Eur. J. Biochem. 237, 424-432 (1996) 0 FEBS 1996

Purification and catalytic properties of two manganese peroxidase isoenzymes from Pleurotus eryngii Maria J. MARTINEZ, Francisco J. RUIZ-DUENAS, Francisco CUILLEN and Angel T. MARTINEZ Centro de Investigaciones Bioi6gicas. Consejo Superior de Investigaciones Cientificas, Madrid, Spain

(Received 26 Octoberi28 December 1995) - EJB 95 1753/4

The ligninolytic basidiomycetes Pleurotus eryngii, Pleurotus ostreatus, Pleurotus pulmonarius and Pleurotus sajor-caju did not exhibit detectable levels of manganese peroxidase (MP) when grown in liquid media with ammonium tartrate as N source. However, after examination of cells grown on different organic N-based media, high M P activity was obtained in peptone medium, up to nearly 3 U/ml in cultures of I? eryngii. Moreover, Mnz+ supplementation was not used to produce MP, since all Mn2+ concentrations assayed (1 -4000 pM) inhibited production of this enzyme in liquid medium.

Two MP isoenzymes were purified to homogeneity from shaken or stationary cultures of I? eryngii grown in peptone medium. The purification process (which included chromatography on Biorad Q-car- tridge, Sephacryl S-200 and Mono-Q) attained 56% activity yield with a purification factor of 25. The isoenzymes differed in PI (3.75 and 3.65), N-terminal sequence and some catalytic properties. They were in some aspects (e.g., molecular mass of 43 kDa) similar to Phanerochaete chrysosporium MP but exhib- ited some distinct characteristics, including Mn”-independent peroxidase activities against 2,6-dime- thoxyphenol and veratryl alcohol, and higher resistance to H,O,. Recent studies have shown that M P are ubiquitous enzymes in ligninolytic fungi, but the results obtained suggest that differences in catalytic properties probably exist between different Mn2 ’ -oxidizing peroxidases produced by these fungi.

Keywords: manganese peroxidase isoenzyme ; Pleurotus eryngii ; manganese-independent activity ; 2,6- dimethoxyphenol ; veratryl alcohol.

Fungi of the genus Pleurotus cause white-rot of lignocellu- losic materials. Some species, including Pleurotus eryngii, have the capacity to remove lignin preferentially (i.e., with limited degradation of cellulose) [ 11, a characteristic relevant to biotech- nological delignification processes for feed production 121 and paper-pulp manufacture [3].

The production of extracellular laccase and aryl-alcohol oxi- dase (AAO) by I? eryngii in liquid medium containing ammo- nium tartrate [4, 51, the usual source of N in lignin biodegrada- tion studies, has been reported [6]. The involvement of R eryngii laccase in lignin degradation ha5 been investigated by means of homoveratric acid, and the results obtained suggested that this non-phenolic substrate is degraded by laccase in the presence of mediators synthesized by the fungus [7]. AAO has been charac- terized [5] as the central enzyme of an H,O,-producing system, based on the redox cycling of anisylic compounds [S, 91. The above system was described in Pleurotus species [lo] but it could operate in other ligninolytic fungi, such as Phanerochaete chrysosporium, in which AAO has been described recently [ l l ] . However, appreciable levels of peroxidases involved in lignin biodegradation, i.e. lignin peroxidase (LP) and manganese per- oxidase (MP) [12], were not detected in cultures of I? eryngii in the above medium [4, 51. Recently, the production of high MP

Correspondence tu A. T. Martinez, Centro de Investigaciones Bio-

Fax: +341 5627528. Abbreviations. AAO, aryl-alcohol oxidase; ABTS, 2,2’-azinobis-(3-

ethylbenzothiazoline-6-sulfonate); LP, lignin peroxidase; MIP, manga- nese-independent peroxidase; MP, manganese peroxidase; PI, isoelectric point.

Enzymes. Aryl-alcohol oxidase (EC 1.1.3.7); laccase (EC 1.10.3.2); diarylpropane peroxidase (EC 1.1 1 . I .14); manganese peroxidase (EC

Ibgicas, CSIC, Velizquez 144, E-28006 Madrid, Spain

1.11.1.13).

activity by this fungus in culture media with peptone as N source was reported [I 31.

In I? ~ ~ ~ y s o s p o r ~ u r n [6] , MP has been considered to play a secondary role in ligniii degradation, compared with LP, due to its incapacity to oxidize non-phenolic lignin substrates. The increasing interest in this enzyme in recent years is due to a number of reasons: its production by most white-rot fungi [14, 151 including lignin-degrading species that lack L P [ 16, 171 ; its capacity to depolymerize synthetic lignin in vitro [18] and to oxidize non-phenolic compounds via peroxidation of lipids [19] ; and its production during fungal degradation of lignocellulose [20, 211, with chelated Mn3+ being a potential lignin-oxidizing mediator for extensive delignification of wood and other ligno- cellulosic materials [22, 231. Recently, it has been reported that an LP isoenzyme from I? chrysosporiurn can oxidize Mnz+, which acts as a better substrate than veratryl alcohol at physio- logical pH values, and it was suggested that generation of che- lated Mn’+ could be the main role of the enzyme in lignin degra- dation [24].

In the present study, growth conditions for the production of Mn2+-oxidizing peroxidases by Pleurotus species are reported. Two isoenzymes were purified and characterized from P. eryn- gii, and their catalytic properties are discussed. Although these enzymes are not strictly dependent on MnZ+ to oxidize phenolic substrates, as reported for MP from P. chrysosporiurn [6], they are considered as manganese peroxidases since Mn2+ is their best substrate.

MATERIALS AND METHODS

Fungal cultures. MP production by I? eryngii CBS 613.91 (= IJFM A1 69), Pleurotus ostreatus CBS 41 1.71, Pleurotus pul-

Martinez et al. (Eur: J . Biochem. 237) 425

monarius CBS 507.85 and Pleurotus sajor-caju MUCL 29757 was investigated in the glucose/ammonium-tartrate and glucose/ peptone/yeast-extract media described below.

To examine the production of MP in different organic N- based media, F! eryngii was grown in six culture media with the following composition (mass/vol.): 2% glucose, 0.5 % peptone (Bacto-Peptone, Difco), 0.2 % yeast extract (Difco) ; 2 % glu- cose, 0.5 % corn-steep liquor (Cerestar), 0.2% yeast extract; 2% glucose, 0.7% yeast extract; 2.7% malt extract; 2% glucose, 0.7 % peptone ; and 2 % glucose, 0.7 % ammonium tartrate. The pH was adjusted to 5.5 after addition of salts (0.1% KH,PO, and 0.05% MgSO, . 7 H,O). The metal content of the different N sources was estimated by atomic-absorption spectroscopy.

The effect of different concentrations of peptone or Mnz+ (as MnSO,) on enzyme production was studied in the glucose/ peptone/yeast-extract medium.

Homogenized pellets (1 g/l) from 6-d-old shaken cultures were used to inoculate 1-1 flasks that contained 200 ml medium (incubated at 180 rpm) or 100 ml medium (incubated under sta- tionary conditions), which were maintained at 28 "C.

Enzymatic activities. MP activity was estimated directly by the formation of Mn3+ . tartrate complex (cZi8, 6500 M-' . cm-I) during oxidation of 0.1 mM MnSO, (in 0.1 M sodium tartrate, pH 5, 0.1 mM H,O,), or indirectly by the Mn"-dependent oxi- dation of 0.1 mM 2,6-dimethoxyphenol (Aldrich) to coerulig- none (3,3',5,5'-tetramethoxy-p,pf-diphenoquinone) under the same conditions. We assumed that two molecules of 2,6-dime- thoxyphenol were oxidized to produce one molecule of the di- meric product [25]. The E,,, of coerulignone was estimated by complete oxidation of different concentrations of 2,6-dime- thoxyphenol with laccase or MP, and a value of 55000 M-' . cm-' was obtained for concentrations in the range of 0-5 pM coerulignone (E,,,, 27500 M-' . cm-I, when referred to 2,6- dimethoxyphenol concentration).

Mn2+-independent peroxidase activities against 0.1 mM 2,6- dimethoxyphenol and 2 mM veratryl alcohol (Aldrich) (the latter measured by formation of the veratraldehyde; E ~ ~ ~ ~ , 9300 M-' . cm-') were also estimated in 0.1 mM H,02, 0.1 M sodium tartrate, pH 5 and pH 3, respectively. The above veratryl alcohol concentration and reaction pH were those used for LP activity estimation [26]. The effect of 1 mM EDTA on Mn2+-indepen- dent peroxidase activities was investigated. AAO activity was determined by the formation of veratraldehyde from 5 mM vera- try1 alcohol in 0.1 M sodium phosphate, pH 6 [5]. Laccase activ- ity was measured with 5 mM 2,2'-azinobis-(3-ethylbenzothiazo- line-6-sulfonate) (ABTS) in 0.1 M sodium acetate, pH 5 ( E , ? ~ of the ABTS cation radical, 29300 M-' . cm-I).

1 U of enzymatic activity was defined as the amount of en- zyme that transforms 1 pmol substrate/min.

MP purification. Purification was carried out from shaken and stationary cultures of P. eryngii grown in medium containing (masshol.) 2% glucose, 0.5% peptone, 0.2% yeast extract and salts (under the conditions described above). After incubation for 16 d (stationary cultures) or 6 d (shaken cultures), the liquids were concentrated 15-fold and dialyzed against 10 mM sodium tartrate, pH 4.5 (Filtron, 5-kDa-cut-off membrane). The concen- trates (approximately 75 nil) were loaded onto a Biorad Q-car- tridge in 10 mM sodium tartrate, pH 4.5 (1 ml/min) and retained proteins were eluted with a NaCl gradient from 0 to 1 M. Frac- tions with MP activity were pooled, concentrated and samples (approximately 1 ml) applied to a Sephacryl S-200 HR (Phar- macia K16/100) column in 10 mM sodium tartrate, pH 4.5 (0.8 mlhnin). Fractions that contained MP activity were pooled, concentrated (Filtron Microsep, 3-kDa cut off), dialyzed against 10 mM sodium tartrate, pH 5, and samples (approximately 1 ml) applied to a Mono-Q anion-exchange column (Pharmacia HR

56). The MP isoenzymes were eluted with a linear gradient from 0 to 0.25 M NaCl in 10 mM sodium tartrate, pH 4.5, over 20 min at 0.8 ml/min. The fractions that contained MP isoen- zymes were collected, concentrated, dialyzed and stored at -20°C.

Enzyme characterization. Protein concentration was deter- mined by means of Bradford reagent (Biorad) with BSA as stan- dard. The carbohydrate content of purified enzymes was deter- mined by means of anthrone reagent [27] with glucose as stan- dard. pH optima for activity were estimated in 0.1 M sodium tartrate.

SDSIPAGE was performed in 12% polyacrylamide gels, using low-molecular-mass standards (Biorad). IEF was per- formed in 5% polyacrylamide gels with a thickness of 1 mm and a pH range of 2.5-5.5 (prepared with Biorad Ampholine, mixing 85% from pH 2.5-5 and 15% from pH 3.5-10) with 1 M H,PO, and 1 M NaOH in anode and cathode, respectively. The pH gradient was measured on the gel by means of a contact electrode. Protein bands after SDS/PAGE were stained with AgNO, (Silver Stain Plus, Biorad), and after IEF with Coomas- sie Blue R-250.

The visible spectra of native isoenzymes were recorded in 10 mM sodium tartrate, pH 5. The heme type and molar content were deduced from the spectrum of the reduced form of the alkaline pyridine hemochrome, prepared by addition of a few crystals of Na,S,O, to 0.6 ml of a solution containing 50 pg en- zyme in 2.1 M pyridine, 75 mM NaOH. The csS7 used for the heme group was 32550 M-' . cm-' [28].

The amino acid composition was determined with a Bio- chrom 20 autoanalyzer (Pharmacia) after hydrolysis of 7 pg pro- tein with 6 M HCI at 110°C for 24 h. N-terminal sequences were obtained by means of automated Edman degradation of 20 pg protein in an Applied Biosystems 477A pulsed-liquid protein sequencer with 120A on-line phenylthiohydantoin analysis.

Kinetic studies. The kinetic constants of Mn'+-dependent peroxidase activities of MP isoenzymes were calculated for: H,O, (estimated by the formation of Mn'+ . tartrate or by the Mn"-dependent oxidation of 2,6-dimethoxyphenol); Mn2+ (alone, estimated as Mn3+ . tartrate, or in the presence of 0.1 mM 2,6-dimethoxyphenol, estimated by coerulignone formation); 2,6-dimethoxyphenol (estimated by coerulignone formation); and vanillylideneacetone [4-(4-hydroxy-3-methoxyphenyl)-3- buten-2-one, Aldrich ; estimated by the decrease of substrate concentration; E,,,, 18300 M-' . cm-'1. Mn''-independent per- oxidase activities were also detected, and the corresponding ki- netic constants were calculated for 2,6-dimethoxyphenol and veratryl alcohol. All reactions were performed under the condi- tions described above.

Both isoenzymes exhibited oxidase activity against NADH (or NADPH), and kinetic constants were obtained for NADH (c,,,,, 6220 M-' . cm- ') in 0.1 M sodium tartrate, pH 5. Superox- ide radical (O;-) generation during MP oxidation of 0.3 mM NADH was evidenced by reduction of 0.4 mM Nitro Blue Tetra- zolium (490 nm) or 30 pM cytochrome-c (550 nm). Mn"-de- pendent oxidation of 2,6-dimethoxyphenol (0.1 mM) was fol- lowed at 469 nm, during NADH (0.3 mM) oxidation (estimated at 340 nm) by MP isoenzymes.

RESULTS

Influence of N sources and effect of Mn2+ on enzyme produc- tion. MP production was not detected in cultures of l? eryngii, F! ostreatus, F! pulrnonarius and F! sajor-cuju grown in glucose/ ammonium-tartrate medium. However, the production of this en- zyme by the four Pleurotus species was found in peptone media,

426 Martinez et al. (Eus J. Biochern. 237)

1000

800

600 c. 2 ' 400

200

0 0 2 4 6 8 10 12 14 16

Incubation time (days)

Fig. 1. Effect of organic N-based media on the production of MP by I? eryngii. Glucose-ammonium, (-D-); glucoselcom-steep liquorlyeast extract, (-V-); malt extract, (-0-); glucose/yeast extract, c-A-1; glu- cose/peptone, (-El--) and glucose/peptone/yeast extract, (-0-1. The corn- position of the culture media is indicated in Materials and Methods. MP activity was estimated by the formation of Mn3+ . tartrate in 0.1 mM MnSO,, 0.1 mM H,O,, 0.1 M sodium tartrate, pH 5. The inset shows maximal MP activity as a function of peptone concen~ation (masslvol.) in glucose/pept~nelyeast-extract medium.

\- + A L 1.0

Laccase

0 - , D l 10 100 1OW 4000

Mn2+ concentration QM)

Fig. 2. Effect of MnZ+ c o n c e n t ~ ~ o n on the p r ~ u ~ t i o n of MP, AAO and laccase by P. eryngii. The fungus was grown in glucoselyeast-ex- ~ a c ~ ~ p t o n e medium with different Mn'+ ~oncen~a t ions and maximal activities are presented. A4P activity was estimated by the formation of Mn3+ 1 tartrate in 0.1 mM MnSO,, 0.1 mM H,O,, 0.1 M sodium tartrate, pH 5. AAO activity was estimated by means of 5 mM veratryl alcohol, 0.1 M sodium phosphate, pH6. Laccase activity was measured with 5 mM ARTS, 0.1 M sodium acetate, pH 5.

the highest levels being found in f? eryngii cultures. MP produc- tion was not detected in media with other organic sources of N (e.g., malt extract and corn-steep liquor; Fig. 1). Low enzyme levels were obtained with yeast extract, but the addition of this compound (which is often used as vitamin source) to glucose/ peptone medium improved the production of MP. Moreover, MP levels increased with increasing concentrations of peptone (Fig. I > .

The influence of Mn2' concentration on the production of extracellular MP, laccase, and AAO activities by shaken cultures of P. eryngii 111 glucoselpeptone/yeast-extract medium is shown in Fig. 2. The highest levels of MP activity were obtained in medium without Mn'+ ; 5 pM Mn2+ produced a strong decrease (approximately 90%) of MP activity and no activity was found at 25 p M Mn" (the results were the same when the samples were dialyzed before activity assessment). Mn2+ positively af-

0 25 50 75 100 125 Elution volume (ml)

0.301 B

0.25

0 20 40 60 80 100 120 140 Elution volume (mi)

Fig. 3. Purification of MP from I? eryngii. (A), Q-cartridge chromatog- raphy. (B), Sephacryl S-200 chromatography. Profiles corresponding to MP (-A-), laccase (-m-) and AAO (-U-) activities, absorbances at 280 nm (-) and 410 nm (. . . .), and the NaCl gradient (---) are shown. After 6-d incubation in glucose/peptone/yeast-extract medium (180 rpm, 28"C), the liquid was concentrated 15-fold, dialyzed against 10 mM sodium tartrate, pH 4.5, and applied to a Riorad Q-cartridge in 10 mM sodium tartrate (1 mllmin). The non-retained fractions that con- tained MP activity were concentrated and c ~ o m a t o ~ a p h e d on Seph- acryl S-200 HR in the same buffer. The enzymatic activities were mea- sured as described in the legend of Fig. 2.

fected levels of AAO activity, although the effect was restricted to very high concentrations and did not affect laccase. Most of the Mn concentrations used had little effect on fungal growth, but a decrease of growth was observed at very high Mn concen- trations (in the millimolar range). No Mn (less than 0.5 kg/g) was detected in any of the N sources assayed (peptone was found to contain 25 yglg Fe, 16 yglg Zn, 21 mglg Na, 5 mglg Ca, 3 mg/g K and 1 mglg P).

Purification of MP isoenzymes. The characteristics and yield of the two isoenzymes were similar when purified froin shaken or stationary cultures (although the maximal MP activity in sta- tionary cultures was attained after 16 d ; data not shown). During purification, MP activity was estimated by the formation of Mn7+ . tartrate, and the enzyme was also detected by heme ab- sorbance at 410 nm.

Fig. 3A shows the results of l o w - p e ~ o ~ a n c e anion-ex- change chromatography ( Q - c ~ t r i d g e ~ of ultrafiltered liquid. MP and laccase did not bind to the gel at pH 4.5, whereas AAO and most of a yellow-orange pigment produced by the fungus, simi- lar to that described in P. osfreatus [29], were retained.

Fig. 3 B shows the results of gei-filtration chromatography (Sephacryl S-200) of the fractions that were not retained by the

Martinez et al. (Eur. J. Biochem. 237)

7 0.5

- 0.4

- -0.3 5. -

(Y

T) ._ ?!

- 0.2 p z

-0.1

-0.0

427

0 5 10 15 20 Elution volume fml)

Fig. 4. Purification of MP from l? eryngii. Mono-Q chromatography and separation of MP-1 and MP-2 isoenzymes. Profiles corresponding to MP activity (-D-), and Mn"-independent peroxidase activities (X5) against 2,6-dimethoxyphenol (-w) and veratryl alcohol (-O-), absor- bances at 280 (-) and 410 nm (-), and the NaCl gradient (- -) are shown. Fractions from Sephacryl S-200 that contained MP were con- centrated, dialyzed and chromatographed on Mono-Q in 10 mM sodium tartrate, pH 5. MP activity was measured by formation of Mn3+ . tartrate in 0.1 mM MnSO,, 0.1 mM H,O,, 0.1 M sodium tartrate, pH 5. Mn2+- independent peroxidase activity against 2,6-dimethoxyphenol was mea- sured with 0.1 mM substrate, 0.1 mM H,O,, 0.1 M sodium tartrate, pH 5 and against veratryl alcohol with 2 mM substrate, 0.1 mM H,O,, 0.1 M sodium tartrate, pH 3.

Table 1. Purification of MP isoenzymes from &! eryngii. The enzy- matic activities in the culture liquid (1 1) and after the different purifica- tion steps were estimated by the formation of Mn" . tartrate (assayed in 0.1 mM MnSO,, 0.1 mM H,O,, 0.1 M sodium tartrate, pH 5). Protein content was estimated by means of Bradford reagent.

Step Protein Activity Purification

total specific 'yield factor

mg U U/mg % -fold

Culture liquid 143 986 7 100 1 Ultrafiltered 27 836 31 85 4 Q-cartridge 14 761 54 77 8 Sephacryl S-200 5 750 139 76 20 Mono-Q MP-1 1 187 187 18 27 Mono-Q MP-2 2 375 170 38 24 Mono-Q Total 3 562 176 56 25

Q-cartridge. The MP activity coincided with a protein peak after laccase activity, at the beginning of a large peak of absorbance at 280 nm (which showed some absorbance at 410 nm due to the presence of the pigment). The high A,,JA,,, ratio of the MP peak suggested the presence of a heme-containing protein.

The purification was completed by high-performance anion- exchange chromatography (Mono-Q) of the MP-containing frac- tions from the previous step (Fig. 4). Two peaks of MP activity that showed high A,,JA,,, ratios (called isoenzymes MP-1 and MP-2), were eluted with a shallow gradient of NaC1. SDS/PAGE and IEF of each of the isoenzymes revealed a single protein band (Fig. 5) . The MP activity of both isoenzymes was stable for at least 72 h at 4"C, and no loss of activity was observed when stored at -20°C. A quantitative outline of the purification procedure is presented in Table 1. The Mn2+-independent activi-

A

Distance (cm)

Fig.5. Estimation of the molecular masses (A) and PI (B) of MP isoenzymes from l? eryngii. (A), SDSPAGE of purified MP-1 (lane b), purified MP-2 (lane c) and Biorad standards (lane a) was performed in 12% polyacrylamide gels (AgNO, staining). (B), IEF of both isoen- zymes after Sephacryl S-200 chromatography (lane a) and of isolated MP-1 (lane b) and MP-2 (lane c) was performed in 5 % polyacrylamide gels with a pH range 2.5-5.5. pH was measured with a contact electrode to obtain a calibration line used to calculate the PI of isoenzymes (Coo- massie blue staining).

ties found at the different purification steps (Table 2) will be discussed below.

Properties of the MP isoenzymes. The molecular masses of both isoenzymes were 43 kDa (Fig. 5 A). Analytical IEF showed that the isoenzymes have slightly different PI, 3.75 for MP-1 and 3.65 for MP-2 (Fig. 5B), which may explain their separation on Mono-Q. Both isoenzymes are glycoproteins, with up to 5% carbohydrate for MP-1 and up to 7% for MP-2.

The visible spectra of native isoenzymes included Soret bands near 406, 502 and 640 nm (Fig. 6), which were shifted after treatment with excess H,O, (the main maximum appeared at approximately 420nm). The A4JAZ8,) ratio was 4 for both isoenzymes. The spectrum of the alkaline pyridine ferrous hemochrome showed maxima at 415, 525 and 556 nm, charac- teristic of protoporphyrin IX, and the intensity of the latter peak indicated a heme content of 1 mol/mol enzyme (calculated from protein content based on a molecular mass of 40 kDa for carbo- hydrate-free MP).

The amino acid compositions (residues/molecule of MP) of both isoenzymes (MP-I, MP-2) were similar (43,45 Asp; 16,17

428 Martinez et al. ( E m J. Biochem. 237)

250 350 450 550 650 Wavelength (nm)

Fig. 6. Ultraviolet-visible absorption spectra of native and H,O,-oxi- dized MP-2 from I? eryngii. The spectra of the native isoenzyme (solid line) and the oxidized form (dashed line), obtained after treatment with excess H,O,, were recorded in 10 mM sodium tartrate, pH 5.

Thr; 24,23 Ser; 41,39 Clu; 25,24 Pro; 43,46 Gly; 51,46 Ala; 22,23 Val; 6,6 Met; 16,16 Ile; 27,29 Leu; 3,3 Tyr; 28,31 Phe; 18,13 Lys; 10,8 His; 10,l l Arg), although some differences (e.g., in Lys content) were detected. The N-terminal sequences of both isoenzymes differed in 3 of the 18 amino acids identified (Fig. 7).

Catalytic properties. Both isoenzymes oxidize Mn’+ to Mn3+ in the presence of H202, and have Mn2+-dependent peroxidase activities against 2,6-dimethoxyphenol, vanillylideneacetone, other phenolic substrates ( e g , phenol red) and ABTS. The K,,, and V,,;,, for Mnz+ were obtained by estimation of the formation of Mn’+ . tartrate and by the Mnz+-dependent oxidation of 2,6- dimethoxyphenol (Table 3). The kinetic constants for peroxidase activity against 2,6-dimethoxyphenol and vanillylideneacetone (in the presence of 0.1 mM Mn2+) are presented.

NADH or NADPH were oxidized by the two isoenzymes (Table 3). The oxidation was partially inhibited by the addition of Mn2+ at the beginning of the reaction. However, when the reaction was carried out by successive addition of NADH, 2,6- dirnethoxyphenol and Mn2 (Fig. 8), oxidation of both NADH and 2,6-dimethoxyphenol was observed. The presence of 0;- during NADH oxidation was demonstrated by the reduction of Nitro Blue Tetrazolium or cytochrome-c.

Mn2+-independent peroxidase activity against 2,6-dime- thoxyphenol was also detected with both isoenzymes (even in

Table 2. Mn2+-independent peroxidase activities against 2,6-dime- thoxyphenol and veratryl alcohol and 2,6-dimethoxyphenol peroxi- dase activity in the presence of Mn” during purification of MP iso- enzymes from I? eiyngii. Enzymatic activities during purification were estimated in 0.1 M sodium tartrate (pH 5 for 2,6-dimentoxyphenol oxi- dation, and pH 3 for veratryl alcohol oxidation) with 0.1 mM 2,6-dime- thoxyphenol, 0.1 mM MnSO,, 0.1 mM H,Oz and 2 mM veratryl alcohol. Relative activities, as percentages of the activity against 2,6-dimethoxy- phenol in the presence of Mn” , are indicated in brackets.

Purification step Activity against

2,6-dimethoxyphenol veratryl alcohol

+ Mn2+ ~ Mn2+

U

Q-Cartridge 200 25 (13%) 17 (9%) Sephacryl S-200 197 28 (14%) 17 (9%) MP- 1 49 5 (10%) 4 (8%) MP-2 98 10 (10%) 9 (9%)

the presence of EDTA). At the different purification steps, this activity represented a similar percentage of the activity against 2,6-dimethoxyphenol in the presence of Mn2+ (Table 2). More- over, activities against Mn2+ and 2,6-dimethoxyphenol (with and without Mn2+) showed similar profiles during Mono-Q chroma- tography (Fig. 4). 2,6-Dimethoxyphenol oxidation was measured at 0. I mM, but the relative extent of Mn’+-independent activity (estimated as the ratio between 2,6-dimethoxyphenol oxidation without Mn2’ and that with Mn’+) increased as a linear function of substrate concentration (in the range 0-0.5 mM 2,6-dime- thoxyphenol). This can be explained if we consider that the K,, for 2,6-dimethoxyphenol was one order of magnitude lower with 0.1 mM Mn2+ than without Mn2+ (Table 3). Mn’+-independent peroxidase activity was observed also with other phenolic sub- strates (e.g., phenol red) and with ABTS. In the absence of Mn2+, the activities of isoenzymes MP-1 and MP-2 towards ABTS represented 60% and 45 %, respectively, of the activities in the presence of Mn2+ (estimated at pH 5, in the presence of 0.1 mM ABTS and 0.1 mM Mn2+).

H,O,-dependent oxidation of veratryl alcohol was detected with both isoenzymes (at pH 3), the activity levels obtained be- ing similar to those of Mn?+-independent oxidation of 2,6-dime- thoxyphenol. The peroxidase activity against veratryl alcohol was not stimulated by Mnz+. As with Mn2+-independent activity against 2,6-dimethoxyphenol, this activity represented a constant percentage of Mn2’ -mediated activity against 2,6-dimethoxy-

A T D M D G R T T A - D A A C C M L F - A T D A D G R T T A - N A A C C V L F -

- x

- P -El - P - x

:& I P I P I P I P I P I P I P I P

Pleurotus eryngii MnP2 Pleurotus eryngii MnPl Pleurotus pulmonarius [55] Pleurotus ostreatus (GeneBank-U21878) Trarnetes versicolor MnPl 1391 Ceriporiopsis subverrnispora MnP6 [17] Ceriporiopsis subvermlspora MnP4 [171 Lentinula edodes MnPl [42] Phanerochaete chrysosporiurn MnPH4 [37] Phanerochaete chrysosporium MnPH5 [46] Phanerochaete chrysosporiurn MnPl [36] Phanerochaete chrysosporiurn [20] Phanerochaete sordida MnPl 1471 Phanerochaete sordida MnPll [47] Phanerochaete sordida MnPlll 1471

Fig. 7. Comparison of N-terminal sequences of the two MP isoenzymes from I? eryngii with MP from other fungi.

429 Martinez et al. ( E m J . BiocCzern. 2 3 3

Table 3. Kinetic constants of MP isoenzymes from F! eryngii. All reactions were performed in 0.1 M sodium tartrate at pH 5, except for veratryl alcohol oxidation which was performed at pH 3. Other components present in reaction mixtures are indicated in brackets.

Substrate Km V,,,

MP-1 MP-2 MP-1 MP-2

PM pmol . min-' mg-'

H,O, (0.1 mM Mn2+) 6 10 170 165 Mn2+ (0.1 mM H,O,) 20 20 190 170

2,6-Dimethoxyphenol (0.1 mM Mn2+, 0.1 mM H,O,)" 10 10 30 30 Vanillylideneacetone (0.1 mM Mn2+, 0.1 mM H,O,)" 5 5 55 60

2,6-Dimethoxyphenol (0.1 mM H,OJ 160 250 20 15

Mn" (0.1 mM 2,6-dimethoxyphenol, 0.1 mM HZOJd 15 15 30 20

NADH 70 60 95 75

Veratryl alcohol (0.1 mM H,O,) 3500 3000 95 45

Estimated from oxidation of the phenolic substrate.

decreased more than 95% when the pH increased from 3 to 4.5

served for the Mn2+-independent oxidation of 2,6-dimethoxy- phenol and ABTS, the latter substrate being easier to oxidize than 2,6-dimethoxyphenol. Oxidation of ABTS (0.1 mM) at pH 3 was completely independent of Mn2+.

No inhibition of peroxidase activity by Mn2' and H,02 was observed in the concentration range 0-0.2 mM. Partial inhibi- tion was produced by higher concentrations of H,O, (25% at 0.5 mM H,O, and 75 % at 1 mM H,O,). Moreover, MnZ+ par- tially inhibited oxidase activity against NADH (97 % inhibition of MP-1 and 72% inhibition of MP-2 by 0.1 mM Mn'+ added to the reaction mixture simultaneously to NADH), but did not affect veratryl alcohol oxidation by MP isoenzymes.

I and was nearly absent at pH 5. A similar effect of pH was ob- 0.6-

DMPoxidation

NADH oxidation

- - - . . . - . . (azide addition)

0 120 240 360 480 0.0 , , 7 , I I

Reaction time (s)

Fig. 8. Oxidation of 2,6-dimethoxyphenol by MP from I? eryngii in the presence of NADH and Mn2+. The purified enzyme (MP-1) was incubated in 0.3 mM NADH, 0.1 M sodium tartrate, pH 5, at room tem- perature, and 0.1 mM 2,6-dimethoxyphenol (DMP) and 0.1 mM MnSO, were added during the incubation. The generation of H,O, during NADH oxidation (followed at 340 nm) by MP-1 required the presence of MnZ+, and enabled 2,6-dimethoxyphenoi oxidation (which forms coerulignone; followed at 469 nm). The latter reaction was inhibited by the addition of 0.1 mM azide with the MnSO, (dotted lines).

phenol during purification steps (Table 2 ) and showed a similar Mono-Q profile (Fig. 4). A characteristic of veratryl alcohol oxi- dation was the high K, values obtained (Table 3). This indicates that the activity was underestimated during enLyme purification (Table 2) since the concentration of veratryl alcohol used (2 mM) was not high enough to saturate the enzyme. Only per- oxidase activity against veratryl alcohol was partially inhibited by sodium metavanadate (at metavanadate concentrations as low as 1 pM). MnZ+ peroxidase activity and Mn2'-independent 2,6- dimethoxyphenol peroxidase activity were not affected, even at the highest concentrations assayed (50 pM). The isoenzymes showed different degrees of inhibition, and the residual activity (14% for MP-1 and 33% for MP-2) remained stable at increas- ing metavanadate concentrations (from 5 pM to 50 pM).

pH optima and substrate inhibition. The pH optima for en- zyme activity on the different substrates were 5 for Mnz+, 4 for 2,6-dimethoxyphenol in the presence of Mn2+, 3.5 for NADH, and 3 for 2,6-dimethoxyphenol or veratryl alcohol without Mn'+. The oxidation of Mnz+ was decreased 90% at pH 3.5 and was nearly absent at pH 3. In contrast, veratryl alcohol oxidation

DISCUSSION The conditions for MP production in liquid cultures of I?

eryngii differ from those reported for P. chrysosporium, the best- known ligninolytic organism, which synthesizes MP with am- monium as N source [6]. However, stimulation of ligninolytic peroxidases by peptone in N-rich medium, as found in I? eryngii, has been reported also in Bjerkundera udustu [30]. Peptone is mainly composed of peptides of different sizes, and it has been suggested that some of them could induce secondary-metabo- lism events (such as the secretion of ligninolytic peroxidases) because they are similar to peptides released during mycelium autolysis at the end of the growth phase 1301. The repression of MP activity by Mn" (in peptone medium) contrasts with the results reported for P. chrysosporium and other ligninolytic fungi [14, 311. Since MnZ+ is the best substrate for P. eryngii MP, the finding that the highest MP activity was in media without Mn (no Mn was detected in peptone) constitutes an apparent biologi- cal contradiction. It may be argued that these enzymes possess high Mnz+-independent activity, but this activity only occurs at relatively high concentrations of phenolic substrates. Recent studies of enzyme production by Pleurotus species under solid- state-fermentation conditions (which are similar to natural growth conditions of ligninolytic fungi) demonstrated that pep- tone and MnZ+ concentrations did not significantly affect MP production during solid-state fermentation of wheat straw 121 1. The authors suggested that some aspects of the physiology of Pleurotus and other white-rot fungi, can be better explained by the information provided by solid-state-fermentation studies than from data obtained under liquid-culture conditions.

MP is produced by more than 50 species of basidiomycetes, and MP isoenzymes have been purified from approximately 15

430 Martinez et al. (Eul: J. Biochern. 237)

of these species [14, 15, 32, 331. Among ligninolytic fungi, MP isoenzymes have been characterized in P. chrysosporium [34- 371, Trametes versicolor [38, 391 and Phlebia radiata [40]. The characteristics of P. eryngii MP differ in some aspects from those reported for these enzymes. The spectra of the isoenzymes from P. eryngii and the corresponding pyridine ferrous hemo- chromes revealed that they contain 1 mol protoporphyrin IX/mol enzyme, as reported for MP from other fungi [40-421. However, the P. eryngii isoenzymes have a lower A,,, JAZ8" ratio than re- ported for P. chrysosporium MP [41], which may be related to the presence of three Tyr residues/molecule. The molecular masses of the two isoenzymes from P. eryngii were the same, being in the same range as those of MP from most ligninolytic fungi (42-45 kDa) except P. radiata [40] and Ceriporiopsis subvermispora [17]. Moreover, the P. eryngii isoenzymes are included in the group of more acidic MP isoenzymes, with PI in the range 3-4. MP isoenzymes with higher pl are produced by F! chrysosporium and other ligninolytic fungi. In addition to small differences in PI and catalytic properties, including lower V,,, (approximately 50%) than reported for P. chrysosporium MP [43], the two isoenzymes from i? eryngii differ in the degree of inhibition by metavanadate. Concentrations of this compound that cause 100% inhibition of veratryl alcohol oxidation by LP from P. chrysosporium [44] only produced 67 % and 86 % inhibi- tion of veratryl alcohol oxidation by MP-2 and MP-1 from P. eryngii, respectively, while oxidation of Mn2+ or 2,6-dimethoxy- phenol (without Mn2+) were not affected. I? eryngii isoenzymes were resistant to H,O, concentrations that affect the activity of MP from P. chrysosporium (strongly inhibited by 0.4 mM H,O,) [41]. This finding may be related to the higher H,O, levels in cultures of P. eryngii due to the existence of an efficient H,O,- producing system based on the enzyme AAO [lo]. H,O, produc- tion via MP is another possibility and, as reported for P. chrysosporium MP [41], P. eryngii MP can oxidize 2,6-dime- thoxyphenol in the absence of exogenous H,02, if NADH and Mn2+ are present. The results obtained suggest the following mechanism: 0;- is generated during' NADH oxidation by MP; Mn2+ is oxidized by O;-, generating H20, [45] ; and 2,6-dime- thoxyphenol is oxidized by MP and Mn'+. The N-terminal se- quences of the MP isoenzymes from F! eryngii show the highest identities with those from I? pulmonarius, F! ostreatus and 7: versicolor (Fig. 7) . The differences between the N-terminal se- quences of P. eryngii isoenzymes (and between the amino acid compositions and PI) suggest that the isoenzymes are encoded by different genes, as occurs for MP isoenzymes from other lig- ninolytic fungi [48].

The most important difference between MP from P. eryngii and MP from P. chrysosporium concerns Mn'+-independent per- oxidase activities against 2,6-dimethoxyphenol and veratryl al- cohol. The ability to oxidize veratryl alcohol has been reported for MP from Lentinula edudes 1491 but was explained by the action of thiyl radicals generated from thiols present in the reac- tion mixture [ S O ] . In the present study, direct oxidation of verat- ryl alcohol by the two isoenzymes from P. eryngii is reported, although the enzyme affinity for veratryl alcohol was two orders of magnitude lower than the affinity for Mn2+. The pH optimum for the veratryl alcohol peroxidase activity was 3, and no activity was found at pH 5. Cation-radical-mediated oxidation of veratryl alcohol at pH 2.5-3 has been reported for a soybean peroxidase, which has unusually high stability at low pH values [51]. Increased redox potential at low pH, as suggested for soybean peroxidase, could enable oxidation of veratryl alcohol by the MP isoenzymes from l? eryngii. It has been reported that MP from P. chrysosporium has very low MnZ +-independent activity against pinacynol [41, 521 and that P. ostreatus MP can oxidize ABTS to some extent without Mn2+ 1531. MP from P. chryosporium

only oxidizes ABTS in the presence of Mnz+ [52], while that from P. eryngii has approximately 50% Mn,+-independent activ- ity (compared with the activity in the presence of Mnz+) at pH 5, and 100% Mn'+-independent activity at pH 3. Moreover, the Mn2+-independent peroxidase activity of P. eryngii MP at pH 5 is more significant at relatively high concentrations of 2,6-dime- thoxyphenol, attaining 50% activity (with respect to that found in presence of Mn2+) when the 2,6-dimethoxyphenol concentra- tion is 0.5 mM. A manganese-independent peroxidase (MIP), first described as Mn'+-inhibited peroxidase, has been reported in B. adusta [54], although it has not been fully characterized. The results presented here show that t? eryngii MP can operate also as MIP of phenolic compounds, when adequate concentra- tions of substrate are available. Contamination of MP isoen- zymes from P. eryngii with other enzymes (e.g., LP or MIP) can be discounted due to the following reasons: the ratios between peroxidase activities against 2,6-dimethoxyphenol, with and without Mnz+, and veratryl alcohol remained nearly constant during purification steps ; the corresponding Mono-Q profiles were similar; SDS/PAGE and IEF of both isoenzymes revealed a single band ; both isoenzymes have similar catalytic properties; peroxidase activity against veratryl alcohol was only partially inhibited by metavanadate concentrations that cause 100% inhi- bition of LP ; and maximal specific activities (V,,,,) obtained were similar with and without Mn2+ (although affinities were very different). In summary, the two peroxidase isoenzymes from P. eryngii exhibit multiple enzymatic activities, being char- acterized by low affinity for veratryl alcohol, medium affinity for phenols (in the absence of Mnz+) and high affinity for Mn'', which results in rapid oxidation of phenolic compounds.

In view of the results obtained in this study and the recent findings concerning Mn2+ oxidation by LP [24] and veratryl al- cohol oxidation by some plant peroxidases [51], it is possible to conclude that the catalytic properties of the peroxidases pro- duced by ligninolytic fungi should be carefully re-evaluated to define different types of enzymes and to establish their participa- tion in lignin biodegradation.

The authors thank A. Garcia-Raso (Department Organic Chemistry, Universidad Mallorca, Spain), for comments on reaction mechanisms, S. Camarero (Centro de Investigaciones Biolo'gicas, Consejo Superior de Investigaciones Cientficas, Madrid, Spain), for information concerning metal contents and discussions about fungal physiology, J. Varela, for amino acid and N-terminal analyses, and M. T. Raposo and M. A. Guij- arro, for skillful technical assistance. This research has been partially funded by the Spanish Biotechnology Programme (BI092-0357) and the Agro-Industry Research Project of the European Union on Biological delignification in paper manufacture (AIR2-CT93-1219).

REFERENCES 1. Valmaseda, M., Almendros, G. & Martinez, A. T. (1990) Substrate-

dependent degradation patterns in the decay of wheat straw and beech wood by ligninolytic fungi, Appl. Microbiol. BiotechnoL

2. Kamra, D. N. & Zadrazil, F. (1986) Influence of gaseous phase, light and substrate pretreatment on fruit-body formation, lignin degradation and in iiifro digestibility of wheat straw fermented with Pleurotus spp, Agric. Wastes 18, 1-17.

3. Martinez, A. T., Camarero, S., GuillCn, F., Gutikrrez, A,, MuRoz, C., Varela, E., Martinez, M. J., Barrasa, J. M., Ruel, K. & Pelayo, M. (1994) Progress in biopulping of non-woody materials: chemi- cal, enzymatic and ultrastructural aspects of wheat-straw deligni- fication with ligninolytic fungi from the genus Pleurotus, FEMS Microbiol. Rev. 13, 265-274.

4. Guilltn, F., Martinez, A. T. & Martinez, M. J. (1990) Production of hydrogen peroxide by aryl-alcohol oxidase from the ligninolytic fungus Pleurotus eryngii, Appl. Microbiol. Biotechnol. 32, 465 - 469.

33, 481 -484.

Martinez et al. (Eur: J. Biochem. 237) 43 1

5. Guillen, F., Martinez, A. T. & Martinez, M. J. (1992) Substrate spec- ificity and properties of the aryl-alcohol oxidase from the lignino- lytic fungus Pleurotus eryngii, Eur: J. Biochem. 209, 603 -61 1,

6. Kuwahara, M., Glenn, J. K.. Morgan, M. A. & Gold, M. H. (1984) Separation and characterization of two extracellular H,O,-depen- dent oxidases from ligninolytic cultures of Phanerochaete chrysosporium, FEBS Lett. 169, 247-250.

7. Martinez, M. J., Muiioz, C., GuillCn, F. & Martinez, A. T. (1994) Studies on homoveratric acid transformation by the ligninolytic fungus Pleurotus eryngii, Appl. Microbiol. Biotechnol. 41, 500- 504.

8. GuillCn, F. & Evans, C. S. (1994) Anisaldehyde and veratraldehyde acting as redox cycling agents for H,O, production by Pleurotus eryngii, Appl. Environ. Microbiol. 60, 281 1-2817.

9. GutiCrrez, A., Caramelo, L., Prieto, A,, Martinez, M. J. & Martinez, A. T. (1 994) Anisaldehyde production and aryl-alcohol oxidase and dehydrogenase activities in ligninolytic fungi from the genus Pleurotus, Appl. Environ. Microbiol. 60, 1783-1788.

10. GuillCn, F., Martinez, A. T., Martinez, M. J. & Evans, C. S. (1994) Hydrogen peroxide-producing system of Pleurotus eryngii involv- ing the extracellular enzyme aryl-alcohol oxidase, Appl. Micro- bid. Biotechnol. 41, 465 -470.

11. Asada, Y., Watanabe, A,, Ohtsu, Y. & Kuwahara, M. (1995) Purifi- cation and characterization of an aryl-alcohol oxidase from the lignin-degrading basidiomycete Phanerochaete chrysosporium, Biosci. Biotechnol. Biochem. 59, 1339- 1341.

12. Kirk, T. K. & Farrell, R. L. (1987) Enzymatic combustion: the mi- crobial degradation of lignin, Annu. Rev. Microbiol. 41,465-505.

13. Martinez, A. T., Ruiz, F. J. & Martinez, M. J. (1994) Influence of Mn concentration and N source on the production of ligninolytic enzymes in liquid cultures of Pleurotus species, (abstract) 5th Int. Mycol. Congr. ; 14-21 August; Vancouver. British Columbia University, p. 134.

14. Hatakka, A. (1994) Lignin-modifying enzymes from selected white- rot fungi - production and role in lignin degradation, FEMS Microbiol. Rev. 13, 125-135.

15. Peliez, F., Martinez, M. J. & Martinez, A. T. (1995) Screening of 68 species of basidiomycetes for enzymes involved in lignin de- gradation, Mycol. Res. 99, 37-42.

16. PeriC, F. H. & Gold, M. H. (1991) Manganese regulation of manga- nese peroxidase expression and lignin degradation by the white rot fungus Dichomitus squalens, Appl. Environ. Microbiol. 57, 2240-2245.

17. Lobos, S., Larrain, J., Salas, L., Cullen, D. & Vicufia, R. (1994) Isoenzymes of manganese-dependent peroxidase and laccase pro- duced by the lignin-degrading basidiomycete Ceriporiopsis sub- vermispora, Microbiology-UK 140, 2691 -2698.

18. Wariishi, H., Valli, K. & Gold, M. H. (1991) In vitro depolymeriza- tion of lignin by inanganese peroxidase of Phanerochaete chrysosporium, Biochem. Biophys. Res. Commun. 176, 269 - 275.

19. Bao, W. L., Fukushima, Y., Jensen, K. A., Moen, M. A. & Hammel, K. E. (1994) Oxidative degradation of non-phenolic lignin during lipid peroxidation by fungal manganese peroxidase, FEBS Lett. 354, 297-300.

20. Datta, A., Bettermann, A. & Kirk, T. K. (1991) Identification of a specific manganese peroxidase among ligninolytic enzymes secreted by Phanerochaete chrysosporium during wood decay, Appl. Environ. Microbiol. 57, 1453 - 1460.

21. Camarero, S. , Martinez, M. J. & Martinez, A. T. (1995) Lignin- degrading enzymes produced by Pleurotus species during solid- state fermentation of wheat-straw, Proc. 11 Int. Workshop on SSF (FMS-95);27-28 February; Montpellier. ORSTOM, p. 33.

22. Barrasa, J. M., Gonzilez, A. E. & Martinez, A. T. (1992) Ultrastruc- tural aspects of fungal delignification of Chilean woods by Ga- noderma australe and Phlebia chrysocrea: a study of natural and in vitro degradation, Holzforschung 46, 1-8.

23. Rios, S. & Eyzaguirre, J . (1992) Conditions for selective degrada- tion of lignin by the fungus Ganoderma australis, Appl. Micro- biol. Biotechnol. 37, 667-669.

24. Khindaria, A., Barr, D. P. & Aust, S. D. (1995) Lignin peroxidases can also oxidize manganese, Biochemistry 34, 7773 -7779.

25. Betts, W. B. & King, J. E. (1991) Oxidative coupling of 2,6-dime- thoxyphenol by fungi and bacteria, Mycol. Res. 95, 526-530.

26. Tien, M. & Kirk, T. K. (1988) Lignin peroxidase of Phanerochaete chrysosporium, Methods Enzymol. 161, 238-248.

27. Trevelyand, W. E. & Harrison, J. S. (1952) Studies of yeast metabo- lism. 1. Fractionation and microdetermination of cell carbohy- drates, Biochem. J. 50, 298-302.

28. Pajot, P. & Groudinsky, 0. (1970) Molecular mass and quaternary structure of yeast L-lactate dehydrogenase (cytochrome b?). Re- vised heme extinction coefficients and minimal molecular weight, Eur: J . Biochem. 12, 158-164.

29. Margraf, W. (1984) Orange/yellow pigments in the basidiomycete Pleurotus ostreatus (Jacq. ex Fr.) Kummer, in Blue light effects in biological systems (Senger, H., ed.) pp. 55 -59, Springer-Verlag, Berlin.

30. Kaal, E. E. J., de Jong, E. & Field, J. A. (1993) Stimulation of ligninolytic peroxidase activity by nitrogen nutrients in the white rot fungus Bjerkandera sp strain BOS55, Appl. Environ. Micro- biol. 59, 4031 -4036.

31. Bonnarme, P. & Jeffries, T. W. (1990) Mn(I1) regulation of lignin peroxidases and manganese-dependent peroxidases from lignin- degrading white rot fungi, Appl. Environ. Microbiol. 56, 210- 217.

32. Buswell, J. A,, Cai, Y. J. & Chang, S. T. (1995) Effect of nutrient nitrogen and manganese on manganese peroxidase and laccase production by Lentinula (Lentinus) edodes, FEMS Microbiol. Lett. 128, 81-87.

33. Orth, A. B. & Tien, M. (1995) Biotechnology of lignin degradation, in The Mycota. II. Genetics and biotechnology (Kiick, U., ed.) pp. 287-302, Springer-Verlag, Berlin.

34. Wariishi, H., Akileswaran, L. & Gold, M. H. (1988) Manganese peroxidase from the basidiomycete Phanerochaete chrysospo- rium: spectral characterization of the oxidized states and the cata- lytic cycle, Biochemistry 27, 5365-5370.

35. Wariishi, H., Dunford, H. B., MacDonald, 1. D. & Gold, M. H. (1 989) Manganese peroxidase from the lignin-degrading basidio- mycete Phanerochaete chrysosporium. Transient state kinetics and reaction mechanism, J. Biol. Chem. 264, 3335-3340.

36. Pribnow, D., Mayfield, M. B., Nipper, V. J., Brown, J. A. & Gold, M. H. (1989) Characterization of a cDNA encoding a manganese peroxidase, from the lignin-degrading basidiomycete Phane- rochaete chrysosporium, J. Biol. Chem. 264, 5036-5040.

37. Pease, E. A,, Andrawis, A. & Tien, M. (1989) Manganese-dependent peroxidase from Phanerochaete chrysosporium. Primary structure deduced from cDNA sequence, J. Biol. Chem. 264, 13 531 - 13535.

38. Johansson, T. & Nyman, P. 0. (1993) Isozymes of lignin peroxidase and manganese(I1) peroxidase from the white-rot basidiomycete Trametes versicolor. 1. Isolation of enzyme forms and character- ization of physical and catalytic properties, Arch. Biochem. BiCJ- phys. 300,49-56.

39. Johansson, T., Welinder, K. G. & Nyman, P. 0. (1993) Isozymes of lignin peroxidase and manganese(I1) peroxidase from the white- rot basidiomycete Trametes versicolor. 2. Partial sequences, pep- tide maps, and amino acid and carbohydrate compositions, Arch. Biochem. Biophys. 300, 57-62.

40. Karhunen, E., Kantelinen, A. & Niku-Paavola, M.-L. (1990) Mn- dependent peroxidase from the lignin-degrading white rot fungus Phlebiu radiata, Arch. Biochem. Biophys. 279, 25-31.

41. Glenn, J. K. & Gold, M. H. (1985) Purification and characterization of an extracellular Mn(I1)-dependent peroxidase from the lignin- degrading basidiomycete Phanerochaete chrysosporium, Arch. Biochem. Biophys. 242, 329-341.

42. Forrester, I. T., Grabski, A. C., Mishra, C., Kelley, B. L., Strickland, W. N., Leatham, G. F. & Burgess, R. R. (1990) Characteristics and N-terminal amino acid sequence of a manganese peroxidase purified from Lentinus edodes cultures grown on commercial wood substrate, Appl. Microbiol. Biotechnol. 33, 359-365.

43. Mayfield, M. B., Kishi, K., Alic, M. & Gold, M. H. (1994) Homolo- gous expression of recombinant manganese peroxidase in Phane- rochaete chrysosporium, Appl. Environ. Microbiol. 60, 4303 - 4309.

44. Archibald, F. S. (1992) Lignin peroxidase activity is not important in biological bleaching and delignification of unbleached Kraft pulp by Trumetes versicolor, Appl. Environ. Microbiol. 58,3101 - 3109.

432 Martinez et al. ( E m J. Biochem. 237)

45. Archibald, F. S . & Fridovich, I. (1982) The scavenging of superox- ide radical by manganous complexes in vitro, Arch. Biochem. Bio- phys. 214, 452-463.

46. Pease, E. A. & Tien, M. (1992) Heterogeneity and regulation of manganese peroxidases from Phanerochaete chrysosporium, J. Bacteriol. 174, 3532-3540.

47. Riittimann-Johnson, C . , Cullen, D. & Lamar, R. T. (1994) Manga- nese peroxidases of the white rot fungus Phanerochaete sordida, Appl. Environ. Microbiol. 60, 599-605.

48. Gold, M. ti. & Alic, M. (1993) Molecular biology of the lignin- degrading basidiomycete Phanerochaete chrysosporium, Micro- biol. Rev. 57, 605-622.

49. Forrester, I. T., Grabski, A. C., Burgess, R. R. & Leatham, G. F. (1988) Manganese, Mn-dependent peroxidases, and the biodegra- dation of lignin, Biochem. Biophys. Res. Commun. 157, 992-999.

SO. Wariishi, H., Valli, K., Renganathan, V. & Gold, M. H. (1989) Thiol- mediated oxidation of nonphenolic lignin model compounds by manganese peroxidase of Phanerochaete chrysosporium, J . Biol. Chem. 264, 14185-14191.

51. McEldoon, J. P., Pokora, A. R. & Dordick, J. S . (1995) Lignin per- oxidase-type activity of soybean peroxidase, Enzyme Microb. Technol. 17, 359-365.

52. Glenn, J. K., Akileswaran, L. & Gold, M. H. (1986) Mn(I1) oxida- tion is the principal function of the extracellular Mn-peroxidase from Phanerochaete chrysosporium, Arch. Biochem. Biophys.

53. Bekker, E. G., Pirtskhalaishvili, D. O., Elisashvili, V. I. & Sinitsyn, A. P. (1993) Manganese peroxidase from Pleurotus ostreatus - isolation, purification, and properties, Biochemistry (Engl. TransZ. Biokhimiya) 57, 863-868.

54. de Jong, E., Field, J. A. & Bont, J. A. M. (1992) Evidence for a new extracellular peroxidase : manganese inhibited peroxidase from the white-rot fungus Bjerkundera sp Bos 55, FEBS Lett.

55. Camarero, S., Bockle, B., Martinez, M. J. & Martinez, A. T. (1996)

251, 688-696.

299, 107-110.

Appl. Env. Microbiol. 62, in the press.