INTRODUCTION

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Lignin, the second most abundant renewable organic polymer on earth, is a major component of wood. Because of the importance of wood and other lignocellulosics as a renewable resource for the production of paper products, feeds, chemicals, and fuels, there has been an increasing research emphasis on the fungal degradation of lignin (5, 30). White rot fungi are believed to be the most effective lignin-degrading microbes in nature. A majority of the previous studies have focused on the lignin-degrading enzymes of Phanerochaete chrysosporium and Trametes versicolor (23, 44). Recently, however, there has been a growing interest in studying the lignin-modifying enzymes of a wider array of white rot fungi, not only from the standpoint of comparative biology but also with the expectation of finding better lignin-degrading systems for use in various biotechnological applications (14, 24, 36, 39).

Three major families of fungal lignin-modifying enzymes (LMEs) are laccases, manganese-dependent peroxidases (MnPs), and lignin peroxidases (LiPs) (5, 24, 30, 50). These LMEs can oxidize phenolic compounds thereby creating phenoxy radicals, while nonphenolic compounds are oxidized via cation radicals (5, 25, 30). LiP and MnP oxidize nonphenolic aromatic compounds with high oxidation-reduction potentials (21), the major components of the lignin polymer. Laccase oxidizes nonphenolic aromatic compounds with relatively low oxidation-reduction potentials (29, 56). In the presence of low-molecular-weight mediators, laccases can also oxidize nonphenolic substrates with high oxidation-reduction potentials (6, 9, 16) as well as certain xenobiotics (27).

Preliminary studies in our laboratory showed the presence of lip gene-homologous sequences in the genomic DNA of several genera of wood rot fungi (13). Subsequent screening on plates containing the polymeric dye poly R-478, decolorization of which is correlated with lignin degradation (22), led to the selection of a strain of Ganoderma lucidum for further studies, based on its rapid growth rate and extensive decolorization of poly R-478 on solid media. G. lucidum is one of the most important and widely distributed white rot fungi in North America and is associated with the degradation of a wide variety of hardwoods (1). Previous studies of G. lucidum have mainly concentrated on the medicinal properties of this fungus (reviewed in reference 28) and, except for two brief preliminary reports (26, 41), little is known about the ligninolytic system of this organism. In this report, we describe the production of LMEs by G. lucidum under different culturing conditions, with emphasis on its laccase.

	MATERIALS AND METHODS

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Fungal strains. G. lucidum (Leysser) Karsten FP-58537-Sp was provided by the U.S. Department of Agriculture USDA Forest Products Laboratory, Madison, Wis. P. chrysosporium BKM-F 1767 (ATCC 24725) was from the American Type Culture Collection, Manassas, Va.

Culture conditions. The fungal cultures were maintained on plates of malt extract (ME) medium which contained, per liter, 1 g of peptone, 20 g each of ME and dextrose, and 20 g of Bacto Agar. Inoculum for liquid cultures was prepared with fungal mycelia from 7-day-old cultures grown in ME liquid medium under static conditions as described previously (14) and homogenized on ice for 5 min with an Omni mixer (Ivan Sorvall, Inc., Newtown, Conn.). Static liquid cultures of G. lucidum were grown in 125-ml Erlenmeyer flasks containing 9 ml of sterile medium plus 1 ml of inoculum, while shaken cultures of G. lucidum were grown in 125-ml Erlenmeyer flasks containing 45 ml of a given medium plus 5 ml of the inoculum. Shaken cultures were oxygenated (with 100% O₂) for 1 minute immediately after inoculation (14) and every day thereafter, whereas static cultures were oxygenated right after inoculation and every third day thereafter. Cultures were grown in previously defined (10) low-nitrogen (LN; 2.4 mM N), high-nitrogen (HN; 24 mM N), or ME media under static or shaken conditions. All cultures were incubated at room temperature, unless mentioned otherwise. To study the effect of various low-molecular-weight aromatic compounds on laccase induction, filter-sterilized ferulic acid, syringic acid, 2,5-xylidine, sinapic acid, veratryl alcohol (VA), or pentachlorophenol was added to 3-day-old static LN cultures to a final concentration of 0.2 mM. LN cultures without added inducers served as controls.

Determination of mycelial dry weight. Mycelial dry weights were determined by vacuum filtering the cultures with preweighed (47-mm-diameter) glass filters. The filters containing the mycelial mass were placed in preweighed 50-mm-diameter aluminum pans and dried at 80°C to constant weight.

Enzyme assays. Samples (100 µl) were collected every day (except where mentioned otherwise), mycelial particles were pelleted at 5,000 × g, and the supernatants were assayed for LiP and MnP as previously described (38, 51). Laccase activity was monitored as described by D'Souza et al. (14), except that the absorbance was measured at 405 nm (extinction coefficient = 35,000 M¹ cm¹) (55). The laccase assays were performed at pH 3.0 with ABTS [2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)] as the substrate, because this was the optimum pH for laccase activity in G. lucidum (15). One unit of LiP activity represents 1 µmol of VA oxidized to veratraldehyde per min, while 1 U of MnP activity represents 1 µmol of Mn(II) oxidized to Mn(III) per min. Laccase activity is expressed as microkatals or nanokatals (micromoles or nanomoles, respectively, of substrate transformed per second) per liter of extracellular culture fluid (ECF).

SDS-polyacrylamide gel electrophoresis (PAGE), IEF, and activity staining of laccase proteins. Sterile HN medium (450 ml in a 2-liter Erlenmeyer flask) was inoculated with 50 ml of a homogenized 7-day-old culture of G. lucidum grown in HN medium. Cultures were oxygenated on the day of inoculation and every day thereafter and incubated with shaking at 200 rpm for 7 days. ECF from these cultures was then concentrated 20-fold by ultrafiltration with a 10-kDa-cutoff membrane (PM-10; Amicon Division, W. R. Grace & Co., Danvers, Mass.). Twenty microliters of the concentrated ECF was mixed with 20 µl of 2× sodium dodecyl sulfate (SDS) loading buffer, loaded onto a SDS-polyacrylamide gel, and electrophoresed at room temperature with Tris-glycine buffer (pH 8.3) at 125 V for 90 min as described previously (31), except that the dye-protein mixture was not boiled before loading.

Southern blot hybridization. Mycelial plugs (5 mm in diameter) of G. lucidum grown on plates of ME agar for 7 days were inoculated into ME liquid medium (three plugs per 100 ml of medium in 500-ml Erlenmeyer flasks). The cultures were allowed to incubate at room temperature for 10 days, mycelia were harvested by filtering the cultures through four layers of cheesecloth, and genomic DNA was isolated as described by Rao and Reddy (43). Southern blots were prepared and probed as described by Sambrook et al. (46). The probe used was the 0.8-kb PstI fragment of the LiP H2-encoding CLG4 cDNA (11). After hybridization at 42°C for 24 h, the blots were washed under high-stringency wash conditions and were then exposed to X-ray film at 70°C for 24 h by using two intensifying screens.

Mineralization of [¹⁴C]DHP. G. lucidum cultures were grown in poplar and HN media. Erlenmeyer flasks (125 ml) containing 9 ml of medium plus 1 ml of inoculum were stoppered with two-holed rubber stoppers fitted with clamped tubes to enable flushing with O₂ and collection of the ¹⁴CO₂ as previously described (18). Poplar cultures were incubated at room temperature under static conditions, while the HN cultures were grown at room temperature under shaken conditions. A positive control was prepared by inoculating 1 ml of an inoculum of P. chrysosporium (BKM-F 1767) into 9 ml of LN medium and incubating the mixture at room temperature under static conditions as previously described (18). The cultures were oxygenated on the day of inoculation and every third day thereafter. After 3 days, [-¹⁴C]propyl side chain-labeled synthetic lignin ([¹⁴C]DHP) having a total activity of ~30,000 dpm was added to each flask. The [¹⁴C]DHP, a gift from Kenneth Hammel (USDA Forest Products Laboratory), had a specific activity of 0.01 mCi/mmol and was passed through a 1- by 30-cm column of Sephadex LH20 (Sigma Chemical Co., St. Louis, Mo.) to remove low-molecular-weight contaminants. At 3-day intervals, the headspace of each culture flask was flushed with CO₂-free air and the evolved ¹⁴CO₂ was trapped in a scintillation mixture containing 50% (vol/vol) scintillation fluid (complete counting cocktail 3a20; Research Products International Corp., Mount Prospect, Ill.), 40% (vol/vol) methanol, and 10% (vol/vol) ethanolamine (Sigma Chemical Co.). The ¹⁴CO₂ was analyzed with a liquid scintillation analyzer (Tri-Carb 2100TR; Packard Instrument Co., Meriden, Conn.).

	RESULTS

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LME production in defined media. Laccase was the only LME produced by G. lucidum in defined media; LiPs or MnPs were not produced. Higher laccase activities were observed in HN shaken cultures than in HN static, LN shaken or static, or ME static cultures (Fig. 1A). HN shaken cultures produced levels of laccase activity that were more than fourfold higher than those produced by the LN shaken cultures, even though the mycelial dry weight of HN cultures was only about twofold higher (2.1 versus 0.95 mg/ml) than that of LN shaken cultures (Fig. 1B) on day 7, when peak laccase activities were observed.

View larger version (26K): [in this window] [in a new window]   FIG. 1.   (A) Laccase production by G. lucidum cultures grown in defined liquid media and in ME media under shaken and static conditions. HN, 24 mM N; LN, 2.4 mM N. (B) Laccase production and mycelial dry weight in G. lucidum cultures grown in LN and HN media under shaken conditions.

View larger version (29K): [in this window] [in a new window]   FIG. 2.   Effect of various low-molecular-weight aromatic compounds on laccase activity of G. lucidum. Each compound was added to 3-day-old LN (without VA) cultures to a final concentration of 0.2 mM. Values plotted represent means of three replicate flasks.

LMEs in wood-containing media. Laccase production in pine cultures (3.7 µkat/liter; Fig. 3A) was much higher than that in poplar cultures (1.4 µkat/liter; Fig. 3B). Cultures containing half the amount of both pine and poplar, on the other hand, produced much higher levels of laccase than those seen in either pine or poplar cultures (14.8 µkat/liter; Fig. 3C). Since syringyl moieties are major constituents of poplar lignin (5), we tested the effect of the addition of syringic acid to pine cultures on laccase and MnP production. Laccase production was threefold higher in pine cultures with added syringic acid than in identical parallel cultures without it (Fig. 4). Addition of syringic acid to HN medium did not increase the production of laccase (results not shown).

View larger version (27K): [in this window] [in a new window]   FIG. 3.   Laccase and MnP production by G. lucidum cultures grown under static conditions in media containing 100 mg of pine wood (A), 100 mg of poplar wood (B), and 50 mg each of pine and poplar woods (C) per flask.

View larger version (21K): [in this window] [in a new window]   FIG. 4.   Effect of syringic acid (0.2 mM) on laccase production by G. lucidum cultures grown in pine wood medium.

View larger version (66K): [in this window] [in a new window]   FIG. 5.   Southern hybridization of restriction enzyme-digested genomic DNA of G. lucidum with random-primed [-32P]dCTP-labeled LiP cDNA CLG4 (11) of P. chrysosporium BKM-F 1767. The sizes of the molecular weight markers are shown on the left. The restriction enzymes used in the digestions are shown above the lanes.

SDS-PAGE and IEF of concentrated ECF. Two laccase activity bands were observed upon nondenaturing SDS-PAGE of the 20-fold-concentrated ECF from HN cultures followed by activity staining (Fig. 6A). The apparent M_r of the bands were 40,000 and 66,000. IEF of 20-fold-concentrated ECF followed by laccase activity staining showed at least five major laccase activity bands (Fig. 6B) with estimated pIs of 3.0, 4.25, 4.5, 4.8, and 5.1. 

View larger version (48K): [in this window] [in a new window]   FIG. 6.   SDS-PAGE (A) and IEF (B) of ECF of G. lucidum cultures grown in HN medium for 7 days. Twenty microliters of 20-fold-concentrated ECF was loaded on an SDS-polyacrylamide gel, while 5 µl was loaded on an IEF gel. Electrophoresis conditions are described in the text. Molecular mass standards for the SDS-PAGE (A) and the IEF marker standards (B) are shown. Both the gels were stained for laccase activity with ABTS as the substrate (34). The arrowheads indicate the laccase activity bands in each gel.

Mineralization of [¹⁴C]DHP. The mineralization of ¹⁴C-labeled synthetic lignin to ¹⁴CO₂ by G. lucidum cultures (Fig. 7) grown under conditions favoring the production of laccase alone (i.e., HN shaken cultures) was relatively low (2.3% mineralization). Mineralization was not much higher (3.3%) in poplar-grown cultures, which produced both laccase and MnP. These consistently low levels of mineralization observed are not the result of low-molecular-weight lignin products because the [¹⁴C]DHP had been purified to remove these (see Materials and Methods). Moreover, the mineralization values shown are net differences between experimental and heat-killed controls. Also, in control experiments, P. chrysosporium showed 19% [¹⁴C]DHP mineralization to ¹⁴CO₂, which is in agreement with previously published reports (4, 18). Addition of "cold" DHP to HN and poplar cultures did not cause increased laccase or MnP production, nor did it elicit LiP production (results not shown).

View larger version (28K): [in this window] [in a new window]   FIG. 7.   Mineralization of [14C]DHP to 14CO2 by G. lucidum (Gl58537) grown in HN and poplar wood media under static conditions. Data for the positive control, P. chrysosporium (Pc) BKM-F 1767 grown in LN medium, are also presented for comparison.

	DISCUSSION

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It has been well documented that nitrogen levels, aeration, and other factors influence LME production by white rot fungi (5, 7, 8, 30, 52). For example, with glucose as the carbon source, LiP and MnP production by P. chrysosporium was seen in LN (2.4 mM N) medium but was completely suppressed in HN (24 mM N) medium (5, 7, 30, 44). Laccase production by P. chrysosporium was not detected in LN or HN medium with glucose as the carbon source but was readily demonstrable when the organism was grown in LN or HN media with cellulose (49) or with glucose and 0.4 mM CuSO₄ (12). The results of this study show that laccase production in G. lucidum is not inhibited in N-rich media with glucose as the carbon source. In fact, the highest levels of laccase are produced by this organism in HN medium. In this respect, our results are in agreement with earlier findings reporting high levels of laccase production under N-rich conditions in Rigidoporus lignosus (20), Ceriporiopsis subvermispora (32), Lentinula edodes (8), and Agaricus bisporus (40, 54).

Our results show that G. lucidum produces much higher levels of laccase in HN shaken than in HN static cultures (Fig. 1B). Stimulation of the production of LMEs, such as LiP, by aeration has also been reported for P. chrysosporium (5, 7, 23, 30). Recently, we have shown that oxygenation had a marked positive influence on laccase production by P. chrysosporium (49). Increasing the oxygen level in the medium has been postulated to lead to increased LME production and increased production of the components of the H₂O₂-producing systems (5, 30). Relatively poor laccase production in LN cultures (shaken or static) is probably due to the fact that levels of growth observed in these cultures were much lower than those in the HN cultures (Fig. 1B). It is also possible that other unknown factors arising from the combined effects of HN and aeration may have contributed to high levels of laccase in HN shaken cultures.

Various low-molecular-weight aromatic compounds have been reported to enhance LME production (3, 5, 7, 30, 47). For example, VA has been shown to enhance laccase production in several white rot fungi (3, 16, 47). This is consistent with our observation that in G. lucidum only VA, among a number compounds tested, showed stimulation of laccase production. 2,5-Xylidine has been reported to give 20-fold stimulation of laccase activity in Trametes villosa (55), 9-fold stimulation in Pycnoporus cinnabarinus (16), and at least a 4-fold stimulation in Irpex lacteus (2) but showed no enhancement of laccase production in G. lucidum. These results suggest that enhancement of laccase production in response to various aromatic compounds differs greatly among different white rot fungi.

Wood is the natural substrate for G. lucidum, which is known to cause extensive delignification of various species of hardwoods worldwide (1). Yet a large majority of the previous studies on the production of LMEs have been carried out with defined media (5, 30), and none has been reported for G. lucidum. Recent results show that LME production when white rot fungi are grown in wood-containing media could be substantially different from that seen in defined media (19, 35, 40, 47-49). For example, the results of this study show that G. lucidum produces laccase only in defined media and in cultures grown with pine but produces both laccase and MnP in cultures grown with poplar. Also, laccase levels were substantially higher in defined media than in wood-grown cultures. These results are consistent with earlier studies which showed that several white rot fungi produce both laccase and MnP in media supplemented with wood but produce only laccase in defined media (26, 32, 47). Furthermore, Galliano et al. (20) and Maltseva et al. (33) reported production of laccase and MnP by R. lignosus and Panus tigrinus, respectively, in sawdust medium and wheat straw medium. However, unlike our study, none of the earlier studies compared LME production in media containing different types of wood. Our finding that MnP is produced only in poplar cultures and not in pine cultures is a unique finding in that not all classes of LMEs are produced consistently even in media containing complex ligninaceous substrates such as wood. Instead, the type of wood substrate appears to determine the types and amounts of LMEs produced by the white rot fungi. The reason for differential MnP production by G. lucidum in pine and poplar cultures is not obvious, but the most likely explanation is that some unique component in poplar triggers MnP production by G. lucidum and that this is lacking in pine. In support of this idea, G. lucidum cultures grown with both pine and poplar (50:50) produced approximately half the amount of MnP as that seen in cultures grown with poplar only. This suggested that the reduced amount of MnP seen in the former cultures is a reflection of the smaller amount of poplar wood (50 mg) in these cultures compared to that in cultures containing poplar only (100 mg). This was further confirmed by growing G. lucidum in cultures containing 50 mg of poplar only, which also produced approximately half the level of MnP as that produced in cultures with 100 mg of poplar (results not shown).

An important finding of this study is that laccase production in G. lucidum cultures containing equal amounts of pine and poplar is 4 times and 10 times higher, respectively, than those seen in cultures containing pine only and poplar only. These results suggest that certain components in these two woods have a synergistic effect on laccase production. While the structure of syringic acid is different from the structure of syringyl moieties of lignin, the ring structures of both are identical. Also, previous researchers have shown that low-molecular-weight aromatic acids, such as syringic acid, that are structurally related to individual phenolic moieties in lignin serve as good inducers of LMEs (16, 55). In this study, we used commercially available syringic acid as a substitute for syringyl moieties of lignin (in the same way as ferulic acid is often used as a substitute for coniferyl alcohol). Addition of syringic acid to pine cultures of G. lucidum resulted in laccase activity (14.7 µkat/liter) comparable to that seen in pine-plus-poplar cultures (14.8 µkat/liter) but was much higher than that produced in the medium with pine alone or poplar alone. These data suggest that the stimulation of laccase production in pine-plus-poplar cultures of G. lucidum is probably due to the syringyl units contributed by poplar lignin.

The molecular masses (40 and 66 kDa) and IEF values (3.0 to 5.1) reported in this study for G. lucidum laccases are in the range observed for laccases isolated from other white rot fungi (32, 37, 53, 55). For example, T. villosa was shown to produce a laccase (two subunits of 63 kDa) that was resolved by IEF into three isoforms with pIs of 3.5, 6 to 6.5, and 5 to 6 (55), while Pleurotus ostreatus was shown to produce three laccases each having a molecular mass of 67 kDa; two had a pI of 4.7, and one had a pI of 2.9 (37). The white rot fungus C. subvermispora was shown to produce four laccase isozymes with pI range of 3.4 to 4.7 (32). In our study, identical laccase isoforms were consistently seen when concentrated ECF from cultures grown in LN, ME, or wood media were used (results not shown), suggesting that the laccase isoforms of G. lucidum are constitutive and that these are not artifacts of the IEF procedure.

Observation of distinct hybridization bands on probing Southern blots of restriction enzyme-digested genomic DNA of G. lucidum with the LiP cDNA (CLG4) of P. chrysosporium suggests the presence of lip-like gene sequences in G. lucidum, but LiP production was not observed in any of the media included in this study. These results are also in agreement with our earlier finding that CLG4 gene homology is widely distributed in white rot fungi (44). Apparently, G. lucidum has the genetic potential to produce LiPs but did not produce LiPs under culture conditions employed in our study. These results are also consistent with the observations by Ruttimann et al. (45) that the Southern hybridization technique utilizing a lip probe from P. chrysosporium shows the presence of lip-like genes in Phlebia brevispora and C. subvermispora but that LiP production could not be demonstrated by either organism. Moreover, Rajakumar et al. (42) demonstrated the presence of lip-like gene sequences in C. subvermispora and P. sordida by employing a PCR procedure but failed to show LiP enzyme activity in liquid cultures. It was suggested that failure to detect LiP production in the above studies could be due to the obscuration of detection by interfering substances, the greater susceptibility of LiPs in these organisms to certain fungal proteases, or simply the lack of adequate culture conditions that favor LiP production by these organisms (36, 42, 45). Perumal and Kalaichelvan (41) published a preliminary report claiming low levels of LiP production ostensibly by a strain of G. lucidum in media amended with lignin isolated from sugarcane bagasse, but this work has not been substantiated further.

The ability of an organism to degrade [¹⁴C]DHP to ¹⁴CO₂ has been widely used to convincingly demonstrate the rate and extent of lignin degradation by various isolates of white rot fungi. G. lucidum mineralizes [¹⁴C]DHP to a very limited extent in media favoring production of laccase only or both laccase and MnP (Fig. 7), suggesting a limited role for laccase (and perhaps MnP) in lignin degradation by this organism. Whether the lignin degradation in wood by G. lucidum (1) is due to laccase or involves MnP and/or LiP as well is not known at this time. However, the contribution of laccase to lignin degradation and its ability to modify nonphenolic-lignin model compounds have been well documented (6, 17, 24, 25).

In summary, our results show that G. lucidum, an important white rot fungus involved in wood decay worldwide, produces laccase as the dominant LME. Laccase production is the result of a marked synergy when G. lucidum is grown with a mixture of pine and poplar woods compared to production with either one alone, and the organism produces at least five isoforms of laccase. This organism produces MnP in poplar but not in pine medium. It fails to produce LiP in defined media or in media containing pine and/or poplar but appears to have the genetic potential to produce LiP, as indicated by positive hybridization of its genomic DNA with the LiP cDNA of P. chrysosporium.