Lentinula edodes produces a multicomponent protein complex containing manganese (II)-dependent peroxidase, laccase and β-glucosidase

FEMS Microbiology Letters 200 (2001) 175^179

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Lentinula edodes produces a multicomponent protein complex containing manganese (II)-dependent peroxidase, laccase and L-glucosidase Randhir S. Makkar a , Akihiko Tsuneda b , Ken Tokuyasu a , Yutaka Mori b

a;

*

a National Food Research Institute, Tsukuba, Ibaraki 305-8642, Japan Northern Forestry Centre, Canadian Forest Service, Edmonton, AB, Canada T6H 3S5

Received 20 January 2001 ; received in revised form 24 March 2001; accepted 26 March 2001 First published online 30 May 2001

Abstract A multicomponent protein complex containing manganese (II)-dependent peroxidase, laccase and L-glucosidase was isolated from culture extracts of the white rot basidiomycete Lentinula edodes. This protein complex showed a single protein band on native polyacrylamide gel electrophoresis (PAGE). On sodium dodecyl sulfate (SDS)^PAGE, however, it displayed three major bands and several additional minor bands ranging in size from 60 kDa to 180 kDa, suggesting it being a complex of six to eight different proteins. The molecular mass of this complex was estimated to be approximately 660 kDa from the elution position of gel filtration. This enzyme complex was effective in transforming environmentally persistent xenobiotics, pentachlorophenol and 2,5-dichlorophenol. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Manganese peroxidase ; Laccase; L-Glucosidase ; Lentinula edodes

1. Introduction The white rot basidiomycete Lentinula edodes (Berk.) Pegler (the edible shiitake mushroom) is the most widely cultivated mushroom in East Asia and its cultivation is regarded as the largest bioconversion process utilizing wood [1]. L. edodes is an active ligno-cellulolytic organism capable of degrading individual components of ligno-cellulose, i.e. lignin, cellulose and hemicellulose, by secreting an array of oxidative and hydrolytic enzymes [2]. Laccases (EC 1.10.3.2) and manganese (II)-dependent peroxidases

* Corresponding author. Japan International Research Center for Agricultural Sciences, Tsukuba, Ibaraki 305-8686, Japan. Tel. : +81 (298) 38-6330; Fax: +81 (298) 38-6316; E-mail : ymori@a¡rc.go.jp Abbreviations : MnP, manganese (II)-dependent peroxidase ; PCP, pentachlorophenol ; DCP, dichlorophenol ; PVP, polyvinylpyrrolidone; TMBZ, 3P,3P,5P,5P-tetramethylbenzidine; ABTS, 2,2P-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid ; pNPG, p-nitrophenyl L-D-glucopyranoside ; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis ; TBP, tribromophenol

(MnPs, EC 1.11.1.7) are one group of enzymes that are responsible for the degradation of lignin by the fungus [3]. Recently these enzymes have been attracting wide attention because of their ability to degrade environmentally persistent xenobiotics such as pentachlorophenol (PCP) and dioxins [4,5]. MnP in particular is considered to be the key enzyme for degradation of these pollutants [6]. Studies by Valli and Gold [7] have shown involvement of MnP in the multistep pathway for dichlorophenol (DCP) degradation. Grabski et al. [8] employed immobilized MnP from L. edodes in a two-stage MnP bioreactor for catalytic generation of chelated Mn3‡ and subsequent oxidation of chlorophenols. Until now MnPs with 44.6 kDa and 59 kDa have been reported from di¡erent strains of L. edodes [1,9], though at least four MnP isozymes have been identi¢ed in the white rot basidiomycetous fungus, Phanerochaete crysosporium [10]. In the course of our search for new MnPs produced by L. edodes, we found out a high molecular mass enzyme complex possessing MnP, laccase and L-glucosidase (EC 3.2.1.21) activities in culture extracts. In this communication we report the isolation and properties of the enzyme complex.

0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 1 5 9 - 8

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2. Materials and methods 2.1. Organism and culture conditions L. edodes dikaryotic strain TMI 800 (Tottori Mycological Institute, Tottori, Japan) was used throughout the study. Cultures, which had been grown at 20³C for 4 weeks in plastic bottles (9U20 cm) containing a commercial medium consisting of rice bran and beech sawdust, were purchased from Kinko Shiitake Agricultural Cooperative, Tottori, Japan. The cultures were further incubated with occasional aeration through the holes on the cap at 25³C for 40 days before use. 2.2. Preparation of extracts Mycelium with the medium in its vicinity was separated from the rest of the medium and extracted with 1:2.5 (w/v) 50 mM triethanolamine^maleic bu¡er, pH 6.0, with stirring. The extraction step was repeated for a total of ¢ve times. Extracts thus obtained were pooled and ¢ltered through a glass ¢lter. Resulting ¢ltrate was centrifuged at 9150Ug for 25 min. Coloring materials mainly consisting of polyphenolic compounds in the supernatant [9] were removed by addition of cross-linked form polyvinylpyrrolidone (PVP) at a ¢nal concentration of 7.7% (w/v) [11]. The extracts thus obtained were ¢ltered through a glass ¢lter and processed for the second PVP treatment followed by centrifugation (9150Ug, 25 min) to remove ¢ne PVP particles. The decolorized extracts were concentrated using PM 10 membrane (Amicon Scienti¢c Systems, Lexington, MA, USA) and ¢ltered through Millipore ¢lters (pore size, 0.22 Wm). Filtered samples were ¢nally ultracentrifuged (60 000Ug, 60 min) in a Beckman XL-70 ultracentrifuge. Samples were processed at 4³C in all steps. 2.3. Enzyme assays Laccase, peroxidase and MnP activities were determined at 37³C by measuring the oxidation of either 3P,3P,5P,5Ptetramethylbenzidine (TMBZ) or 2,2P-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) in the presence or absence of H2 O2 and Mn2‡ . Laccase activity using TMBZ as the substrate was assayed in reaction mixtures (2 ml) containing 1 mM TMBZ and appropriate amount of enzyme in 50 mM McIlvaine bu¡er, pH 4.0. Oxidation of TMBZ was measured by monitoring the increase in absorbance at 650 nm [12]. Laccase activity with ABTS as the substrate was measured by incubating 3 ml of reaction mixture containing 0.03% (w/v) ABTS with appropriate amount of enzyme in the McIlvaine bu¡er and oxidation of ABTS was followed by absorbance increase at 420 nm [1]. Peroxidase activity was determined by adding to the laccase assay solutions H2 O2 to 80 WM ¢nal concentration and subtracting the increase in absorbance caused by laccase activity. MnP activity was measured after adding

both 80 WM H2 O2 and 100 WM MnSO4 W5H2 O to the reaction mixtures, with correction by subtracting the activity without added Mn2‡ . One unit of enzyme activities with TMBZ was de¢ned as the amount of enzyme which catalyzes the oxidation of TMBZ so that the change in A650 is 0.01 per min [12]. When ABTS was used as the substrate, one unit of enzyme activities was expressed as the amount of enzyme required to oxidize 1 Wmol ABTS per min using O420 value for oxidized ABTS of 3.6U104 M31 cm31 [13]. L-Glucosidase activity was assayed by using 2-ml reaction mixtures containing 10 mM p-nitrophenyl L-D-glucopyranoside (pNPG) in 50 mM McIlvaine bu¡er, pH 4.0, and appropriate amount of enzyme. After 30 min of incubation at 37³C, the reaction was stopped by adding 3.0 ml of 1 M sodium carbonate and the p-nitrophenol release was measured with the help of spectrophotometer at 400 nm. One unit of L-glucosidase is de¢ned as the amount that produced 1 Wmol p-nitrophenol per min. 2.4. Puri¢cation of the enzyme complex Ten milliliter (40 mg protein) of the ¢nal extract was applied to a Q Sepharose (Pharmacia) anion exchange column (2.5U18 cm) equilibrated with 50 mM bis-Tris^ HCl, pH 6.8. MnP activity was eluted with the same bu¡er by a stepwise gradient of 0, 0.1, 0.5 and 1 M NaCl concentration at a £ow rate of 3.5 ml min31 and collected in 14-ml fractions. Fractions eluted with 1 M NaCl were pooled and concentrated and loaded onto a preparative TSK 4000 gel ¢ltration column (5.5U60 cm) (Tosoh Corporation, Tokyo, Japan) equilibrated with 50 mM sodium acetate bu¡er, pH 5.0, containing 100 mM Na2 SO4 . Elution was performed with the same bu¡er at a £ow rate of 7 ml min31 and 10.5-ml fractions were collected. The fractions of peak III were pooled and used for further analysis. Protein content was measured by the method of Bradford [14] with bovine serum albumin as the standard. 2.5. Polyacrylamide gel electrophoresis (PAGE) PAGE was carried out in the presence and absence of sodium dodecyl sulfate (SDS) using premade gels (ATTO Co, Tokyo, Japan) according to the supplier's instructions. Gels were stained for protein with the silver staining kit of Bio-Rad (Hercules, CA, USA). 2.6. Transformation of PCP and 2,5-DCP Reaction mixtures (4 ml) to measure the chlorophenol transformation consisted of 50 mM McIlvaine bu¡er, pH 4.0, 50 WM PCP or 2,5-DCP, 100 WM MnSO4 , 80 WM H2 O2 and the enzyme preparation containing 2.9 units of MnP and 1.7 units of laccase (based on TMBZ oxidation). Reaction was initiated by addition of H2 O2 . This mixture was incubated at 30³C for 16 h and the reaction was terminated by addition of 1.4 g of NaCl and 1 ml of

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hexane. Just before terminating the reaction 50 WM of tribromophenol (TBP, an internal standard) was added. The residual chlorophenols and TBP were extracted by mixing vigorously on a Vortex mixer for 30 s, after which the phases were separated by centrifugation (900Ug, 5 min). Chlorophenols in hexane extracts were analyzed by HPLC (Shimadzu LC 10) with a C-18 reverse-phase column (3.2U125 mm) packed with Enviro Sep PP (Phenomenex, Torrance, CA, USA) at 45³C. Chlorophenols were eluted from the column with a gradient of 0.1% acetic acid (A) and 0.1% acetic acid plus methanol (B) as follows (% (v/v) B min31 ): 30/0, 60/10, 100/25, 100/35 and 30/36 at a £ow rate of 0.5 ml min31 . The elution was monitored at 290 nm and quanti¢ed by peak area. 3. Results Majority (70V80%) of MnP activity present in the culture extract was eluted from a Q Sepharose column with the 0.5 M NaCl-containing bu¡er and found to reside in low molecular mass protein(s) (data not shown). In contrast, gel ¢ltration pro¢le of the pooled MnP fractions eluted with the 1 M NaCl-containing bu¡er from Q Sepharose that accounted for approximately 15% of the total activity indicated existence of four species of high molecular mass MnPs in addition to low molecular mass free enzyme(s) as judged by their elution positions (Fig. 1). Peaks I, II and IV formed more than two protein bands on native PAGE, showing those fractions were mixtures of large proteins or protein aggregates involving MnP activity. However, the combined peak III fractions (fractions 76^87 in Fig. 1) that eluted at a position comparable to that of thyroglobulin (669 kDa) exhibited only one protein band on native PAGE (Fig. 2, left), indicating the mole-

Fig. 1. TSK 4000 gel ¢ltration chromatography of the pooled MnP fractions eluted from Q Sepharose column with 1 M NaCl-containing bu¡er. MnP peaks are designated as I^V. Gel ¢ltration calibration proteins used are thyroglobulin (A, 669 kDa), aldolase (B, 158 kDa) and bovine serum albumin (C, 68 kDa). The cellulosome from Clostridium thermocellum strain YM4 (approximately 3.5 MDa) was eluted out in fraction 55. Protein measurement after fraction 99 was in£uenced by coloring materials and is not shown. MnP (b), protein (a).

Fig. 2. Native PAGE (left) and SDS^PAGE (right) of the peak III fractions (fractions 76^87 in Fig. 1 were combined). Native PAGE and SDS^PAGE were carried out in 5% (w/v) and 7.5% (w/v) polyacrylamide gels, respectively. Corresponding migration positions of molecular mass standards (myosin, 200 kDa; L-galactosidase, 116 kDa; bovine serum albumin, 66 kDa; ovalbumin, 45 kDa) are shown.

cule in this fraction is either a pure large protein or a discrete protein aggregate. Indication that L. edodes produces an unknown large mass MnP-containing protein or aggregate prompted us to its characterization since free monomeric enzymes of 44.6 kDa and 59 kDa were the only MnPs reported in L. edodes and no enzyme aggregates nor large mass proteins have been known for MnP. On subjecting the combined peak III fractions to SDS^PAGE, three major bands (72, 115, 130 kDa) and several additional minor protein bands ranging in size from 60 kDa to 180 kDa were obtained (Fig. 2, right). This suggests the molecule is a protein aggregate comprising six to eight subunits. According to the manufacturer of the silver staining kit, the white bands formed at the positions corresponding to 72 kDa and 60 kDa imply the presence of proteins containing few cysteine residues (if any). The enzyme activities of the protein aggregate were assayed on di¡erent substrates. As shown in Table 1, it oxidized TMBZ and ABTS, and with addition of H2 O2 and Mn2‡ the activity to oxidize TMBZ and ABTS increased, suggesting coexistence of laccase, peroxidase and MnP in the aggregate. However, oxidation of Veratryl alcohol, a good substrate for lignin peroxidase, was not observed even in the presence of H2 O2 . The complex had a high activity to hydrolyze pNPG (Table 1) but did not hydrolyze pNP L-D-cellobiopyranoside, pNP L-D-xylopyranoside and carboxymethylcellulose. As shown in Fig. 3, maximum activities were observed between pH 3.5 and 4.5 for laccase and at pH 5.0 for MnP. Laccase activity fell to

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Table 1 Enzyme activities of the 660-kDa complexa Substrateb

Co-substrate

Activity type

Units mg31c

TMBZ TMBZ TMBZ ABTS ABTS ABTS pNPG

^ H 2 O2 H2 O2 , Mn‡2 ^ H 2 O2 H2 O2 , Mn‡2 ^

laccase peroxidase manganese peroxidase laccase peroxidase manganese peroxidase L-glucosidase

39.1 23.3 66.6 11.3 0.6 10.2 240

a

Activities were assayed in 50 mM McIlvaine bu¡er, pH 4.0. No activities were observed towards veratryl alcohol, pNP L-D-cellobiopyranoside, pNP L-D-xylopyranoside and carboxymethylcellulose. c Units of enzymes were de¢ned as described in Materials and Methods. b

zero at pH 6.0, whereas MnP was active in a broad pH range. L-Glucosidase had a relatively sharp pH optimum of 4.0. We tried to assign laccase, MnP and L-glucosidase activities to the subunits separated on SDS^PAGE by activity staining using TMBZ, ABTS and 4-methylumbelliferyl L-D-glucopyranoside as the substrates but no activities were detected even with the sample treated with SDS at 60³C presumably due to the inactivation of the subunit enzymes. It was demonstrated that PCP and, to a lesser extent, 2,5-DCP in the McIlvaine bu¡er were decreased by incubation with the 660-kDa enzyme complex for 16 h at 30³C (Table 2). 4. Discussion In this study, it was shown that at least four large mass protein aggregates (approximately 300 kDaV4 MDa) possessing MnP activity are formed in the L. edodes culture grown on commercial wood substrate for the ¢rst time. One of such aggregates with a molecular mass of approximately 660 kDa was isolated and found to be an

enzyme complex consisting of six to eight subunits having four types of enzyme activities, i.e. laccase, peroxidase, MnP, and L-glucosidase. However, in some cases it is not possible to make a clear-cut distinction between laccases and peroxidases based solely on their ability to use H2 O2 as an oxygen donor [15]. In fact our data show that stimulation in oxidative activity by H2 O2 with ABTS as the substrate is almost negligible (Table 1). Therefore, we judge that the complex most probably contains L-glucosidase, MnP and laccase that is stimulated by H2 O2 when TMBZ is the substrate. Although these enzyme activities are maximal at di¡erent pH values, they seem to be considerably active at the physiological pH (about 4.5) of the extracellular mycelium £uids. In£uence of pH on laccase and MnP activities in the 660-kDa complex is consistent with the results reported for the free monomeric enzymes [9,16]. The existence of large mass MnP-containing aggregates with molecular masses from approximately 300 kDa^4 MDa as indicated by TSK 4000 gel ¢ltration is in good agreement with our preliminary electron microscopic observations of spherical particles of di¡erent sizes having diameters of 10^60 nm in culture extracts of this fungus (unpublished data). It is well known that several species of anaerobic bacteria produce the cellulase/hemicellulase complexes termed the cellulosomes [17]. The cellulosomes possess extremely strong hydrolyzing activity toward crystalline cellulose that exhibits stubborn resistance to the enzymatic attack. The 660-kDa enzyme complex isolated in this study can be considered as a similar enzyme complex to the cellulosome but it di¡ers from the cellulosomes in that it contains both the hydrolytic and oxidative enTable 2 Treatment of PCP and 2,5-DCP with the 660-kDa complexa Compound

Fig. 3. E¡ects of pH on laccase (O), MnP (a) and L-glucosidase (E) activities in the 660-kDa complex. Activities were measured in 50 mM McIlvaine bu¡er at di¡erent pH values with ABTS (for laccase and MnP) and pNPG (for L-glucosidase) as the substrates. The levels of 100% activities of laccase, MnP and L-glucosidase correspond to 11.6, 11.8 and 214, respectively.

PCP DCP a

Amount (Wg ml31 ) Initial

Residual

13.3 8.2

6.4 þ 0.3 6.3 þ 0.3

Reduction (%) b

52 23

Reactions were carried out as described in Section 2. Values represent means þ the standard deviations for triplicate experiments.

b

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zymes. Organizing the complex consisting of both hydrolytic and oxidative enzymes is thought to have advantage in the degradation of obstinate ligno-cellulosic materials if it facilitates cooperative actions of these enzymes, and might be contributing to the prominent ligno-cellulose degrading ability of L. edodes. The enzyme complex was shown to have capability of transforming PCP and 2,5-DCP and thus seems to be a promising agent for bioremediation of the environment polluted with those xenobiotics, but it is necessary to identify the transformation products and con¢rm their safety before being put to practical use. In order to elucidate the function and signi¢cance of the complex, enzymatic activities and the structural role of the individual subunits and the e¡ect of the complex formation should be ¢rst clari¢ed through dissociation and reassociation studies. Acknowledgements This work was supported in part by a grant (Bio Design Program) from the Ministry of Agriculture, Forestry and Fisheries of Japan. References [1] Buswell, J.A., Cai, Y. and Chang, S.T. (1995) E¡ect of nutrient nitrogen and manganese on manganese peroxidase and laccase production by Lentinula (Lentinus) edodes. FEMS Microbiol. Lett. 128, 81^88. [2] Leatham, G.F. (1985) Extracellular enzymes produced by the cultivated mushroom Lentinus edodes during degradation of lignocellulosic medium. Appl. Environ. Microbiol. 50, 859^867. [3] Hatakka, A. (1994) Lignin-modifying enzymes from selected whiterot fungi: production and role in lignin degradation. FEMS Microbiol. Rev. 13, 125^188. [4] Bumpus, J.A., Tien, M., Wright, D. and Aust, S.D. (1985) Oxidation of persistent environmental pollutants by a White Rot Fungus. Science 228, 1434^1436.

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[5] Valli, K., Wariishi, H. and Gold, M.H. (1992) Degradation of 2,7dichlorodibenzo-p-dioxin by lignin-degrading basidiomycete Phanerochaete chrysosporium. J. Bacteriol. 174, 2131^2137. [6] Reddy, C.A. (1995) The potential for white-rot fungi in the treatment of pollutants. Curr. Opin. Biotechnol. 6, 320^328. [7] Valli, K. and Gold, M.H. (1991) Degradation of 2,4-dichlorophenol by the lignin-degrading fungus Phanerochaete chrysosporium. J. Bacteriol. 173, 345^352. [8] Grabski, A.C., Grimek, H.J. and Burgess, R.R. (1998) Immobilization of manganese peroxidase from Lentinula edodes and its biocatalytic generation of Mn(III)-chelate as a chemical oxidant of chlorophenols. Biotechnol. Bioeng. 60, 204^215. [9] Forrester, I.T., Grabski, A.C., Mishra, C., Kelly, B.D., Strickland, W.N., Leatham, G.F. and Burgess, R.R. (1990) Characteristics and N-terminal amino acid sequence of a manganese peroxidase puri¢ed from Lentinus edodes cultures grown on a commercial wood substrate. Appl. Micrbiol. Biotechnol. 33, 359^365. [10] Kirk, T.K., Croan, S., Tien, M., Murtagh, K.E. and Farrell, R.L. (1985) Production of multiple ligninases by Phanerochaete chrysosporium: e¡ect of selected growth conditions and use of a mutant strain. Enzyme Microb. Technol. 8, 27^32. [11] Loomis, W.D. (1969) Removal of phenolic compounds during the isolation of plant enzymes. Methods Enzymol. 13, 555^563. [12] Okeke, B.C., Paterson, A., Smith, J.E. and Watson-Craik, I.A. (1994) The relationship between phenol oxidase activity, soluble protein and ergosterol with growth of Lentinus species in oak sawdust logs. Appl. Micrbiol. Biotechnol. 41, 28^31. [13] Bourbonnais, R. and Paice, M.G. (1988) Veratryl alcohol oxidases from the lignin-degrading basidiomycete Pleurotus sajor-caju. Biochem. J. 255, 445^450. [14] Bradford, M.M. (1976) A rapid and sensitive method for the quanti¢cation of microgram quantities of protein utilizing the principle of the protein dye binding. Anal. Biochem. 72, 248^254. [15] Jong, E.D., Field, J.A. and de Bont, J.A.M. (1992) Evidence for a new extracellular peroxidase :manganese-inhibited peroxidase from the white-rot fungus Bjerkandera sp. BOS 55. FEBS Lett. 299, 107^ 110. [16] Kofujita, H., Ohta, T., Asada, Y. and Kuwahara, M. (1991) Puri¢cation and characterization of laccase from Lentinus edodes. Mokuzai Gakkaishi 37, 562^569. [17] Bayer, E.A., Chanzy, H., Lamed, R. and Shoham, Y. (1998) Cellulose, cellulases and cellulosomes. Curr. Opin. Struct. Biol. 8, 548^557.

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