et al. (1997) and by Yadav and Loper (2000) showed that P-450 monooxygenase genes exist in this fungus. Substantial evidence for P-450 mediated hydroxylation of benzo(a)pyrene was presented by Masaphy et al. (1996) who showed that microsomal and soluble fractions from P. chrysosporium exhibited characteristic reduced carbon monoxide difference spectra. Benzo(a)pyrene hydroxylation was shown to be dependent on NADPH and was inhibited by carbon monoxide. Furthermore benzo(a)pyr-ene caused a type I spectral shift (indicative of substrate binding) when added to soluble and microsomal preparations. A cytochrome P-450 monooxygenase is also thought to be responsible for phenanthrene oxidation during biodegradation of this compound by Pleurotus ostreatus (Bezalele et al. 1996).
Most recently, the genome of P. chrysosporium has been sequenced at the U.S. Department of Energy's facility at Walnut Hill, California. This fungus is the first basidiomycete whose genome has been sequenced. The genome is about 30 Mb and is comprised of 10 chromosomes. Of interest is the observation that this fungus may have the genetic potential to produce over one-hundred P-450 monooxygenases (Nelson 2001). Clearly, elucidation of the contribution of P-450 monooxygenases to azo dye degradation and xenobiotic metabolism, in general, will be an area of considerable research interest.
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