Eur. J. Biochcm.

209,603 - 61 1 (1992)
FEBS 1992

Substrate specificity and properties of the aryl-alcohol oxidase

from the ligninolytic fungus Pleurotus eryngii
Francisco GUILLEN, Angel T. MARTINEZ and Maria Jesus MARTINEZ
Centro de Investigaciones Biologicas, Consejo Superior de Invcstigacioncs Cientificas, Madrid, Spain

(Received May 4/July 22, 1992) - EJB 92 0613

The production in a 5-1 fermenter of the extracellular enzymes laccase and aryl-alcohol oxidase
by the fungus Pleurotus eryngii was studied. The latter enzyme has been purified 50-fold by Sephacryl
S-200 and Mono Q chromatography. Purified aryl-alcohol oxidase is a unique flavoprotein with 15%
carbohydrate content, a molecular mass of 72.6 kDa (SDS/PAGE) and a p l of 3.9. The enzyme
presents wide specificity, showing activity on benzyl, cinnamyl, naphthyl and aliphatic unsaturated
alcohols. Neither activity nor inhibition of veratryl alcohol oxidation was found with saturated
alcohols, but competitive inhibition was produced by aromatic compounds which were not aryl-
alcohol oxidase substrates, such as phenol or 3-phenyl-1-propanol. From these results, it was apparent
that a double bond conjugated with a primary alcohol is necessary for substrate recognition by aryl-
alcohol oxidase, and that activity is increased by the presence of additional conjugated double
bonds and electron donor groups. Both affinity and maximal velocity during enzymic oxidation of
methoxybenzyl alcohols were affected in a similar way by ring substituents, increasing from benzyl
alcohol ( K , = 0.84 mM, V,,, = 52 Ujmg) to 4-methoxybenzyl alcohol ( K , = 0.04 mM, V,,, =
208 U/mg). Aryl-alcohol oxidase presents also a low oxidase activity with aromatic aldehydes, but
the highest activity was found in the presence of electron-withdrawing groups.

It is well known that lignin biodegradation is an oxidative
process carried out mainly by white-rot fungi, but our actual
knowledge of the ligninolytic system still reveals large gaps.
To date, three types of extracellular ligninolytic enzymes have
been involved in this process : lignin peroxidases, Mn-depen-
dent peroxidases and laccases (Kirk and Farrell, 1987;
Higuchi, 1990). These enzymes catalyze the one-electron oxi-
dation of lignin units resulting in various non-enzymic reac-
tions which include bond cleavage. Whereas Mn-dependent
peroxidase and laccase oxidize only phenolic units, which
represent 10-20% in grass lignin and lower percentages in
wood (Lapierre and Monties, 1989), lignin peroxidase is able
to attack the non-phenolic units, and it is considered the
most effective lignin-degrading enzyme. However, the actual
implication of these enzymes in lignin biodegradation is still
ambiguous (Lewis and Yamamoto, 1990; Sarkanen et al.,
1991).
The activity of ligninolytic peroxidases requires the pres-
ence of extracellular H 2 0 2 .This compound has been detected
in cultures of white-rot fungi (Faison and Kirk, 1983; Veness
and Evans, 1989) and its production ascribed to several oxi-
dases (Shimada and Higuchi, 1991) whose physiological roles
are not well known. H202produced by intracellular enzymes
acting on glucose (Eriksson et al., 1986; Kelley and Reddy,
1986), methanol (Nishida and Eriksson, 1987) or fatty-acyl-
CoA (Greene and Gould, 1984) requires a transport system
Correspondence to M. J. Martinez, Ccntro de Investigaciones Bio-
logicas, Consejo Superior de Investigaciones Cientificas, Velizquez
144, E-28006 Madrid, Spain
Enzymes. Aryl-alcoholoxidase(EC 1.1.3.7); laccase (EC 1.10.3.2);
lignin peroxidase (EC 1.11.1.7); Mn-dependent peroxidase (EC
1.11.1.7).
to the external medium that has not been reported. From
this viewpoint, extracellular enzymes such as glyoxal oxidase
(Kersten and Kirk, 1987) and aryl-alcohol oxidase discussed
below, present a clear advantage.
Information about the synergistic action of fungal enzymes
involved in lignin degradation is very scarce, and their simul-
taneous production has been reported only in some species.
The ligninolytic system of Phanerochaete chrysosporiurn was
the first investigated, and it includes several isoenzymic forms
of lignin peroxidase and Mn-dependent peroxidase (Kirk and
Farrell, 1987). Other species, like Trametes versicolor (Dodson
et al., 1987; Evans, 1985; Johansson and Nyman, 1987),
Trametes gihhosa, Trarnetes hirsuta (Nerud et al., 1991),
Phlebia radiata (Karhunen et al., 1990; Niku-Paavola et al.,
1988), Phkebia hrevispora (Pirez and Jeffries, 1990) and
Lentinula edodes (Bonnarme and Jeffries, 1990; Leatham,
1986) also produce laccase, but in many ligninolytic fungi only
laccase has been reported. Studies on the H202-producing
enzymes are less numerous because their importance depends
on the true role of peroxidases in lignin biodegradation. It has
been reported that P. chrysosporium produces all the above-
mentioned oxidases except aryl-alcohol oxidase. This enzyme
was first detected in the culture liquid of T. versicolor
(Polystictus versicolor; Farmer et al., 1960) and more recently
in three species of the genus PEeurotus (Bourbonnais and Paice,
1988, 1989; GuillCn et al., 1988, 1990; Sannia et al., 1991), in
which neither lignin peroxidase nor Mn-dependent peroxidase
have been found, although some of them cause preferential
degradation of lignin (Valmaseda et al., 1990). However, the
simultaneous production of aryl-alcohol oxidase and lignin
peroxidase by Bjerkandera adusta (Kimura et al., 1990;
604
Muheim et al., 1990a) increased the interest in the study of
aryl-alcohol oxidase.
In this context, we present results concerning the simul-
taneous production of aryl-alcohol oxidase and laccase by
Pleurotus eryngii, as well as the purification and characteristics
of aryl-alcohol oxidase. Special attention was given to the
specificity of aryl-alcohol oxidase, and we present here, for
the first time, the structural requirements of alcohol substrates
of the enzyme, a kinetic study of the oxidation of
methoxybenzyl alcohols and the activity of P . eryngii aryl-
alcohol oxidase with some aromatic aldehydes. Finally, the
properties of aryl-alcohol oxidase from different fungi and its
cventual role in lignin degradation are discussed.
MATERIALS AND METHODS
Fungal strain and culture conditions
P . eryngii A169 was isolated from mycelium of a fruit-
body cap and kept on 2% malt-extract agar in our fungal
collection. It was cultivated in a 5-1 fermenter (Microferm
MF-105, New Brunswick ScientificCo., Inc.) with 3 1 medium,
containing 10 g/1 glucose, 2 g/1 ammonium tartrate, 1 g/l
KH2P04, 1 g/1 yeast extract, 0.5 g/l MgS04 . 7 H 2 0 and
0.5 g/l KC1, and 1 ml mineral solution [lo0 mg/l B40,Na2
. 10HzO, 70 mg/l ZnS04 7 H z 0 , SO mg/l FeS04 . 7H20,
10 mg/l CuSO, . 5 H z 0 , 10 mg/l MnS04 . 4 H 2 0 , 10 mg/l
(NH4)6M07024. 4H20], at 26"C, 600 rpm and 1 l/min aer-
ation. Mycelium from 10-day-old cultures in 1-1 Roux flasks,
containing 100 ml of the same medium, was washed with
water, homogenized and used as inoculum at 1 g/1.
Analysis of protein, reducing sugars and carbohydrate
Protein concentration was determined by the method of
Bradford (1976), with bovine serum albumin as standard.
Reducing sugars in culture liquid were assayed by the
method of Somogyi-Nelson (Somogyi, 1945) and carbo-
hydrate content of purified aryl-alcohol oxidase by the
anthrone procedure (Trevelyand and Harrison, 1952), using
glucose as standard.
Enzyme assays
H 2 0 z concentration was determined by the peroxidasel
phenol-red assay described by Pick and Keisari (1980), using
0.1 M sodium phosphate, pH 6.0. A standard curve of H z 0 2
was prepared with dilutions of Perhydrol 30% (Merck) pro-
cessed in the same way. The H 2 0 2concentration of the com-
mercial solution was calculated from its absorbance at 230 nm
( ~ 2 3 0= 81 M-' . cm-').
Aryl-alcohol oxidase activity was assayed spectrophoto-
metrically as the oxidation of veratryl (3,4-dimethoxybenzyl)
alcohol to veratraldehyde ( E =~ 9 300 ~ ~ - ' . cm-'). The
M Amino acid composition and sequence analysis
reaction mixture contained 0.1 M sodium phosphate, pH 6.0,
and 10 mM veratryl alcohol.
Laccase activity was assayed in 100 mM sodium acetate,
pH 5.0, with 10 mM guaiacol. Oxidation of guaiacol was fol-
lowed by absorbance increase at 465 nm ( E =~ 12 100 ~ M~ -
. cm-').
Lignin peroxidase activity was measured as the HzOz-
dependent oxidation of veratryl alcohol according to Tien
and Kirk (1984) and Mn-dependent peroxidase activity under
conditions described by Paszcynsky et al. (1988) using 0.01YO
phenol red as substrate.
1 U enzyme activity is expressed as the amount of enzyme
releasing 1 pmol oxidized productimin, at 25 "C.
Aryl-alcohol oxidase purification
To obtain crude enzyme, 3 1 culture liquid from a 14-
day-old culture were concentrated 400-fold by ultrafiltration
(Amicon, 10-kDa-cut-off membrane) and polysaccharide was
removed as described elsewhere (GuillCn et al., 1990). This
crude preparation can be stored at - 20 "C for several months
without loss of aryl-alcohol oxidase activity.
Crude enzyme samples of 1 ml were loaded onto a column
(Pharmacia, K16/100) of Sephacryl S-200 equilibrated in
10 mM sodium tartrate, pH 3.0, at a flow rate of 20 ml/h.
Fractions containing aryl-alcohol oxidase activity were
pooled, concentrated at reduced pressure (30°C) to 2 ml and
dialysed at 4°C against 10 mM sodium phosphate, pH 5.5.
The enzyme was applied to a Mono Q anion-exchange
column (Pharmacia, HR S / S ) equilibrated in 10 mM sodium
phosphate, prepared with 10 mM phosphoric acid adjusted to
pH 5.5 with NaOH, and eluted with a linear gradient of 0 -
500mM NaCl in the same buffer, over a 20min period at
1 ml/min flow rate. Fractions containing aryl-alcohol oxidase
activity were pooled and dialysed against 10 mM sodium
phosphate, pH 6.0, and stored at - 70 'C. Purification was
carried out at room temperature unless otherwise stated.
Electrophoresis and isoelectric focusing
SDSjPAGE was performed by the method of Laemmli
(1970), with 7.5% polyacrylamide gels. Myosin (200 kDa),
/?-galactosidase (116 250 Da), phosphorylase b (97.4 kDa),
bovine serum albumin (66.2 kDa) and ovalbumin (45 kDa;
all Bio-Rad) were used as standards.
IEF was performed on 5% polyacrylamide gels with a
thickness of 1 mm and pH of 2.5 - 5.0 (Pharmacia Ampho-
line). The anode and cathode solutions were 1 M phosphoric
acid and 0.4 M Hepes, respectively.
The gels were stained for protein by the silver technique
(Morrisey, 1981).
Determination of molecular mass
The molecular mass of native aryl-alcohol oxidase was
determined by gel filtration on Superose 6 (Pharmacia, HR 10/
30). The column was eluted with SO mM sodium phosphate,
pH 5.5, containing 150 mM NaC1, and the eluate was
monitored at 280 nm and 460 nm (flow rate 0.5 ml/min). The
column was calibrated with bovine thyroglobulin (670 kDa),
bovine y-globulin (158 kDa), ovalbumin (44 kDa), horse
myoglobin (17 kDa) and vitamin B-12 (1350 Da) as standards
(Bio-Rad).
Amino acid composition was determined with an Bio-
tronik Photometer BT 7025 autoanalyzer after hydrolysis of
33 pg protein in 6 M HCI. The N-terminal sequence of aryl-
alcohol oxidase was determined by automated Edman degra-
' dation in an Applied Biosystems 477A pulsed-liquid protein
sequencer with 120A on-line phenylthiohydantoin analysis.
Substrate specificity of aryl-alcohol oxidase
The 43 compounds listed in Tables 2 and 4 were tested as
electron-donor substrates of aryl-alcohol oxidase, at 2 mM.

The specificity of aryl-alcohol oxidase was first qualitatively
explored by changes in the absorption spectrum of the reaction
mixtures. With alcohols whose corresponding aldehydes do
not have carbonyl groups conjugated with double bonds (com-
pounds nos 17-20 and 26-29), and with dimers and the
dehydrogenation polymer of coniferyl alcohol (nos 23 - 25),
thc method used was H 2 0 zproduction. measured by coupling
the peroxidase/phenol-red procedure mentioned above.
The activity of purified aryl-alcohol oxidase towards the
different substrates was compared by measuring O 2consump-
tion with a Clark-type electrode. As this general method was
less sensitive than HzOzproduction, aryl-alcohol oxidase ac-
tion on compounds with low substrate activity (nos 2,30,32 -
34,36,40,42 and 43) was determined by this procedure, except
for phenolic compounds that are substrates of the peroxidase
present in the reaction mixture.
As a consequence of the low solubility of several sub-
strates, the V,,, and K , were determined only for five meth-
oxylated benzyl alcohols (nos 1 - 3, 5 and 6). The initial velo-
cities were measured spectrophotometrically at the wave-
lengths shown in Table 3. E of the corresponding aldehydes
was calculated under the conditions of the activity assays.
Inhibition studies were carried out with veratryl alcohol as
the variable-concentration substrate, using 5 mM and 50 mM
alcohol. Kinetic constants were estimated from straight lines
fitted to Lineweaver-Burk plots by the least-squares method.
The assays above were performed in 100 mM sodium
phosphate, pH 5.0, at room temperature.
Chemicals
All the compounds used in the specificity study were pur-
chased from Aldrich Chemical Co. except those indicated
below. 1-(3,4-Dimethoxyphenyl)-l-ethanolwas obtained by
3,4-dimethoxyacetophenonereduction.
Two lignin models were prepared by the procedure de-
scribed by Nakatsubo (1988): 1-(3,4,5-trirnethoxyphenyl)-
glycerol-f&guaiacyl ether and 3-(3,4,5-trimethoxyphenyl)-2-
(3,4-dimethoxyphenyl)-1,3 -propanediol. Synthetic guaiacyl-
type dehydrogenation polymer was prepared by enzymic poly-
merization of coniferyl alcohol using horseradish peroxidase
from Boehringer (grade I) and H2OZ(Faix et al., 1985).
RESULTS
Enzyme production
In a previous study of P.eryngii oxidase, the highest levels
of aryl-alcohol oxidase activity were found in C-limited cul-
tures supplemented with yeast extract (Guillen et al., 1990),
and the same medium was used to scale up aryl-alcohol oxi-
dase production.in a 5-1 fermenter.
The evolution of extracellular aryl-alcohol oxidase activity
(Fig. 1A) showed a pattern similar to that previously de-
scribed. The enzyme was produced during growth, and maxi-
mal activity was detected after glucose depletion, when the
extracellular protein level was still relatively low (Fig. 1B).
Among ligninolytic enzymes, laccase activity attained the
highest level during the autolysis phase, although a peak was
observed after 8 days, but neither lignin peroxidase nor Mn-
dependent peroxidase activities were found over 50 days, Ex-
tracellular HzOz was observed only during primary growth
(Fig. 1A).
605
100, I10
n
I\ 6 '
-e
.-c
W
IOOC
I
I
\
\
a \
50 1 ,A
I -4\y-
,LL1 \
L-
0 5 10 15 20 25 30 35 40 45 50
Culture t i m e (days)
Fig. 1. Evolution of different parameters in culture liquid of P. eryngii.
(A) Aryl-alcohol oxidase (AAO), laccase and HzOz production. (B)
Levels of protein and reducing sugars.
Purification of aryl-alcohol oxidase
Table 1 summarizes the procedure used for aryl-alcohol
oxidase purification. Two chromatographic steps were re-
quired to obtain enzyme that showed electrophoretic hom-
ogeneity. Although the resolving capacity of size-exclusion
chromatography is moderate, a very high degree of purifi-
cation was achieved on Sephacryl S-200, exploiting its adsorp-
tion capacity (depending on mobile-phase pH) previously de-
scribed by Belew et al. (1978). The choice of 10 mM sodium
tartrate, pH 3.0, as the eluent buffer on the Sephacryl S-200
column was based on the stability of unpurified aryl-alcohol
oxidase at acidic pH (GuillCn et al.. 1990) and selective protein
adsorption at different pH. This was demonstrated by a pre-
vious assay of enzyme adsorption onto Sephacryl S-200, show-
ing a 1&fold increase in aryl-alcohol oxidase specific activity
on reducing the pH from 7.0 to 3.0 (Fig. 2). In this step,
aryl-alcohol oxidase was purified 1Mold by combining both
adsorption and filtration properties of the gel. Some contami-
nating proteins, like laccase, were found to bind strongly to
the gel and were washed out from the column with 500 mM
NaCl (data not shown). Fig. 3 shows the low number of pro-
tein peaks obtained after the Mono Q step and the large
area of the peak containing aryl-alcohol oxidase activity in
comparison with the other proteins.
Properties of purified aryl-alcohol oxidase
Based on gel-filtration chromatography on Superose-6,
the molecular mass of native aryl-alcohol oxidase, that eluted
as a sharp peak, was estimated to be 66.7 kDa. The denatured
molecular mass, determined by SDS/PAGE, was estimated
to be 72.6 kDa (Fig. 4A), suggesting that the enzyme is a
monomeric protein. Analytical IEF showed a homogeneous
protein with p I o f pH 3.9 (Fig. 4B).
606
Table 1. Purification of aryl-alcohol oxidase.

Purification Total Yield Total Specific Purification

step activity protein activity factor

U 9 mg U/mg -fold

Culture liquid 218.5 100.0 135.0 1.6 1.0

Crude enzyme 218.0 99.8 48.2 4.5 2.8
Sephacryl S - 200 191.8 87.8 3.3 58.1 36.3
Mono Q 166.9 76.4 2.1 79.5 49.7

loo i A
4
200 000

-: 12 + 116 260
97 400
e
a
6 6 200

4 5 000

a b
--I-- I --- 190
2 3 4 5 6 7 8
PH B
Fig. 2. Selective protein adsorption on Sephacryl S-200. Aryl-alcohol
oxidase (AAO), protein yields and specific activity in supernatant
after mixing 20 ~1 dialysed crude enzyme containing 150 mU aryl-
alcohol oxidase activity with 4 mlgel, equilibrated with 10 mM buffers
of different pH.

0.5 r
I
1300

2.0' '
0 2 4 6 8 10 12
Distance migrated (cm)

Fig. 4. Molecular mass and isoelectric point. (A) SDS/PAGE of purified

aryl-alcohol oxidase (a) and standards (b). (B) pZ of aryl-alcohol
oxidase (AAO) was determined after obtaining the pH-gradient pro-
file in the IEF gel with a contact electrode.

0 4 8 12 18 20 24 moiety, and a 100: 17 protein/carbohydrate ratio was calcu-

Retention time ( m i d
Fig.3. Elution profile of aryl-alcohol oxidase and other proteins on
Mono Q chromatography.
The spectrum of the native enzyme demonstrated the pres-
ence of a flavin group with absorption peaks at 384 nm and
460 nm. Enzyme reduction caused a loss of peaks and reoxi-
dation restored the spectral properties (Fig. 5). Anthrone
analysis revealed that aryl-alcohol oxidase has a carbohydrate
lated.
The effect of pH on initial velocities of veratryl alcohol
oxidation was studied at pH 2.0-12.0 using 100 mM borate/
citrate/phosphate buffer, and the highest velocity was
obtained at pH 5.0. The enzyme was stable in solution at
pH 6.0 - 9.0 when kept at 25 "C for 24 h. The effect of tem-
perature on aryl-alcohol oxidase activity was determined at
p H 6 over the range 20-75"C, using 5°C increments for
3 min, obtaining the maximal value at 55 "C. Thermal Stdbihty
was determined at fixed intervals during a 30-min incubation
 , - Natlve
Reduced
-. Reoxidized
substituents (e.g. 4-methoxy group in nos 4-6 and 10) prod-
607

tives (nos 2 - 13) were strongly affected by the nature, position

and number of ring substituents. In general, electron-donor

uced favourable effects on enzyme activity, whereas electron-

withdrawing substituents (e.g. 4-nitro group in no. 12) prod-
uced the opposite effect. However, compounds containing a
4-hydroxy group (nos 9 and 11) were oxidized much slower
than benzyl alcohol. The negative effect of the hydroxy group
on aryl-alcohol oxidase activity was also observed in y-alco-
hols: whereas cinnamyl alcohol was oxidized 4.5-times faster
than benzyl alcohol, coniferyl alcohol had no appreciable
substrate activity. Of the aromatic alcohols, 2-naphtha-
lenemethanol was the compound most readily oxidized by
the enzyme. Although an extremely low aryl-alcohol oxidase
activity was observed with monounsaturated, aliphatic ally1
~ alcohol (0.4% estimated as H 2 0 2production), the oxidation
300 350 400 450 500 550
rate of 2,4-hexadien-l-o1 was third in the extent of all the
Wavelength (nm) substrates tested.
Fig. 5. Absorption spectra of aryl-alcohol oxidase. Native enzyme (ap- The kinetic constants of purified aryl-alcohol oxidase cor-
proximately 0.5 mg/ml) in 20 mM sodium phosphate, pH 6 , was re- responding to benzyl alcohol and the methoxylated derivatives
duced with sodium dithionite and reoxidized with 02. with highest substratc activity are listed in Table 3. The values
obtained showed that, except for 3-methoxybenzyl alcohol,
the position of the substituents on the aromatic ring affected
of an enzyme solution at 30 -70 "C. Aryl-alcohol oxidase was Km and ,fI in the same Nay. The highest enzyme affinity and
stable up to 5O"C, had a half-life of 5 min at 60 "C and efficiency ( VmaX/Km) of the oxidation reaction were observed
exhibited total inactivation after 5 min at 65°C. with 4-methoxybenzyl alcohol.
The number of amino acid residues in each aryl-alcohol To complete the study of the substrate structures involved
oxidase molecule was estimated (on the basis of a molecular in the attachment to the enzyme, different alcohols were tested
mass of 72.6 kDa) to be 87 Asx, 48 Thr, 56 Ser, 52 Glx, 60 as inhibitors. Competitive inhibition were produced by phenol
Gly, 66 Ala, 54 Val, 11 Met, 38 Ile, 41 Leu, 14 Tyr, 36 Phe, (K, = 1.92 mM) and 3-phenyl-1-propanol (Ki = 4.48 mM),
13 His, 13 Lys, 33 Arg and 61 Pro (Cys and Trp were not whereas no inhibition was observed with methanol, propanol,
determined). 3-hydroxybenzyl and 4-hydroxybenzyl alcohols. The very low
The single N-terminal sequence obtained was as fol- activity on several primary alcohols containing a phenolic
lows: Ala-Asp-Phe-Asp-Tyr-Val-Val-Val-Gly-Ala-Gly-Asn- group (nos 8 - 2 1 and 22) was caused by the apparent lack of
Ala-Gly-Asn. aryl-alcohol oxidase affinity, shown by the absence of enzyme
inhibition with this type of compound. When considering the
competitive inhibition produced by phenol, the existence of
Substrate specificity of aryl-alcohol oxidase intermolecular hydrogen bonds between phenolic and primary
A preliminary study showed that a crude enzyme prep- hydroxy groups of the phenolic alcohols could explain the low
aration from P. eryngii oxidized primary aromatic a-alcohols activity obtained.
such as bcnzyl, 4-methoxybenzyl and vcratryl alcohol, and the Aryl-alcohol oxidase from P.eryngii showed some activity
a$-unsaturated coniferyl alcohol (GuillCn et al., 1990). In on aromatic aldehydes, but the highest value obtained, corre-
order to determine the specificity rcquirements of the purified sponding to 4-nitrobenzaldehyde, did not reach 5%' of benzyl
enzyme, alcohols listed in Table 2 were evaluated as substrates. alcohol activity (Table 4). This activity is not due to contami-
Benzyl alcohol was the simplest aromatic structure oxidized nation by an aldehyde oxidase because the ratio of alcohol/
by aryl-alcohol oxidase and, except for 4-phenylbenzyl aldehyde activities is unchanged during the whole purification
alcohol, all the benzyl derivatives assayed (nos 2- 13) were process, and a unique protein was observed after IEF. Con-
oxidized l o a variable extent. Spectral changes in the reaction trary to the observation with alcohols, electron-withdrawing
mixture were also observed with 2-naphthalenemethanol. substituents promote aldehyde oxidation by aryl-alcohol oxi-
However, the secondary a-alcohol 1-(3,4-dimethoxyphenyI)- dase, while electron donors produce the oppoite effect.
1-ethanol and the primary b-alcohol2-phenyl-1-ethanolwere
not activc as substrates. Of the primary y-alcohols (nos 18 --
22), only those containing the E-B double bond (i.e. cinnamyl DISCUSSION
and coniferyl alcohols) were oxidized by aryl-alcohol oxidase.
In reaction mixtures containing lignin model dimers (nos 23 Aryl-alcohol oxidase of P. eryngii was first reported by
and 24), which are y-alcohols but are not a$-unsaturated, or GuillCn et al. (1988, 1990). In Pfeuvotus specics, this enzyme
dehydrogenation polymer (no. 25), neither H 2 0 2production appears to be constitutive, as it is produced in different growth
nor O2 consumption was detected, even after 24 h. With the phases and culture conditions (Guilltn et al., 1990; Valmaseda
series of saturated aliphatic alcohols (nos 26 - 29), H 2 0 2was et al., 1991), and it is not inducible by compounds enhancing
not produced, but spectral changes were observed in the reac- lignin peroxidase production (Sannia et al., 1991). In B.
tion mixtures containing unsaturated aliphatic alcohols (nos adusta, similar aryl-alcohol oxidase activities were obtained
30 and 31). in different culture media, whereas lignin peroxidase activity
The rates of aryl-alcohol oxidase oxidation of substrates was strongly influenced by medium composition (Kimura et
are compared in Table 2 as percentages of the activity observed al., 1990). Since lignin peroxidase activity is assayed as the
with benzyl alcohol. Oxidation rates of benzyl alcohol deriva- H2O2-dependent oxidation of veratryl alcohol, and aryl-
608

Table 2. Aryl-alcohol oxidase activity with different alcohols. New maxima observed during alcohol oxidation to aldehyde, and aryl-alcohol
oxidase activity relative to benzyl alcohol. 8-0-4 dimer, 1-(3,4,5-trimethoxyphenyl)glycerol-j?-guaiacyl ether; p-1 dimer, 3-(3,4,5-
trimethoxyphenyl)-2-(.3,4-dimethoxyphenyl)-l,3- propanediol; DHP, dehydrogenation polymer of coniferyl alcohol; n.d., not detected.

Compound Compound Wavelength 0,

no. cons umption

nm %

1 Benzyl alcohol 250 100.0

2 2-Methoxybenzyl alcohol 325 < 5.0"
3 3-Methoxybenzyl alcohol 313 100 * 0
4 4-Methoxybenzyl alcohol 2 85 571.4
5 2,4-Dimethoxybenzyl alcohol 314 177.5
6 Veratryl alcohol 308 326.1
7 3,4,5-Trimethoxybenzyl alcohol 286 5.3
8 3-Hydroxybenzyl alcohol 313 < 5.0
9 4-Hydroxybenzyl alcohol 286 < 5.0
10 3-Hydroxy-4-methoxybenzyl alcohol 310 319.1
11 4-Hydroxy-3-methoxybenzyl alcohol 310 < 5.0
12 4-Nitrobenzyl alcohol 264 9.1
13 3-Phenoxybenzyl alcohol 308 78.0
14 4-Phenylbenzyl alcohol n.d. n.d.
15 2-Naphthalenemethanol 250 745.7
16 1-(3,4-Dimethoxyphenyl)-l-ethanol n.d. n.d.
17 2-Phenyl-1-ethanol - b
n.d.
18 3-Phenyl-1-propanol - n.d.
19 3-(3,4-Dimethoxyphenyl)-l-propanol - n.d.
20 3,3-Diphenyl-l-propanol - n.d.
21 Cinnamyl alcohol 293 451.1
22 Coniferyl alcohol 338 < 5.0
23 p-0-4 dimer - < 5.0
24 p-1 dimer - < 5.0
25 DHP - < 5.0
26 Methanol - n.d.
27 Ethanol - n.d.
28 Propanol - n.d.
29 Butanol - n.d.
30 Ally1 alcohol 212 < 5.0"
31 2,4-Hexadien-l-ol 280 531.0

a Low activities of 2-methoxybenzyl and ally1 alcohol were estimated as 1.2% and 0.4%, respectively, by HLOzproduction.
I, The oxidation of these compounds produce no changes in the ultraviolet spectrum, but absence of aryl-alcohol oxidase activity was
demonstrated by the H202-production test.
 609

Table 3. Kinetic constants for aryl-alcohol oxidase oxidation of methoxylated benzyl alcohols.

Alcohol Wavelength &

nm

4-Methoxybenzyl 285 16 980 0.04 208.3 5207.5

3-Methoxybenzyl 314 2540 0.22 29.4 133.6
Vera t ry 1 310 9300 0.41 125.9 304.9
2,4-Dimethoxybenzyl 314 8840 0.79 102.0 129.1
Benzyl 250 13 800 0.84 51.8 61.7

Table 4. Aryl-alcohol oxidase activity with different aldehydes. Maxima disappearing during aldehyde oxidation to acids, and aryl-alcohol
oxidase activity relative to benzyl alcohol (100%). n.c., no change.

Compound Compound Wavelength H A

no product ion

nm

32 Benzaldehyde 250 0.86

33 3-Methoxybenzaldehyde 314 0.85
34 4-Methoxybenzaldehyde 2 85 0.03
35 2,4-Dimethoxybenzaldehyde n.c. -
36 Veratraldehyde 308 0.01
37 4-Hydroxybenzaldehyde n.c. -
38 4-Hydroxy-3-methoxybenzaldehyde n.c.
39 4-Hydroxy-3,5-dimethoxybenzaldehyde n.c. -
40 4-Nitrobenzaldehyde 2 64 4.77
41 2-Naphthaldehyde n.c.
42 Cinnamaldehyde 2 93 0.38
43 4-Methoxycinnamaldehyde 338 0.01

alcohol oxidase catalyzed the same reaction without 1-1202,
the latter activity could be confused with lignin peroxidase.
The aryl-alcohol oxidase from P. eryngii, P. sajor-caju, P.
ostreatus and B. adusta are glycoproteins containing a flavin
prosthetic group identified as FAD by Sannia et al. (1991).
This flavin group acts as coenzyme of different oxidases, and
other dehydrogenases (Dixon 1971; Bright and Porter 1975).
The different aryl-alcohol oxidases have molecular mass 71 -
83 kDa and pl3.8 - 4.5, and the main difference between aryl-
alcohol oxidase from these fungi concerns the number of
isoenzymes. P. sajor-cuju and B. adusta contain two proteins
with aryl-alcohol oxidase activity (Bourbonnais and Paice,
1988; Muheim et al., 1990b) after IEF, while the aryl-alcohol
oxidase from P. eryngii is similar to that of P. ostrcatus. In
both species, a unique protein with electrophoretic and IEF
homogeneity, and similar N-terminal sequences (14 of the 15
amino acid residues matched in both enzymes) are produced
(Sannia et al., 1991). These sequences include the Gly-Xaa-
Gly-Xaa-Xaa-Gly consensus sequence which is characteristic
of flavoenzymes and other dinucleotide binding proteins
(Wierenga et al., 1985).
Although aryl-alcohol oxidase have been purified from the
above-mentioned fungi, specificity studies only consisted in
determinations of reaction velocity at constant concentration
of several substrates. In the present study, the information
on enzyme specificity acquired from activity assays, using a
variety of aromatic and aliphatic alcohols, was completed
with competitive inhibition studies. Moreover, the influence of
610
aromatic-ring substituents on the aryl-alcohol oxidase kinetic
constants were investigated, establishing their effect on both
enzyme affinity and reaction velocity.
The aryl-alcohol oxidase from P. eryngii presents a wide
substrate specificity, as demonstrated by the high activities
obtained with primary alcohols with very different chemical
structures, such as 2-naphthalenemethanol, 4-methoxybenzyl
alcohol and 2,4-hexadien-l-01. The high activity observed with
the latter compound, not reported in studies of aryl-alcohol
oxidase from other fungi, indicates that aromaticity is not
required for aryl-alcohol oxidase activity. In addition, com-
petitive inhibition by 3-phenyl-1-propanol and the absence
of inhibition by propanol, indicated the participation of the
aromatic ring in the recognition of the former compound.
From the results obtained, it was evident that at least one
double bond conjugated with a primary alcohol group was
necessary for aryl-alcohol oxidase activity. The enzyme was
named aromatic-alcohol oxidase by Farmer (1960) and
veratryl alcohol oxidase by Bourbonnais and Paice (1988),
Kimura et al. (1990) and Sannia et al. (1991), whereas aryl-
alcohol oxidase was the name used by Guilltn et al. (1988,
1990)and Muheim et al. (1990b), and accepted by the Enzyme
Nomenclature Committee. Since aryl-alcohol oxidase from
P . eryngii shows a certain activity on allyl alcohol, and
polyunsaturated alcohols are very good substrates, unsatu-
rated-alcohol oxidase could also be a suitable name for the
enzyme.
The results from the study of the different aryl-alcohol
oxidase substrates showed the influence on enzyme activity of
two structural characteristics of these compounds: the number
of conjugated double bonds ; the presence of electron-donor
groups. The first effect is well illustrated by the strong increase
in aryl-alcohol oxidase activity from allyl alcohol to 2,4-hexa-
dien-1-01, and from benzyl alcohol to cinnamyl alcohol or 2-
naphthalenemethanol. Of the electron-donor substituents, the
methoxy group in the para position produced significant in-
creases in aryl-alcohol oxidase activity with substituted benzyl
alcohols, Both substrate characteristics influence electron
availability at the alcohol group and affect its enzymic oxi-
dation, which consists of the transfer of two electrons from
an alcohol to 02.A measure of this electron availability is
provided by the oxidation potential (Eliz)that presented the
lowest values in some of the substrates with high aryl-alcohol
oxidase activity (cinnamyl alcohol = 1.36 V, 4-methoxybenzyl
alcohol = 1.22 V and 2-naphthalenemethanol = 1.25 V),
when compared with benzyl(2.10 V) or allyl alcohol (2.65 V;
Lund 1957; Parker et al., 1978). The most obvious effect of
low oxidation potentials on enzyme activity is the increase in
reaction velocity with electron availability. To examine their
influence on enzyme activity, kinetic constants with several
substrates were calculated. Unfortunately, this study was lim-
ited by the partial solubility in aqueous media of some sub-
strates. The results obtained show that the activity increase is
the result of a parallel increase in enzyme velocity (Vm,,)
and affinity (l/Km).The role of substrate-molecule electron
distribution in modification of enzyme affinity cannot be es-
tablished at the present, but it is possible to conceive that the
same polarization of the electron cloud that increases the
velocity of the oxidative reaction can facilitate enzyme/sub-
strate recognition.
The activity of aryl-alcohol oxidase on aromatic aldehydes
is described here for the first time. Similar dud activity had
also been described in other alcohol oxidases, e.g. in methanol
oxidase (Hopkins, 1985). The fact that electron-withdrawing
substituents increase enzyme activity with aromatic aldehydes
could be explained by assuming that the first step on the
oxidation reaction is nucleophilic attack at the hydroxy group
on the primary carbon.
Deobald et al. (1989) reported lignocellulose oxidation
by H202-producing aryl-aldehyde oxidase described in
Streptumvces viridusporus. However, in the case of aryl-
alcohol oxidase the activity requirements described above
suggest that HzOz generation from oxidation of lignin
hydroxy groups could be discarded, since these hydroxy
groups are generally secondary or unconjugated with double
bonds. The absence of aryl-alcohol oxidase activity with the
dehydrogenation polymer and the lignin dimers confirmed
this assumption. Anyway, this does not preclude the eventual
participation of aryl-alcohol oxidase in lignin degradation as
a H 2 0 2 source. Effectively, some aryl-alcohol oxidase sub-
strates (mainly aromatic alcohols) can be formed during lignin
depolymerization, via enzymic reduction (Muheim et al.,
1991)of the aromatic aldehydes released by fungal breakdown
of intermonomer linkages and Ca-CP cleavage (Kirk and
Farrell, 1987), or they can be directly synthesized by the lig-
ninolytic fungi (Shimada et al., 1981; Gallois et al., 1990).
We thank Dr F. Reyes, Dr A. E. Gonzalez (CIB, CSIC) and Dr
B. Rodriguez (Instituto dt. Quimicu Orgunicu Generul, CSIC) for their
valuable suggestions, and J. Amezaga for preparation of lignin dimers
and synthesis of dchydrogenation polymer. This investigation has
been supported by the Spanish Biotechnology Program, the ECLAIR
Program of the European Community (Biopulping and Biobleaching
Project) and by Minus de Almugrevu (Jnstituto Nacionul de Industriu
Group, Spain).
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