APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2001, p.

26102616
0099-2240/01/$04.000 DOI: 10.1128/AEM.67.6.26102616.2001
Copyright 2001, American Society for Microbiology. All Rights Reserved.

Vol. 67, No. 6

Cloning of a Phenol Oxidase Gene from Acremonium murorum

and 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 reactions
under mild conditions. We isolated a phenol oxidase from the fungus Acremonium murorum var. murorum that
was capable of decolorizing plant chromophores (such as anthocyanins). This enzyme is of interest in laundrycleaning products because of its broad specificity for chromophores. We expressed an A. murorum cDNA library
in Saccharomyces cerevisiae and subsequently identified enzyme-producing yeast colonies based on their ability
to decolor a plant chromophore. The cDNA sequence contained an open reading frame of 1,806 bp encoding
an enzyme of 602 amino acids. The phenol oxidase was overproduced by Aspergillus awamori as a fusion protein
with glucoamylase, cleaved in vivo, and purified from the culture broth by hydrophobic-interaction chromatography. The phenol oxidase is active at alkaline pH (the optimum for syringaldazine is pH 9) and high
temperature (optimum, 60C) and is fully stable for at least 1 h at 60C under alkaline conditions. These
characteristics and the high production level of 0.6 g of phenol oxidase per liter in shake flasks, which is
equimolar with the glucoamylase protein levels, make this enzyme suitable for use in processes that occur
under alkaline conditions, such as laundry cleaning.
able. Consequently, applications to produce consumer goods
need redox enzymes, especially those that can be produced
easily by recombinant strains.
We identified a fungus, Acremonium murorum, which secretes an unknown phenol oxidase capable of decolorizing
chromophores such as cyanidin and pelargonidin. Our objectives in this study were (i) to clone the corresponding phenol
oxidase gene, (ii) to express the gene at high levels in Aspergillus awamori, a fungus which is used in industry for the production of proteins (8, 23), and (iii) to characterize the enzyme
with respect to its suitability for laundry cleaning.

Blue oxidases are a subfamily of multicopper enzymes, including laccases, ascorbate oxidases, and vertebrate ceruloplasmin, that are produced by a large number of plants and fungi
(20). These enzymes catalyze the four-electron reduction of
molecular oxygen to water with the concurrent one-electron
oxidation of a substrate, usually a polyphenolic compound
(16). Relatively little is known about the physiological role of
these enzymes in nature. Laccases, for example, are implicated
in a number of processes such as conidial pigmentation, lignin
degradation, pathogenicity, and fruiting-body formation (reviewed in reference 22).
Fungal multicopper oxidases are receiving increasing interest as potential industrial enzymes in applications such as detoxification of toxic phenolic compounds and azo dyes (reviewed in reference 12), enzymatic bleaching of kraft pulp (2),
and delignification (30) because these oxidases catalyze the
oxidation of phenols. Also, it is often desirable to convert
compounds under mild conditions to create new product properties or to maintain other properties of a beverage or food
product in other processes, e.g., food processing. In the area of
laundry cleaning, enzymatic bleach might be a good alternative
to current chemical bleaches.
Blue oxidase genes have been cloned from a number of
species, mainly plants, white-rot basidiomycetes, and some
plant pathogens (for a review, see references 4 [and references
therein] and 20). However, in most fungi, oxidases (mainly
laccases) are produced at levels that are too low for commercial purposes, even when cloned genes are expressed in heterologous hosts (14, 17). For any of these potential applications
to become reality, an inexpensive oxidase source must be avail-

MATERIALS AND METHODS

Bacterial and fungal strains. For standard bacterial cloning, Escherichia coli
DH5 (9) was used. For cloning of a cDNA library, E. coli XL1-Blue MRF
{(mcrA)183 (mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac
(F proAB lacIqZ M15 Tn10 [Tetr])} (Invitrogen, Carlsbad, Calif.) was used.
Saccharomyces cerevisiae strain VW-K1 (MATa leu2) was used for expression of
the cDNA library. Acremonium murorum var. murorum CBS 157.72 was obtained
from the Centraal Bureau voor Schimmelcultures (CBS), Baarn, The Netherlands. 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 CBS
115.52.
Cultivation of A. murorum. A shake flask containing 100 ml of potato dextrose
broth (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 25C. The culture
was grown for 3 days at 25C in a rotary shaker (250 rpm), and then it was
transferred to 100 ml of minimal medium (1), enriched with 0.5% yeast extract,
and grown for another 3 days at 25C.
Extraction of total RNA and isolation of poly(A) RNA. Total RNA was
prepared by extraction with Trizol (Life Technologies, Inc., Rockville, Md.). The
RNA concentration was determined by measuring absorbance at optical densities of 260 and 280 nm (OD260/280). Purification of poly(A) mRNA from total
RNA 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 synthesis
kit (Stratagene, La Jolla, Calif.) according to the manufacturers protocol, except
that reverse transcriptase Superscript II (Life Technologies, Inc.) was used instead of Moloney murine leukemia virus reverse transcriptase.

* Corresponding author. Mailing address: Biotechnology Group,

Unilever Research Vlaardingen, Olivier van Noortlaan 120, 3133 AT
Vlaardingen, The Netherlands. Phone: 31 104605263. Fax: 31
104605383. E-mail: robin.gouka@unilever.com.
2610

VOL. 67, 2001

PHENOL OXIDASE GENE FROM ACREMONIUM MURORUM

Construction of a cDNA library. cDNA was cloned as EcoRI/XhoI fragments

into plasmid pYES2.0 (Invitrogen). For large-scale ligations, approximately 200
ng of cDNA was ligated to 1.5 g of EcoRI/XhoI-digested pYES2.0, in a total
volume of 7.5 l with 1 U of T4 DNA ligase for 5 h at room temperature.
Aliquots of 2.5 l were used to transform 50 l of electrocompetent E. coli
XL1-Blue MRF cells (Stratagene) (conditions, 1,700 V, 200 , and 25 F).
After addition of 1 ml of SOC (per liter: 20 g of Bacto-tryptone [Difco Laboratories], 5 g of Bacto-yeast extract [Difco], 0.58 g of NaCl, 0.18 g of KCl, 2.0 g of
MgCl2 6 H2O, 2.46 g of MgSO4 7 H2O, 3.6 g of glucose) to each mix, the cells
were regenerated for 1 h at 37C, plated on Luria-Bertani medium (LB) (1%
Bacto-tryptone [Difco], 0.5% Bacto-yeast extract [Difco], 10 g of NaCl liter1,
pH 7.0) with ampicillin (100 g/ml), and grown at 37C for another 16 h.
Dilutions were plated to calculate the titer of the library. To each plate was
added 3 ml of capable of decolorizing plant chromophores (such as anthocyanins), and bacteria were scraped off, pooled, and stored in small aliquots. Largescale DNA was prepared from 200- to 500-ml cultures of LB inoculated with an
aliquot of transformants and propagated overnight.
Transformations. Lithium acetate-mediated transformations of S. cerevisiae
VW-K1 were carried out by the method of Gietz and Woods (5). Cells were
washed with 1 M sorbitol and finally plated onto selective medium [0.67% yeast
nitrogen base minimal medium (without amino acids and with ammonium sulfate)] and 2% glucose and incubated for 3 to 5 days at 30C. Transformation of
A. awamori was carried out as described previously (6).
Screening of an A. murorum cDNA library in S. cerevisiae for decolorization of
cyanidin. Approximately 50,000 colonies of an A. murorum cDNA expression
library 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 minimal
medium, 0.1 M sodium phosphate (pH 7.2), and 120 mg of cyanidin liter1 to
yield approximately 3,000 colonies per plate, and incubated at 30C. The plates
were screened daily for halo-producing transformants.
Isolation of plasmid DNA from yeast. Plasmid DNA was isolated as described
by Hoffman and Winston (11), using a mixture of lysis buffer, phenol, and glass
beads followed by centrifugation, transformation of E. coli with a small sample of
the supernatant, and isolation of the plasmid from E. coli.
DNA sequence analysis. DNA sequence analysis was carried out on an LKB
automated 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 expression
vector pAWGLA2 (7). The oxidase gene was fused to the 3 end of the Aspergillus niger glucoamylase gene. The genes are separated by a DNA sequence
encoding a KEX2-type recognition site (Asn-Val-Ile-Ser-Lys-Arg). The expression signals (promoter and transcription terminator) are derived from the A.
awamori -1,4-endoxylanase A gene. Since the N-terminal amino acid sequence
of wild-type AMO could not be determined due to the low production levels by
Acremonium, the fusion was based on cleavage of AMO by the rules of Von
Heijne (25, 26) (cleavage between signal peptide and protein). Based on this
hypothesis, the glucoamylase was fused to amino acid 22 (Met) of AMO.
For a correct fusion at the 3 end of the phenol oxidase gene, pUR7876 was
digested with XhoI and XbaI and ligated with two annealed oligonucleotides
(5-TCGAGCTTAAGT-3 and 5-CTAGACTTAAGC-3), thereby introducing
an AflII site, resulting in pUR7880. For a correct fusion at the 5 end, pUR7880
was modified by replacing an EcoRV/NarI fragment with a 170-bp EcoRV/NarI
PCR-derived DNA fragment, giving plasmid pUR7890. This vector contains part
of the KEX2 recognition site starting at an EcoRV site and the 5 part of the A.
murorum gene up to the NarI site. The PCR fragment was obtained with the
primers Acr06 (5-GAGAGAGATATCCAAGCGCATGCCCAAGTTCGAGC
TGGACATTCCTGAGG-3) and Acr02 (5-GCTTGATCTCGATCTCATAGT
AGT-3) on plasmid pUR7876 as a template. From pUR7890, pUR7891 was
constructed by inserting the A. murorum gene, present on a 2-kb EcoRV/AflII
fragment, into the Aspergillus expression vector pAWGLA2, which was also
digested with EcoRV and AflII. Finally, pUR7891 was digested with NotI and
ligated with the Aspergillus nidulans amdS and the A. awamori pyrG doubleselection marker from pAW10S-4 (24), resulting in pUR7893 (Fig. 1).
Construction of recombinant A. awamori strains. Strain A. awamori AWC4.20
was transformed with pUR7893, and transformants were selected in two ways,
either by restored growth on minimal medium (1) due to the integration of the
wild-type pyrG gene or by growth on minimal medium with 10 mM acetamide as
a sole nitrogen source, due to the integration of the amdS gene. The latter
selection method usually results in transformants containing multiple copies of
the plasmid, since these transformants grow better and faster on acetamidecontaining medium. Transformants were purified twice on Aspergillus minimal

2611

FIG. 1. Plasmid pUR7893. Open bars, A. awamori 5 and 3 regulatory sequences; filled arrows, coding sequences. Abbreviations and
designations: glaA, glucoamylase gene; amdS, acetamidase gene; pyrG,
orotidine 5-monophosphate decarboxylase gene; amp, -lactamase
gene, exlA, -1,4-endoxylanase gene; ori, origin of replication. Only
relevant restriction sites are indicated.

medium (1). Conidia were obtained by growing mycelium on potato dextrose

agar plates for 5 to 7 days at 30C.
Shake flask induction experiments with A. awamori. Production in shake flasks
of the AMO by A. awamori under control of the -1,4-endoxylanase A transcription 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 separated
from the mycelium by filtration through Miracloth (Calbiochem-Behring, La
Jolla, Calif.). Concentration of the proteins in the medium was carried out by
ammonium sulfate precipitation (80% saturation). The precipitate was kept at
4C for 16 h and pelleted by centrifugation for 45 min at 25,000 g The protein
pellet was dissolved in 2.5 ml of 30 mM sodium phosphate (pH 8.5) and subsequently desalted using a Sephadex G25 column (Amersham Pharmacia Biotech).
Proteins were eluted with 3.5 ml of 30 mM sodium phosphate (pH 8.5). Enzyme
analysis was carried out by polyacrylamide gel electrophoresis (PAGE), plate
assays, and enzyme activity assays. For PAGE, samples were boiled in 1%
sodium dodecyl sulfate (SDS) without reducing agent. Glucoamylase was detected as previously described (7).
Plate assays. Fungal conidia were inoculated onto agar plates, containing
minimal medium (1) with 1.5% agar and a substrate, either an anthocyanidin
(120 mg of cyanidin liter1 [Fluka; Sigma-Aldrich Corp., St. Louis, Mo.] or 240
mg of pelargonidin liter1 [Roth, Karlsruhe, Germany]) or 2 mM ABTS [2,2azinobis(3-ethylbenzthiazolinesulfonic acid)]. The plates were incubated at 25C
and screened daily for the presence of clearing zones (anthocyanidins) or green
halos (ABTS) as a result of extracellular enzyme activity. To detect enzyme
production by recombinant strains containing the phenol oxidase gene fused to
the exlA promoter, D-xylose was added to the plates at a 5% concentration.
Enzyme activity assays. Decolorization of anthocyanidins was measured in 1
ml of solution containing 100 mM sodium phosphate (pH 7.5), 0.1 mM cyanidinchloride or pelargonidinchloride, and 20 to 200 l of enzyme sample. The change
in absorbance (per minute) was measured during 5 min in a UV-VIS spectrum
(wavelength, 200 to 700 nm; absorbance peak, 579 nm). Standard ABTS oxidation assays were carried out by adding the appropriate amount of enzyme to 50
mM sodium phosphate (pH 6.0)2 mM ABTS solution (final volume, 1 ml) and
monitoring the absorbance increase at 414 nm (extinction coefficient, 35 mM
cm1). One unit of enzyme activity was defined as the amount of enzyme that
oxidizes 1 mol of ABTS per min per ml at 20C.
For determination of the AMO activity as a function of pH, standard amounts
of AMO (1,500 U) were added to 1 ml of Britton and Robinson buffer (B&R
buffer) (3) (pH range, 3.0 to 11.0)2 mM ABTS solution, and the activity was
measured at 414 nm. Similarly, syringaldazine (SGZ) oxidation activity was
determined in B&R buffer100 M SGZ by monitoring the absorbance change
at 530 nm. B&R buffer was prepared by mixing a 100-ml solution of 28.6 mM
citric acid, 28.6 mM KH2PO4, 28.6 mM boric acid, and 28.6 mM diethylbarbituric

2612

GOUKA ET AL.

APPL. ENVIRON. MICROBIOL.

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

Enzyme

Amur

A. murorum polyphenol oxidase

M. verrucaria bilirubin oxidase
T. villosa laccase (lcc1)
T. versicolor laccase (lcc4)
P. ostreatus laccase (pox2)
P. radiata laccase
A. bisporus laccase
N. crassa laccase
M. thermophila laccase
S. thermophilum laccase
B. cinereus laccase
C. parasitica laccase
A. nidulans laccase

Mver

Tvil

Tvers4

Pos2

Prad

Abis

Ncras

Mther

Sther

Bcin

Cpara

Anid

66

15
15

15
16
68

14
12
62
64

15
14
63
64
38

14
13
47
47
48
43

12
12
25
27
27
23
26

11
15
28
27
25
24
25
60

14
12
27
26
25
25
27
56
63

14
11
30
29
26
26
22
35
32
29

13
17
31
26
24
24
23
57
56
51
37

11
6
16
17
19
17
17
18
18
18
20
19

a
Enzyme amino acid sequences (numbers in parentheses below are accession numbers) are the polyphenol oxidase from A. murorum (this paper), bilirubin oxidase
from 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), and
A. 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.

acid, which was set at the appropriate pH with 0.2 M sodium hydroxide and then
diluted with water to 200 ml. AMO activity as a function of temperature was
determined by measuring the activity of standard amounts of AMO (1,500 U) in
B&R buffer (pH 4.5)2 mM ABTS at various temperatures (without preincubation of AMO). Stability as a function of pH was measured by incubating standard
amounts of AMO in B&R buffer (pH range 3.0 to 9.6) at 4C and determining
the residual activity at different times using the standard ABTS oxidation assay
described above. Stability as a function of temperature was measured by incubating standard amounts of AMO in B&R buffer (pH 8.5) at different temperatures and determining the residual activity at different times using the standard
ABTS oxidation assay.
Purification of A. murorum phenol oxidase. Hydrophobic-interaction chromatography was used to purify AMO from the fermentation broth. A phenylSepharose 6 fast-flow column (Amersham Pharmacia Biotech) was equilibrated
with 50 mM sodium phosphate30% (1.3 M) ammonium sulfate (pH 6.0). Ammonium sulfate was added to the enzyme sample to a final concentration of 1.3
M. Proteins were eluted with a linear decreasing-salt gradient. Enzyme activity in
the different fractions was measured at pH 6.0 using 2 mM ABTS as substrate.
Those samples containing phenol oxidase were pooled and dialyzed against 20
mM sodium phosphate (pH 8.5) and stored at 20C.
Determination of the amino-terminal sequence of AMO. Protein samples were
analyzed using an apparatus consisting of a Porton LF3000 sequencer and online
phenylthiohydantoine analysis (Beckman Instruments Inc., Fullerton, Calif.)
with a Beckman high-performance liquid chromatograph type 125S and a Beckman detector type 168. The phenylthiohydantoine derivatives were analyzed
online using a C18 Microbore RP column (Beckman). Separation was achieved
by using a gradient of 5% tetrahydrofuran in water and acetonitrile. Detection
was done at 268 nm.
Nucleotide sequence accession number. The sequence data for AMO have
been submitted to the EMBL database under accession number no. AJ271104.

RESULTS
Isolation, cloning and characterization of AMO. The fungus
A. murorum var. murorum CBS 157.72 produced a clearing
zone when cultured on solidified media containing either cyanidin or pelargonidin, and it secreted a phenol oxidase into the
medium when grown in shake flask cultures.
From an A. murorum cDNA expression library in S. cerevisiae VW-K1, we identified a transformant that produced a
clearing zone (halo) around the colony on cyanidin-containing
plates. This transformant was purified, and plasmid DNA, designated pUR7876, was isolated. Retransformation of S. cerevisiae with pUR7876 DNA again resulted in halo-forming colo-

nies. The DNA sequence of the EcoRI/XhoI cDNA insert in

pUR7876 was determined by subcloning fragments in pUC19
(29). The insert consisted of 2,120 nucleotides, including 5 and
3 nontranslated sequences and a poly(A) tail. The DNA sequence, with the ATG codon at position 135, comprised an
open reading frame of 1,806 nucleotides, encoding an enzyme
of 602 amino acids. The 3 nontranslated region was 165 bases
and was followed by a poly(A) tail. An in-frame ATG codon
located 63 bp downstream of the first one also could be used to
start translation, resulting in a protein of 581 amino acids.
Comparison of the deduced amino acid sequence with the
sequences of proteins in the databases (Table 1) showed an
identity of 66% with a bilirubin oxidase isolated from the
fungus Myrothecium verrucaria (13) (see Table 1). Furthermore, the A. murorum phenol oxidase was similar to the consensus sequences of the four copper-binding sites present in
laccases (4) (Fig. 2). The homology with laccases was restricted
to those consensus areas, and the overall identity of AMO with
laccases was 15% (Table 1).
Heterologous production of AMO by A. awamori. We transformed A. awamori AWC4.20 with pUR7893 to obtain transformants that overproduced AMO. In a cyanidin plate assay,
all transformants produced large halos around the fungal colony, indicating that cyanidin was converted into a colorless
compound by the secreted enzyme. Similar plates, in which
cyanidin 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 in
submerged cultivation and had similar activities on cyanidin.
These activities corresponded to a decrease in the OD579 (absorbance peak of cyanidin at pH 7.5) of approximately 0.06 to
0.07/l of medium sample in 1 min, which is 2 to 3 orders of
magnitude more than the production levels obtained with culture medium of A. murorum.
The amounts of extracellular AMO activity, as determined
with an ABTS activity assay, reached up to 25 U/ml after 30 h
of induction. Based on specific activity of 40 U/mg (see below),

VOL. 67, 2001

PHENOL OXIDASE GENE FROM ACREMONIUM MURORUM

2613

FIG. 3. SDS8 to 18% gradient PAGE from supernatants of AWC7893 transformants cultivated in shake flasks. The samples were boiled
without reducing agent. The gel was stained with Coomassie brilliant
blue. The arrows indicate the position of the phenol oxidase. Lane 1,
AWC-7893-5p; lane 2, AWC-7893-10A; lane 3, AWGLA, which contains a single copy of the A. niger glucoamylase gene (7). M, molecular
size marker (kDa).

the level of recombinant enzyme secreted in these shake flask

cultures was approximately 600 mg per liter.
Medium samples of two transformants, AWC-pUR7893-5p
and -10A, also were analyzed on SDS-PAGE (8 to 18% gradient gel), stained with Coomassie brilliant blue (Fig. 3). Both
samples contained high levels of recombinant enzyme, visible
as a main band of about 67 kDa. In addition, a second, minor
band of about 40 kDa was visible. Apparently, this smaller
band represented a faster-migration form of denatured AMO,
since both proteins have the same amino-terminal sequence
(SPLSPAYTLF) and, under nondenaturing conditions, only a
single band is visible (see below). The amounts of AMO were
almost equimolar with the amounts of glucoamylase (visible as
a band at approximately 80 kDa), indicating that degradation
was minimal. The identity of glucoamylase also was confirmed
by Western blot analysis (data not shown).
Determination of the amino-terminal sequence gave the sequence SPLSPAYTLF, indicating that the enzyme was processed after amino acid 61. Thus, although the fusion was
actually based on the theoretical cleavage site of a signal peptide between amino acids 21 and 22 (the fusion of glucoamylase was made with amino acid 22 of AMO), AMO is cleaved
after amino acid 61. This means that the glucoamylase-AMO
FIG. 2. Amino acid sequence alignment of AMO with other blue
copper enzymes. Only those areas that contain the types I, II, and III
copper ligands (marked in bold as 1, 2, and 3) are shown. Amino acid
sequence data were obtained as described in Table 1. For abbreviations on left, see Table 1, footnote b. T. tsunodae bilirubin oxidase has

GenBank accession number AB006824. A consensus sequence is given

below each 13-row set. An amino acid identity of 100% among all
enzymes is shown in uppercase, and an identity between 80% and
100% is in lowercase.

2614

GOUKA ET AL.

fusion molecule contained two consecutive propeptides: the

prosequence of glucoamylase (NVISKR, containing the KEX2
cleavage site) and the prosequence of AMO (theoretically 40
amino acids). A zymogram containing cyanidin as a substrate
showed that only AMO was responsible for the decolorization
of cyanidin.
Characterization of recombinant AMO. We purified the enzyme from the culture broth by hydrophobic-interaction chromatography. The major peak in enzyme activity, which eluted
at approximately 45% salt, was clearly visible as a blue band on
the column during elution. The specific activity of the purified
enzyme was about 40 U/mg of protein.
From SDS-PAGE, the molecular size of AMO was estimated to be 67 kDa, whereas the calculated molecular size was
60 kDa. The 7-kDa difference could be explained by N-glycosylation at two putative sites (Asn-X-Thr). Furthermore, the
40-kDa band that was observed in the medium samples of the
transformants also was present in purified AMO. When AMO
was not boiled, a single band of approximately 32 kDa was
visible on SDS-PAGE (data not shown). Apparently, the presence of 1% SDS is not sufficient to fully denature the protein.
This was confirmed by analysis of the activity after incubation
of AMO in 1% SDS, which showed no decrease in AMO
activity. Boiling completely destroyed the activity.
On an isoelectric focusing gel, a band with a pI that was near
3.5 was enzymatically active when the gel was incubated with
ABTS. This pI was lower than the calculated pI of 4.3.
Recombinant AMO had an optimal pH of 4 to 4.5 with
ABTS as substrate (Fig. 4A). With SGZ as a substrate, the
maximum activity was observed at pH 8.5 to 9. At 60C, the
highest AMO activity was measured, and it was approximately
2.5-fold higher than the activity observed at 20C (Fig. 4B).
The stability of the purified enzyme was measured as a
function of pH and temperature. The enzyme was highly unstable at low pH, whereas at alkaline pH the enzyme retained
full activity for at least 280 h when incubated at 4C (Fig. 5A).
Thermostability analysis showed that the enzyme was stable at
50C for 3 h and almost fully stable for 20 min at 60C. After
prolonged incubation or incubation at higher temperatures,
the activity decreased (Fig. 5B).
DISCUSSION
We isolated and characterized a phenol oxidase from the
fungus A. murorum. Amino acid sequence comparison shows a
high degree of identity (66%) with a bilirubin oxidase (BOX)
isolated from the fungus Myrothecium verrucaria (13). As with
other blue copper enzymes, e.g., laccases and ascorbate oxidase, four consensus domains for all types (I, II, and III) of
copper ligands are present in AMO (Fig. 2). Three other fungal bilirubin oxidases have been reported, from Trachyderma
tsunodae (10), Penicillium janthinellum (18), and Pleurotus ostreatus (15). However, these enzymes differ from both AMO
and Myrothecium bilirubin oxidase. The T. tsunodae bilirubin
oxidase amino acid sequence is very similar to the sequences of
laccases (Fig. 2) with, for example, 74% identity with the
amino acid sequence of T. villosa laccase encoded by the lcc4
gene. The identity of T. tsunodae bilirubin oxidase with AMO
and M. verrucaria bilirubin oxidase is only 12 and 14%, respectively. Similarly, the P. ostreatus bilirubin oxidase appears to be

APPL. ENVIRON. MICROBIOL.

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 () and SGZ (100 M) as substrate
(). Assays were performed in B&R buffer at the indicated pH at
30C. (B) AMO activity as a function of temperature (normalized to
the activity at 20C) with ABTS (2 mM) as substrate in B&R buffer, pH
4.5. Both experiments were carried out in duplicate; standard errors
were 10%.

identical to P. ostreatus laccase POX2 (15) and has only 13%

identity with the amino acid sequence of AMO and M. verrucaria bilirubin oxidase. The P. janthinellum enzyme, which contains copper, zinc, and iron atoms, also is very different from
AMO and M. verrucaria bilirubin oxidase, since these proteins
contain only copper.
The amino-terminal part of the protein shows the characteristics of a signal sequence. The predicted (26) signal peptide
cleavage site for AMO is between amino acids 21 (Ala) and 22
(Met). The protein also contains two dibasic amino acid sequences, residues 51/52 and 60/61 (both Arg-Arg), which might
be cleaved by a KEX2-like protease and which could indicate
that AMO is initially produced as a proenzyme (21). The first
residue of the mature recombinant enzyme is Ser-62 (although
the N-terminal sequence of AMO produced by A. murorum
could not be determined exactly, this sequence was not in
contradiction with the sequence obtained from the recombinant form). Based on these results, residues 22 through 61
probably comprise a propeptide whose proteolytic removal
occurs during maturation of AMO. Consequently, the recombinant fusion molecule probably contains two consecutive

VOL. 67, 2001

PHENOL OXIDASE GENE FROM ACREMONIUM MURORUM

FIG. 5. The effect of pH and temperature on AMO stability. (A)

AMO stability as a function of pH. AMO was incubated at different
pHs (310) in B&R buffer at 4C, and the residual activity (normalized
to t 0 h) was analyzed after 3 (), 20 (), 168 (), and 280 (E) h
in B&R buffer (pH 6) with 2 mM ABTS. (B) AMO stability as a
function of temperature and time. AMO was incubated at different
temperatures in B&R buffer (pH 8.5), and the residual activity (normalized to t 0 h) was analyzed after 0.3 (), 1 (), 3 (), and 21 (E)
h in B&R buffer (pH 6) with 2 mM ABTS. Both experiments were
carried out in duplicate; standard errors were 10%.

propeptides: the glucoamylase prosequence NVISKR and the

prosequence of AMO. Based on the amino-terminal-sequence
data, processing occurs correctly after the AMO prosequence.
If translation begins at the second in-frame ATG codon, the
resulting protein does not contain a theoretical site for cleavage of a signal peptide, suggesting that the first ATG is the
translation initiation codon that is used in vivo.
To make the application of multicopper enzymes feasible in
industrial processes or products, the production levels in shake
flasks should be at least approximately 1 g per liter. However,
the amounts that have been reported are usually low. In a
homologous system the amounts can range from a few milligrams per liter up to 80 mg per liter for Botrytis cinereus laccase
(19). However, these levels are still low for commercial purposes, and cultivation of these fungi is often difficult. Although
laccases have been isolated from a large number of ascomycetes (e.g., A. nidulans, Neurospora crassa, and Podospora anserina), 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 recombinant phenol oxidase from A. awamori transformants grown in

2615

shake flasks was high (600 mg per liter). As normally the

production yield is improved when shake flask experiments are
replaced by fed-batch fermentation processes, AMO has potential commercial utility for industrial purposes or consumer
products.
AMO had an optimal activity for ABTS and SGZ as substrates at pH from 4 to 4.5 and 8.5 to 9, respectively. These
optima are at least equal and often higher than described for
other polyphenol oxidases such as laccases (27, 28) and indicate that AMO has potential to be used under alkaline conditions. Furthermore, under alkaline conditions the enzyme is
fully stable for at least 3 h at 50C and loses only 15% activity
after 20 min at 60C. This is close to the stability observed for
the thermophilic fungi Myceliophthora thermophila and
Scytalidium thermophilum (28) and higher than for laccases
isolated from Polyporus pinsitus and Rhizoctonia solani (28).
In conclusion, we isolated a new phenol oxidase derived
from the fungus A. murorum var. murorum CBS 157.72. The
enzyme converted anthocyanidins to colorless compounds and
could be applied as a mild alternative for chemical bleaching,
such as in laundry cleaning. This enzyme forms an attractive
alternative to other polyphenol oxidases due to (i) its potential
to be produced at high levels (at least 0.6 g/liter) by cultivation
of a recombinant strain of A. awamori, (ii) its high stability
under alkaline conditions and high temperatures, (iii) its high
activity at 50 to 60C, and (iv) its activity under even extreme
alkaline conditions (pH 9 to 10).
ACKNOWLEDGMENTS
We thank John Chapman and Maarten Egmond for critical reading
of the manuscript and Han van Brouwershaven for amino-terminal
sequence analysis.
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