Báo cáo khoa học: Detection and characterization of a novel extracellular fungal enzyme that catalyzes the specific and hydrolytic cleavage of lignin guaiacylglycerol b-aryl ether linkages pdf

Detection and characterization of a novel extracellular fungal enzyme
that catalyzes the specific and hydrolytic cleavage of lignin
guaiacylglycerol b-aryl ether linkages
Yuichiro Otsuka
1
, Tomonori Sonoki
1
, Seiichiro Ikeda
1
, Shinya Kajita
1
, Masaya Nakamura
2
and Yoshihiro Katayama
1
1
Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo,
Japan;
2
Forestry and Forest Products Research Institute, Microbial Technology Laboratory, Tsukuba, Norin Kenkyu Danchi-nai,
Ibaraki, Japan
Cleavage of the arylglycerol b-aryl ether linkage is the most
important process in the biological degradation of lignin.
The bacterial b-etherase was described previously and shown
to be tightly associated with the cellular membrane. In this
study, we aimed to detect and isolate a new extracellular
function that catalyses the b-aryl ether linkage cleavage of
high-molecular lignin in the soil fungi. We screened and
isolated 2BW-1 cells by using a highly sensitive fluorescence
assay system. The b-aryl ether cleavage enzyme was pro-
duced by a newly isolated fungus, 2BW-1, and is secreted

into the extracellular fraction. The b-aryl ether cleavage
enzyme converts the guaiacylglycerol b-O-guaiacyl ether
(GOG) to guaiacylglycerol and guaiacol. It requires the Ca
alcohol structure and p-hydroxyl group and specifically
attacks the b-aryl ether linkage of high-molecular mass
lignins with addition of two water molecules at the Ca and
Cb positions.
Keywords: lignin biodegradaion; b-aryl ether linkage; fungi;
guaiacylglycerol b-O-guaiacyl ether; extracellular enzyme.
Lignins are the most abundant high-molecular mass
aromatic compounds in plants. In trees, high levels of
lignin are synthesized in wood and account for 15–36% of
the dry weight of wood. Lignins are complex phenolic
polymers that reinforce the walls of certain cells in the
vascular tissues of higher plants. Lignin plays an import-
ant role in mechanical support, water transport and
pathogen resistance. The lignification process encompasses
the biosynthesis of monolignols such as p-coumaryl,
coniferyl and synapyl alcohols, and polymerization into
the final molecule. Polymerization is thought to result
from oxidative (radical-mediated) coupling between a
monolignol and the growing oligomer/polymer. The
oxidative coupling between monolignols can result in the
formation of several different interunit linkages. In native
lignins, b-O-4-linkages are the most abundant and b-b-,
b-5-, 5-5- and 5-O-4-linkages are also found. Therefore,
lignins have very complicated structures with C-C and
C-O-C linkages, and it is difficult for living organisms to
degrade them. However, many soil microorganisms can
easily digest lignins to fulfill important roles in the earth’s

carbon cycle.
Lignin-biodegradation systems in nature can be sum-
marized as follows. Initially, basidiomycetes secrete peroxi-
dases and/or laccases and degrade the aromatic polymer
lignin [1–7]. The role of each enzyme in this complicated
process is an active area of research and debate. Thus far,
mainly white rot fungi, Phanerochaete chrysosporium and
Trametes (Coriolus) versicolor, have been studied regarding
these peroxidases. P. chrysosporium produces two types of
peroxidases, manganese peroxidase (MnP) and lignin per-
oxidase (LiP) and T. versicolor generally produces laccase.
Laccase reacts with polyphenols including lignin, and other
lignin-derived aromatic compounds, that, in turn, can be
both polymerized and depolymerized. MnP can oxidize
Mn
2+
to Mn
3+
;Mn
3+
, in turn, is able to oxidize a wide
range of phenolic substrates including phenolic lignin. LiP
can directly oxidize a variety of phenolic and nonphenolic
aromatic compounds. These peroxidases remove an elec-
tron and a proton from phenolic hydroxyl, aromatic amino
groups or other aromatic side chains to form free radicals.
Although this acts to cleave Ca-Cb linkages and b-O-4
linkages in the lignin structure, the free radicals cause
random depolymerization of lignin. The various low-
molecular mass lignins produced by these peroxidases

and/or laccase are decomposed to carbon dioxide and
water by specific bacterial enzymes, such as ring-fission
enzymes [8,9], demethylases [10,11] and b-etherases [12–14].
Correspondence to Y. Otsuka, Graduate School of Bio-Applications
and Systems Engineering, Tokyo University of Agriculture and
Technology, Koganei, Tokyo, Japan.
Fax: + 81 42 388 7364, Tel.: + 81 42 388 7364,
E-mail:
Abbreviations: GOU, guaiacylglycerol-b-O-4-methylumbelliferone;
GOG, guaiacylglycerol b-O-guaiacyl; GOU aO, a-O-methylumbel-
liferyl-b-hydroxyl-propiovanillone; GOG aO, a-O-guaiacyl-
b-hydroxyl-propiovanillone; GOUbz, O-benzyl-guaiacylglycerol-
b-O-4-methylumbelliferone; GOGbz, O-benzyl-guaiacylglycerol-
b-O-guaiacyl; DHP-GOU, reduced and polymerized form of GOU;
DHP-GOU, fluorescent-labeled synthetic lignin; 4MU, 4-methyl-
umbelliferone; MnP, manganese peroxidase; LiP, peroxidase;
SYK-6, Sphingomonas paucimobilis SYK-6.
(Received 1 October 2002, revised 18 December 2002,
accepted 27 February 2003)
Eur. J. Biochem. 270, 2353–2362 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03545.x
In our current studies, we have focused on b-aryl ether
linkages in lignin. Such linkages account for more than 50%
of the intermolecular linkages in lignin. In Shingomonas
paucimobilis SYK-6 (SYK-6), we have already identified the
enzyme that specifically cleaves lignin dimmers with b-aryl
ether linkages [12]. This enzyme belongs to the family of
glutathione S-transferases (GSTs) and reductively cleaves
the b-aryl ether bond [14]. However, this enzyme does not
attack b-aryl ether linkages in high-molecular mass mate-
rials in vivo because it acts at the intramolecular level and

cannot gain access to the b-aryl ether linkages in high-
molecular mass lignins [12].
If we could identify and characterize a secretory enzyme
that cleaves b-aryl ether bonds in high-molecular mass
lignins, we would uncover a new field of lignin degradation
in nature. In addition, such an enzyme would convert high-
molecular mass lignins into low-molecular mass lignins that
retain benzene rings. Such lignins would have enormous
biomass and be useful as an industrial material. Therefore in
this study, we tried to detect and characterize such an
enzyme in various fungi.
As described below, we succeeded in characterizing the
production and reaction mechanism of a novel secretory
enzyme that cleaves b-aryl ether bonds.
Materials and methods
Chemicals
Guaiacylglycerol-b-O-4-methylumbelliferone (GOU),
guaiacylglycerol b-O-guaiacyl (GOG), a-O-methylumbel-
liferyl-b-hydroxyl-propiovanillone (GOU aO) and a-O-gua-
iacyl-b-hydroxyl-propiovanillone (GOG aO) were prepared
as described previously [12]. O-Benzyl-guaiacylglycerol-b-
O-4-methylumbelliferone (GOUbz) and O-benzyl-guaiacyl-
glycerol-b-O-guaiacyl (GOGbz) were synthesized by as
described in a previous report [15]. A reduced and
polymerized form of GOU (DHP-GOU) was synthesized
as follows. Acetone (50 mL) containing 0.2% GOU aO
and 0.2% conyferyl alcohol, and 20 mL of 3% H
2
O
2

were
dropped into 430 mL of 100 m
M
potassium phosphate
[pH 6.0, containing 30% acetone and 6 mg horseradish
peroxidase (44 UÆmg
)1
, Sigma)] and stirred for 14 h at
20 °C. After the additions, 4 mg of peroxidase was added
and the reaction mixture was stirred for additional 12 h.
The resultant precipitate was collected and washed three
times with 10 mL water and centrifuged at 4000 g for
10 min. The precipitate was dried completely on the
phosphorus (V) oxide. Dioxane/water (9 : 1) solution (used
to dissolve the crude DHPs) was poured into 300 mL of
diethyl ether for recrystallization. The precipitate was
washed four times with diethyl ether and dispersed in
distilled water. After lyophilization, the fluorescent-labeled
synthetic lignin (DHP-GOU Ca carbonyl type) was pro-
duced. DHP-GOU (Ca carbonyl type) was fractionated by
gel permeation chromatography on an Asahipack GS310
column (500 mm in length · 7.6 mm diameter). N,N-
Dimethylformamide containing 0.1
M
lithium chloride was
used as the eluant at a flow rate 0.5 mLÆmin
)1
.Relative
molecular mass was estimated by calibration with polysty-
lene standard (M

r
¼ 175 000, 9000, 4000, 2000, 800)
(Fig. 1). The 1H-NMR spectra of DHP-GOU (Ca carbonyl
type) were analysed using a JEOL-GX270 (solvent dimeth-
ylsulfoxide-d6) (Fig. 2). For the Ca position reduction of
GOU aO in DHP-GOU (Ca carbonyl type), 1 g each
of DHP-GOU and sodium borohydride were dissolved
in dioxane/methanol (4 : 1) and stirred for 12 h at 4 °C.
A large excess of water was added and the resultant
precipitate was collected as DHP-GOU.
Isolation of the fungi and enzyme
Activity of the b-aryl ether cleavage enzyme was assayed as
described in a previous report [12]. Soil samples were
collected from several sites in Futyunomori Park in Tokyo.
Each soil sample was suspended in 2 mL of Vogel’s medium
(VM) [16]. After 7 days of stationary culture at 28 °C, 10 lg
of GOU were added and incubation was continued for 12 h.
The fluorescence of each sample was examined under a UV
illuminator (model TDS-15, Upland, Japan). Fluorescence-
emitting samples were streaked onto VM plates for the
isolation of single colonies. After incubation at 28 °Cfor
3 days, 1400 colonies were selected randomly from the
plates and suspended separately in liquid VM. After
stationary culture for 7 days at 28 °C, 10 lgofGOUwere
added to each sample. After further incubation for 12 h at
28 °C, the fluorescence of cultures was examined.
Activity of the b-aryl ether cleavage enzyme was assayed
as described previously [12]. Cultures of fluorescing cells
were centrifuged at 15 000 g for 10 min. Supernatants
(1 mL) were added to 1.0 mL of 200 m

M
glycine/NaOH
buffer (pH 10.0). Fluorescence of 4-methylumbelliferone
released from GOU was measured with excitation at
360 nm and emission at 450 nm with a fluorophotometer
(Shimadzu, Japan RF-1200).
The production of b-aryl ether cleavage enzyme
by 2BW-1 cells
2BW-1 cells were suspended in VM and cultured without
agitation at 28 °C. Pieces of hyphae were collected with
1.0 mL of culture solution at 24 h intervals, and 10 lgof
GOU were added to each collected sample. After incubation
for 12 h at 28 °C, b-aryl ether cleavage activity was
measured as described above.
Localization of b-aryl ether cleavage activity
A 14-day-old culture (4 mL) of 2BW-1 cells was separated
into supernatant and residue by centrifugation at 4000 g for
15 min at 20 °C. Additional supernatant was removed from
the cell debris by ultra centrifugation at 60 000 g for 60 min
at 4 °C. The resulting extracellular fraction (EC) was used
for the assay of b-aryl ether cleavage activity. One gram of
residue was washed twice with 100 mL of 0.8% (w/v) Nail
solution and centrifuged at 4000 g for 15 min. Half of the
residue was designated the hyphae fraction (HP) and the
remainder was homogenized with mortar and pestle for
5 min in liquid nitrogen and suspended in 15 mL of 10 m
M
of Tris buffer (pH 7.5) at 4 °C for 60 min. The resulting
suspension was separated into supernatant and residue by
centrifugation at 60 000 g for 60 min at 4 °C. The resultant

supernatant and residue were designated the cytoplasm
fraction (CY) and the membrane fraction (M), respectively.
2354 Y. Otsuka et al. (Eur. J. Biochem. 270) Ó FEBS 2003
For EC and CY, reactions were initiated by addition of
0.1 mL of EC or CY to 1.0 mL of VM containing 10 lgof
GOU. For HP and M, reactions were initiated by addition
of0.1gofHPorMto1.0mLofVMcontaining10lgof
GOU. After incubation for 12 h at 28 °C, the reaction
mixture was centrifuged at 15 000 g for 10 min. Reactions
were terminated by addition of 1.0 mL of 200 m
M
glycine/
NaOH buffer (pH 10.0). Fluorescence was measured with
excitation at 360 nm and emission at 450 nm.
Purification of the b-aryl ether cleavage enzyme
b-aryl ether cleavage activity was measured by using a GOU
fluorometric assay of b-aryl ether cleavage. One unit of
enzyme activity was defined as the amount of b-aryl ether
cleavage enzyme that released 1 ng of 4-methylumbellifer-
one (4MU) per hour. Two hundred millilitres of EC were
concentrated with an Ultrafree 30k system (Millipore,
Tokyo, Japan) to 5 mL. The concentrated solution was
purified by gel filtration through a Sephadex G-75 column
(0.5 cm · 30 cm, Pharmacia, Tokyo, Japan) with 10 m
M
of
Tris/HCl buffer as the mobile phase. The most active
fractions were further purified by ion-exchange chromato-
graphy on a Mono Q column (0.5 cm · 0.5 cm, Pharmacia)
and eluted with a gradient of 0 m

M
to 1
M
(NH
4
)
2
SO
4
in
water. Proteins eluted from the column were detected by
monitoring the absorbance at 280 nm and examined by
SDS/PAGE.
Fig. 1. A highly sensitive assay system for b-aryl ether cleavage function. (A) Scheme of 4-methylumbelliferone (4MU) released from guaiacylglycerol
b-O-4-methylumbelliferone (GOU) and DHP-GOU. Upon cleavage of the b-aryl ether linkage, 4-methylumbelliferone is released and emits
powerful fluorescence. Fluorescence of 4MU was measured with excitation at 360 nm and emission at 450 nm. (B) Gel filtration chromatogram of
DHP-GOU (Ca alcohol type). Fractions used in this study are shaded and substrate molecular mass is more than 1000. Relative molecular mass was
calibrated using polystylene standard series (175 000, 9000, 4000, 2000, 800). (C) 1H-NMR spectrum of acetylated DHP-GOU (Ca alcohol type).
Ó FEBS 2003 The fungal b-aryl ether cleavage enzyme (Eur. J. Biochem. 270) 2355
SDS/PAGE
SDS/PAGE was performed with a stacking gel of 7.5%
(w/v) acrylamide and a separating gel of 12.5% (w/v) acryl
amide, as described by Laemmli [17]. The molecular mass
and subunit composition of b-aryl ether cleavage enzyme
were determined by electrophoresis under reducing condi-
tions. An MW-Marker (SDS) kit (Oriental Yeast Industry
Co., Japan) was used as the source of standard proteins.
Protein bands were visualized with a Coomassie/Brilliant
Blue-staining procedure.
Enzymic reaction and analysis of metabolites

Reactions were initiated by the addition of 100 llofa
solution of enzyme (200 lgÆmL
)1
) to 0.9 mL of a solution of
50 lg of substrate in 10% (v/v) dimethyl sulfoxide and
incubated for 12 h at 28 °C. Then, reaction mixtures were
acidified to pH 2 by the addition of 12
M
HCl and extracted
three times with 300 lL of ethyl acetate. The extract was
then dried on a rotary evaporator (REN-1, Iwaki Glass. Co.
Ltd, Iwaki, Japan) with a vacuum controller (FTP-10;
Asahi Techno Glass, Japan). The residue was dissolved in
20 lg of pyridine and treated with N,O-bis(trimethyl-
silyl)trifluoroacetamide (BSTFA; Tokyo Kasei Co., Tokyo,
Japan) to prepare trimethylsilyl derivatives. Then 1 lgof
the solution of these derivatives was subjected to gas
chromatography (model 390, GL Science, Tokyo, Japan)
and gas chromatography-mass spectrometry (GC-MS;
Auto Mass System II; JEOL, Tokyo, Japan). A fused silica
capillary column (CP-Sil 5CB; 25 m · 0.32 mm; i.d.,
0.25 lm; Chrompack, the Netherlands) was used as the
stationary phase. The temperature of the eluant was raised
at 5 °CÆmin
)1
from 100–300 °C. The eluant was detected
by a flame ionization detector.
To clarify whether the enzymatic reaction was a hydro-
lytic reaction or an oxidative reaction, the enzymic activity
was measured under oxygen-saturated (100%; 7.5 mgÆO

)1
)
conditions or low-oxygen (17%; 1.3 mgÆO
)1
) conditions
with surrounding argon gas. The method for measurement
of activity is described above. To examine the enzymatic
incorporation of
18
O
2
and
18
O-labeled water into GG,
reactions were initiated by the addition of 20 lLofa
Fig. 2. Phylogenetic tree of 2BW-1 based on 18S rDNA sequence comparisons of sequences of 18S rDNA and drawn using
GENETYX
version 10.1
software. The numbers on some branches refer to confidence levels estimated by bootstrap analysis (100 replications).
Fig. 3. Localization of b-aryl ether cleavage function and assay for high-
molecular mass lignin structure. (A) Localization of b-aryl ether clea-
vage enzyme in 2BW-1. HP, hyphae fraction; EC, extracellular frac-
tion; CY, cytoplasmic fraction. Control, GOU added to 1 mL of VM.
(B) Assay of b-aryl ether cleavage by the extracellular fraction with a
model compound that resembles high-molecular mass lignin.
2356 Y. Otsuka et al. (Eur. J. Biochem. 270) Ó FEBS 2003
solution of enzyme (1 mgÆmL
)1
)and20lL of a solution of
GOG in dimethyl sulfoxide (50 lgÆmL

)1
)to160lL Tris/
HCl buffer (pH 7.5) with bubbling
18
O
2
gas for 5 min
(Nippon Sanso Co., Kawasaki, Japan) or
18
O-labeled water
(94%
18
O atom; Nippon Sanso Co., Kawasaki, Japan) and
incubatedfor12hat28°C. After the incubation, the
mixture was analysed as described above.
Results and discussion
In our previous study, we characterized the b-etherase and
the nucleotide sequences of the ligE and ligF genes of
S. paucimobilis SYK-6 [13]. b-Etherase is a member of the
glutathione-S-transferase superfamily [14]. It catalyzes the
reductive cleavage of the b-aryl ether linkage of GOU aO
(Fig. 7, structure V) to produce b-hydroxypropiovanillone
and 4MU [12,14]. However, the b-etherase is associated
tightly with cell membranes and was not secreted into the
extracellular fraction [12]. Therefore, this b-etherase cannot
cleave the b-aryl ether linkages of high-molecule-mass
lignins.
Isolation of microorganisms that can cleave b-aryl
ether linkages
A very sensitive assay was necessary for isolation of

microorganisms that can cleave b-aryl ether linkages, so
we used guaiacylglycerol b-O-4-methylumbelliferone
(GOU) for our screening tests (Fig. 1A). In addition, we
synthesized DHP-GOU as a fluorescent-labeled synthetic
lignin to assay activity for high-molecular mass lignin (see
Materials and methods). The synthesized DHP-GOU
(Ca carbonyl type) was fractionated by gel permeation
chromatography (Fig. 1B). We used the mixture that was
contained from 9000–17 000 M
r
as DHP-GOU (Ca car-
bonyl type) in this study. The 1H-NMR spectrum of
acetylated DHP-GOU (Ca carbonyl type) was analysed
(Fig. 1C). A signal at 3.8 p.p.m. was assigned to the
methoxyl group (OCH
3
) of the guaiacyl structure in
conyferyl alcohol and GOU aO. The signal at 2.2 p.p.m.
was assigned to the methyl group (CH
3
) originating from
the CH
3
of 4MU in GOU aO(Ca carbonyl type). The area
ratio between the signals at 3.8 p.p.m and 2.2 p.p.m. was
calculated as 10 : 1. It was considered that DHP-GOU
(Ca carbonyl type) contained conyferyl alcohol and
GOU aO at the ratio of 9 : 1 by chemical structure. To
prepare DHP-GOU we used the reduction of the Ca
position of GOU aO in DHP-GOU (Ca carbonyl type; see

Materials and methods). When the b-aryl ether linkage of
GOU structure is cleaved, the 4-methylumbelliferone
(4MU) generated can be detected with high sensitive
because of its strong fluorescence.
Using GOU, we isolated six fungi from soil samples with
enzymes able to cleave b-aryl ether linkages. One isolate,
2BW-1, generated the strongest fluorescence and therefore,
it appeared that 2BW-1 cleaved the b-aryl ether linkage
efficiently. 2BW-1 also cleaved the b-aryl ether linkages of
DHP-GOU (data not shown), a result that suggested that
2BW-1 might cleave b-aryl ether linkages in high-molecular-
mass materials.
Table 1. Purification of b-aryl ether cleavage enzyme in 2BW-1.
Step Vol (mL) Protein (mg) Activity (UÆmg
)1
) Purification (fold) Recovery
Culture medium 200 5520 3 1 100
Ultrafiltration (30 kDa) 5 3254 3.9 1.3 115
Sephadex G-75 8 591 31.3 10.27 38
Mono Q 2 426 49.4 16.2 34
Fig. 4. Analysis by SDS/PAGE of the purified b-aryl ether cleavage
enzyme from 2BW-1.
Fig. 5. The time course of cleavage the b-aryl ether linkage by purified
b-aryl ether cleavage enzyme in various condition. GOU was used as
substrate and measured of fluorescence intensity.
Ó FEBS 2003 The fungal b-aryl ether cleavage enzyme (Eur. J. Biochem. 270) 2357
Taxonomic position of 2BW-1
2BW-1 did not produce spores under any tested condi-
tions. Therefore, to determine the taxonomic position of
this novel fungus, we determined the nearly complete

sequence of its 18S rDNA. The sequence of the 18S
rDNA of 2BW-1 was strongly homologous to that of the
ascomycetes, Chaetomium elatum, Podospora anserina,
Sordaria fimicola and Neurospora crassa (Fig. 2). We
had considered that 2BW-1 would be a member of
basidiomycetes. However, these results indicate that
2BW-1 belongs rather to ascomycetes. The sequence from
2BW-1 was very similar to that from C. elatum and
C. globosum (more than 99% homology). These results
suggest that 2BW-1 is a member of the genus Chaeto-
mium. C. globosum and C. elatum have been studied as
wood-rotting fungi. They are able to grow on wood chips
and decompose wood via the degradation of cellulose [22].
However, in Chaetomium sp., the degradation system of
lignin or lignin related compounds have not been studied.
Similarly Chaetomium sp. 2BW-1 was able to grow on
wood chips as the sole carbon source. In addition, 2BW-1
also grew in the lignin-related compounds, p-hydroxy-
benzoic acid, gallic acid and vanillic acid, as a sole source
of carbon.
Cell growth and the production of the b-aryl ether
cleavage enzyme
We cultured 2BW-1 in stationary test tubes at 28 °C. To
observe the expression of b-aryl ether cleavage activity, we
collected the culture solution that contained hyphae at 24 h
intervals during a 3 week incubation. The enzymatic activity
in the culture was determined as emitted fluorescence
generated by cleavage of a b-aryl ether linkage. The
enzymatic activity was not detected until cultures were
6-days-old. In 7-day-old cultures, we detected weak activity.

The activity increased for 7 more days and then decreased
(data not shown). This result suggested that production of
the b-aryl ether cleavage enzyme might be induced under
specific conditions. Analysis of the products of the reaction
revealed the presence of guaiacylglycerol (GG) and 4MU
(data not shown).
Localization of enzymatic activity
To confirm the localization of the b-aryl ether cleavage
activity, we prepared a hyphae fraction (HP), an extracel-
lular fraction (EC), a cytoplasmic fraction (CY) and a
membrane fraction (M) from cultures of 2BW-1 (see
Materials and methods). Enzymatic activity was determined
Fig. 6. GC and GC-MS analysis of TMS-derivatives of metabolites produced from GOG by the purified enzyme. See text for details.
2358 Y. Otsuka et al. (Eur. J. Biochem. 270) Ó FEBS 2003
as the emitted fluorescence from GOU incubated with each
fraction. We detected strong fluorescence only with EC
(Fig. 3A). These results indicated that the b-aryl ether
cleavage enzyme accumulated and was stable in the
extracellular fraction. The extracellular fraction of 2BW-1
generated abundant GG and 4MU from GOU by cleaving
the ether linkage. In addition, this extracellular enzyme
cleaved the b-aryl ether linkages of DHP-GOU, a high-
molecular mass compound (Fig. 3B).
Purification and characterization of the b-aryl ether
cleavage enzyme
To characterize in further detail the b-aryl ether cleavage
enzyme, we tried to purify it from the extracellular fraction,
as summarized in Table 1. The EC was concentrated by
ultrafiltration and applied to a gel-filtration column of
Sephadex G-75. The fractions with the highest activity were

collected and subjected to anion-exchange chromatography.
The active fraction yielded only a single band after SDS/
PAGE (Fig. 4), indicating that the b-aryl ether cleavage
enzyme had been purified to homogeneity. The overall
purification factor was about 16.2-fold, and the final yield
was 34%. The final product had a specific activity of about
49.4 UÆmg
)1
(Table 1). The molecular mass of the purified
enzyme was estimated to be about 65 kDa. The time course
of cleavage of the b-aryl ether linkage by the purified
enzyme (20 lgÆmL
)1
)wasfollowedwithGOUasthe
substrate and by measurement of fluorescence intensity
(Fig. 5). From this result, we used an enzymatic reaction
time of 12 h in further experiments.
Reaction mechanism and substrate specificity
To identify the reaction mechanism of the enzyme, we
analysed the reaction products by GC and GC-MS.
4-Methylumbelliferone (m/z 248) and guaiacylglycerol
(m/z 502) were detected as major reaction products by
GC-MS analysis, indicating that the enzyme cleaved the
b-aryl ether linkage in GOU specifically to produce GG and
4MU. In addition, the enzyme also cleaved the b-aryl ether
bond in GOG to produce GG and guaiacol (Fig. 6).
To clarify the substrate specificity of the b-aryl ether
cleavage enzyme, we synthesized the substrates GOUbz
Fig. 7. Substrate specify of the b-aryl ether
cleavage activity of 2BW-1. Structure I, gua-

iacylglycerol b-O-4-methylumbelliferone; II,
guaiacylglycerol; III, O-benzyl-guaiacylgly-
cerol b-O-4-methylumbelliferone; IV,
O-benzyl-guaiacylglycerol b-O-guaiacyl;
V, a-O-(b-methylumbelliferyl)-b-(hydroxy)
propriovanillone; VI, a-O-(b-guaiacyl)-
b-(hydroxy)propriovanillone.
Ó FEBS 2003 The fungal b-aryl ether cleavage enzyme (Eur. J. Biochem. 270) 2359
(structure III) and GOGbz (structure IV) by replacing the
p-hydroxyl group of GOU (structure I) and GOG
(structure II) by a benzyl group (Fig. 7). The b-aryl ether
cleavage enzyme could not cleave the b-aryl ether linkages
of GOUbz (III) and GOGbz (IV). In addition, the b-aryl
ether cleavage enzyme failed to cleave the b-aryl ether
linkage of GOU aO (Fig. 7 structure V). Thus, the b-aryl
ether cleavage enzyme required a p-hydroxyl group and a
Ca alcohol structure for activity. Despite the high speci-
ficity of the Ca structure and p-hydroxyl group, the b-aryl
ether cleavage enzyme could react with DHP-GOU. This
result indicates reactivity for the structure that retained the
Ca alcohol and p-hydroxyl group that exists on the surface
of DHP, as shown in Fig. 1. Therefore, the enzyme activity
for DHP-GOU was lower than GOU. However, this
enzyme did not act on substrates such as guaiacol and
a-andb-naphthol (data not shown).
The enzyme produced GG and 4MU from GOU,
suggesting that the cleavage of the b-aryl ether bond
might be a hydrolytic reaction. We examined whether this
enzyme catalyzed an oxidative or a hydrolytic reaction. If
the enzyme catalysed an oxidative reaction, its activity

should reflect the level of oxygen in the atmosphere.
Therefore, we measured the activity in low-oxygen
(1.3 mg O) atmosphere. In the absence of oxygen, the
enzyme reaction was very slow or none-existent (Fig. 5).
Therefore, the reaction seemed to resemble the mono-
oxygenase reaction of P
450
. We examined the incorpor-
ation of oxygen using GOG in an atmosphere of
18
O
2
and
analysed the reaction mixture by GC-MS. Figure 8 shows
the mass spectrum of GG; no
18
O were found in the
reaction products. Therefore, we examined the incorpor-
ation of the water molecule using GOG in
18
O-labeled
water. The mass spectrum revealed that two molecules
Fig. 9. The proposed mechanism of catalysis of the b-aryl ether cleavage enzyme.
Fig. 8. Mass spectra of guaiacylglycerol and guaiacol, products of the b-aryl ether cleavage reaction. The reaction products of GOG generated by the
b-aryl ether cleavage enzyme in
18
O-labeledwater,inanatmosphereof
18
O
2

and in a control reaction (no radiolabel).
2360 Y. Otsuka et al. (Eur. J. Biochem. 270) Ó FEBS 2003
of
18
O-labeled water were incorporated into GG. In
addition, we found the radiolabeled oxygen at the Ca and
Cb positions in a comparison of the mass spectrum with
that of the products of the reaction with GG and
unlabeled water. The incorporation of
18
Ofromradio-
labeled water was not observed with guaiacol.
It was clear that the b-aryl ether cleavage enzyme
catalyzed the addition of two molecules of H
2
O(atCa and
Cb positions) and cleavage the b-aryl ether bond. In
addition, under differing enzyme conditions, although the
fluorescent segregation quantity was proportional to the
enzyme amount, the reaction rate remained mainly
constant (Fig. 5). If this enzyme reaction was only a
onestep reaction, the reaction rate must be faster where
more enzyme exists. Therefore, we considered that the
enzyme reaction was a twostep reaction where 4MU
was released at the second step. Figure 9 shows a model
of the reaction mechanism of this novel hydrolytic
enzyme [18–21,23–25]. Enzymatic dehydration generates
the quinonemethide from GOG (structure II). The reaction
mixture turned yellow as a result of formation of the
quinonemethide. The scheme is consistent with the fact

that the b-aryl ether cleavage enzyme requires a hydroxyl
group and a Ca alcohol structure. Probably, at this
time, molecular oxygen affects the formation of the
quinonemethide. Then, water attacks the Ca position in
the quinonemethide and the b-aryl ether linkage is cleaved.
Another water molecule then attacks the Ca position to
generate GG. As the result, GOG (structure II) is
converted to GG and guaiacol. In addition, this reaction
model is consistent with the fact that the initial reaction
rate of this enzyme was very slow (Fig. 5). There are no
reports of similar enzymatic reactions, to our knowledge.
In this report, we have described a new secretory
enzyme that specifically cleaves the b-aryl ether linkage
of the major intramolecular bond in lignins. The b-aryl
ether cleavage enzyme was produced by a newly isolated
fungus, 2BW-1 and is secreted into the extracellular
fraction. It attacks the b-aryl ether linkage of high-
molecular mass lignins with the addition of two water
molecules at positions, Ca and Cb. In addition, 2BW-1
did not belong to the Basidiomycetes (known as lignin
degradation fungi) but to the Ascomycetes (known
mainly as cellulolytic fungi. Therefore, further charac-
terization of this enzyme and isolation of its gene should
contribute to improved utilization of high-molecular
mass lignins and provide a new perspective on the
evolutionary history of fungal lignin-degradation
systems.
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