Purification and characterization of peroxidases from the dye-decolorizing fungus Bjerkandera adusta

FEMS Microbiology Letters 165 (1998) 43^50

Puri¢cation and characterization of peroxidases from the dye-decolorizing fungus Bjerkandera adusta Annette Hein£ing a; *, Mar|èa Jesuès Mart|ènez b , Angel T. Mart|ènez b , Matthias Bergbauer a , Ulrich Szewzyk a a

FG Microbial Ecology, Technical University Berlin, Franklinstr. 29, D-10587 Berlin, Germany b Centro de Investigaciones Bioloègicas, CSIC, Velaèzquez 144, E-28006 Madrid, Spain Received 5 May 1998; revised 8 June 1998 ; accepted 8 June 1998

Abstract A peroxidase oxidizing Mn2‡ (MnP) is described for the first time in Bjerkandera adusta, a fungus efficiently degrading xenobiotic compounds. The MnP appeared as two isoenzymes, which were purified to homogeneity together with two lignin peroxidases (LiP). Their N-terminal sequences were identical, but the MnP isoenzymes showed more basic isoelectric points and differences in amino acid composition and catalytic properties. The B. adusta LiP is similar to LiP from Phanerochaete chrysosporium. However, the interest of the MnP described here is related to its ability to catalyze Mn2‡ -mediated as well as Mn2‡ -independent reactions on aromatic compounds, which may be of use for applications in biotechnology and environmental technology. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Manganese peroxidase; Lignin peroxidase; White-rot fungus ; Bjerkandera adusta

1. Introduction Bjerkandera adusta is a wood-rotting basidiomycete belonging to the white-rot fungi. White-rot fungi are the most e¤cient lignin degraders in nature [1] and they are also able to oxidize xenobiotic compounds including some environmental pollutants [2,3]. Lignin degradation by white-rot fungi is cata-

* Corresponding author. Tel.: +49 (30) 31426827; Fax: +49 (30) 31473461; E-mail: [email protected]

lyzed by secreted oxidases and peroxidases [4]. Among these enzymes are laccases and the peroxidases lignin peroxidase (LiP) and manganese peroxidase (MnP), the latter ¢rst described in Phanerochaete chrysosporium [5^7]. LiP catalyzes the oxidation of non-phenolic aromatic compounds like veratryl alcohol. MnP oxidizes Mn2‡ to Mn3‡ , which is able to oxidize many phenolic compounds [8]. Some phenols are also able to reduce compound I of MnP but not compound II. Therefore Mn2‡ is necessary for completion of the catalytic cycle of P. chrysosporium MnP [9]. The biotechnological interest of Bjerkandera species arises from their ability to degrade aromatic

0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 2 5 5 - 9

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xenobiotics [10,11] as well as lignin and extractives on Eucalyptus wood [12]. However, little is known about ligninolytic enzymes produced by B. adusta, the most prevalent Bjerkandera species in Europe. This study reports puri¢cation and characterization of MnP and LiP isoenzymes from a B. adusta strain e¤ciently degrading dyes [11], the former enzyme being described for the ¢rst time in this fungal species.

2. Materials and methods 2.1. Organism and culture conditions B. adusta DSM 11310 was grown in glucose-ammonium medium with the azo dye reactive violet 5 [11]. The cultures (450 ml in 2-l £asks) were inoculated (4% v/v) with washed and homogenized mycelium from 10-day-old cultures grown in 1% malt extract at 28³C and 140 rpm. 2.2. Peroxidase puri¢cation The supernatant was separated from the mycelium (1.2 Wm ¢lter) and Q Sepharose (Pharmacia) was added (2.5 ml l31 ). The suspension was shaken for 1 h at 4³C, ¢ltered and the procedure repeated until peroxidase activity in the ¢ltrate was below 2%. The adsorbed proteins were eluted in 20 mM phosphate, 0.5 M NaCl (pH 6), concentrated by ultra¢ltration (10 kDa cut-o¡) and dialyzed against 20 mM L-histidine (pH 6). The concentrated proteins were applied to a Mono Q HR5/5 column (Pharmacia) equilibrated with the above bu¡er, and those retained were eluted with a 0^0.3 M NaCl gradient (180 ml, 1 ml min31 ). Pools of LiP and MnP were concentrated and dialyzed against the bu¡er used in the next puri¢cation step. Then they were chromatographed on a Mono Q column using 20 mM histidine bu¡er of lower pH than in the previous step (pH 4.6 for LiP, pH 4.8 for MnP), and a 0^0.2 M NaCl gradient (80 ml, 1 ml min31 ). Puri¢cation of MnP was completed by Mono Q chromatography in 10 mM tartrate (pH 4.5) using a 0^0.05 M NaCl gradient (25.6 ml, 0.8 ml min31 ). The di¡erent isoenzymes were concentrated, dialyzed and stored at 320³C.

2.3. Enzymatic activities The following reaction conditions were used to assay enzyme activities during the puri¢cation procedure: manganese-independent peroxidase (MIP) activity was measured with 1 mM 2,6-dimethoxyphenol (DMP) in 0.1 M sodium tartrate (pH 4.5) using 0.1 mM H2 O2 . MnP activity was measured as above but adding 1 mM MnSO4 (and subtracting MIP activity). LiP activity was determined with 2 mM veratryl alcohol in 0.1 M sodium tartrate at pH 3 and 0.1 mM H2 O2 . Laccase activity was investigated by oxidation of 1 mM DMP or ABTS (2,2Pazino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) in 0.1 M sodium tartrate (pH 4.5). Dye-decolorizing activities were determined with 50 mg l31 CI reactive violet 5 or CI reactive blue 38 (both from DyStar, Frankfurt, Germany), and 0.1 mM H2 O2 in 0.1 M tartrate at pH 3.5 [11]. After enzyme puri¢cation, steady-state kinetic constants were determined for di¡erent substrates in 0.1 M tartrate at the pH values indicated below. Activities were calculated from the linear phase of the reaction using molar absorbances of the oxidation products of Mn2‡ (O238 = 6500 M31 cm31 ), DMP (O469 = 27 500 M31 cm31 , referred to DMP concentration [13]), veratryl alcohol (O310 = 9300 M31 cm31 ) and ABTS (O420 = 36 000 M31 cm31 ). One activity unit was de¢ned as the amount enzyme transforming 1 Wmol of substrate per minute. 2.4. Enzyme characterization Protein concentration was determined with Bradford reagent. The content of N-linked carbohydrate was estimated after deglycosylation of 0.5 Wg protein with 40 mU N-glycosidase F from Boehringer. SDSPAGE of native and deglycosylated proteins was performed in 12% polyacrylamide gels. Isoelectric focusing (IEF) was performed in 5% polyacrylamide gels with a thickness of 1 mm prepared with Pharmalyte (Pharmacia) (pH 2.5^5) or with Servalyt (Serva) (pH 2^4). 1 M H3 PO4 and 0.5 M NaOH were used as cathode and anode £uids respectively. Protein bands were stained with AgNO3 after SDSPAGE, and with Coomassie blue R-250 after IEF. The amino acid composition was determined with a

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Biochrom 20 autoanalyzer (Pharmacia) after hydrolysis of 20 Wg protein with 6 M HCl at 110³C for 24 h. N-terminal sequences were obtained by automated Edman degradation of 7 Wg protein in an Applied Biosystems 477A pulsed liquid protein sequencer with 120A on-line phenylthiohydantoin analysis.

45

enzymes was con¢rmed by SDS-PAGE and IEF (Fig. 2). A summary of the puri¢cation process, in-

3. Results 3.1. Enzyme detection during fungal growth During cultivation of B. adusta on dye-containing glucose-ammonium medium, MnP and MIP-type activities rose simultaneously and reached a maximum (23 U l31 MnP and 11 U l31 MIP) after 5 days. LiP activity could not be detected in the culture supernatant, but it appeared during enzyme puri¢cation. Laccase activity was not found. Dyedecolorizing peroxidase activity with the azo dye reactive violet 5 and the phthalocyanine dye reactive blue 38 reached a maximum simultaneously with MnP and MIP-type activity at day 5. At this time point approximately 60% of the added dye was decolorized. 3.2. Puri¢cation of B. adusta LiP and MnP The cultures were harvested at day 5 after inoculation, extracellular proteins were concentrated by adsorption to Q Sepharose, and fractionated by anion exchange chromatography as shown in Fig. 1A. Fractions containing most LiP (86^109 ml) and MnP (110^125 ml) activities were pooled separately for further puri¢cation. Fractions between 45 and 75 ml contained MIP activity with DMP, but no activity with veratryl alcohol. Most of the dye-decolorizing activity with reactive violet 5 and reactive blue 38 was found at 110^125 ml, together with MnP activity. The puri¢cation of LiP isoenzymes was completed by Mono Q chromatography at pH 4.6, as shown in Fig. 1B. MnP isoenzymes could not be separated using similar chromatographic conditions, but it was accomplished using an extremely shallow gradient, as shown in Fig. 1C. Puri¢cation was completed by rechromatography under the same conditions (Fig. 1D). The homogeneity of the iso-

Fig. 1. Puri¢cation of LiP and MnP isoenzymes from Bjerkandera adusta DSM 11310. A: Anion exchange chromatography of the extracellular proteins (Mono Q, 20 mM histidine bu¡er, pH 6). B: Puri¢cation of LiP1 and LiP2 isoenzymes from LiP-containing fractions in A (Mono Q, 20 mM histidine bu¡er, pH 4.6). C: Puri¢cation of MnP1 and MnP2 isoenzymes from MnPcontaining fractions in A (Mono Q, 10 mM tartrate bu¡er, pH 4.6). D: Mono-Q rechromatography of isoenzyme MnP1 from C. Absorbances at 405 or 410 nm (solid line), pro¢les of LiP (b), MnP (E) and MIP activity (O), and NaCl gradient (dotted line) are shown.

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N-deglycosylation the mass of MnP1 was reduced to 42.7 kDa, revealing 3% N-linked carbohydrate. Isoelectric points of 3.45 for MnP1, and 3.35 for MnP2 were estimated from analytical IEF (Fig. 2B). The molecular mass of the two LiP isoenzymes was 48 kDa for the native enzymes and 45.5 kDa for the N-deglycosylated proteins, corresponding to 5% N-linked carbohydrate. The isoelectric points were estimated to be 3.1 for LiP1 and 3.0 for LiP2 (Fig. 2C). 3.4. Amino acid composition and N-terminal sequence The following amino acid compositions (residues/ molecule) were obtained for the B. adusta peroxidases (MnP1, LiP2): Asx (49, 56), Thr (26, 41), Ser (30, 19), Glx (39, 39), Gly (37, 39), Ala (47, 60), Cys (6, 6), Val (24, 22), Met (6, 10), Ile (19, 21), Leu (27, 30), Phe (28, 36), His (9, 9), Lys (8, 7), Arg (10, 11), Pro (35, 35) and Tyr (0, 0) (Trp was not determined). No appreciable di¡erences were found between isoenzymes of LiP or MnP. The N-terminal sequences (25 residues) for LiP1, LiP2 and MnP1 from B. adusta were identical (and no di¡erences were found after partial sequencing, nine amino acids, of MnP2). The sequences are shown in Table 2, together with those of MnP and LiP isoenzymes from other fungi. The residues in positions 3, 15 and 16 probably correspond to cysteines, not detected during sequencing, since three Cys residues are conserved in other MnP and LiP isoenzymes at these positions. Fig. 2. Estimation of molecular masses (A) and isoelectric points (B, C) of Bjerkandera adusta DSM 11310 MnP and LiP isoenyzmes. A: SDS-PAGE of puri¢ed MnP1 (lane 1), MnP2 (lane 2), LiP1 (lane 3), LiP2 (lane 4) and Pharmacia LMW markers (M). B : IEF of MnP1 and MnP2 (pH range 2.5^5, measured with a contact electrode). C: IEF of LiP1 and LiP2 (pH range 2^4). The calibration lines used for isoelectric point calculation are shown below the gels.

cluding isoenzyme yield at the di¡erent steps is shown in Table 1. 3.3. Physical properties The molecular mass of the two MnP isoenzymes was estimated by SDS-PAGE to be 44 kDa. After

3.5. Catalytic properties The MnP isoenzymes exhibited both Mn2‡ -oxidizing and Mn2‡ -independent activities on the substrates DMP and veratryl alcohol. The pH optima for MnP1 enzymatic activities were 5 for oxidation of Mn2‡ , 4.5 for Mn2‡ -dependent oxidation of DMP, and 3 for Mn2‡ -independent oxidation of DMP or veratryl alcohol. The catalytic constants with various substrates are shown in Table 3. Because of the high Km for veratryl alcohol oxidation by MnP, the LiP-type activity of this enzyme was underestimated during the puri¢cation, using a substrate concentration of 2 mM.

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Table 1 A summary of LiP and MnP puri¢cation from Bjerkandera adusta DSM 11310 Protein (mg)

MnP-type activitya Total activity (U)

Culture liquid Q Sepharose adsorption Mono Q (pH 6) : LiP pool MnP pool From LiP pool: LiP1 LiP2 From MnP pool: MnP1 MnP2

^c 10.2

211 172

LiP-type activityb

Speci¢c activity (U mg31 )

Yield (%)

^ 16.9

100 81.5

9.53 73.4

^ 59.1

Total activity (U)

Speci¢c activity (U mg31 )

Yield (%)

0d 51.6

^

^ 100

25 7

14.7 4.12

48.5 13.6

5.06

1.7 1.7

16.2 124.8

0.34 0.22

^ ^

6 0.8 6 0.8

^ ^

8.2 6.1

24.2 27.7

15.9 11.8

0.43 0.11

34.3 8.1

79.8 76.4

16.2 3.8

1.55 0.33

3.6 3.1

3 0.6

a

Estimated by Mn2‡ -dependent oxidation of DMP at pH 4.5 (corrected for MIP activity). Estimated by oxidation of veratryl alcohol at pH 3. c Dashes: not determined. d LiP activity was not detectable in the culture. b

The pH optima for oxidation of veratryl alcohol by LiP1 and LiP2 were 3.25 and 3.1, respectively. With ABTS the optimum of both isoenzymes was at pH 3. The catalytic constants are given in Table 3. The Km and Vmax values obtained for the two isoenzymes of LiP were very similar. However, LiP2 had a lower Km for H2 O2 and a higher Vmax with ABTS than LiP1.

4. Discussion This is the ¢rst report describing the isolation and characterization of a MnP from B. adusta. After its description in P. chrysosporium [7] MnP isoenzymes were characterized from several white-rot fungi including Trametes versicolor [14,21,22] and Pleurotus species [13,23]. With isoelectric points of 3.45 and

Table 2 Comparison of N-terminal sequences of MnP (A) and LiP (B) isoenzymes from Bjerkandera adusta DSM 11310 and other fungi A

B

5 10 15 20 25 VAXPDGVNTATNAAXXALFAVRDDI VACPDGVNTATNAACCQLFAVRIDD ATCADGRTTA-NAACCVLFPILDDI ATCDDGRTTA-NAACCILFPILDDI AVCPDGTRV-SHAACCAFIPLAQDL AVGSDGTVVP-DSVQYDFIPLAQDL VTXSDGTAVP-

Bjerkandera adusta DSM 11310 MnP1 Trametes versicolor MPG 1 [14] Pleurotus eryngii MnPL1 (GenBank AF007221) Pleurotus eryngii MnPL2 (GenBank AF007222) Phanerochaete chrysosporium MnP1 [15] Lentinula edodes MnP [16] Poria subvermispora MnP [17]

VAXPDGVNTATNAAXXXLFAVRDDI VACPDGKNTAINAACCSLFTARDDI VACPDGRNTAINA VACPDGVNTATNAACCQLFAVREDL VACPDGVHTASNAACCAWFPVLDDI ATCSNGK-VVPAASCCTWFNVLSDI ATCPDGT-QLMNAECCALLAVRDDL

Bjerkandera adusta DSM 11310 LiP1/LiP2 Bjerkandera adusta IFO 5307 LiP1 [18] Bjerkandera adusta IFO 5307 LiP2 [18] Trametes versicolor LiPG IV [14] Phanerochaete chrysosporium LiPH2 [19] Phanerochaete chrysosporium LiPH8 [19] Phlebia radiata LiP [20]

Identical residues at the same position are indicated in bold type (dashes indicate gaps introduced to maximize alignment).

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Table 3 Kinetic constants of MnP and LiP isoenzymes from Bjerkandera adusta DSM 11310a pH

Vmax (Wmol min31 mg31 )

Vmax /Km

2

1

2

1

2

5.5 20 15 8.5 160 4000

4 22 ^b ^ 180 4500

140 180 45 50 28 18

120 180 ^ ^ 18 16

^ 9 ^ ^ 0.18 0.0045

^ 8.2 ^ ^ 0.1 0.0036

58 170 210

38 150 190

38 32 37

39 33 47

^ 0.19 0.18

^ 0.22 0.25

Km (WM) 1

A: MnP isoenzymes H2 O2 (0.1 mM Mn2‡ ) Mn2‡ (0.1 mM H2 O2 ) Mn2‡ (0.1 mM DMP, 0.1 mM H2 O2 ) DMP (0.1 mM Mn2‡ , 0.1 mM H2 O2 ) DMP (0.1 mM H2 O2 ) Veratryl alcohol (0.1 mM H2 O2 ) B: LiP isoenymes H2 O2 (2 mM veratryl alcohol) Veratryl alcohol (0.1 mM H2 O2 ) ABTS (0.1 mM H2 O2 ) a b

5 5 5 5 3 3 3.25 3.25 3

Estimated in 0.1 M tartrate at the pH indicated (other reaction components shown in parentheses). Dashes: not determined.

3.35, the two B. adusta MnP are more acidic than most MnP isoenzymes (showing isoelectric points between 2.9 and 5.3). Their molecular mass is in the range of most fungal MnP. In contrast to the wellstudied MnP isoenzymes from P. chrysosporium [9], the B. adusta MnP1 and MnP2 exhibit manganeseindependent activity on aromatic substrates, such as veratryl alcohol and DMP. However, the catalytic e¤ciency (Vmax /Km ) is much higher with Mn2‡ than with the aromatic substrates. In the case of veratryl alcohol the low catalytic e¤ciency is caused by a high Km value compared to LiP. On the other hand, with DMP the catalytic e¤ciency of B. adusta MnP is in the same order of magnitude as described for LiP and aromatic substrates. With respect to these unusual catalytic properties B. adusta MnP is similar to recently described MnP from Pleurotus species [13,23]. LiP isoenzymes also have been characterized from several white-rot fungi [5,6,18,21,22]. The two LiP isoenzymes puri¢ed here are similar to LiP isoenzymes from P. chrysosporium [24] with respect to their catalytic properties and molecular mass. However, with isoelectric points of 3.1 and 3.0, the B. adusta LiP isoenzymes are more acidic than those from other fungi (showing isoelectric points between 3.1 and 4.7). Moreover, a lower isoelectric point and di¡erent N-terminal sequences indicate that the isoenzymes isolated here are di¡erent from the LiP isoenzyme with isoelectric point of 4.2 isolated by Ki-

mura et al. [18,25] from another B. adusta strain grown on glucose-peptone medium. The MnP and LiP isoenzymes from B. adusta differed signi¢cantly in their a¤nity to H2 O2 resulting in Km values of 4^5.5 WM (MnP) or 38^58 WM (LiP). N-terminal sequencing (25 residues) revealed that B. adusta MnP and LiP have high similarity to MnP (1^2 di¡erent residues) and LiP (4^5 di¡erent residues) from T. versicolor [14,22] (see Table 2). The Nterminal sequence of B. adusta MnP1 di¡ers in 10 residues from the catalytically similar P. eryngii MnPL1 and MnPL2, but the N-termini of P. chrysosporium MnP isoenzymes are even less related. On the other hand, P. chrysosporium LiP H2 has a relatively similar N-terminal sequence to that of B. adusta LiP, whereas that of LiP H8 is extremely different. In spite of the limited sequence information it can be noted that similarities of the N-termini of peroxidases are in accordance with the classi¢cation in di¡erent taxa of the genera producing them. Bjerkandera, Pleurotus and Trametes belong to Poriales, whereas Phanerochaete and Phlebia belong to Stereales and Lentinula to Agaricales [26]. MnP and LiP have been proposed to form two separate families within the plant peroxidase superfamily [27] due to the low amino acid identities (below 50%) encountered between P. chrysosporium MnP and LiP genes. Surprisingly, the 25 N-terminal amino acids of B. adusta MnP1 and LiP isoenzymes isolated here were identical, indicating exceptionally

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high similarity between the two types of peroxidases in B. adusta. However, these LiP and MnP di¡ered signi¢cantly in other properties, such as isoelectric point, serine and threonine content and catalytic properties. In conclusion, we have identi¢ed and characterized the peroxidases produced by B. adusta under the culture conditions used for dye decolorization [11]. The relevance of these peroxidases for the versatile degrading abilities of B. adusta remains to be investigated. In this respect the MnP isoenzymes are of special interest. Their ability to catalyze Mn2‡ -mediated as well as Mn2‡ -independent reactions may serve for di¡erent functions than those described for P. chrysosporium MnP both in lignin biodegradation and biotechnological applications.

[8]

[9]

[10]

[11]

[12]

Acknowledgments [13]

The authors thank R. Opitz (TU Berlin) for skilful technical assistance and J. Varela (CIB, Madrid) for amino acid and N-terminal analyses. This work has been partially funded by the Deutsche Forschungsgemeinschaft, Sfb 193 and the Gesellschaft von Freunden der TU Berlin e.V.

[14]

[15]

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[21] Johansson, T. and Nyman, P.O. (1993) Isoenzymes of lignin peroxidase and manganese(II) peroxidase from the white-rot basidiomycete Trametes versicolor. 1. Isolation of enzyme forms and characterization of physical and catalytical properties. Arch. Biochem. Biophys. 300, 49^56. [22] Johansson, T., Welinder, K.G. and Nyman, P.O. (1993) Isoenzymes of lignin peroxidase and manganese(II) peroxidase from the white-rot basidiomycete Trametes versicolor. 2. Partial sequences, peptide maps, and amino acid and carbohydrate compositions. Arch. Biochem. Biophys. 300, 57^62. [23] Mart|ènez, M.J., Boëckle, B., Camarero, S., Ruiz-Duenìas, F.J., Guilleèn, F.A. and Mart|ènez, A.T. (1996) MnP Isoenzymes produced by two Pleurotus species in liquid culture and during wheat-straw solid-state fermentation. ACS Symp. Ser. 655, 183^196.

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