Applied Microbiology Biotechnology

Appl Microbiol Biotechnol (1990) 32:436-442

© Springer-Verlag 1990

Screening of basidiomycetes for lignin peroxidase genes using a D NA probe Yoshio Kimura, Yasuhiko Asada, and Masaaki Kuwahara Department of Bioresource Science, Kagawa University, Miki-Cho, Kagawa 761-07, Japan -;

Summary. Basidiomycetes were screened for lignin peroxidase (LPO) genes using a DNA probe prepared from the LPO restriction fragment of Phanerochaete chrysosporium. Southern blot analysis showed restriction fragments of chromosomal DNA of Bjerkandera adusta and Coriolus consors hybridized with the probe. Bjerkandera adusta produced LPO in a glucose-peptone medium. Ion-exchange chromatography showed that. this fungus produced multiple molecular forms of LPO. One of the enzymes, LPO-2, was purified and characterized. The molecular weight of LPO2 was 41 000 with a pI of 4.2. Spectral analysis demonstrated that LPO-2 is a haem protein. The enzyme cleaved lignin model dimers mainly at the Ca-Cfl position of the side chain. The LPO-2 exhibited close similarity to LPOs of P. chrysosporium with respect to their basic properties.

Introduction Lignin occurs as one of the major components of woody and herbaceous plants and is the most abundant renewable aromatic polymer an earth. Biological degradation of lignin has aroused interest in its potential application to industrial processes such as biomechanical pulping, bleaching of paper, production of feedstock from wood and agricultural wastes and conversion of lignocellulosic materials to fuel and chemicals. The most potent lignin-degrading organisms are white-rot fungi which are taxonomically members of the basidiomycetes. Since Phanerochaete chrysosporium has been found to degrade lignin more extensively than other fungi, studies on the Offprint requests to: M. Kuwahara

lignin-degrading process have been mainly focused on this fungus (for a recent review see Kirk and Farrell 1987). Lignin degradation of this fungus is a secondary metabolic event which is initiated by nitrogen, carbon or sulphur starvation (Jeffries et al. 1981). This fungus has been found to produce extracellular peroxidases, now called lignin peroxidase (LPO) or ligninase, which appear in response to nutrient depletion (Tien and Kirk 1984; Gold et al. 1984; Faison and Kirk 1985). This enzyme is a glycosylated haem protein which contains protoporphyrine IX as a prothetic group (Gold et al. 1984; Tien and Kirk 1984). The -existence of multiple molecular forms of LPO has been demonstrated (Kirk et al. 1986; Paszczynski et al. 1986; Leisola et al. 1987). Lignin peroxidase catalyses the H202-dependent oxidation of a variety of lignin model compounds by a one-electron oxidation mechanism followed by a series of non-enzymatic reactions to yield numerous products (Kirk et al. 1986; for a recent review see also Gold et al. 1989). Although the properties of the enzyme and its reaction mechanism are well characterized in P. chrysosporium, information on this enzyme from other wood-rotting fungi is limited to Coriolus versicolor (Dodson et al. 1987) and Phlebia radiata (NikuPaavola et al. 1988) at present. Recently, the LPO gene has been cloned and sequenced either from eDNA (Tien and Tu 1987; de Boer et al. 1987) or chromosomal DNA (Smith et al. 1988; Asada et al. 1988; Walther et al. 1988) of Phanerochaete chrysosporium in some laboratories including ours. These experiments also showed that there are many LPO-related genes in this fungus which constitute a gene family. The sequence homology at the distal histidine of this enzyme with that of peroxidases of other origins has also been shown (Tien and Tu 1987). Based

437

Y. Kimura et al.: Screening for lignin peroxidase genes

mmol, New England Nuclear, Boston, USA) using a nick translation kit (Takara Shuzo) to give a specific activity of over 107 cpm/txg).

on these results the LPO gene of P. chrysosporium is expected to retain sufficient D N A sequence homology to hybridize to the corresponding gene of other wood-rotting fungi. In this study, we screened several fungi for the LPO gene using the gene of P. chrysosporium as a hybridization probe. Production of LPO by Bjerkandera adusta, which was selected by the gene hybridization, was examined and the enzyme was purified and characterized.

Southern blot hybridization. The DNA fragments of restriction digests were electrophoresed in a 0.7% agarose gel and transferred to Gene-Screen Plus membranes (New England Nuclear). Hybridization was carried out in 4 x standard salinecitrate (SSC) containing 1% sodium dodecyl sulphate (SDS) at 48 ° C for 40 h. The gels were autoradiographed with RX X-ray film (Fuji Film, Tokyo, Japan). Enzyme assay. The LPO activity was assayed spectrophotometrically as described by Tien and Kirk (1984) using a reaction mixture containing 0.8 m M veratryl alcohol, 0.1 M sodium tartrate (pH 3.0), 0.25 M H202 and enzyme solution in a final volume of 0.5 ml. Veratryl alcohol oxidase activity was assayed using a mixture containing 0.8 m M veratryl alcohol, 0.1 M Naphosphate buffer (pH 7.0) and enzyme solution in a final volume of 0.5 ml. One unit of each enzyme activity was defined as 1 nmol veratryl alcohol oxidation to veratraldehyde per minute per millilitre assay mixture. Specific activity was expressed as units per milligram protein. Mn(II)-dependent peroxidase activity was assayed according to a method described previously (Kuwahara et al. 1984). Protein was measured by the method of Bradford (1976) using bovine serum albumin (Sigma, St. Louis, USA) as standard.

Materials and methods Strains and culture conditions. Phanerochaete chrysosporium ME 446 (ATCC 34541), B. adusta (IFO 4983), C. consors (IFO 6512), Grifola froudosa (IFO 7040), Pholiota nameko (IFO 7041), Irpex lacteus (IFO 5367), Lenzites betulina (IFO 4963), Flammulina velutipes (IFO 30244) and Pleurotus ostreatus (IFO 6515) were used in this study. The fungi were cultured in a glucose-peptone medium containing (per litre: 20 g glucose, 5g polypeptone, 2 g yeast extract, 1 g KH2PO4, 0.5g MgSO4.7H20; pH 5.5) or Kirk's media (Kirk et al. 1978) supplemented with 1% glucose as a carbon source and 1.2 mM (low-nitrogen medium) or 30 mM (high-nitrogen medium) ammonium tartrate as a nitrogen source. Polypeptone and yeast extract were the products of Nihon Pharmaceutical Co. (Osaka, Japan) containing 12.5%-14.5% and 6%-9% nitrogen, respectively, on a dry matter basis. Stationary cultures were carried out at 37°C in 200-ml erlenmeyer flasks containing 15 ml medium.

Gel electrophoresis. Molecular sizes and purity of the purified LPO were examined by SDS-polyacrylamide gel electrophoresis (PAGE) employing a 10% gel containing 0.1% SDS. For molecular weight determinations, gels were calibrated with a mixture of proteins of known molecular weight using Pharmacia (Uppsala, Sweden) low- and high-molecular-weight calibration kits as standard proteins. Purity and pIs of purified LPOs was assayed by isoelectric focusing (IEF) on an LKB (Uppsala, Sweden) Amphorine PAGplate (pH 4.0-5.0). For determination of pIs, ge]ls were calibrated with a low pI calibration kit (Pharmacia). Protein bands on gels were stained with Coomassie brilliant blue R-250.

Preparation of genomic DNA and enzymatie digestion. Fungal DNA was extracted from fresh mycelia harvested from a 6-day culture grown in a glucose-peptone medium as described previously (Asada et al. 1988). Digestion of DNA with HindIII or PstI (Takara Shuzo, Kyoto, Japan) was carried out according to the manufacturer's manual.

Spectroscopy. Ultraviolet/visible (UV/VIS) absorption spectra of LPO were recorded on a Hitachi (Tokyo, Japan) 340 spectrophotometer. Spectra were obtained at room temperature in 1-cm cuvettes containing enzyme (0.13 mg/ml protein) dissolved in 20 m M succinate buffer (pH 4.5). Other conditions for the modification of LPO were described by Gold et al. (1984).

Preparation of a DNA probe. The 1.8 kb NaeI fragment of the LPO gene (H8) prepared as described previously (Asada et al. 1988) was double-digested with XhoI and SmaI (Takara Shuzo) and the digests were electrophoresed in low-melting-temperature agarose (Takara Shuzo) gels. Based on the sequence of the gene the fragment obtained was expected to be 0.59 kb in size as shown in Fig. 1. The DNA corresponding to the 0.59 kb fragment was extracted from the gel with 20 m M TRIS (pH 8.0) containing 10 m M ethylene diamine tetraacetate (EDTA) and purified by precipitation in 70% ethanol. The fragment was labelled with [c~-32p]dCTP (specific activity, 111 TBq/ 0 I

0.5 I

1.0 ~

Haell

Exon Intron

No. No.

1

Xho I

2 1

3 2

4 3

Degradation of lignin model compounds. A r-l-type model compound, 1-(3',4'-dimethoxyphenyl)-1,3-dihydroxy-2-(4"methoxyphenyl) propane, and a fl-O-4-type model compound, 4-ethoxy-3-methoxyphenylglycerol-fl-guaiacylether, were used

5 4

6 5

Kpn

1.5 kb ~ I I

Sma

7 6

I

8 7

9 8

Fig. l. Restriction map of the lignin peroxidase (LPO) gene (Hs) of Phanerochaete chrysosporium: m , intron; r~, exon; ~ - - ~ , restriction fragment used for preparation of the probe

438

Y. Kimura et al. : Screening for lignin peroxidase genes taining 0.5 m M model, 0.5 m M HzO2 and 50 ~g LPO-2 for 6 h (/3-1 model) or 15 h (/3-0-4 model) at 30°C. Gas chromatographic mass/spectrophotometric (GC/MS) analyses of substrate and products, acetylated with anhydrous acetic acid-pyridine were carried out using a Hitachi M-80B G C / M S equipped with a CBP1 capillary column (0.25 mm internal diameter x 25 m long, Shimazu, Kyoto, Japan).

Results Hybridization of restriction digests of funoal DNA with the lignin peroxidase DNA probe The HindlII or PstI fragments of chromosomal DNA of the fungi listed in Materials and methods were electrophoresed and the gels were hybridized with the XhoI-SmaI fragment of the LPO gene of Phanaerochaete chrysosporium. As shown in Fig. 2, the HindlII fragment of B. adusta (lane 1) and the PstI fragment of C. consors (lane 3) revealed complex hybridization patterns, as observed in P. chrysosporium (lanes 2 and 4). The pattems indicated the presence of multiple LPO genes or LPO-related genes in the chromosomal DNAs of these fungi. Restriction fragments of other fungi gave only faint hybridization signals,

Fig. 2. Southern hybridization of restriction fragments of fun. gal DNAs to the LPO D N A probe: DNAs from P. chrysosporium (lanes 2 and 4), Bjerkandera adusta (lane 1) and Coriolus consors (lane 3) were cut with HindlII (lanes I and 2) or PstI (lanes 3 and 4). The experimental conditions are described in the text in this study. These compounds were kindly provided by Professor A. Enoki, Kinki University, Osaka, Japan. The reactions were carried out in 0.3 ml of 10 m M Na-acetate (pH 3.8) con300

Lignin peroxidase

Veratryl alcohol oxidase

20(;

7

E 10(3

0

2

4

Incubation

6 time

ol

8

2

4

6

8

(days)

Fig. 3. Effect of culture media on production of LPO and veratryl alcohol oxidase: A, glucose-peptone; O, Kirk's high nitrogen; (D, Kirk's low nitrogen. Data points are means of four or five replicate cultures

439

Y. K i m u r a et al.: S c r e e n i n g for lignin p e r o x i d a s e g e n e s

insufficient to suggest the presence of LPO-related genes in these fungi.

Production of lignin peroxidase by B. adusta As the hybridization experiment suggested the possibility of production of LPO by B. adusta and C. consors, extracellular production of this enzyme by B. adusta was examined. Figure 3 shows the changes in the levels of LPO and veratryl alcohol oxidase (VAO) under various culture conditions. Maximum activity of LPO was reached after 6 days in a glucose-peptone medium. However, the activity was much lower in Kirk's lownitrogen medium, in which P. chrysosporium produced a high level of LPO. The activity of VAO was found to be much lower than that of LPO under the conditions tested. The stimulative effect of veratryl alcohol on the production of LPO, established in the culture of P. chrysosporium, was not observed except for a slight stimulation in Kirk's lOW- and high-nitrogen media (data not shown). Mn(II)-dependent peroxidase activity was not detected in any of these cultures.

2 2o ,-~m ~ .-_ r~ ~ .~ -; zo ~ ~10 ~ I~ 2 k~ *~ Ix .-~ = I~ ~

~ 20

Fraction

Step 1. Culture filtrates (total 260 ml) of B. adusta grown in glucose-peptone medium for 6 days were concentrated 30-fold by ultrafiltration using Amicon PM-10 membranes. The concentrate was dialysed against 20 m M succinate buffer (pH 4.5). The dialysate contained 20.6 mg protein with a specific activity of 7300 and total units of 150700. Step 2. The dialysed enzyme solution (9.5 ml) was applied to a DEAE-Sepharose CL-6B (Sigma) column (20 mm internal diameter x 45 mm long) previously equilibrated with the same buffer. After the column was washed with the buffer, elution was carried out with a stepwise gradient of NaCl and 3-ml fractions were collected. The elution of protein was monitored by the absorbances at 280 nm and 407 nm based on the spectrum of a haem protein, and LPO and VAO activity was measured. Figure 4 illustrates elution profiles of proteins and LPO activity. The highest activity was eluted in Peak 2 which represented 58% of the total activity recovered by the chromatography. The rest of the activity was distributed in Peaks 1 (13%), 3 (10%) and 4 (19%). Although 56% of VOA activity (390 units) appeared in peak 2, the activity was

4

3

0 I----- 0 t--a----~

Purification of lignin peroxidase of B. adusta

III

II

40

60

80

100

number

Fig. 4. D E A E - S e p h a r o s e c o l u m n c h r o m a t o g r a p h y o f t h e culture filtrate o f B. adusta g r o w n in a g l u c o s e - p e p t o n e m e d i u m for 6 days: /~, lignin p e r o x i d a s e activity; © , a b s o r b a n c e at

280 nm; O, absorbance at 407 nm. Elution buffer: /, 20 mM succinate buffer (pH 4.5); 11, +0.1M NaC1; III, +0.5 M NaC1 negligibly low compared to that of LPO. Active fractions of Peak 2 protein (LPO-2); fractions 4144, containing 93% of the total activity eluted in Peak 2) were pooled and concentrated sixfold through a Centricon-10 microconcentrator (Amicon, Denver, USA) followed by dialysis against 20 m M succinate buffer (pH 4.5). The dialysate contained 4.0mg protein with total units of 62 000. Step 3. The dialysate was subjected to ion exchange H P L C with a TSK-GEL DEAE-5PW column (7.5 mm internal diameter×75 mm long; Tosoh, Tokyo, Japan). The elution was carried out at a flow rate of 0:8 ml/min using 20 m M succinate buffer (pH 4.5) by a linear gradient of NaC1 of 0-1 M. The runs were repeated to fractionate the whole volume of the dialysate. After excluding a small peak of VAO, which appeared prior to LPO, active fractions of the eluate were

440

Y. Kimura et al.: Screening for lignin peroxidase genes

Fig. 5. Polyacrylamide gel electrophoresis of purified lignin peroxidase (LPO-2) of B. adusta. The purified enzyme (10 txg) was applied to a 7.5% gel column and run at pH 8.0. The direction of electrophoresis was from the cathode (top) to the anode. The protein was stained with Coomassie brilliant blue R250

pooled, concentrated 25-fold with a Centricon-10 concentrator and dialysed against 20 m M succinate buffer (pH 4.5). The dialysate of LPO-2 contained 1.7 mg protein with total units of 31 000. Step 4. The dialysate was subjected to gel filtration HPLC with a TSK-GEL G3000SW column (7.5 mm internal diameter × 300 mm long; Tosoh). The elution was carried out at a flow rate of 0.2 m l / m i n using 20 m M succinate buffer (pH 4.5) as a mobile phase. The runs were repeated to fractionate the whole volume o f the dialysate. Active fractions of the eluate were pooled and dialysed against 20 m M succinate buffer (pH 4.5). The LPO-2 preparation finally obtained contained 0.87 mg protein with total units of 26000 and a specific activity of 29900. The preparation was found to be purified to give a single band in PAGE (Fig. 5).

Characterization of lionin peroxidase of B. adusta The SDS-PAGE of LPO-2 against standard proteins of known molecular weight demonstrated a molecular weight of 41000; IEF gave a pI of 4.2. The UV/VIS spectra indicated that the enzyme was a haem protein since native LPO-2 showed strong absorption at 407 nm and weak peaks at 508 and 638 nm, the peaks of the reduced-CO form of the enzyme were at 424, 542 and 571 nm, and treatment of the enzyme with K C N or NaN3 shifted the maxima in the spectrum to 423 or 411 nm, respectively. These spectral properties are compatible with those of LPOs of P. chrysosporium (Gold et al. 1984). The characteristics of LPO-2 were analysed using veratryl alcohol as a substrate. The maximum activity of LPO-2 was observed at pH 3.0

and at 30 ° C, while the K,~ value for veratryl alcohol was 230 p~M. Activity of LPO-2 was completely inhibited by 1 m M NAN3. Addition of 1 m M EDTA and KCN resulted in an approximately 10% decrease in activity, whereas dithiothreitol gave a less inhibitory effect. Divalent cations did not affect the enzyme activity at a concentration of 1 m M except that MnC12 and CuSO4 revealed 13% and 5% inhibition, respectively. The G C / M S analyses demonstrated that LPO2 cleaved lignin model dimers mainly at the CotC/3 bond of the side chain. The diaryl propane (ill) model gave veratraldehyde (3,4-dimethoxybenzaldehyde), 1,2-dihydroxy- 1-(4'-methoxyphenyl) ethane and its ketone. The/3-0-4 model yielded 4-ethoxy-3-methoxybenzaldehyde and 4-ethoxy3-methoxyphenyl glycerol, suggesting that the model was also cleaved at the ether bond. These cleavage patterns are similar to those shown in P. chrysosporium (Gold et al. 1984).

Discussion

Since LPO was isolated from a culture of P. chrysosporium (Tien and Kirk 1984; Gold et al. 1984), studies on lignin degrading enzymes have been extensively focused on this enzyme. However, the presence of this enzyme has been demonstrated only in P. chrysosporium, C. versicolor (Dodson et al. 1987) and Phlebia radiata (Niku-Paavola et al. 1988). Operation of an unclarified nitrogen-based regulatory system in LPO synthesis (Faison and Kirk 1985) seems to depress expression of the LPO gene, resulting in difficulty in detecting this enzyme activity in fungi. Sequence homology has been demonstrated with respect to various fungal genes so far; Asper9illus niger trpC to A. nidulans trpC and Neurospora crassa trp-1 ( K o s et al. 1985), A. nidulans aroB to mammalian and bacterial aroB (Upshall et al. 1986), .4. niger oliC to that of N. crassa (Ward et al. 1988) and others. However, these experiments were carried out mainly to develop a transformation system in fungal cells. Our Southern hybridization experiments suggested the presence of D N A sequences homologous to the LPO gene of Phanaerochaete chrysosporium in several fungi including B. adusta and C. consors (Fig. 2). The actual extent of the homology between the LPO genes of P. chrysosporiurn and those of other fungi can only be determined by D N A sequence comparisons. We think this hybridization method is a useful tool for screening LPO-producing fungi, because the gene is detected directly even

Y. Kimura et al.: Screening for lignin peroxidase genes

when expression of the gene is repressed by the regulatory system. Further screening using this technique is currently being carried out. Our experiments demonstrated that B. adusta produced LPO in a high-nitrogen medium enriched with peptone as a nitrogen source (Fig. 3), whereas other fungi produce this enzyme only in nitrogen-limited cultures (Tien and Kirk 1984; Gold et al. 1984; Faison and Kirk 1985). Others (Waldner et al. 1988) have found that B. adusta grown under low-nitrogen conditions produced aryl-alcohol oxidase, whereas LPO activity was not detected in the enzyme preparation. It can be presumed that some components of peptone or yeast extract added to promote growth of the fungus stimulated more extensive production of LPO than of VAO. Another possibility is that this fungus is not susceptible to nitrogen-mediated regulation of LPO synthesis, as shown in one of the strains of P. chrysosporium (Buswell et al. 1984) or mutants of the fungus (Kuwahara et al. 1987). Production of LPO in nitrogen-rich media and higher productivity of the enzyme than P. chrysosporium were intriguing characteristics of this fungus for the possibility of large-scale production of LPO. Lignin peroxidase was purified from a culture of B. adusta. The LPO properties of this fungus were demonstrated to have close similarity with those of P. chrysosporium (Kirk et al. 1986; Leisola et al. 1987) and Phlebia radiata enzymes (Niku-Paavola et al. 1988). Multiple enzymes were separated by ion exchange chromatography (Fig. 4). Although they differed in pI (data not shown), a high degree of homology seems to exist among these LPOs with minor differences, as shown for Phanerochaete chrysosporium enzymes (Leisola et al. 1987). Preliminary amino acid analysis revealed that the amino acid sequence of the N-terminal region of LPO-2 differed from those of H2 and H8 of P. chrysosporium (data not shown). Characterization of LPOs other than LPO-2 is currently being carried out. Acknowledgements. This research was supported in part by the Grant-in-Aid of Scientific Research "Research on Energy" of the Ministry of Education, Science and Culture, Japan. We are grateful to Dr. Shingo Kawai, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan, for conducting the GC/MS analysis.

References Asada Y, Kimura Y, Kuwahara M, Tsukamoto A, Koide K, Oka A, Takanami M (1988) Cloning and sequencing of a ligninase gene from a lignin-degradingbasidiomycete, Pha-

441

nerochaete chrysosporium. Appl Microbiol Biotechnol 29: 469-473 Boer HA de, Zhang YZ, Collins C, Reddy CA (1987) Analysis of nucleotide sequences of two ligninase cDNAs from a white-rot filamentous fungus, Phanerochaete chrysosporium. Gene 60:93-102 Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254 Buswell JA, Mollet B, Odier E (1984) Ligninolytic enzyme production by Phanerochaete chrysosporium under conditions of nitrogen sufficiency. FEMS Microbiol Lett 25: 295-299 Dodson PJ, Evans CS, Harvey PJ, Palmer JM (1987) Production and properties of an extracellular peroxidase from Coriolus versicolor which catalyses Ca-Cfl cleavage in a lignin model compound. FEMS Microbiol Lett 42:17-22 Faison BD, Kirk TK (1985) Factors involved in the regulation of a ligninase activity in Phanerochaete chrysosporium. Appl Environ Microbiol 49:299-304 Gold MH, Kuwahara M, Chiu AA, Glenn JK (1984) Purification and characterization of an extracellular H202-requiring diarylpropane oxygenase from the white rot basidiomycete, Phanerochaete chrysosporium. Arch Biochem Biophys 234:353-362 Gold MH, Wariishi H, Valli K (1989) Extracellular peroxidases involved in lign~n degradation by the white rot basidiomycete Phanerochaete chrysosporium. In: Whitaker JR, Sonnet PE (eds) Biocatalysis in agricultural biotechnology, ACS Symposium Series nb. 389. American Chemical Society, Washington DC, USA, pp 127-140 Jeffries TW, Choi S, Kirk TK (1981) Nutritional regulation of lignin.degradation by Phanerochaete chrysosporium. Appl Environ Microbiol 42:290-296 Kirk TK, Schultz E, Connors WJ, Lorenz LF, Zeikus JG (1978) Influence of culture parameters on lignin metabolism by Phanerochaete chrysosporium. Arch Microbiol 117:277-285 Kirk K, Croan S, Tien M~ Murtagh KE, Farrell RL (1986) Production of multiple ligninases by Phanerochaete chrysosporium : effect of selecte,d growth conditions and use of a mutant strain. Enzyme Microb Technol 8:27-32 Kirk TK, Kersten PJ, Mozuch MD, Kalyanaraman B (1986) Ligninase of Phanerochaete chrysosporium. Mechanism of its degradation of the non-phenolic arylglycerol fl-aryl ether substructure of lignin. Biochem J 236:279-287 Kirk TK, Farrell RL (1987) Enzymatic "combustion": the microbial degradation of lignin. Ann Rev Microbiol 4t :465505 Kos A, Kuijvenhoven J, Wernars K, Bos CJ, Broek HWJ van den, Pouwels PH, Hondel CAMJJ van den (1985) Isolation and characterization of the Aspergillus niger trpC gene. Gene 39:231-238 Kuwahara M, Glenn JK, Morgan MA, Gold MH (1984) Separation and characterization of two extracellular H202-dependent oxidases from ligninolytic cultures of Phanerochaete chrysosporium. FEBS Lett 169:247-250 Kuwahara M, Asada Y, Kimura Y, Aokage M (1987) Isolation of mutants of a lignin-degrading basidiomycete, Phanerochaete chrysosporium, that produce lignin peroxidase in high nitrogen cultures. Mokuzai Gakkaishi 33:821-823 Leisola MSA, Kozulic B, Meussdoerffer F, Fiechter A (1987) Homology among multiple extracellular peroxidases from Phanerochaete chrysosporium. J Biol Chem 262:419-424 Niku-Paavola M-L, Kar]hunen E, Salola P, Raunio V (1988) Ligninolytic enzymes of the white-rot fungus Phlebia radiata. Biochem J 254:877-884

442 Paszczynski A, Huynh V-B, Crawford R (1986) Comparison of ligninase-I and peroxidase-M2 from the white-rot fungus Phanerochaete chrysosporium. Arch Biochem Biophys 244:750-765 Smith TL, Schalch H, Gaskell J, Covert S, Cullen D (1988) Nucleotide sequence of a ligninase gene from Phanerochaete chrysosporium. Nucleic Acids Res 16:1219 Tien M, Kirk TK (1984) Lignin-degrading enzyme from Phanerochaete chrysosporium: purification, characterization, and catalytic properties of a unique H202-requiring oxygenase. Proc Natl Acad Sci USA 81:2280-2284 Tien M, Tu C-PD (1987) Cloning and sequencing of a cDNA for a ligninase from Phanerochaete chrysosporium. Nature 326:520-523 Upshall A, Gilbert T, Saari G, O'Hara PJ, Weglenski P, Berse B, Miller K, Timberlake WE (1986) Molecular analysis of

Y~ Kimura et al.: Screening for lignin peroxidase genes the aroB gene of Asperoillus nidulans. Mol Gen Genet 204:349-354 Waldner R, Leisola MSA, Fiechter A (1988) Comparison of ligninolytic activities of selected white-rot fungi. Appl Microbiol Biotechnol 29:400-407 Walther I, Kalin M, Reiser J, Suter F, Fritsche B, Saloheimo M, Leisola M, Teeri T, Knowles JKC, Fiechter A (1988) Molecular analysis of a Phanerochaete chrysosporium lignin peroxidase gene. Gene 70:127-137 Ward M, Wilson LJ, Carmona CL, Turner G (1988) The oliC3 gene of Asperyillus niger: isolation, sequence and use as a selectable marker for transformation. Curt Genet 14:3742 Received 5 July 1989/Accepted 5 September 1989