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APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/01/$04.0010 DOI: 10.1128/AEM.67.6.2610–2616.2001

June 2001, p. 2610–2616 Vol. 67, No. 6

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Cloning of a Phenol Oxidase Gene from Acremonium murorumand Its Expression in Aspergillus awamori

ROBIN J. GOUKA,* MONIQUE VAN DER HEIDEN, TON SWARTHOFF, AND C. THEO VERRIPS

Biotechnology Group, Unilever Research Vlaardingen, 3133 AT Vlaardingen, The Netherlands

Received 16 November 2000/Accepted 6 March 2001

Fungal multicopper oxidases have many potential industrial applications, since they perform reactionsunder mild conditions. We isolated a phenol oxidase from the fungus Acremonium murorum var. murorum thatwas capable of decolorizing plant chromophores (such as anthocyanins). This enzyme is of interest in laundry-cleaning products because of its broad specificity for chromophores. We expressed an A. murorum cDNA libraryin Saccharomyces cerevisiae and subsequently identified enzyme-producing yeast colonies based on their abilityto decolor a plant chromophore. The cDNA sequence contained an open reading frame of 1,806 bp encodingan enzyme of 602 amino acids. The phenol oxidase was overproduced by Aspergillus awamori as a fusion proteinwith glucoamylase, cleaved in vivo, and purified from the culture broth by hydrophobic-interaction chroma-tography. The phenol oxidase is active at alkaline pH (the optimum for syringaldazine is pH 9) and hightemperature (optimum, 60°C) and is fully stable for at least 1 h at 60°C under alkaline conditions. Thesecharacteristics and the high production level of 0.6 g of phenol oxidase per liter in shake flasks, which isequimolar with the glucoamylase protein levels, make this enzyme suitable for use in processes that occurunder alkaline conditions, such as laundry cleaning.

Blue oxidases are a subfamily of multicopper enzymes, in-cluding laccases, ascorbate oxidases, and vertebrate ceruloplas-min, that are produced by a large number of plants and fungi(20). These enzymes catalyze the four-electron reduction ofmolecular oxygen to water with the concurrent one-electronoxidation of a substrate, usually a polyphenolic compound(16). Relatively little is known about the physiological role ofthese enzymes in nature. Laccases, for example, are implicatedin a number of processes such as conidial pigmentation, lignindegradation, pathogenicity, and fruiting-body formation (re-viewed in reference 22).

Fungal multicopper oxidases are receiving increasing inter-est as potential industrial enzymes in applications such as de-toxification of toxic phenolic compounds and azo dyes (re-viewed in reference 12), enzymatic bleaching of kraft pulp (2),and delignification (30) because these oxidases catalyze theoxidation of phenols. Also, it is often desirable to convertcompounds under mild conditions to create new product prop-erties or to maintain other properties of a beverage or foodproduct in other processes, e.g., food processing. In the area oflaundry cleaning, enzymatic bleach might be a good alternativeto current chemical bleaches.

Blue oxidase genes have been cloned from a number ofspecies, mainly plants, white-rot basidiomycetes, and someplant pathogens (for a review, see references 4 [and referencestherein] and 20). However, in most fungi, oxidases (mainlylaccases) are produced at levels that are too low for commer-cial purposes, even when cloned genes are expressed in heter-ologous hosts (14, 17). For any of these potential applicationsto become reality, an inexpensive oxidase source must be avail-

able. Consequently, applications to produce consumer goodsneed redox enzymes, especially those that can be producedeasily by recombinant strains.

We identified a fungus, Acremonium murorum, which se-cretes an unknown phenol oxidase capable of decolorizingchromophores such as cyanidin and pelargonidin. Our objec-tives in this study were (i) to clone the corresponding phenoloxidase gene, (ii) to express the gene at high levels in Aspergil-lus awamori, a fungus which is used in industry for the produc-tion of proteins (8, 23), and (iii) to characterize the enzymewith respect to its suitability for laundry cleaning.

MATERIALS AND METHODS

Bacterial and fungal strains. For standard bacterial cloning, Escherichia coliDH5a (9) was used. For cloning of a cDNA library, E. coli XL1-Blue MRF9{(mcrA)183 (mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac(F9 proAB lacIqZ M15 Tn10 [Tetr])} (Invitrogen, Carlsbad, Calif.) was used.Saccharomyces cerevisiae strain VW-K1 (MATa leu2) was used for expression ofthe cDNA library. Acremonium murorum var. murorum CBS 157.72 was obtainedfrom the Centraal Bureau voor Schimmelcultures (CBS), Baarn, The Nether-lands. A. awamori AWC4.20 is a pyrG mutant strain derived from A. awamori 40(described in World Patent 91/19782, p.13) and a derivative of A. awamori CBS115.52.

Cultivation of A. murorum. A shake flask containing 100 ml of potato dextrosebroth (Difco Laboratories, Detroit, Mich.) was inoculated with spores of A.murorum obtained from a culture growing on a potato dextrose agar (Oxoid,Ogdensburg, N.Y.) plate that had been incubated for 1 week at 25°C. The culturewas grown for 3 days at 25°C in a rotary shaker (250 rpm), and then it wastransferred to 100 ml of minimal medium (1), enriched with 0.5% yeast extract,and grown for another 3 days at 25°C.

Extraction of total RNA and isolation of poly(A)1 RNA. Total RNA wasprepared by extraction with Trizol (Life Technologies, Inc., Rockville, Md.). TheRNA concentration was determined by measuring absorbance at optical densi-ties of 260 and 280 nm (OD260/280). Purification of poly(A)1 mRNA from totalRNA was carried out with the Oligotex mRNA kit (Qiagen, Valencia, Calif.)according to the protocol provided by the supplier.

cDNA synthesis. cDNA synthesis was carried out by using a cDNA synthesiskit (Stratagene, La Jolla, Calif.) according to the manufacturer’s protocol, exceptthat reverse transcriptase Superscript II (Life Technologies, Inc.) was used in-stead of Moloney murine leukemia virus reverse transcriptase.

* Corresponding author. Mailing address: Biotechnology Group,Unilever Research Vlaardingen, Olivier van Noortlaan 120, 3133 ATVlaardingen, The Netherlands. Phone: 31 104605263. Fax: 31104605383. E-mail: [email protected].

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Construction of a cDNA library. cDNA was cloned as EcoRI/XhoI fragmentsinto plasmid pYES2.0 (Invitrogen). For large-scale ligations, approximately 200ng of cDNA was ligated to 1.5 mg of EcoRI/XhoI-digested pYES2.0, in a totalvolume of 7.5 ml with 1 U of T4 DNA ligase for 5 h at room temperature.Aliquots of 2.5 ml were used to transform 50 ml of electrocompetent E. coliXL1-Blue MRF9 cells (Stratagene) (conditions, 1,700 V, 200 v, and 25 mF).After addition of 1 ml of SOC (per liter: 20 g of Bacto-tryptone [Difco Labora-tories], 5 g of Bacto-yeast extract [Difco], 0.58 g of NaCl, 0.18 g of KCl, 2.0 g ofMgCl2 z 6 H2O, 2.46 g of MgSO4 z 7 H2O, 3.6 g of glucose) to each mix, the cellswere regenerated for 1 h at 37°C, plated on Luria-Bertani medium (LB) (1%Bacto-tryptone [Difco], 0.5% Bacto-yeast extract [Difco], 10 g of NaCl liter21,pH 7.0) with ampicillin (100 mg/ml), and grown at 37°C for another 16 h.Dilutions were plated to calculate the titer of the library. To each plate wasadded 3 ml of capable of decolorizing plant chromophores (such as anthocya-nins), and bacteria were scraped off, pooled, and stored in small aliquots. Large-scale DNA was prepared from 200- to 500-ml cultures of LB inoculated with analiquot of transformants and propagated overnight.

Transformations. Lithium acetate-mediated transformations of S. cerevisiaeVW-K1 were carried out by the method of Gietz and Woods (5). Cells werewashed with 1 M sorbitol and finally plated onto selective medium [0.67% yeastnitrogen base minimal medium (without amino acids and with ammonium sul-fate)] and 2% glucose and incubated for 3 to 5 days at 30°C. Transformation ofA. awamori was carried out as described previously (6).

Screening of an A. murorum cDNA library in S. cerevisiae for decolorization ofcyanidin. Approximately 50,000 colonies of an A. murorum cDNA expressionlibrary in S. cerevisiae VW-K1 were plated on medium containing 4% (wt/vol)galactose, 0.5% (wt/vol) glucose, 0.67% (wt/vol) yeast nitrogen base minimalmedium, 0.1 M sodium phosphate (pH 7.2), and 120 mg of cyanidin liter21 toyield approximately 3,000 colonies per plate, and incubated at 30°C. The plateswere screened daily for halo-producing transformants.

Isolation of plasmid DNA from yeast. Plasmid DNA was isolated as describedby Hoffman and Winston (11), using a mixture of lysis buffer, phenol, and glassbeads followed by centrifugation, transformation of E. coli with a small sample ofthe supernatant, and isolation of the plasmid from E. coli.

DNA sequence analysis. DNA sequence analysis was carried out on an LKBautomated laser fluorescent DNA sequencer (Amersham Pharmacia Biotech,Inc., Piscataway, N.J.).

Plasmid construction. For production of A. murorum oxidase by A. awamori,the gene encoding the A. murorum oxidase (AMO) was inserted into expressionvector pAWGLA2 (7). The oxidase gene was fused to the 39 end of the Aspergil-lus niger glucoamylase gene. The genes are separated by a DNA sequenceencoding a KEX2-type recognition site (Asn-Val-Ile-Ser-Lys-Arg). The expres-sion signals (promoter and transcription terminator) are derived from the A.awamori b-1,4-endoxylanase A gene. Since the N-terminal amino acid sequenceof wild-type AMO could not be determined due to the low production levels byAcremonium, the fusion was based on cleavage of AMO by the rules of VonHeijne (25, 26) (cleavage between signal peptide and protein). Based on thishypothesis, the glucoamylase was fused to amino acid 22 (Met) of AMO.

For a correct fusion at the 39 end of the phenol oxidase gene, pUR7876 wasdigested with XhoI and XbaI and ligated with two annealed oligonucleotides(59-TCGAGCTTAAGT-39 and 59-CTAGACTTAAGC-39), thereby introducingan AflII site, resulting in pUR7880. For a correct fusion at the 59 end, pUR7880was modified by replacing an EcoRV/NarI fragment with a 170-bp EcoRV/NarIPCR-derived DNA fragment, giving plasmid pUR7890. This vector contains partof the KEX2 recognition site starting at an EcoRV site and the 59 part of the A.murorum gene up to the NarI site. The PCR fragment was obtained with theprimers Acr06 (59-GAGAGAGATATCCAAGCGCATGCCCAAGTTCGAGCTGGACATTCCTGAGG-39) and Acr02 (59-GCTTGATCTCGATCTCATAGTAGT-39) on plasmid pUR7876 as a template. From pUR7890, pUR7891 wasconstructed by inserting the A. murorum gene, present on a 2-kb EcoRV/AflIIfragment, into the Aspergillus expression vector pAWGLA2, which was alsodigested with EcoRV and AflII. Finally, pUR7891 was digested with NotI andligated with the Aspergillus nidulans amdS and the A. awamori pyrG double-selection marker from pAW10S-4 (24), resulting in pUR7893 (Fig. 1).

Construction of recombinant A. awamori strains. Strain A. awamori AWC4.20was transformed with pUR7893, and transformants were selected in two ways,either by restored growth on minimal medium (1) due to the integration of thewild-type pyrG gene or by growth on minimal medium with 10 mM acetamide asa sole nitrogen source, due to the integration of the amdS gene. The latterselection method usually results in transformants containing multiple copies ofthe plasmid, since these transformants grow better and faster on acetamide-containing medium. Transformants were purified twice on Aspergillus minimal

medium (1). Conidia were obtained by growing mycelium on potato dextroseagar plates for 5 to 7 days at 30°C.

Shake flask induction experiments with A. awamori. Production in shake flasksof the AMO by A. awamori under control of the b-1,4-endoxylanase A transcrip-tion control sequences was carried out according to the method of Gouka et al.(6). The induction medium was supplemented with 0.5 mM CuCl2.

Protein analysis. For analysis of secreted proteins, the medium was separatedfrom the mycelium by filtration through Miracloth (Calbiochem-Behring, LaJolla, Calif.). Concentration of the proteins in the medium was carried out byammonium sulfate precipitation (80% saturation). The precipitate was kept at4°C for 16 h and pelleted by centrifugation for 45 min at 25,000 3 g The proteinpellet was dissolved in 2.5 ml of 30 mM sodium phosphate (pH 8.5) and subse-quently desalted using a Sephadex G25 column (Amersham Pharmacia Biotech).Proteins were eluted with 3.5 ml of 30 mM sodium phosphate (pH 8.5). Enzymeanalysis was carried out by polyacrylamide gel electrophoresis (PAGE), plateassays, and enzyme activity assays. For PAGE, samples were boiled in 1%sodium dodecyl sulfate (SDS) without reducing agent. Glucoamylase was de-tected as previously described (7).

Plate assays. Fungal conidia were inoculated onto agar plates, containingminimal medium (1) with 1.5% agar and a substrate, either an anthocyanidin(120 mg of cyanidin liter21 [Fluka; Sigma-Aldrich Corp., St. Louis, Mo.] or 240mg of pelargonidin liter21 [Roth, Karlsruhe, Germany]) or 2 mM ABTS [2,29azi-nobis(3-ethylbenzthiazolinesulfonic acid)]. The plates were incubated at 25°Cand screened daily for the presence of clearing zones (anthocyanidins) or greenhalos (ABTS) as a result of extracellular enzyme activity. To detect enzymeproduction by recombinant strains containing the phenol oxidase gene fused tothe exlA promoter, D-xylose was added to the plates at a 5% concentration.

Enzyme activity assays. Decolorization of anthocyanidins was measured in 1ml of solution containing 100 mM sodium phosphate (pH 7.5), 0.1 mM cyanidin-chloride or pelargonidinchloride, and 20 to 200 ml of enzyme sample. The changein absorbance (per minute) was measured during 5 min in a UV-VIS spectrum(wavelength, 200 to 700 nm; absorbance peak, 579 nm). Standard ABTS oxida-tion assays were carried out by adding the appropriate amount of enzyme to 50mM sodium phosphate (pH 6.0)–2 mM ABTS solution (final volume, 1 ml) andmonitoring the absorbance increase at 414 nm (extinction coefficient, 35 mMcm21). One unit of enzyme activity was defined as the amount of enzyme thatoxidizes 1 mmol of ABTS per min per ml at 20°C.

For determination of the AMO activity as a function of pH, standard amountsof AMO (1,500 U) were added to 1 ml of Britton and Robinson buffer (B&Rbuffer) (3) (pH range, 3.0 to 11.0)–2 mM ABTS solution, and the activity wasmeasured at 414 nm. Similarly, syringaldazine (SGZ) oxidation activity wasdetermined in B&R buffer–100 mM SGZ by monitoring the absorbance changeat 530 nm. B&R buffer was prepared by mixing a 100-ml solution of 28.6 mMcitric acid, 28.6 mM KH2PO4, 28.6 mM boric acid, and 28.6 mM diethylbarbituric

FIG. 1. Plasmid pUR7893. Open bars, A. awamori 59 and 39 regu-latory sequences; filled arrows, coding sequences. Abbreviations anddesignations: glaA, glucoamylase gene; amdS, acetamidase gene; pyrG,orotidine 59-monophosphate decarboxylase gene; amp, b-lactamasegene, exlA, b-1,4-endoxylanase gene; ori, origin of replication. Onlyrelevant restriction sites are indicated.

VOL. 67, 2001 PHENOL OXIDASE GENE FROM ACREMONIUM MURORUM 2611

acid, which was set at the appropriate pH with 0.2 M sodium hydroxide and thendiluted with water to 200 ml. AMO activity as a function of temperature wasdetermined by measuring the activity of standard amounts of AMO (1,500 U) inB&R buffer (pH 4.5)–2 mM ABTS at various temperatures (without preincuba-tion of AMO). Stability as a function of pH was measured by incubating standardamounts of AMO in B&R buffer (pH range 3.0 to 9.6) at 4°C and determiningthe residual activity at different times using the standard ABTS oxidation assaydescribed above. Stability as a function of temperature was measured by incu-bating standard amounts of AMO in B&R buffer (pH 8.5) at different temper-atures and determining the residual activity at different times using the standardABTS oxidation assay.

Purification of A. murorum phenol oxidase. Hydrophobic-interaction chroma-tography was used to purify AMO from the fermentation broth. A phenyl-Sepharose 6 fast-flow column (Amersham Pharmacia Biotech) was equilibratedwith 50 mM sodium phosphate–30% (1.3 M) ammonium sulfate (pH 6.0). Am-monium sulfate was added to the enzyme sample to a final concentration of 1.3M. Proteins were eluted with a linear decreasing-salt gradient. Enzyme activity inthe different fractions was measured at pH 6.0 using 2 mM ABTS as substrate.Those samples containing phenol oxidase were pooled and dialyzed against 20mM sodium phosphate (pH 8.5) and stored at 220°C.

Determination of the amino-terminal sequence of AMO. Protein samples wereanalyzed using an apparatus consisting of a Porton LF3000 sequencer and onlinephenylthiohydantoine analysis (Beckman Instruments Inc., Fullerton, Calif.)with a Beckman high-performance liquid chromatograph type 125S and a Beck-man detector type 168. The phenylthiohydantoine derivatives were analyzedonline using a C18 Microbore RP column (Beckman). Separation was achievedby using a gradient of 5% tetrahydrofuran in water and acetonitrile. Detectionwas done at 268 nm.

Nucleotide sequence accession number. The sequence data for AMO havebeen submitted to the EMBL database under accession number no. AJ271104.

RESULTS

Isolation, cloning and characterization of AMO. The fungusA. murorum var. murorum CBS 157.72 produced a clearingzone when cultured on solidified media containing either cya-nidin or pelargonidin, and it secreted a phenol oxidase into themedium when grown in shake flask cultures.

From an A. murorum cDNA expression library in S. cerevi-siae VW-K1, we identified a transformant that produced aclearing zone (halo) around the colony on cyanidin-containingplates. This transformant was purified, and plasmid DNA, des-ignated pUR7876, was isolated. Retransformation of S. cerevi-siae with pUR7876 DNA again resulted in halo-forming colo-

nies. The DNA sequence of the EcoRI/XhoI cDNA insert inpUR7876 was determined by subcloning fragments in pUC19(29). The insert consisted of 2,120 nucleotides, including 59 and39 nontranslated sequences and a poly(A) tail. The DNA se-quence, with the ATG codon at position 135, comprised anopen reading frame of 1,806 nucleotides, encoding an enzymeof 602 amino acids. The 39 nontranslated region was 165 basesand was followed by a poly(A) tail. An in-frame ATG codonlocated 63 bp downstream of the first one also could be used tostart translation, resulting in a protein of 581 amino acids.Comparison of the deduced amino acid sequence with thesequences of proteins in the databases (Table 1) showed anidentity of 66% with a bilirubin oxidase isolated from thefungus Myrothecium verrucaria (13) (see Table 1). Further-more, the A. murorum phenol oxidase was similar to the con-sensus sequences of the four copper-binding sites present inlaccases (4) (Fig. 2). The homology with laccases was restrictedto those consensus areas, and the overall identity of AMO withlaccases was ,15% (Table 1).

Heterologous production of AMO by A. awamori. We trans-formed A. awamori AWC4.20 with pUR7893 to obtain trans-formants that overproduced AMO. In a cyanidin plate assay,all transformants produced large halos around the fungal col-ony, indicating that cyanidin was converted into a colorlesscompound by the secreted enzyme. Similar plates, in whichcyanidin was substituted for the oxidase substrate ABTS,showed that ABTS also was oxidized by this enzyme. Four A.awamori AWC4.20-pUR7893 transformants were analyzed insubmerged cultivation and had similar activities on cyanidin.These activities corresponded to a decrease in the OD579 (ab-sorbance peak of cyanidin at pH 7.5) of approximately 0.06 to0.07/ml of medium sample in 1 min, which is 2 to 3 orders ofmagnitude more than the production levels obtained with cul-ture medium of A. murorum.

The amounts of extracellular AMO activity, as determinedwith an ABTS activity assay, reached up to 25 U/ml after 30 hof induction. Based on specific activity of 40 U/mg (see below),

TABLE 1. Percent identity (calculated by the ClustalW method) of A. murorum polyphenol oxidase to other blue copper oxidasesa

EnzymeIdentity (%) with polyphenol oxidase fromb:

Amur Mver Tvil Tvers4 Pos2 Prad Abis Ncras Mther Sther Bcin Cpara Anid

A. murorum polyphenol oxidase 66 15 15 14 15 14 12 11 14 14 13 11M. verrucaria bilirubin oxidase 15 16 12 14 13 12 15 12 11 17 6T. villosa laccase (lcc1) 68 62 63 47 25 28 27 30 31 16T. versicolor laccase (lcc4) 64 64 47 27 27 26 29 26 17P. ostreatus laccase (pox2) 38 48 27 25 25 26 24 19P. radiata laccase 43 23 24 25 26 24 17A. bisporus laccase 26 25 27 22 23 17N. crassa laccase 60 56 35 57 18M. thermophila laccase 63 32 56 18S. thermophilum laccase 29 51 18B. cinereus laccase 37 20C. parasitica laccase 19A. nidulans laccase

a Enzyme amino acid sequences (numbers in parentheses below are accession numbers) are the polyphenol oxidase from A. murorum (this paper), bilirubin oxidasefrom M. verrucaria (Q12737), and laccases from T. villosa (lcc1) (Q99044), Trametes versicolor (lcc4) (Q12719), P. ostreatus (pox2) (Q12739), Phlebia radiata (Q01679),A. bisporus (Q12542), N. crassa (P10574), M. thermophila (AR023901), S. thermophilum (AR007280), Botrytis cinerea (Q12570), Cryphonectria parasitica (Q03966), andA. nidulans (P17489). When neccessary, DNA sequences were translated into amino acid sequences.

b Abbreviations: Amur, A. murorum; Mver, M. verrucaria; Tvil, T. villosa; Tvers4, T. versicolor (lcc4); Pos2, P. ostreatus (pox2); Prad, P. radiata; Abis, A. bisporus; Ncras,N. crassa; Mther, M. thermophila; Sther, S. thermophilum; Bcin, B. cinereus; Cpara, C. parasitica; Anid, A. nidulans.

2612 GOUKA ET AL. APPL. ENVIRON. MICROBIOL.

the level of recombinant enzyme secreted in these shake flaskcultures was approximately 600 mg per liter.

Medium samples of two transformants, AWC-pUR7893-5pand -10A, also were analyzed on SDS-PAGE (8 to 18% gradi-ent gel), stained with Coomassie brilliant blue (Fig. 3). Bothsamples contained high levels of recombinant enzyme, visibleas a main band of about 67 kDa. In addition, a second, minorband of about 40 kDa was visible. Apparently, this smallerband represented a faster-migration form of denatured AMO,since both proteins have the same amino-terminal sequence(SPLSPAYTLF) and, under nondenaturing conditions, only asingle band is visible (see below). The amounts of AMO werealmost equimolar with the amounts of glucoamylase (visible asa band at approximately 80 kDa), indicating that degradationwas minimal. The identity of glucoamylase also was confirmedby Western blot analysis (data not shown).

Determination of the amino-terminal sequence gave the se-quence SPLSPAYTLF, indicating that the enzyme was pro-cessed after amino acid 61. Thus, although the fusion wasactually based on the theoretical cleavage site of a signal pep-tide between amino acids 21 and 22 (the fusion of glucoamy-lase was made with amino acid 22 of AMO), AMO is cleavedafter amino acid 61. This means that the glucoamylase-AMO

FIG. 2. Amino acid sequence alignment of AMO with other bluecopper enzymes. Only those areas that contain the types I, II, and IIIcopper ligands (marked in bold as 1, 2, and 3) are shown. Amino acidsequence data were obtained as described in Table 1. For abbrevia-tions on left, see Table 1, footnote b. T. tsunodae bilirubin oxidase has

FIG. 3. SDS–8 to 18% gradient PAGE from supernatants of AWC-7893 transformants cultivated in shake flasks. The samples were boiledwithout reducing agent. The gel was stained with Coomassie brilliantblue. The arrows indicate the position of the phenol oxidase. Lane 1,AWC-7893-5p; lane 2, AWC-7893-10A; lane 3, AWGLA, which con-tains a single copy of the A. niger glucoamylase gene (7). M, molecularsize marker (kDa).

GenBank accession number AB006824. A consensus sequence is givenbelow each 13-row set. An amino acid identity of 100% among allenzymes is shown in uppercase, and an identity between 80% and100% is in lowercase.


fusion molecule contained two consecutive propeptides: theprosequence of glucoamylase (NVISKR, containing the KEX2cleavage site) and the prosequence of AMO (theoretically 40amino acids). A zymogram containing cyanidin as a substrateshowed that only AMO was responsible for the decolorizationof cyanidin.

Characterization of recombinant AMO. We purified the en-zyme from the culture broth by hydrophobic-interaction chro-matography. The major peak in enzyme activity, which elutedat approximately 45% salt, was clearly visible as a blue band onthe column during elution. The specific activity of the purifiedenzyme was about 40 U/mg of protein.

From SDS-PAGE, the molecular size of AMO was esti-mated to be 67 kDa, whereas the calculated molecular size was60 kDa. The 7-kDa difference could be explained by N-glyco-sylation at two putative sites (Asn-X-Thr). Furthermore, the40-kDa band that was observed in the medium samples of thetransformants also was present in purified AMO. When AMOwas not boiled, a single band of approximately 32 kDa wasvisible on SDS-PAGE (data not shown). Apparently, the pres-ence of 1% SDS is not sufficient to fully denature the protein.This was confirmed by analysis of the activity after incubationof AMO in 1% SDS, which showed no decrease in AMOactivity. Boiling completely destroyed the activity.

On an isoelectric focusing gel, a band with a pI that was near3.5 was enzymatically active when the gel was incubated withABTS. This pI was lower than the calculated pI of 4.3.

Recombinant AMO had an optimal pH of 4 to 4.5 withABTS as substrate (Fig. 4A). With SGZ as a substrate, themaximum activity was observed at pH 8.5 to 9. At 60°C, thehighest AMO activity was measured, and it was approximately2.5-fold higher than the activity observed at 20°C (Fig. 4B).

The stability of the purified enzyme was measured as afunction of pH and temperature. The enzyme was highly un-stable at low pH, whereas at alkaline pH the enzyme retainedfull activity for at least 280 h when incubated at 4°C (Fig. 5A).Thermostability analysis showed that the enzyme was stable at50°C for 3 h and almost fully stable for 20 min at 60°C. Afterprolonged incubation or incubation at higher temperatures,the activity decreased (Fig. 5B).

DISCUSSION

We isolated and characterized a phenol oxidase from thefungus A. murorum. Amino acid sequence comparison shows ahigh degree of identity (66%) with a bilirubin oxidase (BOX)isolated from the fungus Myrothecium verrucaria (13). As withother blue copper enzymes, e.g., laccases and ascorbate oxi-dase, four consensus domains for all types (I, II, and III) ofcopper ligands are present in AMO (Fig. 2). Three other fun-gal bilirubin oxidases have been reported, from Trachydermatsunodae (10), Penicillium janthinellum (18), and Pleurotus os-treatus (15). However, these enzymes differ from both AMOand Myrothecium bilirubin oxidase. The T. tsunodae bilirubinoxidase amino acid sequence is very similar to the sequences oflaccases (Fig. 2) with, for example, 74% identity with theamino acid sequence of T. villosa laccase encoded by the lcc4gene. The identity of T. tsunodae bilirubin oxidase with AMOand M. verrucaria bilirubin oxidase is only 12 and 14%, respec-tively. Similarly, the P. ostreatus bilirubin oxidase appears to be

identical to P. ostreatus laccase POX2 (15) and has only 13%identity with the amino acid sequence of AMO and M. verru-caria bilirubin oxidase. The P. janthinellum enzyme, which con-tains copper, zinc, and iron atoms, also is very different fromAMO and M. verrucaria bilirubin oxidase, since these proteinscontain only copper.

The amino-terminal part of the protein shows the charac-teristics of a signal sequence. The predicted (26) signal peptidecleavage site for AMO is between amino acids 21 (Ala) and 22(Met). The protein also contains two dibasic amino acid se-quences, residues 51/52 and 60/61 (both Arg-Arg), which mightbe cleaved by a KEX2-like protease and which could indicatethat AMO is initially produced as a proenzyme (21). The firstresidue of the mature recombinant enzyme is Ser-62 (althoughthe N-terminal sequence of AMO produced by A. murorumcould not be determined exactly, this sequence was not incontradiction with the sequence obtained from the recombi-nant form). Based on these results, residues 22 through 61probably comprise a propeptide whose proteolytic removaloccurs during maturation of AMO. Consequently, the recom-binant fusion molecule probably contains two consecutive

FIG. 4. Dependence of AMO activity on pH and temperature. (A)AMO activity as a function of pH (normalized to the optimum activity)with ABTS (2 mM) as substrate (M) and SGZ (100 mM) as substrate(e). Assays were performed in B&R buffer at the indicated pH at30°C. (B) AMO activity as a function of temperature (normalized tothe activity at 20°C) with ABTS (2 mM) as substrate in B&R buffer, pH4.5. Both experiments were carried out in duplicate; standard errorswere ,10%.


propeptides: the glucoamylase prosequence NVISKR and theprosequence of AMO. Based on the amino-terminal-sequencedata, processing occurs correctly after the AMO prosequence.If translation begins at the second in-frame ATG codon, theresulting protein does not contain a theoretical site for cleav-age of a signal peptide, suggesting that the first ATG is thetranslation initiation codon that is used in vivo.

To make the application of multicopper enzymes feasible inindustrial processes or products, the production levels in shakeflasks should be at least approximately 1 g per liter. However,the amounts that have been reported are usually low. In ahomologous system the amounts can range from a few milli-grams per liter up to 80 mg per liter for Botrytis cinereus laccase(19). However, these levels are still low for commercial pur-poses, and cultivation of these fungi is often difficult. Althoughlaccases have been isolated from a large number of ascomy-cetes (e.g., A. nidulans, Neurospora crassa, and Podospora an-serina), deuteromycetes (Botrytis cinereus), and basidiomycetes(e.g., Coriolus hirsutus, Trametes villosa [or Polyporus pinsitus],Agaricus bisporus, Polyporus versicolor, and Pleurotus ostreatus),their production levels in heterologous hosts are also usually,100 mg per liter (14, 17). In contrast, the yield of the recom-binant phenol oxidase from A. awamori transformants grown in

shake flasks was high (600 mg per liter). As normally theproduction yield is improved when shake flask experiments arereplaced by fed-batch fermentation processes, AMO has po-tential commercial utility for industrial purposes or consumerproducts.

AMO had an optimal activity for ABTS and SGZ as sub-strates at pH from 4 to 4.5 and 8.5 to 9, respectively. Theseoptima are at least equal and often higher than described forother polyphenol oxidases such as laccases (27, 28) and indi-cate that AMO has potential to be used under alkaline condi-tions. Furthermore, under alkaline conditions the enzyme isfully stable for at least 3 h at 50°C and loses only 15% activityafter 20 min at 60°C. This is close to the stability observed forthe thermophilic fungi Myceliophthora thermophila andScytalidium thermophilum (28) and higher than for laccasesisolated from Polyporus pinsitus and Rhizoctonia solani (28).

In conclusion, we isolated a new phenol oxidase derivedfrom the fungus A. murorum var. murorum CBS 157.72. Theenzyme converted anthocyanidins to colorless compounds andcould be applied as a mild alternative for chemical bleaching,such as in laundry cleaning. This enzyme forms an attractivealternative to other polyphenol oxidases due to (i) its potentialto be produced at high levels (at least 0.6 g/liter) by cultivationof a recombinant strain of A. awamori, (ii) its high stabilityunder alkaline conditions and high temperatures, (iii) its highactivity at 50 to 60°C, and (iv) its activity under even extremealkaline conditions (pH 9 to 10).

ACKNOWLEDGMENTS

We thank John Chapman and Maarten Egmond for critical readingof the manuscript and Han van Brouwershaven for amino-terminalsequence analysis.

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