白色腐朽菌による木材分解の酵素・組織化学的研究

誌名誌名宇都宮大学農学部演習林報告 = Bulletin of the Utsunomiya UniversityForests

ISSNISSN 02868733

著者著者吉澤, 伸夫川上, 晴代砂川, 政英

巻/号巻/号 26号

掲載ページ掲載ページ p. 19-34

発行年月発行年月 1990年3月

農林水産省 農林水産技術会議事務局筑波産学連携支援センターTsukuba Business-Academia Cooperation Support Center, Agriculture, Forestry and Fisheries Research CouncilSecretariat

[Bull.Utsunomiya Univ.Foγ26， P. 19-3.1 (1990)( Original Article) J

Enzymatic and Histochemical Study

of W ood Degradation by White胸 RotFungi* 1

Nobuo YOSHIZAWA*Z， Haruyo KAWAKAMI*2， Masahide SUNAGAWA吋

Shinso YOKOT A* 2， and Toshinaga IDEI* 2

白色腐粍菌による木材分解の酵素・

組織化学的研究*1

吉j畢伸夫*2 .川上晴代“・砂川政英*B

横田信三*2・出井利長叫

要ヒ己日

ヒラタケ(Pleurotusostreαtus)，ナメコ (Pholiotanαmeko)及びマイタケ(Grifolαfrondosα)

について，腐朽過程における木材細胞壁の形態的・組織化学的変化を畿光顕微鏡を用いて観察した。

また，木材成分の分解に関与する主要酵素(ペルオキシダーゼ，ラッカーゼ及びセルラーゼ)の腐朽

期間中における活性変化を調べ，細胞壁中の 1)クニンと多糖類の分解様式の違いを考察した。

三種の白色腐朽爵において異なる腐朽型が認められた。ナメコによる木材腐朽は，セルロースとリ

グニンを問時に分解する典型的な白色腐朽を示し，細胞愛の薄化が内腔側からりグニンの分解と並行

的に進行した。ヒラタケ及びマイタケ腐朽材では，腐朽初期に局所的な二次愛での選択的説リグニン

が認められた。これらの菌では，細胞壁の薄化がおこる前に 1)ク守ニンのほとんどが二次壁から分解さ

れた。ナメコ及びマイタケ腐朽材では，腐朽初期から多震のヘミセルロースが分解された。三穫の麓

いずれにおいてもへミセルロースがセルロースよりも早く分解された。

観察したりグニン分解酵素のうち，ナメコではラッカーゼが， ヒラタケ及びマイタケでは，ペルオ

キシダーゼとラッカーゼ、の両酵素が主にリグニン分解に関与しているものと考えられる。三種の萄い

ずれにおいても，セルラーゼ活性は腐朽初期に比較的高く，次いで念、滅後，腐朽の進行と共に徐々に

増加するという変化を示した。

キーワード:白色腐朽菌，脱リグニン，ペルオキシダーゼ，ラッカーゼ，セルラーゼ

SUMMARY

Micromorphological and histochemical changes that occur in wood decayed by white-rot

fungi were examined mainly using fluorescence microscopy. The activities of the main en-

zymes (peroxidase， laccase， and cellulase) involved in wood degradation were measured

during various periods of decay and were followed histochemically. Differences in the pat-

terns of lignin and carbohydrate removal from the cell walls are discussed.

Different patterns of wood d巴cayand ligninolytic時 cellulolyticactivity were found in wood

dec且yedby three white-rot fungi. Pholiotαnαmeko， a simultaneous white-rotter， caused

* I Received August 21， 1989. This report was presented at the 39th Annual Meeting of

the Japan Wood Rese且rchSociety， April， 1989， Naha， Okinawa.

*2 Wood Products， Departmentof Forestry， Faculty of Aguriculture， Utsunomiya Uni・

versity， Utsunomiya 321

* 3 Present address : Faculty of Agriculture， Hokkaido University， Sapporo 060

19

20 Nobuo YOSJIlt:AWA， ct al [F¥ul1. Utsunomiya Univ. 1"0γ.

concomitant degradation of all cellも.vallcomponents. Progr巴ssivethinning of th巴 cellwalls

occurred from the inside outward shortly after lignin was removed in preference to cellu

10se in the second且rywa11. Pleurotus ostreαtus and Grifolαfrondosα， white-rotters that s巴ー

1ective1y delignify wood， caused extensiv巴 ligninremova1 in 10calized ar巴asof the decayed

wood. In the s巴condarywall， lignin was removed from the 1umen side outward before ex-

tensive thinning， resulting from cellu10se degradation， occurred. In the early stag巴sof de-

cay by P. ostreαtus and G. frondosα， lignin was removed from every part of the secondary

wall but not from the midd1e 1amella. Throughout the decay periods， P. nαmeko and

G. frondosαremoved 1arge amounts of hemicellu10se from the secondary wall. In wood de-

cayed by P. ostreatus， hemicellu10se degradation a1so occurred in preference to that of ce1-

1u1ose， although the extent was small

Pheno1-oxidizing enzymes of both peroxidase and 1accase types are consider巴dto be

mainly invo1ved in the delignification of wood decayed by P. ostreαtus and G. frondosα.In

decay by P. nαmeko， enzyme of the 1accase type appears to p1ay an important ro1e in lig-

nin degradation. Patterns of changes in cellulase activity were a1most the same且mongthe

thre巴fungi.Histochemica1 assay of peroxidas巴isconsider巴dto be usefu1 in detecting the

chang巴sin activity during decay

Keywords: white-rot fungi， de1ignification， peroxidase， laccas巴， cellu1ase

INTRODUCTION

The whit巴-rotfungi have in common the

ability to degrade lignin as well as the other

wood components. There are some morphoω

logically different types of white“rot that occur

in wood due to the variation in the way lignin

and carbohydrates ar巴 removed(Wilcox， 1970;

Liese， 1970; Fukazawa et al.， 1976; Otjen and

Blanchette， 1986; B1anchette et al.， 1988). Som

日 white-rotfungi have been found to simultane

ous1y remove all of the wood cell wall compon

ents， while others have been found to se1ectivel

y degrade lignin in preference to cellu10s巴(Otj

en and Blanchette， 1986; Blanchette et al.， 198

8; Yoshizawa et al.， 1989). They a1so have in

common the ability to produce extracellu1ar

pheno1-oxidizing enzymes， including 1accase

and peroxidase (Eriksson， 1981; Higuchi，

1985; Kirk and Shimada， 1985).

Th巴 se1ectiveremova1 of 1ignin from wood

by white-rot fungi is expected to catalyze a

new technique for the bioconversion of lig-

nocellulosic materials， such as biopu1ping，

production of sugar and ethano1， treatment of

1ignin-derived wastes， and livestock feed

(Kawase， 1958; Eriksson， 1981; Kirk and

Ch旦ng，1981; Myers巴tal.， 1988). To obtain the

best delignifier for possible use in a new bio-

technologica1 process， a large numb日rof sp巴-

cies of white-rot fungi h旦veb巴enscreened for

potentially industria1 use (B1anchette， 1984a

and b; Shimizu et al.， 1986; Objen et al.， 1987;

Blanchette et al.， 1988; Nishibe et al.， 1988).

Mutant strains of some white-rot fungi that

1ack the ability to degrade cellu10se also have

been deve10ped (Eriksson et al.， 1980a and b).

A biodegradative assay of the 1abe1ed [14C]

lignin dev日loped by Kirk and others (1975)

was app1ied to decayed wood-mea1s of Betula

ermαni for the screening of white-rot fungi

with the abi1ity to se1ectively degrade lignin，

by a group of severa1 investigators (Hiroi

et al.， 1986) who concluded that Grifola

frondosα， Lentinus edodes and Gαnodermα

lucidum preferentially degraded lignin. More

recently， Otjen and others (1987) have r巴port-

ed from th巴irextensive examination of the

lignin-d巴grading ability of thirty wood-

inhabiting basidiomycetes， that Phellinus

pini-2， Pholiota mutαbilis， Phlebiαbreuispore-

1 and Phαnerochαete chrysosporium were the

best delignifiers for preferentia1 lignin degra-

¥'01.26，1990J Enzynlatic dcgradation 01' ¥¥'0口dby whilc-口，ll'ungi 21

dation. L. edodes and Cryptoderma pini also

were confirmed from histochemical examina-

tions by Id巴iand others (1983) and Yoshizawa

and others (1989) to have the ability to selec叩

tively degrade lignin in the early stages of

decay. They suggested that it is necessary to

consider the extent of lignin degradation in

the cell wall when these fungi are applied for

various industrial use because the specificities

of these fungi for lignin degradation changes

depending on the stage of decay.

The anatomy of the host cell types also has

been shown to be factors in recognizing decay

patterns among fungi that can degrade lignin

selectively (Otjen and Blanchette， 1986). In

addition， culture conditions also influence the

specificities of fungi for lignin removal (Kirk

et al， 1978; Yamamoto et al.， 1986). Concern‘

ing the degradation process of cell walls

caused by numerous white-rot fungi， more

detailed histochemical and enzymatic examト

nations are required to screen the species of

fungi that selectively delignify wood for indus-

trial applications.

The purpose of our study was 1) to observe

the micromorphological and histochemical

changes that occur in wood decayed by three

white-rot fungi・ namely，Pleurotus ostreαtus，

Pholiotαnαmeko， and Grifola frondosα， 2) to

examine the changes in the巴nzymeactlvltles

mainly involoved in wood degradation during

the varied periods of decay， and 3) to histo

chemically comp且re the enzymatic changes

caused by d巴cayin the cell walls. Histochemト

cal examination of the cell wall degradation

was made mainly using fluorescence micro司

scopy.

MA TERIALS AND METHODS

W ood samples and decay

Sapwood blocks (10 x 10 x 10 m田) were cut

from freshly harvested J apanese cypress

(Chαmα官cypαrisobtusαEndl.γ1 dried for 72

hrs at 70'C， and dry weight determined. After

the blocks were saturat巴dwith distilled water，

they were sterilized by autoclaving for 30 min

at 120'C. Blocks were then placed in a single

lay日ron mycelium-covered sea sand in grass

vials (500ml). Fungi were grown on the sea

sand for three we巴ksprior to inoculating the

wood blocks in th巴 glassvials containing 100

ml malt-yeast medium (l5g of Difco malt

extract， 2g of Difco yeast extract， and 1000ml

of distilled water).

W ood blocks were decayed by the three

white-rot fungi， Pleurotus ostreatus (Fr.)

Quel， Pholiotα凡αmeko(T.Ito)S.， and Grifola

frondosα(Fr.) S.F.Gray， for various periods

to obtain samples with v且riousextents of de-

cay. Perc巴ntageweight同 losseswere calculated

from the weight of the decayed blocks collect-

ed from the culture vials at regular intervals.

After varied periods of decay， blocks with

weight losses approximately equal to the

mean of ten replicatons were chosen for both

chemical and histochemical analyses.

Chemical analysis

Decayed wood blocks with the same weight

losses were combined for chemical analysis

Decayed and sound wood fractions being sub

jected to extraction of the crude enzymes were

ground to pass a 40同 mesh screen， and the

wood meals were analyzed for sulfuric acid

lignin and holocellulose.

Enzyme extraction and assays

After incubation at regular intervals (20-100

days)， blocks were collect巴dfrom the culture

Vl且ls，and the mycelia were removed carefully

from the wood blocks. Immediately， the

dec旦yedblocks were cut into small pieces and

homogenized in ice-cold distilled water with a

homogenizer. The homogenized samples were

kept in a refrigerator for 2 hrs to extract

crude enzymes. After extraction， the mixtures

were filtrated through Celite powder (N 0.503，

John-Manville) in a Buchner funnel. Enzyme

protein was precipitated from the filtrates in

* 1 J apanese cypress is a dominant speci巴S

planted in Japan， and the effective utiliza-

tion of thinnings is being explored from

the aspect of biomass conversion.

22 Nobuo YOSHIZ!¥¥VA et aI

an aqueous solution of ammonium sulfate (0.9

saturation) for 4 hrs at 4 T. The sediment

was separated by filtration， collected on Celit巴

powder and dialyz巴d against distilled water

with dialysis membrane (Seamless cellulose

tubing， Viskase) for 12 hrs丘t4 'C. The crude

enzyme solution obtained was subject巴d to

enzymatic analysis. An ultraviolet spectro-

photometer (Shimadzu UV-2100) was used to

measure th巴 absorbancefor ass且yingenzyme

achvIty.

Protein content

Protein content was m巴asuredaccording to

the method of Lowry and others (1951). The

final reaction mixture contained O.lml of the

enzyme preparation， 1ml of alkaline copper

reagent， and O.lml of 1N phenol reagent. Ab

sorbance was measur巴d at 660 nm with a

cuvette h且vinga 1 cm light path. The protein

content obtained was used for correction of

the enzyme activity.

PeroxidαseαctwLty

Peroxidase activity was assayed according

to the method of Minocha and Halperin

(1976). The reaction mixture consisting of 1ml

of O.lM guaiacol， 1ml of O.lM phosphate

buffer (pH7.4)， and 1ml of crude enzyme

solution was kept旦t20 'C. The reaction was

initiated by the addition of 27μ1 of 30mM

hydrogen peroxide. Peroxidase activity was

followed by recording the increase in ab-

sorbance at 470 nm of the reaction mixture.

LαccαseαCUULty

Laccase activity was assayed according to

th邑 methodof Leonowicz 旦nd Grzywnowicz

(1981). The reaction mixture contained 0.2ml

of 0.5mM syringaldazine in ethanol， 1.5ml of

O.lM acet且tebuffer (pH5.3)， and enzyme soluω

tion in a final volume of 1.8ml. The reaction

was initiated at 20T by the addition of crude

enzyme solution， and the increas巴 in ab-

sorbance at 525 nm was recorded

[BuIl. UlsunomIya UnIv. 1'01'.

CellulαseαcuuLty

Cellulase activity was assayed by measur-

ing the increase in absorbance resulting from

the increase of reducing sugar in the reaction

mixture containing sodium carboxymethyl

cellulose (CMC) as a substrat日 (Samejima，

1985). The reaction mixture consisted of 0.5ml

of 0.1% CMC， 1ml of O.lM ac日tatebuffer， and

0.5ml of enzyme solution. The amount of

reducing sugar liberated was determined by

measuring the absorbance at 660 nm using

Nelson-Somogyi reagent. The unit of the en-

zyme activity was defined as 1 micromoles of

glucose liberated per min.

Histochemical observation

After incubation， one representative wood

block decayed by each fungus was cut into

halves目 Onehalf-cut block was fixed with 2%

glutalaldehyde，巴mb巴ddedin a low-viscosity

epoxy resin formulation (Spurr， 1969)， and

sectioned with a glass knife. After removing

resin， transverse sections 10μm thick， with or

without acridine orange (AO) staining， were

observed with a fluorescence microscope

(Olympus BH2-RFK)， using excitation with

blue and UV light， respectively， to detect

the histochemical and micromorphological

changes of the cell walls， as described pre-

viously (Idei et al.， 1983; Y oshiza wa et al.，

1989). Other sections were stained with

phloroglucinol姐 HClto detect the pr巴senceof

lignin. Other representative blocks were used

for observing the changes in activity of perox

idase and polyphenol oxidase during decay.

For observing peroxidase activity， trans-

verse sections， 10μm thick， were incubated

for 5min at 20 T in 且n incubation medium

containing 20mM lead nitrate， 14mM homo

vanl日icacid， O.lmM rhodamine B， and 0.02%

of freshly diluted hydrogen peroxide in 0.2M

acetate buffer (pH6.0)， according to the metlト

od of Papadimitriou and others (1976). After

dehydr且tion in graded solutions of ethanol，

sections were immersed in xylen， embedded

in Eukitt， and then observed with a fluores-

cenc巴 microscopeusing excitation with green

Vol.26.1990] Enχymatic dcgmuation 01' wood by white-ro( I'ungi

light. The excitation filter was an IF 545， and

an R 610 served as the barrier filter. Control

sections were boiled and then incubated in the

test solution lacking hydrogen peroxide

Polyphenol oxidase activity was observed by

ordinary ligh t microscopy after the sections

were incubated in 10ml of 0.067M Sorensen's

phosphate buffer at pH 7.0 containing 50mg of

DL-β-(3， 4-Dihydroxyphenyl) alanine (DOP

A) for 1 hr at 37 'c (Gahan， 1984). Control

sections were incubated in the same medium

containing 0.02M sodium diethyldithiocarba

mate.

decay

Degradation of wood compon巴nts during

RESULTS

Changes in weight losses during decay by

the three white-rot fungi ar巴 shownin Figure

1. In the incipient stage of d日cay(20 days)， no

difference in weight losses of decayed wood

was found among the fungi. of the three

whit巴ーrotfungi tested， however， P. nαmelw

caused greater weight losses with further ad-

vances of decay. P. ostreαtus and G. frondosa

had almost similar ch乱ngesto each other in

weight losses. After 40 days of culture， both

fungi c且used gradually increasing weight

losses.

o P.ostreαtus A P. nameko

15 ロG.(rondosα

ム/(

¥。芝〉叩間的O

主切一ω〉〉

20 40 60 80 100

Culture period (daY5)

Fig. 1. Changes in weight losses during

decay by three white-rot fungi

23

Figures 2 and 3 show the rates of decrease

in holocellulose and lignin， respectively， by

decay. P. ostreαtus exhibit日dthe least ability

to degrade wood to the extent of a 10% w巴ight

loss compared to the other two fungi. Holocell-

ulose degradation was greater in wood decay-

ed by P. nαmeko and G. frondosαthan in that

decayed by P. ostreαtus. In wood decayed by

G. frondosαholocellulose degradation gr且du-

ally increased with increas巴sin weight loss，

wh巴reasit rapidly increased beyond about a

5% weight loss in wood decayed by P. nαmeko.

In wood decayed by P. ostreαtus， holocellulose

removal from the cell walls was relatively

small， but it gradually increased with the in-

creases in weight loss

No differences in lignin degrad且tlOn were

not r巴cognizedup to the 2% weight loss point

in wood decayed by the three white同 rotfungi

(Fig. 3). With progress of decay， greatest

lignin removal from the cell walls occurred

in wood decayed by G. frondosα， whereas

lignin degradation gradually increased in

P. ostreαtus and P. nαmeko. In wood decayed

by P. nαmeko， the rate of lignin removal was

somewhat greater than that of P. 6streαtus

until the 8% weight loss point， but the amount

of lignin degradation by the latter exceeded

that of the former in dec且yedblocks with 10%

weight losses.

20

~ c

Eh15

c

(l) 同町田、司

s 10 Tコ

s

t

M

4

0

町

出

北

b

el-G

r明

H

n

M

副

官

o

n

h

p

p

G

O

A

ロ

半

O

由一宇田

α

10

Weight 1055(%)

Fig. 2. Rates of holocellulose losses in wood

decayed by three white-rot fungi目

24

~ 15

"" )

由同

O

コ由O o o iこ

.~ 10 ω 師団由」

U Q)

℃

恥

O

由司ト」

叩

0:: 5

。

Fig.3.

2

Nobuo YOSHIZA W A e¥ al

O P.ostreαtus

A P. nam伽

ロG.frondosα

4 68

Weight 1055(%) 10

wood Rates of lignin losses in

decayed by three white-rot fungi.

Changes in enzyme activities during decay

As shown in Figure 4， peroxidase and lac-

case activities detected in wood rotted by

P. ostreatus both increased up to thepoint of

decay at 40 days， then both grad ually de-

creased until 80 days， and then tended to in

crease again to the point of a 100-d旦y-culture

The reincreasing in peroxidase activity was

especially remarkable. Peroxidase activity

was greater than laccase activity throughout

the progress of decay by P. ostreαtus.

In wood decayed by P. nαmeko， Figure 5 in

dicates considerably geat laccase activity in

the incipient stage of decay， but thereafter it

rapidly decreased. On the other hand， perox

idase activity was considerably little， and sig-

-6 X10

(

ε '0.0 ε

、¥.... 国

3.0

三 2.0h .... 〉

.... U 国

由回司U

詰 1.0」

。

Fig.4.

[Bull. Utsunomiya Univ

P. ostreatus

@

o Laccase e Peroxidase

@ー-e、

、⑧

20 40 60 80 100

Culture period(daY5)

F'or

-6 ，10

3.0 ~ー、、

ε '0.0 ε 、¥

“ 時

X )

h .... 2.0 ・5

.... O 国

由師団方

× O L

1.0 ~

Changes in the activities of perox-

idas巴且ndlaccase in wood decayed

by P. ostreαtus.

nificant changes in it w巴renot found during

the entire cultur巴 period.

In G. frondosa rotted wood， as shown in

Figure 6， peroxidase activity gradu呂lly in伊

creased， reaching a peak at 60 days of culture，

and thereafter decreased. During 60-to 80“

day-culture periods， peroxidase activity was

gre且terthan laccase activity. G. frondosαhad

changes in laccase activity almost similar to

P. nαmeko， that is， in the incipient stage of

decay great activity of laccase occurred and

then it rapidly decr巴asedwith the advance of

d巴cay

¥:01.26，1990J Enzymatic dcgradation 01' ¥¥'ood by wbitc-rot I'ungi 25

6 x10 -6

x10 P. nαmeko

15.0 15.0

(

ε tJj)

E 、¥..... 国

X

O o Laccase • Peroxidase

(

ε tJj)

E 、¥→j

国

X )

10.0 .e 〉

..... O 町

三 10.0吋トb

〉

H

O 回

世田町O U 国」 5.0

。20

Fig.5.

40 60 80

Culture period(days)

100

①

田町てコx 0

5.0包a

Changes in the activities of perox

idase and laccase in wood decayed

by P.ηαyηeko

A comparison of the intensities of the perox-

idase and laccase activities during decay

among the thre巴 white-rot fungi show that

peroxidase activity was relatively great in

wood decayed by P. ostreαtus and G. frondosα，

and that laccase acativity was great in the

P. nαmelw and G. frondosa rotted woods，

especially in the incipient stages of decay in

both fungi (Figs. 1-3)

The patterns of changes in cellulase activi-

ties were almost the same among the three

fungi as shown in Figure 7. Relatively great

cellu11ase activities were detected in woods de-

cayed for 20 days. After the ce11ulase activities

decreased， they grad ua11y increased toward

th巴 cultureperiod of 100 days. Ce11ulase activi-

ty was greater in G. frondosa rotted wood

than those in the other two fungi at且11times

during culture. Ce11ulase activity was the least

in wood decayed by P. nαmeko among the

three white-rot fungi.

S x10

6.0 (

E tJj)

E 、¥寸トJ

国

~ )

.e 4.0 〉..... ()

ro 由u) 出。O 悶

-1 2.0

。

Fig.6.

〉、

“ 〉

.....

1.5

詰 1.0

由山田

コ由

O

0.5

Fig.7.

G. frondosα

o Laccase O

• Peroxidase

20 40 60 80

Culture period(days)

100

一6x10

6.0 ~ E tJj)

ε 、¥...， 国

~ )

〉、..... 4.0 .::: .....

O 回

。的問てコx o 」

申

2.0 n..

Changes in the activities of perox-

idase and laccase in wood decayed

by G. frondosα.

口

20

o p.ostreαtU8 ム P.nameko

口 G.frondosα

40 60 80 100

Culture period(days)

Changes of cellulase activities in

woods decayed by three white-rot

fungi.

26 Nobuo YOSf-II%AWA el al

Histochem istry of cell wall degradation

Histochemical studies showed that P.

ostreαtus and G. frondosαcaused selecti ve

delignification， whereas P. 7W7ηelw caused

simultaneous rot (Figs. 8-11)

When decayed wood was observed with a

fluorescence microscope， the secondary wa11s

gradually varied in color with the progress of

decay. When sections wer巴 stainedwith AO，

the discoloration change from dark green into

reddish brown first occurred in the inside of

the secondary wa11 in the incipient stage of de-

cay， where bluish white autofluorescence of

lignin disappeared， and then expanded out-

ward with the further ad vance of decay， as

shown in Figures 8 and 9. These changes in

color of the secondary wa11s were common in

the early stages of decay in the three white-rot

fungi

Fig. 8. Fluorescence photomicrographs of

wood (weight loss 1%) decayed by

P. nameko

Note: Asterisk indicates the same ce11

A: AO stained， B: unstained. Bar:

30μm.

[slIll. UlslIl1ullliya Unir. I.'or

As shown in Figure 9， with the further ad-

vance of decay， wood decayed by P. nα1ηelw

had a typica11y simultaneous white rot con-

comitantly degrading lignin and ce11ulose

which resulted in the extensive thinning of the

secondary wa11 from the inside outward. On

the other hand， P. ostreatus and G. frondosa

caused selective delignifications of woods res-

ulting in the preferential removal of lignin

from localized areas of the decayed wood in

advanced stages of decay. where dark green

fluoresc巴nce appeared inside the secondary

wa11 (Fig. 10A). In the darkly fluoresced areas

inside the secondary wa11， a phloroglucinol-

HCl test showed no color reaction (Fig. 10B)

indicating the alteration or absence of lignin，

whereas lignin sti11 remained in the middle

lamellae. Such lignin removal from the c巴11

walls also was found in wood decayed by G.

frondosαas shown in Figure 11. In woods de-

cayed by P. ostreαtllS and G. frondosa， the

thinning of the ce11 wa11s did not occur， at

least before the positive reaction in color by a

phloroglucinol-HCl test disappeared in the

secondary wa11

Fig. 9. Fluorescence photomicrograph of

wood (Weight loss 4%) decayed by

P. namelw and stained with AO

Bar: 30μm

¥.'0¥.26，1990] ¥';nヌyl11alicdcgradalioll 01' w()od by whilc-n>l I'ungi 27

Fig.l0. Photomicrographs of wood (weight

loss 6%) decayed by P. ostreatus

and showing that lignin was re-

moved from the secondary walls in

localiz巴d areas but not from the

intercellular layers. A: AO stained，

B: phloroglucinol-HCl stained

Note: Asterisks indicate the same cell.

Bar: 30μm.

Histochemical assays of peroxidase and poly-

phenol oxidase activities

Histochemical demonstration of peroxidase

was conducted by fluorescence microscopy

Peroxidase activity was visualized directly，

since reaciton products emitted a red fluores

cence when exited with a green light

In woods decayed by P. ostreαtus and G.

frondosα， peroxidase was detected first in the

pit membranes in the incipient stages of decay

(Fig. 12A). Peroxidase activities in the cell

walls were limited at first to localized areas

of earlywood. Peroxidase activities first ap-

p巴aredinside the secondary walls， then pro-

gressively expanded toward the intercellular

layers prior to the disappearance of color

reactions by phloroglucinol-HCl， indicating

the absence of lignin. With the progress of d巴-

cay， strong red-fluorescence indicating great

peroxidase activiti巴sspread in entire areas of

the decayed wood (Figs. 12B and 13). In wood

decayed by P. nαmeko， however， only a little

peroxidase activity was detected (Fig. 14).

Ray parenchyma cells had gr巴aterp巴roxidase

activity than did the cell walls of tracheids.

Polyphenol oxidase was detected extracel-

lularly on the surface of the hyphal walls

under an ordinary light microscope (Fig. 15)

A weak polyphenol oxidase was observed as

having a light brown color in the mycelium

aggregates in the early stages of decay， and

then the hyphal walls became stained deeply

with the increase in the growth of mycelia.

However， the polyphenol-oxidase acitivity de-

tected in the cell walls of tracheids was consi

d-erably weaker compared with that on the

hyphal walls. Distinct differences in polyphen

ol-oxidase activity could not be detected in the

cell walls having various extents of degrada-

tion throughout the entire decay by the three

white-rot fungi (Fig. 15)

Fig. 11. Photomicrograph of wood (weight

loss 8%) decayed by G. frondosαand

stained with phloroglucinol-HCI.

Bar: 30μm.

28 Nobuo YOSIIIZ八Wr'¥ el al， [sull. Ulsunomiya Uni¥'， for

Fig. 12. Fluoresecence photomicrographs showing pexidase activity in wood decayed by P.

ostreatus with the red color indcating the peroxidase activity in the cell walls. A: wood

in the incipient "stages of decay (weigh t loss 1%). B: wood with 6% weight loss. showing

that cells with great peroxidase activity increased， Bar: 30μm

Note: Arrowheads indicate the pit membranes with great peroxidase activity

Fig. 13. Fluorescence photomicrograph show-

ing peroxidase activity m wood

(weight loss 4%) decayed by G

frondosαBar: 30μm. Fig. 14. Fluorescence photomicrograph show

mg peroxidase activity m wood

(weight loss 6%) decayed by P.

nameko

Note: The peroxidase activity is relatively

greater in ray parenchyma cells

than in tracheids， Bar: 30μm

Vol.26，1990J Enχymalic dcg-rudation o!" wood hy while-rol fung'i 29

Fig. 15. Photomicrographs showing poly

phenol-oxidase activities in woods

(weight loss 2%) decayed by P

nαmeko. Asterisks indicate the

same cell. A: control， B: DOPA

stained

Note Polyphenol oxidase was detected in

the hyphae bu t not in the cell walls

of tracheids. Bar: 30μm

DISCUSSION

In this study， histochemical and micromor

phological changes that occur in wood decay-

ed by three white.rot fungi， P. ostreαtus， P.

nαmeko， and G. frondosa， were examined

mainly using fJuorescence microscopy， and

the activities of peroxidase， laccase， and cellu

lase mainly in volved in wood degradation

W 巴remeasured during various periods of de-

cay， and followed in part， histochemically.

Histochemical tests well demonstrated that

P. ostreαtus and G. frondosa caused a selec-

tive delignification in wood. The decay pat

terns were almost similar to that of C. pini

(Yoshizawa et al.， 1989). Based on phloro

glucinol-HCl staining， lignin is alt巴red or

removed from the secondary walls of localized

areas in wood blocks decayed by these fungi，

but not from the intercellular layers at least

up to the first 10% of the weight loss. G.

frondosαdegrades both holocellulose and lig-

mn even in the early stage of decay (Figs. 2

and 3). Especially， delignification rapidly

increased beyond the point of 6% weight loss.

(Fig. 3)， indicating that lignin removal pre-

ceded holocellulose degradation. P. ostreαtus

has a relatively small ability in degrading

carbohydrates dur・ing decay in comparison

with other fungi used in this experiment. P.

ostreatus and G. frondosa did not cause thin-

ning of the secondary walls， at least until

lignin removal from the secondary walls

almost was completed. This fact indicates that

a selective delignification occurred in pre-

ference to the removal of holocellulose. On the

other hand， P. nαmeko caused an extensive

thinning of the secondary walls from the

inside outward shortJy after lignin removal

occurred in small areas on the lumen side， a

decay pattern commonly observed in wood

degraded by white-rot fungi causing concomi-

tant degradation of both c巴lluloseand lignin

(Kirk and Moore， 1972; Blanchette et al.， 1985;

Yoshizawa et al.， 1989). Actually， P. nαmeko

caused greater weight losses and appears to

d巴grade more carbohydrates than it does

lignin

It has been well-demonstrated that white-rot

fungi， which can degrade all the major wood

components， have at least two different

types of selecti ve delign山 cation(Liese， 1970;

Blanchette et al.， 1985; Otjen and Blanchette，

1986， 1987; Yoshizawa et al.， 1989). The first

type results in extensive lignin removal from

localized areas leaving only white cellulose

fibers within wood blocks， a characteristic of

P. pini-2 (Otjen and Blanchette， 1987; Otjen

et al.， 1988). The second type results in a

more uniform loss throughout wood blocks，

but a less extensive loss from individual cell

walls， such as caused by P. mutαbilis，

P. breuisporαー1， and P. chrysosporium

(Objen and Blanchette， 1987; Objen et al.，

1988). P. ostreαtus and G. frondosαexamined

in this study show characteristics of the

first type of decay， at least up to the weight

loss of 10% under the conditions provided.

However， these fungi caused an extensive

thinning of the secondary wall in a later stage

30 トJobuoYOSHIZA W八 ctal

of decay in the manner of decay similar to P.

nameko. This also seems to be similar for

Dichomitus squales and Heterobαsidion

αnnosum (Blanchette et al.， 1987). Similar

evidence was reported in wood decayed by C.

pini (Y oshizawa et al.， 1989) which caused

complete lignin removal even from the middle

lam巴llaeas well as from the secondary walls.

It also caused a dentate thinning of the sec-

ondary walls after the ext巴nsive delignifica

tion. These facts observed in the progressive

degradation of cell walls may be related to the

changes in a complex ligninolytic-cellulolytic

system during decay. It is very important to

note the specificity of th巴sefungi for lignin

degradation in earlier stages of decay，

considering the potenti旦1of white-rot fungi to

selectively delignify、、Toodfor industrial appli

cations. More res巴archis needed to further

define the differences in enzyme activities of

fungi in vol ved in selecti v巴 delignification.

Histochemical observations apparently

showed the changes in the peroxidase activity

associated with d巴cayby the thre日 fungi.In P

ostreαtus and G. frondosαrotted woods， red

fluorescence showing peroxidase activity was

detected in the cell walls and increased with

the progress of decay， suggesting that perox

idase plays an important role in d巴grading

lignin. On the contrary， the fact that only

little peroxidase activity was observed in P.

nameko rotted wood app巴arsto show the in-

volvement of other phenol-oxidizing enzymes，

such as laccase， rather than peroxidase. On

the other hand， distinct differences in polyphe

nol oxidase activity were not observed in the

cell walls of decayed tracheids， although the

activity detected on the mycelia walls in

creased with the progress of decay in wood

decayed by the three fungi. Further ultra‘

structural research is need日don this point

Degradation of wood components observed

by histochemical tests was follow巴d by the

measurement of some enzyme activities in-

volved in wood degradation. In woods decayed

by P. nαmeko and G. frondosα， laccase acti vト

ty w旦svery great in the incipient stage of dι

[Bull. Ulsunol1liya Uni¥". For.

cay and then rapidly decreased， whereas in P

ostreαtus rotted wood there was relatively

little during decay (Figs. 4， 5， and 6). On

the other hand， peroxidase activities of P.

ostreαtus and G. frondosαwer日 relatively

great， but that of P. nameko was little and

did not vary during decay. These facts also

were confirmed bv histochemical tests for

detecting peroxidase activity. The patterns of

changes in cellulase activities during decay

were almost the same among the three fungi

(Fig. 7)司 Changesin enzyme activities we ob-

tained were almost in agreement with eearlier

results of lignin biodegradation (Oki et al.，

1981; Morohoshi巴tal.， 1985). It is considered

that diff日r巴ntenzyme activities caused differ-

ent decay patt日rns.

Based on the ch且ngesin phenol-oxidizing

enzyme activities during dec且y， and the

results of histochemical observations， perox-

idase and laccase are consider巴dto be involv-

ed in lignin degradation by the three white-rot

fungi we used. Phenol-oxidizing enzymes of

both peroxidas巴 andlaccase types appear to

be involved in delignifying wood during decay

by P. ostreαtus and G. frondosαOn the other

hand， in decay by P. nαmelw， the laccase type

appears to play an important role in lignin

degradation.

In woods decayed by P. nameho and G.

frondosa， holocellulose removal occurred fast-

er in the incipient stage of decay， and was

much greater throughout the period of decay

than was that of P. ostreatus. Considering

that hemicellulose tends to be degrad日dfaster

than cellulose in removing lignin (Kirk

and Moore， 1972; Kirk and Highley， 1973;

Ander and Eriksson， 1977; Blanchette， 1984b;

Blanchett巴 et且1.， 1985; Blanchette and Abad，

1988)， the fact that G. frondosa caused greater

hemicellulose degradation may be caused by

the relatively great activities of lacc且seand

cellulase during decay. Hemicellulose appears

to be degraded faster than cellulose as a

requirement for a carbohydrate source for

lignin degradation (Kirk and Highley， 1973).

Blanchette and others (1985) later demon-

Vo1.26，1990J Enzyrnalic dcgradalion 01' wood by whilcωrol fungi 31

strated， using some white-rot fungi， that the

percent losses of xylose and mannose were

always much greater than the loss of glucose，

and suggested that lignin and hemicellulose

were the major components removed from the

localized， white tissues， The great activiti巴sln

th巴 incipientstage of decay may be involved in

the gr巴ater hemicellulose degradation

However， cellulase activity of P ηαmeko was

not necessarily great compar巴dto that of the

other two fungi， irrespective of the greater de-

gradation of holocellulose. This suggests that

in decay by P. 叩 melwother enzymes， such

as hemicellulases， are involved in hemicel-

lulos巴 removal，and also that a more complex

ligninolytic-cellulolytic system made by the

secreted enzymes is involved in the progresω

sive thinning of the second且rywalls

Woods decayed by white-rot fungi have

two patterns of hemicellulose degradation

(Blanch巴tteand Abad， 1988). Coriolus uersi-

color progressively degraded hemic巴llulosein

the wall from the lumen toward the middle

lamella. whereas Phlebia tremellosus and P.

pini caused extensive loss of hemicellulose

throughout the entire cell wall. These facts

suggest that the pattern of hemicellulose re‘

moval from the cell wall appears to be almost

the same as that of lignin removal in these

fungi. Of the three white-rot fungi used her巴，

P. nαmeko appears to hav巴 characteristicsof

the first typ巴 ofhemicellulose degradation，

whereas P. ostreαtus and G. frondosa shows

that of the second type. However， further reω

search is needed for recognizing the patterns

of hemicellulose removal in the three white-rot

fungi we studi巴d.

Extracellular phenol-oxidizing enzyme ac-

ti vity has be巴ndetected in virtually all white

rot fungi examined (Kirk and Shimada，

1985). Phenol-oxidizing enzymes hav日 beenes

tablished as playing a role in degrading lignin

(Ander and Eriksson， 1977; Eriksson， 1981;

Oki et al.， 1981; Higuchi， 1985; Kirk and

Shimada， 1985; Morohashi et al.， 1985; Kirk，

1987). Garci且 andothers (1987) demonstrat日d，

using P. chrysospor山 m，the accumulation of

extracellular peroxidase in both the hyphal

walls and the area of wood degradation， but

not ligninase. On the other hand， Srebotnik

and others (1988) detected the localization of

ligninase in th日 cytoplasm of hyphae of P.

chrysosporium by immunogold labelling.

More recently， Daniel and others (1989 a and

b) demonstrated， using double immunola-

belling techniqu日s， that lignin degrading

peroxidas巴 was localized in charact巴ristic

zones of degradation produced within the

secondary cell wall regions in both 巴arly

and late stages of wood degradatin by P.

chrysosporium 旦nd L. edodes. Activity of

peroxidase fluoresced by rhodamine B in our

study appar巴ntly was localized in the deg司

radation sit日s in the secondary walls of

tracheids. These facts suggest旦 closecorrela-

tion between changes in the micromorphology

of decay and peroxidase distribution.

Histochemical and 日nzymaticresults that

we obtained also indicate the involvement of

phenol守 oxidizing enzymes (peroxidase and

laccase) in the degradation of lignin as well

as did the earlier reports， although an accu-

rate relationship between the rate of lignin

degr且dationand the changes in activities of

these enzymes during decay was not estab

lished necessarily. Further research concern-

ing th日 biodegradationof lignin is needed to

develop the bioconversion of lignocellulosic

materials

ACKNOWLEDGMENT

The authors thank Dr. Koichi Yamamoto

and Dr. Tadakazu Hiroi， Forestry and Forest

Products Research Institute， for providing the

strain of G. frondosα.

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