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

Detection and characterization of a novel extracellular fungal enzyme that catalyzes the specific and hydrolytic cleavage of lignin

guaiacylglycerol b-aryl ether linkages

Yuichiro Otsuka1, Tomonori Sonoki1, Seiichiro Ikeda1, Shinya Kajita1, Masaya Nakamura2

and Yoshihiro Katayama1

1

Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo, Japan;2Forestry 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

Mn2+to Mn3+; Mn3+, 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: y-otuka@cc.tuat.ac.jp

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,

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)

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

paucimobilisSYK-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-high-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% H2O2were

dropped into 430 mL of 100 mM potassium phosphate

[pH 6.0, containing 30% acetone and 6 mg horseradish

peroxidase (44 UÆmg)1, Sigma)] and stirred for 14 h at

20C 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 0.1Mlithium 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 (Mr¼ 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 4C

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 28C, 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 28C for

3 days, 1400 colonies were selected randomly from the plates and suspended separately in liquid VM After stationary culture for 7 days at 28C, 10 lg of GOU were added to each sample After further incubation for 12 h at

28C, 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 mM 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 28C Pieces of hyphae were collected with 1.0 mL of culture solution at 24 h intervals, and 10 lg of GOU were added to each collected sample After incubation for 12 h at 28C, 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 20C Additional supernatant was removed from the cell debris by ultra centrifugation at 60 000 g for 60 min

at 4C 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 mM

of Tris buffer (pH 7.5) at 4C for 60 min The resulting suspension was separated into supernatant and residue by centrifugation at 60 000 g for 60 min at 4C The resultant supernatant and residue were designated the cytoplasm fraction (CY) and the membrane fraction (M), respectively

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 lg of

GOU For HP and M, reactions were initiated by addition

of 0.1 g of HP or M to 1.0 mL of VM containing 10 lg of

GOU After incubation for 12 h at 28C, the reaction

mixture was centrifuged at 15 000 g for 10 min Reactions

were terminated by addition of 1.0 mL of 200 mMglycine/

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 mMof 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 mMto 1M(NH4)2SO4in 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 forb-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).

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 ll of a

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 28C Then, reaction mixtures were

acidified to pH 2 by the addition of 12MHCl 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 lg of

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 5CÆmin)1from 100–300C 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 18O2 and 18O-labeled water into GG, reactions were initiated by the addition of 20 lL of a

Fig 2 Phylogenetic tree of 2BW-1 based on 18SrDNA sequence comparisons of sequences of 18SrDNA 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.

solution of enzyme (1 mgÆmL)1) and 20 lL of a solution of

GOG in dimethyl sulfoxide (50 lgÆmL)1) to 160 lL Tris/

HCl buffer (pH 7.5) with bubbling 18O2 gas for 5 min

(Nippon Sanso Co., Kawasaki, Japan) or18O-labeled water

(94%18O atom; Nippon Sanso Co., Kawasaki, Japan) and

incubated for 12 h at 28C 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 paucimobilisSYK-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 Mr 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 (OCH3) of the guaiacyl structure in

conyferyl alcohol and GOU aO The signal at 2.2 p.p.m

was assigned to the methyl group (CH3) originating from

the CH3of 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.

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.

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 28C 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-MSanalysis of TMS-derivatives of metabolites produced from GOG by the purified enzyme See text for details.

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) was followed with GOU as the 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.

(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- and b-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 P450 We examined the incorpor-ation of oxygen using GOG in an atmosphere of18O2and analysed the reaction mixture by GC-MS Figure 8 shows the mass spectrum of GG; no 18O were found in the reaction products Therefore, we examined the incorpor-ation of the water molecule using GOG in 18O-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-labeled water, in an atmosphere of 18 O 2 and in a control reaction (no radiolabel).

of 18O-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 18O from

radio-labeled water was not observed with guaiacol

It was clear that the b-aryl ether cleavage enzyme

catalyzed the addition of two molecules of H2O (at Ca 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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