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Expression of Pleurotus eryngii aryl-alcohol oxidase in Aspergillus nidulans: purification and characterization of the recombinant enzyme
Biochimica et Biophysica Acta 1546 (2001) 107^113 www.bba-direct.com
Expression of Pleurotus eryngii aryl-alcohol oxidase in Aspergillus nidulans: puri¢cation and characterization of the recombinant enzyme è ngel T. Mart|¨nez, Mar|¨a Jesu¨s Mart|¨nez * Elisa Varela, Francisco Guille¨n, A Centro de Investigaciones Biolo¨gicas, Consejo Superior de Investigaciones Cient|¨¢cas, Vela¨zquez 144, E-28006 Madrid, Spain Received 11 August 2000; received in revised form 19 December 2000; accepted 20 December 2000
Abstract Aryl-alcohol oxidase (AAO) is an extracellular flavoenzyme involved in lignin biodegradation by some white-rot fungi. The enzyme catalyzes the extracellular oxidation of aromatic alcohols to the corresponding aldehydes. The electron acceptor is molecular oxygen yielding H2 O2 as the product. Herein we describe, for the first time, the expression of AAO from Pleurotus eryngii in the ascomycete Aspergillus nidulans. The activity of the recombinant enzyme in A. nidulans cultures is much higher than found in the extracellular fluid of P. eryngii. The recombinant enzyme showed the same molecular mass, pI and catalytic properties as that of the mature protein secreted by P. eryngii. The enzymic properties are also similar to those reported from other Pleurotus and Bjerkandera species. ß 2001 Elsevier Science B.V. All rights reserved. Keywords: Aryl-alcohol oxidase; Pleurotus; Heterologous expression; Aspergillus
1. Introduction Lignin biodegradation is an oxidative process carried out predominantly by white-rot fungi. These fungi are able to degrade this polymer to CO2 and H2 O [11,22]. Of practical signi¢cance is the use of these fungi in lignocellulose utilization. Pleurotus eryngii and related species have been characterized mainly for their ability to degrade wheat lignin preferentially with a limited attack to cellulose [24]. This is an important characteristic for biotechnological applications related to feed production [21] and paper manufacturing [10]. White-rot fungi produce dif-
* Corresponding author. Fax: +34-91-562-7518; URL address: http://www.cib.csic.es/Vlignina/lignina_es.html ; E-mail: [email protected]
ferent extracellular ligninolytic enzymes, which include laccases and peroxidases, and oxidases [19]. Pleurotus species have been shown to secrete laccase, Mn-oxidizing peroxidase and aryl-alcohol oxidase (AAO) under liquid and solid state fermentation conditions [6,24,25]. Pleurotus Mn-oxidizing peroxidase is a new type of ligninolytic enzyme which shares catalytic properties with the two ligninolytic peroxidases ¢rst reported in Phanerochaete chrysosporium, lignin peroxidase (LiP) and Mn-dependent peroxidase (MnP) [26]. AAO has been isolated and characterized from di¡erent Pleurotus species [3,16,31], Bjerkandera adusta [29] and P. chrysosporium [1]. The P. eryngii enzyme has been most extensively characterized and exhibits a wide substrate speci¢city, being capable of oxidizing benzyl, cinnamyl, naphthyl and aliphatic unsaturated primary alcohols [16]. AAO is involved
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in redox cycling where this enzyme oxidizes extracellularly aromatic alcohols to the corresponding aldehydes yielding H2 O2 . The aldehyde is then reduced by intracellular enzymes again yielding the alcohol substrate for AAO [14,17]. In accord with this is the ¢nding that many fungi synthesize aryl compounds; this has been reported in P. chrysosporium [20], B. adusta [23] and Pleurotus species [18]. One aldehyde that is produced is p-anisaldehyde with high levels reported in B. adusta and Pleurotus species. The high levels of p-anisaldehyde in Pleurotus and Bjerkandera is related with the high AAO a¤nity for p-anisyl alcohol [16,29]. In addition to AAO, intracellular aryl-alcohol dehydrogenase, ¢rst characterized from P. chrysosporium [28] and later reported from Bjerkandera and Pleurotus species [8,14,39], is also involved in these redox cycling reactions. Two roles have been proposed for AAO in lignin degradation. The ¢rst is for H2 O2 production. The H2 O2 can be used by peroxidases. Such is the case for Pleurotus pulmonarius where the high rate of lignin mineralization of wheat straw correlates with the high level expression of both AAO and Mn-oxidizing peroxidase [5]. Alternatively, H2 O2 has also been suggested to be the precursor to hydroxyl radical, also proposed to be involved in the initial attack of lignin [2,12]. The cooperation of AAO and laccase for the production of hydroxyl radical has been recently demonstrated [15].The second role proposed for AAO is for the reduction of phenoxy radicals. This in turn prevents the polymerization of lignin during its biodegradation by ligninolytic enzymes, as reported in Pleurotus ostreatus [27]. Recently, gene cloning of AAO from P. eryngii and P. pulmonarius have been reported [38,40]. The aim of the present work is to express the fungal AAO in the ascomycete Emericella nidulans (conidial state Aspergillus nidulans). We have utilized the strong alcA promoter for high level expression which is required for di¡erent biotechnological applications. 2. Materials and methods 2.1. Oligonucleotides Oligos ENt and EACt were synthesized based on the reported sequences of AAO of P. eryngii [40]
(GenBank sequences 64069). They were prepared with a Beckman-oligo 1000M synthesizer, and used as primers in automatic sequencing and for polymerase chain reaction (PCR) ampli¢cation. ENt (5PATGTCGTTTGGTGCACTT-3P) contained the ATG codon, followed by the ¢ve initial codons of the AAO cDNA. EACt (5P-CCCTACTGATCAGCC-3P) contained the complementary sequence of the ¢ve last codons of the AAO cDNA. 2.2. DNA ampli¢cation and plasmid construction A 1779 bp fragment of AAO cDNA was ampli¢ed by PCR using oligos ENt and EACt. One Wg of template, consisting of pBSK þ containing AAO cDNA cloned into the EcoRI site, 40 pmol of each oligo and 2.5 U of Pfu (Stratagene) were used for DNA ampli¢cation using Perkin^Elmer Gene Amp System 2400. A ¢rst cycle of 5 min at 94³C was followed by 30 cycles of 1 min at 94³C, 1 min at 58³C, 2 min at 72³C and a ¢nal step of 10 min at 72³C. 2.3. Recombinant plasmid construction For subcloning, the 1779 bp DNA fragment obtained by PCR was phosphorylated with T4 polynucleotide kinase (Biolabs). This DNA fragment was cloned into the SmaI site of pALCA vector containing the alcohol dehydrogenase promoter of Aspergillus nidulans. The vector also contained the agrB gene as a selectable marker and the trpC transcriptional terminator [13]. The resulting plasmid is referred as pALAAO. 2.4. Expression hosts and transformation procedures A. nidulans biA1, metG1, argB2 [13] was transformed with pALAAO for high-level expression. Protoplasts, used for transformation, were obtained from shaking cultures. Brie£y, A. nidulans was grown in minimal medium [7], supplemented with 10 Wg/l Dbiotin, 74.5 mg/l L-methionine and 530 mg/l L-arginine, at 25³C and 180 rpm. Minimal medium contained 10 g/l glucose, 0.92 g/l ammonium tartrate and 20 ml/l of a 50U salts solution (which included 26 g/l KCl, 26 g/l MgSO4 c7H2 O, 76 g/l KH2 PO4 , and 50 ml/l of a trace elements solution containing 40 mg/l
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Na2 B4 O7 c10H2 O, 400 mg/l CuSO4 c5H2 O, 800 mg/l Fe2 (SO4 )3 c2H2 O, 800 mg/l Na2 MoO4 c2H2 O and 8 mg/l ZnSO4 c7H2 O) (pH 6.8). The medium was inoculated with 5U106 conidia/ml (¢nal) and incubated 12 h at 30³C and 180 rpm. The mycelia were then harvested by ¢ltration, washed with 600 mM MgSO4 and protoplast obtained and transformed as described by Tilburn et al. [36]. Hybridizations were carried out according with the method of Southern [34]. A 3.9 kb SalI^PstI DNA fragment was used as a probe for the aao gene [40] and a 1.4 kb HindIII DNA fragment (argB probe) in Southerns to determine the site of integration in the A. nidulans genome. Probes were labeled using the Rediprime DNA labeling kit (Amersham). 2.5. Production puri¢cation and characterization of recombinant AAO A. nidulans harboring pALAAO was grown in minimal medium (described above) or complete medium [7], supplemented with 10 Wg/l D-biotin and 74.5 mg/l L-methionine. Complete medium contained 10 g/l glucose, 2 g/l bactopeptone, 1 g/l yeast extract, 1.5 mg/l casamino-acids, and 20 ml/l 50U salts solution (pH 6.8). After autoclaving, 10 ml of a vitamin solution (containing 50 mg/l HCl^thiamine, 10 mg/l
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biotin, 100 mg/l nicotinic acid, 200 mg/l calcium Dpanthotenate, 50 mg/l HCl-piridoxin, 100 mg/l ribo£avin and 100 mg/l p-aminobutyric acid) was added. Cultures were incubated at 28³C and 180 rpm for 24 h. The mycelia were then ¢ltered, washed and transferred to identical media except containing less glucose (0.05%) and 100 mM threonine for induction. After additional incubation (48 h) the mycelia were removed by ¢ltration and the culture liquid was concentrated by ultra¢ltration (Filtron, 5 kDa cuto¡ membrane) and then dialyzed against 10 mM sodium tartrate, pH 3. Recombinant AAO was puri¢ed using the same protocol described for the native enzyme from P. eryngii [16]. Brie£y, the concentrated preparation was ¢rst subjected to gel ¢ltration chromatography on Sephacryl S-200 and then to anion exchange chromatography on Mono-Q. Puri¢ed concentrated recombinant AAO was stored at 380³C. Recombinant AAO was N-deglycosylated with 125 mU/ml of endo-L-N-acetylglucosaminidase (endo-H from Boehringer). Sodium dodecyl sulfate^polyacrylamide gel electrophoresis (SDS^PAGE) of native and deglycosylated proteins was performed in 7.5% polyacrylamide gels. Isoelectrofocusing (IEF) was performed on 5% polyacrylamide gels (1 mm thickness) in a pH range of 2.5^5.5, prepared by mixing Bio-
Fig. 1. (A) Map of plasmid pALAAO. This vector contains the alcohol dehydrogenase promoter of A. nidulans (alcA), argB as selection marker and trpC as transcriptional terminator. (B) Southern blot hybridization of restricted DNA from wild-type (1) and transformed (2) A. nidulans with AAO probe from P. eryngii.
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Rad Ampholine from 2.5^5.0 (85%) and 3^10 (15%). The anode and cathode solutions were 0.5 M phosphoric acid and sodium hydroxide, respectively. The SDS^PAGE gels were stained with Silver Stain Plus (Bio-Rad), and those from IEF with Coomassie blue R-250. 2.6. Analysis of protein, reducing sugars and enzyme assays Protein concentration was determined by the method of Bradford [4] using albumin as standard, and reducing sugars were assayed by the method of Somogyi-Nelson [33]. AAO activity was assayed spectrophotometrically as the oxidation of veratryl alcohol to veratraldehyde (O310 = 9300 M31 cm31 ). The kinetic constants of recombinant AAO from A. nidulans were determined by measuring the rate of oxidation of veratryl, benzyl, m-anisyl (3-methoxybenzyl) and p-anisyl (4-methoxybenzyl) alcohols to the corresponding aldehydes (O310 = 9300 M31 cm31 , O250 = 13 800 M31 cm31 , O314 = 2540 M31 cm31 and O285 = 16 950 M31 cm31 , respectively) [16].
Fig. 3. Puri¢cation of recombinant AAO from A. nidulans. The ¢gure shows the elution pro¢le from the Mono-Q column equilibrated in 10 mM sodium phosphate (pH 6), and eluted with a NaCl gradient (0^500 mM in 20 min). OD at 280 nm (continuous line) and 465 nm (dotted line) are shown.
3. Results 3.1. Expression of P. eryngii AAO in A. nidulans Fig. 1A shows the orientation of the AAO cDNA in plasmid pALCA1 which was used for expression in A. nidulans. Southern blot analysis of restricted genomic DNA from wild-type and recombinant A. nidulans, using gene argB as a probe, showed that the plasmid containing aao cDNA was integrated in the genome of A. nidulans at the argB locus (data not shown). The AAO probe only hybridized to DNA from the recombinant strain (Fig. 1B). 3.2. Production and puri¢cation of recombinant AAO from A. nidulans
Fig. 2. Production of recombinant AAO from A. nidulans. The recombinant strain (a) was grown at 28³C and 180 rpm for 24 h, in minimal or complete media (continuous and dashed lines, respectively). Then, washed mycelia were transferred to the induction media (0.05% glucose and 100 mM threonine). Controls were carried out with wild-type A. nidulans (E). Means and 95% con¢dence limits are shown.
Expression levels of recombinant AAO was studied in both minimal or complete media to study AAO production. In both media, expression was induced by addition of threonine (with lowered glucose). As shown in Fig. 2, AAO activity was found in both culture media. AAO ¢rst appeared at approximately 10 h and reached maximal activity (400^500 mU/ml) at around 48 h post-induction. No AAO activity was detected in wild-type A. nidulans. The activity of the recombinant enzyme was
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Fig. 4. Molecular mass and isoelectric point determination. (A) SDS^PAGE of AAO from P. eryngii and recombinant AAO from A. nidulans glycosylated (lanes 2 and 4, respectively), treated with endo-H (lanes 3 and 5) and Bio-Rad standards (lane 1). (B) pI of both glycosylated enzymes.
much higher than that found in fungal cultures of P. eryngii [37]. Even when AAO activity from P. eryngii was 5-fold increased by addition of peptone to glucose-tartrate medium, this activity, near 50 mU/ml [37], was still much lower than that obtained with the recombinant strain. Recombinant AAO from A. nidulans was puri¢ed
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by methods used for native enzyme from P. eryngii [16]. Using low salt conditions of 10 mM sodium tartrate bu¡er (pH 3), many proteins, but not AAO, bind to Sephacryl S-200 via unspeci¢c interactions. The Mono-Q ion exchange column was necessary to remove minor impurities. The puri¢ed recombinant AAO showed a high ratio between 465 nm and 280 nm absorbances due to the FAD prosthetic group (Fig. 3). The homogeneity of this recombinant protein was con¢rmed on Superdex-75 column, SDS^PAGE and IEF. The molecular mass of recombinant AAO from A. nidulans was determined to be 70 kDa on Superdex-75 and 72.5 kDa by SDS^PAGE. These values are similar to those found for AAO from P. eryngii (Fig. 4A). The recombinant protein, similar to the wild-type enzyme from P. eryngii, contained 14% N-linked carbohydrate as estimated by SDS^PAGE after treatment with endo-H. Both proteins showed also the same pI of 3.8 (Fig. 4B). The kinetic constants of recombinant AAO were calculated using benzyl, m-anisyl, p-anisyl and veratryl alcohols and compared with those of P. eryngii AAO (Table 1). No signi¢cant di¡erences were observed between the results concerning the native enzyme and those reported by Guille¨n et al [16]. Comparison of the kinetic constants for the methoxysubstituted benzyl alcohols revealed that the position and number of methoxyl substituents showed a similar relationship for the enzymes isolated from A. nidulans and P. eryngii. 4. Discussion In the present study we report for ¢rst time the expression of recombinant AAO from the basidiomy-
Table 1 Kinetic constants for recombinant AAO from A. nidulans and the mature protein produced by P. eryngii AAO from P. eryngii Km (mM) Benzyl m-Anisyl p-Anisyl Veratryl
0.85 0.22 0.04 0.41
Vmax (U/mg) 52 29 208 135
AAO* from A. nidulans Km /Vmax (mM/(U/mg)) 61 132 5200 305
Km (mM) 0.63 0.30 0.03 0.56
Vmax (U/mg) 27 20 139 104
Km /Vmax (mM/(U/mg)) 43 68 4344 185
The reaction mixtures were performed in 100 mM phosphate bu¡er, pH 6. Means of three replicates are shown (standard deviations were less than 9% of the means).
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cete P. eryngii, an enzyme involved in lignin biodegradation, in A. nidulans. High-level expression of this enzyme was achieved by the use of the A. nidulans alcA gene promoter. This system is one of the best characterized expression system in ¢lamentous ascomycetes [13]. The expression resulted in 10^50 times higher levels of activity than that found in P. eryngii cultures. The recombinant AAO produced by A. nidulans showed the same molecular mass and pI as that of the mature protein secreted by P. eryngii [16]. These properties are also similar to those reported in other Pleurotus species [3,16,31,32] and Bjerkandera adusta [29]. All of these enzymes are FAD-containing glycoprotein with similar molecular mass (70^88 kDa) and pI (3.8^4.3). The AAO reported in P. chrysosporium has a similar molecular mass (78 kDa) but has a less acidic pI of 5.3 [1]. The kinetic study of the recombinant AAO showed the highest a¤nity for p-anisyl alcohol. This is similar to results reported for AAO from P. eryngii, P. pulmonarius and B. adusta [16,29,32]. The recombinant enzyme, however, showed a slightly increase in Vmax /Km for oxidation of these substrates. The recombinant enzyme, as well as the AAO from P. eryngii and P. pulmonarius, showed higher a¤nity for p-anisyl and veratryl alcohols than B. adusta AAO. The interest for ligninolytic enzymes is related to their potential application in biotechnological processes principally connected to paper industry. The expression of fungal peroxidases in Escherichia coli has been reported [9,41]. However, low yield of refolding protocols to obtain active proteins constitutes an important limitation. In these situations, fungal expression system may provide a promising alternative. Heterologous expression of peroxidases has been reported in di¡erent Aspergillus species [30,35]. Our work has shown that the recombinant AAO produced from A. nidulans is essential identical to the native enzyme produced by P. eryngii. Thus, heterologous expression in this fungal system is a promising alternative to providing the high enzyme levels required for biotechnological applications. Acknowledgements The authors thank Dr M.A. Pen¬alva for providing
the pALCA vector and the A. nidulans strain used in this study and Dr F.J. Ruiz-Duen¬as for her valuable suggestions and Dr M. Tien for critical reading of the manuscript. This research has been funded by the BIO95-337, BIO96-393 and BIO99-908 projects of the Spanish Biotechnology Programs. References [1] Y. Asada, A. Watanabe, Y. Ohtsu, M. Kuwahara, Biosci. Biotechnol. Biochem. 59 (1995) 1339. [2] S. Backa, J. Gierer, T. Reitberger, T. Nilsson, Holzforschung 47 (1993) 181. [3] R. Bourbonnais, M.G. Paice, Biochem. J. 255 (1988) 445. [4] M.M. Bradford, Anal. Biochem. 72 (1976) 248. [5] S. Camarero, B. Bo«ckle, M.J. Mart|¨nez, A.T. Mart|¨nez, Appl. Environ. Microbiol. 62 (1996) 1070. [6] S. Camarero, M.J. Mart|¨nez, A.T. Mart|¨nez, Proceedings of the Second International Workshop on SSF (FMS-95), Montpellier, 27^28 February, 1995. [7] D.J. Cove, Biochim. Biophys. Acta 113 (1966) 51. [8] E. de Jong, A.E. Cazemier, J.A. Field, J.A.M. de Bont, Appl. Environ. Microbiol. 60 (1994) 271. [9] W.A. Doyle, A.T. Smith, Biochem. J. 315 (1996) 15. [10] K.-E.L. Eriksson, Wood Sci. Technol. 29 (1990) 79. [11] K.-E.L. Eriksson, R.A. Blanchette, P. Ander, Microbial and Enzymatic Degradation of Wood Components, Springer, Berlin, 1990. [12] C.S. Evans, M.V. Dutton, F. Guille¨n, R.G. Veness, FEMS Microbiol. Rev. 13 (1994) 235. [13] J.M. Ferna¨ndez-Can¬o¨n, M.A. Pen¬alva, Mol. Gen. Genet. 246 (1995) 110. [14] F. Guille¨n, C.S. Evans, Appl. Environ. Microbiol. 60 (1994) 2811. [15] F. Guille¨n, V. Go¨mez-Toribio, C. Mun¬oz, M.J. Mart|¨nez, A.T. Mart|¨nez, Arch. Biochem. Biophys. 382 (2000) 142. [16] F. Guille¨n, A.T. Mart|¨nez, M.J. Mart|¨nez, Eur. J. Biochem. 209 (1992) 603. [17] F. Guille¨n, A.T. Mart|¨nez, M.J. Mart|¨nez, C.S. Evans, Appl. Microbiol. Biotechnol. 41 (1994) 465. [18] A. Gutie¨rrez, L. Caramelo, A. Prieto, M.J. Mart|¨nez, A.T. Mart|¨nez, Appl. Environ. Microbiol. 60 (1994) 1783. [19] A. Hatakka, FEMS Microbiol. Rev. 13 (1994) 125. [20] K.A. Jensen Jr, K.M.C. Evans, T.K. Kirk, K.E. Hammel, Appl. Environ. Microbiol. 60 (1994) 709. [21] D.N. Kamra, F. Zadrazil, Agric. Wastes 18 (1986) 1. [22] T.K. Kirk, R.L. Farrell, Annu. Rev. Microbiol. 41 (1987) 465. [23] C. Lapadatescu, C. Ginie©s, J.-L. Le Que¨re¨, P. Bonnarme, Appl. Environ. Microbiol. 66 (2000) 1517. [24] A.T. Mart|¨nez, S. Camarero, F. Guille¨n, A. Gutie¨rrez, C. Mun¬oz, E. Varela, M.J. Mart|¨nez, J.M. Barrasa, K. Ruel, M. Pelayo, FEMS Microbiol. Rev. 13 (1994) 265. [25] M.J. Mart|¨nez, B. Bo«ckle, S. Camarero, F. Guille¨n, A.T.
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Mart|¨nez, MnP isoenzymes produced by two Pleurotus species in liquid culture and during wheat straw solid-state fermentation, in: T.W. Je¡ries, L. Viikari (Eds.), Enzymes for Pulp and Paper Processing, Vol. 655, Washington, DC, 1996, pp. 183^196. M.J. Mart|¨nez, F.J. Ruiz-Duen¬as, F. Guille¨n, A.T. Mart|¨nez, Eur. J. Biochem. 237 (1996) 424. L. Marzullo, R. Cannio, P. Giardina, M.T. Santini, G. Sannia, J. Biol. Chem. 270 (1995) 3823. A. Muheim, M.S.A. Leisola, Bioconversion plant raw materials, Biotechnol. Adv. (VTT Symp.) 122 (1991) 30. A. Muheim, R. Waldner, M.S.A. Leisola, A. Fiechter, Enzyme Microb. Technol. 12 (1990) 204. F.J. Ruiz-Duen¬as, M.J. Mart|¨nez, A.T. Mart|¨nez, Appl. Environ. Microbiol. 65 (1999) 4705. G. Sannia, P. Limongi, E. Cocca, F. Buonocore, G. Nitti, P. Giardina, Biochim. Biophys. Acta 1073 (1991) 114. S. Sarkar, A.T. Mart|¨nez, M.J. Mart|¨nez, Biochim. Biophys. Acta 1339 (1997) 23.
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[33] M. Somogyi, J. Biol. Chem. 160 (1945) 61. [34] E.M. Southern, J. Mol. Biol. 98 (1975) 503. [35] P. Stewart, R.E. Whitwam, P.J. Kersten, D. Cullen, M. Tien, Appl. Environ. Microbiol. 62 (1996) 860. [36] J. Tilburn, C. Scazzocchio, G.G. Taylor, J.H. Zabicky-Zissman, R.A. Lockington, R.W. Davies, Gene 26 (1983) 205. [37] E. Varela, Deteccio¨n De Genes Que Codi¢can Lignina Peroxidasa y Aril-Alcohol Oxidasa (AAO) En Hongos Ligninol|¨ticos. Caracterizacio¨n Bioqu|¨mica y Molecular De La AAO De Pleurotus, Ph.D. Thesis, Universidad Auto¨noma, Madrid, 1998. [38] E. Varela, B. Bo«ckle, A. Romero, A.T. Mart|¨nez, M.J. Mart|¨nez, Biochim. Biophys. Acta 1476 (2000) 129. [39] E. Varela, F. Guille¨n, A.T. Mart|¨nez, M.J. Mart|¨nez, Res. I Congr. Hispano-Luso Biotecnol., 1992, p. 190. [40] E. Varela, A.T. Mart|¨nez, M.J. Mart|¨nez, Biochem. J. 341 (1999) 113. [41] R. Whitwam, M. Tien, Arch. Biochem. Biophys. 333 (1996) 439.
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Expression of Pleurotus eryngii aryl-alcohol oxidase in Aspergillus nidulans: puri¢cation and characterization of the recombinant enzyme è ngel T. Mart|¨nez, Mar|¨a Jesu¨s Mart|¨nez * Elisa Varela, Francisco Guille¨n, A Centro de Investigaciones Biolo¨gicas, Consejo Superior de Investigaciones Cient|¨¢cas, Vela¨zquez 144, E-28006 Madrid, Spain Received 11 August 2000; received in revised form 19 December 2000; accepted 20 December 2000
Abstract Aryl-alcohol oxidase (AAO) is an extracellular flavoenzyme involved in lignin biodegradation by some white-rot fungi. The enzyme catalyzes the extracellular oxidation of aromatic alcohols to the corresponding aldehydes. The electron acceptor is molecular oxygen yielding H2 O2 as the product. Herein we describe, for the first time, the expression of AAO from Pleurotus eryngii in the ascomycete Aspergillus nidulans. The activity of the recombinant enzyme in A. nidulans cultures is much higher than found in the extracellular fluid of P. eryngii. The recombinant enzyme showed the same molecular mass, pI and catalytic properties as that of the mature protein secreted by P. eryngii. The enzymic properties are also similar to those reported from other Pleurotus and Bjerkandera species. ß 2001 Elsevier Science B.V. All rights reserved. Keywords: Aryl-alcohol oxidase; Pleurotus; Heterologous expression; Aspergillus
1. Introduction Lignin biodegradation is an oxidative process carried out predominantly by white-rot fungi. These fungi are able to degrade this polymer to CO2 and H2 O [11,22]. Of practical signi¢cance is the use of these fungi in lignocellulose utilization. Pleurotus eryngii and related species have been characterized mainly for their ability to degrade wheat lignin preferentially with a limited attack to cellulose [24]. This is an important characteristic for biotechnological applications related to feed production [21] and paper manufacturing [10]. White-rot fungi produce dif-
* Corresponding author. Fax: +34-91-562-7518; URL address: http://www.cib.csic.es/Vlignina/lignina_es.html ; E-mail: [email protected]
ferent extracellular ligninolytic enzymes, which include laccases and peroxidases, and oxidases [19]. Pleurotus species have been shown to secrete laccase, Mn-oxidizing peroxidase and aryl-alcohol oxidase (AAO) under liquid and solid state fermentation conditions [6,24,25]. Pleurotus Mn-oxidizing peroxidase is a new type of ligninolytic enzyme which shares catalytic properties with the two ligninolytic peroxidases ¢rst reported in Phanerochaete chrysosporium, lignin peroxidase (LiP) and Mn-dependent peroxidase (MnP) [26]. AAO has been isolated and characterized from di¡erent Pleurotus species [3,16,31], Bjerkandera adusta [29] and P. chrysosporium [1]. The P. eryngii enzyme has been most extensively characterized and exhibits a wide substrate speci¢city, being capable of oxidizing benzyl, cinnamyl, naphthyl and aliphatic unsaturated primary alcohols [16]. AAO is involved
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in redox cycling where this enzyme oxidizes extracellularly aromatic alcohols to the corresponding aldehydes yielding H2 O2 . The aldehyde is then reduced by intracellular enzymes again yielding the alcohol substrate for AAO [14,17]. In accord with this is the ¢nding that many fungi synthesize aryl compounds; this has been reported in P. chrysosporium [20], B. adusta [23] and Pleurotus species [18]. One aldehyde that is produced is p-anisaldehyde with high levels reported in B. adusta and Pleurotus species. The high levels of p-anisaldehyde in Pleurotus and Bjerkandera is related with the high AAO a¤nity for p-anisyl alcohol [16,29]. In addition to AAO, intracellular aryl-alcohol dehydrogenase, ¢rst characterized from P. chrysosporium [28] and later reported from Bjerkandera and Pleurotus species [8,14,39], is also involved in these redox cycling reactions. Two roles have been proposed for AAO in lignin degradation. The ¢rst is for H2 O2 production. The H2 O2 can be used by peroxidases. Such is the case for Pleurotus pulmonarius where the high rate of lignin mineralization of wheat straw correlates with the high level expression of both AAO and Mn-oxidizing peroxidase [5]. Alternatively, H2 O2 has also been suggested to be the precursor to hydroxyl radical, also proposed to be involved in the initial attack of lignin [2,12]. The cooperation of AAO and laccase for the production of hydroxyl radical has been recently demonstrated [15].The second role proposed for AAO is for the reduction of phenoxy radicals. This in turn prevents the polymerization of lignin during its biodegradation by ligninolytic enzymes, as reported in Pleurotus ostreatus [27]. Recently, gene cloning of AAO from P. eryngii and P. pulmonarius have been reported [38,40]. The aim of the present work is to express the fungal AAO in the ascomycete Emericella nidulans (conidial state Aspergillus nidulans). We have utilized the strong alcA promoter for high level expression which is required for di¡erent biotechnological applications. 2. Materials and methods 2.1. Oligonucleotides Oligos ENt and EACt were synthesized based on the reported sequences of AAO of P. eryngii [40]
(GenBank sequences 64069). They were prepared with a Beckman-oligo 1000M synthesizer, and used as primers in automatic sequencing and for polymerase chain reaction (PCR) ampli¢cation. ENt (5PATGTCGTTTGGTGCACTT-3P) contained the ATG codon, followed by the ¢ve initial codons of the AAO cDNA. EACt (5P-CCCTACTGATCAGCC-3P) contained the complementary sequence of the ¢ve last codons of the AAO cDNA. 2.2. DNA ampli¢cation and plasmid construction A 1779 bp fragment of AAO cDNA was ampli¢ed by PCR using oligos ENt and EACt. One Wg of template, consisting of pBSK þ containing AAO cDNA cloned into the EcoRI site, 40 pmol of each oligo and 2.5 U of Pfu (Stratagene) were used for DNA ampli¢cation using Perkin^Elmer Gene Amp System 2400. A ¢rst cycle of 5 min at 94³C was followed by 30 cycles of 1 min at 94³C, 1 min at 58³C, 2 min at 72³C and a ¢nal step of 10 min at 72³C. 2.3. Recombinant plasmid construction For subcloning, the 1779 bp DNA fragment obtained by PCR was phosphorylated with T4 polynucleotide kinase (Biolabs). This DNA fragment was cloned into the SmaI site of pALCA vector containing the alcohol dehydrogenase promoter of Aspergillus nidulans. The vector also contained the agrB gene as a selectable marker and the trpC transcriptional terminator [13]. The resulting plasmid is referred as pALAAO. 2.4. Expression hosts and transformation procedures A. nidulans biA1, metG1, argB2 [13] was transformed with pALAAO for high-level expression. Protoplasts, used for transformation, were obtained from shaking cultures. Brie£y, A. nidulans was grown in minimal medium [7], supplemented with 10 Wg/l Dbiotin, 74.5 mg/l L-methionine and 530 mg/l L-arginine, at 25³C and 180 rpm. Minimal medium contained 10 g/l glucose, 0.92 g/l ammonium tartrate and 20 ml/l of a 50U salts solution (which included 26 g/l KCl, 26 g/l MgSO4 c7H2 O, 76 g/l KH2 PO4 , and 50 ml/l of a trace elements solution containing 40 mg/l
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Na2 B4 O7 c10H2 O, 400 mg/l CuSO4 c5H2 O, 800 mg/l Fe2 (SO4 )3 c2H2 O, 800 mg/l Na2 MoO4 c2H2 O and 8 mg/l ZnSO4 c7H2 O) (pH 6.8). The medium was inoculated with 5U106 conidia/ml (¢nal) and incubated 12 h at 30³C and 180 rpm. The mycelia were then harvested by ¢ltration, washed with 600 mM MgSO4 and protoplast obtained and transformed as described by Tilburn et al. [36]. Hybridizations were carried out according with the method of Southern [34]. A 3.9 kb SalI^PstI DNA fragment was used as a probe for the aao gene [40] and a 1.4 kb HindIII DNA fragment (argB probe) in Southerns to determine the site of integration in the A. nidulans genome. Probes were labeled using the Rediprime DNA labeling kit (Amersham). 2.5. Production puri¢cation and characterization of recombinant AAO A. nidulans harboring pALAAO was grown in minimal medium (described above) or complete medium [7], supplemented with 10 Wg/l D-biotin and 74.5 mg/l L-methionine. Complete medium contained 10 g/l glucose, 2 g/l bactopeptone, 1 g/l yeast extract, 1.5 mg/l casamino-acids, and 20 ml/l 50U salts solution (pH 6.8). After autoclaving, 10 ml of a vitamin solution (containing 50 mg/l HCl^thiamine, 10 mg/l
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biotin, 100 mg/l nicotinic acid, 200 mg/l calcium Dpanthotenate, 50 mg/l HCl-piridoxin, 100 mg/l ribo£avin and 100 mg/l p-aminobutyric acid) was added. Cultures were incubated at 28³C and 180 rpm for 24 h. The mycelia were then ¢ltered, washed and transferred to identical media except containing less glucose (0.05%) and 100 mM threonine for induction. After additional incubation (48 h) the mycelia were removed by ¢ltration and the culture liquid was concentrated by ultra¢ltration (Filtron, 5 kDa cuto¡ membrane) and then dialyzed against 10 mM sodium tartrate, pH 3. Recombinant AAO was puri¢ed using the same protocol described for the native enzyme from P. eryngii [16]. Brie£y, the concentrated preparation was ¢rst subjected to gel ¢ltration chromatography on Sephacryl S-200 and then to anion exchange chromatography on Mono-Q. Puri¢ed concentrated recombinant AAO was stored at 380³C. Recombinant AAO was N-deglycosylated with 125 mU/ml of endo-L-N-acetylglucosaminidase (endo-H from Boehringer). Sodium dodecyl sulfate^polyacrylamide gel electrophoresis (SDS^PAGE) of native and deglycosylated proteins was performed in 7.5% polyacrylamide gels. Isoelectrofocusing (IEF) was performed on 5% polyacrylamide gels (1 mm thickness) in a pH range of 2.5^5.5, prepared by mixing Bio-
Fig. 1. (A) Map of plasmid pALAAO. This vector contains the alcohol dehydrogenase promoter of A. nidulans (alcA), argB as selection marker and trpC as transcriptional terminator. (B) Southern blot hybridization of restricted DNA from wild-type (1) and transformed (2) A. nidulans with AAO probe from P. eryngii.
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Rad Ampholine from 2.5^5.0 (85%) and 3^10 (15%). The anode and cathode solutions were 0.5 M phosphoric acid and sodium hydroxide, respectively. The SDS^PAGE gels were stained with Silver Stain Plus (Bio-Rad), and those from IEF with Coomassie blue R-250. 2.6. Analysis of protein, reducing sugars and enzyme assays Protein concentration was determined by the method of Bradford [4] using albumin as standard, and reducing sugars were assayed by the method of Somogyi-Nelson [33]. AAO activity was assayed spectrophotometrically as the oxidation of veratryl alcohol to veratraldehyde (O310 = 9300 M31 cm31 ). The kinetic constants of recombinant AAO from A. nidulans were determined by measuring the rate of oxidation of veratryl, benzyl, m-anisyl (3-methoxybenzyl) and p-anisyl (4-methoxybenzyl) alcohols to the corresponding aldehydes (O310 = 9300 M31 cm31 , O250 = 13 800 M31 cm31 , O314 = 2540 M31 cm31 and O285 = 16 950 M31 cm31 , respectively) [16].
Fig. 3. Puri¢cation of recombinant AAO from A. nidulans. The ¢gure shows the elution pro¢le from the Mono-Q column equilibrated in 10 mM sodium phosphate (pH 6), and eluted with a NaCl gradient (0^500 mM in 20 min). OD at 280 nm (continuous line) and 465 nm (dotted line) are shown.
3. Results 3.1. Expression of P. eryngii AAO in A. nidulans Fig. 1A shows the orientation of the AAO cDNA in plasmid pALCA1 which was used for expression in A. nidulans. Southern blot analysis of restricted genomic DNA from wild-type and recombinant A. nidulans, using gene argB as a probe, showed that the plasmid containing aao cDNA was integrated in the genome of A. nidulans at the argB locus (data not shown). The AAO probe only hybridized to DNA from the recombinant strain (Fig. 1B). 3.2. Production and puri¢cation of recombinant AAO from A. nidulans
Fig. 2. Production of recombinant AAO from A. nidulans. The recombinant strain (a) was grown at 28³C and 180 rpm for 24 h, in minimal or complete media (continuous and dashed lines, respectively). Then, washed mycelia were transferred to the induction media (0.05% glucose and 100 mM threonine). Controls were carried out with wild-type A. nidulans (E). Means and 95% con¢dence limits are shown.
Expression levels of recombinant AAO was studied in both minimal or complete media to study AAO production. In both media, expression was induced by addition of threonine (with lowered glucose). As shown in Fig. 2, AAO activity was found in both culture media. AAO ¢rst appeared at approximately 10 h and reached maximal activity (400^500 mU/ml) at around 48 h post-induction. No AAO activity was detected in wild-type A. nidulans. The activity of the recombinant enzyme was
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Fig. 4. Molecular mass and isoelectric point determination. (A) SDS^PAGE of AAO from P. eryngii and recombinant AAO from A. nidulans glycosylated (lanes 2 and 4, respectively), treated with endo-H (lanes 3 and 5) and Bio-Rad standards (lane 1). (B) pI of both glycosylated enzymes.
much higher than that found in fungal cultures of P. eryngii [37]. Even when AAO activity from P. eryngii was 5-fold increased by addition of peptone to glucose-tartrate medium, this activity, near 50 mU/ml [37], was still much lower than that obtained with the recombinant strain. Recombinant AAO from A. nidulans was puri¢ed
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by methods used for native enzyme from P. eryngii [16]. Using low salt conditions of 10 mM sodium tartrate bu¡er (pH 3), many proteins, but not AAO, bind to Sephacryl S-200 via unspeci¢c interactions. The Mono-Q ion exchange column was necessary to remove minor impurities. The puri¢ed recombinant AAO showed a high ratio between 465 nm and 280 nm absorbances due to the FAD prosthetic group (Fig. 3). The homogeneity of this recombinant protein was con¢rmed on Superdex-75 column, SDS^PAGE and IEF. The molecular mass of recombinant AAO from A. nidulans was determined to be 70 kDa on Superdex-75 and 72.5 kDa by SDS^PAGE. These values are similar to those found for AAO from P. eryngii (Fig. 4A). The recombinant protein, similar to the wild-type enzyme from P. eryngii, contained 14% N-linked carbohydrate as estimated by SDS^PAGE after treatment with endo-H. Both proteins showed also the same pI of 3.8 (Fig. 4B). The kinetic constants of recombinant AAO were calculated using benzyl, m-anisyl, p-anisyl and veratryl alcohols and compared with those of P. eryngii AAO (Table 1). No signi¢cant di¡erences were observed between the results concerning the native enzyme and those reported by Guille¨n et al [16]. Comparison of the kinetic constants for the methoxysubstituted benzyl alcohols revealed that the position and number of methoxyl substituents showed a similar relationship for the enzymes isolated from A. nidulans and P. eryngii. 4. Discussion In the present study we report for ¢rst time the expression of recombinant AAO from the basidiomy-
Table 1 Kinetic constants for recombinant AAO from A. nidulans and the mature protein produced by P. eryngii AAO from P. eryngii Km (mM) Benzyl m-Anisyl p-Anisyl Veratryl
0.85 0.22 0.04 0.41
Vmax (U/mg) 52 29 208 135
AAO* from A. nidulans Km /Vmax (mM/(U/mg)) 61 132 5200 305
Km (mM) 0.63 0.30 0.03 0.56
Vmax (U/mg) 27 20 139 104
Km /Vmax (mM/(U/mg)) 43 68 4344 185
The reaction mixtures were performed in 100 mM phosphate bu¡er, pH 6. Means of three replicates are shown (standard deviations were less than 9% of the means).
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cete P. eryngii, an enzyme involved in lignin biodegradation, in A. nidulans. High-level expression of this enzyme was achieved by the use of the A. nidulans alcA gene promoter. This system is one of the best characterized expression system in ¢lamentous ascomycetes [13]. The expression resulted in 10^50 times higher levels of activity than that found in P. eryngii cultures. The recombinant AAO produced by A. nidulans showed the same molecular mass and pI as that of the mature protein secreted by P. eryngii [16]. These properties are also similar to those reported in other Pleurotus species [3,16,31,32] and Bjerkandera adusta [29]. All of these enzymes are FAD-containing glycoprotein with similar molecular mass (70^88 kDa) and pI (3.8^4.3). The AAO reported in P. chrysosporium has a similar molecular mass (78 kDa) but has a less acidic pI of 5.3 [1]. The kinetic study of the recombinant AAO showed the highest a¤nity for p-anisyl alcohol. This is similar to results reported for AAO from P. eryngii, P. pulmonarius and B. adusta [16,29,32]. The recombinant enzyme, however, showed a slightly increase in Vmax /Km for oxidation of these substrates. The recombinant enzyme, as well as the AAO from P. eryngii and P. pulmonarius, showed higher a¤nity for p-anisyl and veratryl alcohols than B. adusta AAO. The interest for ligninolytic enzymes is related to their potential application in biotechnological processes principally connected to paper industry. The expression of fungal peroxidases in Escherichia coli has been reported [9,41]. However, low yield of refolding protocols to obtain active proteins constitutes an important limitation. In these situations, fungal expression system may provide a promising alternative. Heterologous expression of peroxidases has been reported in di¡erent Aspergillus species [30,35]. Our work has shown that the recombinant AAO produced from A. nidulans is essential identical to the native enzyme produced by P. eryngii. Thus, heterologous expression in this fungal system is a promising alternative to providing the high enzyme levels required for biotechnological applications. Acknowledgements The authors thank Dr M.A. Pen¬alva for providing
the pALCA vector and the A. nidulans strain used in this study and Dr F.J. Ruiz-Duen¬as for her valuable suggestions and Dr M. Tien for critical reading of the manuscript. This research has been funded by the BIO95-337, BIO96-393 and BIO99-908 projects of the Spanish Biotechnology Programs. References [1] Y. Asada, A. Watanabe, Y. Ohtsu, M. Kuwahara, Biosci. Biotechnol. Biochem. 59 (1995) 1339. [2] S. Backa, J. Gierer, T. Reitberger, T. Nilsson, Holzforschung 47 (1993) 181. [3] R. Bourbonnais, M.G. Paice, Biochem. J. 255 (1988) 445. [4] M.M. Bradford, Anal. Biochem. 72 (1976) 248. [5] S. Camarero, B. Bo«ckle, M.J. Mart|¨nez, A.T. Mart|¨nez, Appl. Environ. Microbiol. 62 (1996) 1070. [6] S. Camarero, M.J. Mart|¨nez, A.T. Mart|¨nez, Proceedings of the Second International Workshop on SSF (FMS-95), Montpellier, 27^28 February, 1995. [7] D.J. Cove, Biochim. Biophys. Acta 113 (1966) 51. [8] E. de Jong, A.E. Cazemier, J.A. Field, J.A.M. de Bont, Appl. Environ. Microbiol. 60 (1994) 271. [9] W.A. Doyle, A.T. Smith, Biochem. J. 315 (1996) 15. [10] K.-E.L. Eriksson, Wood Sci. Technol. 29 (1990) 79. [11] K.-E.L. Eriksson, R.A. Blanchette, P. Ander, Microbial and Enzymatic Degradation of Wood Components, Springer, Berlin, 1990. [12] C.S. Evans, M.V. Dutton, F. Guille¨n, R.G. Veness, FEMS Microbiol. Rev. 13 (1994) 235. [13] J.M. Ferna¨ndez-Can¬o¨n, M.A. Pen¬alva, Mol. Gen. Genet. 246 (1995) 110. [14] F. Guille¨n, C.S. Evans, Appl. Environ. Microbiol. 60 (1994) 2811. [15] F. Guille¨n, V. Go¨mez-Toribio, C. Mun¬oz, M.J. Mart|¨nez, A.T. Mart|¨nez, Arch. Biochem. Biophys. 382 (2000) 142. [16] F. Guille¨n, A.T. Mart|¨nez, M.J. Mart|¨nez, Eur. J. Biochem. 209 (1992) 603. [17] F. Guille¨n, A.T. Mart|¨nez, M.J. Mart|¨nez, C.S. Evans, Appl. Microbiol. Biotechnol. 41 (1994) 465. [18] A. Gutie¨rrez, L. Caramelo, A. Prieto, M.J. Mart|¨nez, A.T. Mart|¨nez, Appl. Environ. Microbiol. 60 (1994) 1783. [19] A. Hatakka, FEMS Microbiol. Rev. 13 (1994) 125. [20] K.A. Jensen Jr, K.M.C. Evans, T.K. Kirk, K.E. Hammel, Appl. Environ. Microbiol. 60 (1994) 709. [21] D.N. Kamra, F. Zadrazil, Agric. Wastes 18 (1986) 1. [22] T.K. Kirk, R.L. Farrell, Annu. Rev. Microbiol. 41 (1987) 465. [23] C. Lapadatescu, C. Ginie©s, J.-L. Le Que¨re¨, P. Bonnarme, Appl. Environ. Microbiol. 66 (2000) 1517. [24] A.T. Mart|¨nez, S. Camarero, F. Guille¨n, A. Gutie¨rrez, C. Mun¬oz, E. Varela, M.J. Mart|¨nez, J.M. Barrasa, K. Ruel, M. Pelayo, FEMS Microbiol. Rev. 13 (1994) 265. [25] M.J. Mart|¨nez, B. Bo«ckle, S. Camarero, F. Guille¨n, A.T.
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