ELSEVIER Journal of Biotechnology 41 (1995) 1-17

Biotechnolom

Review

Enzymes of white-rot fungi involved in lignin degradation and ecological determinants for wood decay

U. Tuor a,1, K. Winterhalter b, A. Fiechter a** a Swiss Federal Institute of Technology, Instirute of Biolechnology, ETH-Hiinggerberg, CH-8093 Ziirich, Switzerland b Swiss Federal hstitule of Technology, Department of Biochemistry, ETH-Zenlrum, CH-8092 Zikich, Switzerland

Received 12 November 1994; accepted 17 March 1995

Abstract

White-rot fungi preferably degrade wood from deciduous trees, whilst wood decay by brown-rot fungi is predominant on coniferous substrates. A compilation of recent publications on ligninolytic fungal species and their substrate preference is presented. These organisms can be classified on the basis of their enzyme systems, but an unambiguous allocation to specific hosts proved to be difficult. Environmental conditions may be crucial in governing the selectivity of fungal biodegradation of wood components. Possible mechanisms of primary attack of the wood cell wall by hemicellulose degradation are discussed. Furthermore, a hypothetical scheme for lignin biodegradation involving oxidative cleavage of phenol& C,-oxo-substituted substructures is presented.

Keywords: White-rot fungi; Ecology; Wood biodegradation; Microbial population; Lignin degradation; Hemicellulose degradation; Ligninase; Manganese peroxidase; Lactase

1. Introduction

The stems of higher plants obtain mechanical strength and rigidity by the structural elements cellulose, hemicellulose and lignin, which are syn- thesized and deposited in the plant cell walls. Under microgravity conditions aboard the space shuttle, lignification in pine, oats and mung beans seedling was decreased by 6-24% (Cowles et al., 1989). In recent years the enzymatic machinery of

* Corresponding author. 1 Present address: Helbling Ingenieurunternehmung,

Hohlstr. 610, CH-8048 Ziirich, Switzerland.

selected wood degrading white-rot fungi has been elucidated to a large extent. The discovery of ligninase (lignin peroxidase (Lip) Glenn et al. (1983), Tien and Kirk, 1983) and Mn-peroxidase (MnP; Kuwahara et al., 1984) from Phane- rochaete chrysosporium triggered biochemical re- search on lignin biodegradation. Here we present the available information on ecological determi- nants of white-rot wood decay and relate it to the present knowledge of the enzymology of white-rot fungi and other microorganisms. Based on pre- sent knowledge, the biochemistry and physiology of white-rot wood decay can not easily be corre- lated with environmental conditions because of the variability of the fungi and their weak speci-

016%1656/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDZ 0168-1656(95)00042-9

2 U. Tuor et al. /Journal of Biotechnology 41 (1995) 1 -I 7

ficity towards their hosts. As research proceeds, our perception of lignin degradation is changed from an oxidative depolymerization process caused by a single enzyme to a process of con- certed oxidative and reductive conversions in which different classes of enzymes participate. The emerging hypothetical schemes also involve cleavage of lignin-carbohydrate complexes by hemicellulases as crucial steps in the overall lignin biodegradation.

2. Host specificity of wood-rotters

Hardwood and softwood are distinguished by structural elements building the phenylpropane backbone of the lignin component. Lignin is a threedimensional, optically inactive phenyl- propanoid polymer randomly synthesized from coniferyl, p-coumaryl and sinapyl alcohol precur- sors (Sarkanen and Ludwig, 1971; Fig. 1). Soft- wood lignin is referred to as guaiacyl lignin, con- taining more than 95% coniferyl alcohol (4-hy- droxy-3-methoxy-cinnamyl alcohol) units. A struc- tural model of spruce lignin was proposed by Adler (1977). The remaining elements are mainly p-coumaryl (6hydroxycinnamyl) alcohols with trace amounts of sinapyl (3,5-dimethoxy-4-hy- droxy-cinnamyl alcohol) alcohols. Typical hard- wood lignins, also classified as guaiacyl-syringyl lignins, contain coniferyl- and sinapyl alcohol-de- rived subunits. The relative amount of the latter

Table 1 Number of functional groups in lignin per 100 C,C, units

Functional group Spruce lignin Birch lignin

Methoxyl 92-96 139-158 Phenolic hydroxyl (free) 15-30 9-13 Benzyl alcohol 15-20 Noncyclic benzyl ether 7-9 Carbonyl 20

Their content may vary depending on the origin of the lignin (e.g., middle lamella or secondary cell wall lignin). The values are compiled from Adler (1977) and Sarkanen and Ludwig (1971).

varies from 26 to 60% (Chang and Sarkanen, 1973). Hardwood lignins typically contain 1.2-1.5 methoxyl groups per phenylpropane unit (Sarkanen and Hergert, 1971). A structural repre- sentation was proposed by Nimz (1974). Although classified as guaiacyl-syringyl lignin, grass lignin also contains additional small amounts of p- coumaryl alcohol-derived subunits. The men- tioned differences in chemical composition are reflected in the frequency of functional groups (Table 1): spruce (softwood) lignin contains 92-96 methoxyl and 15-20 free phenolic functional groups per 100 phenylpropane units, whereas the corresponding numbers for birch (hardwood) lignin are 139-158 and 9-13, respectively (Sarkanen and Ludwig, 1971; Adler, 1977).

According to macroscopic differences of their substrate utilization, wood-rotters are classified into three specific decay groups: white-rot,

OH OH OH

p-coumaryl alcohol coniferyl alcohol sinapyl alcohol

Fig. 1. Precursor alcohols of lignin.

U. Tuor et al. /Journal of Biotechnology 41 (1995) I -I 7 3

brown-rot and soft-rot fungi. Most wood-rotting fungi are able to degrade both cellulose and lignin in wood, but have different degradation rates for cellulose, hemicellulose and lignin. Brown-rot fungi have an obvious preference for coniferous substrates (gymnosperm& A survey of substrate relationships reported that 19% of North American polypores are brown-rot fungi, among which 60 out of 71 (85%) occur primarily on conifers, and that they are mainly soft-wood degraders. They develop predominantly in conif- erous forest regions as they are more efficient in gaining energy from wood for growth and repro- duction than white-rot fungi, favouring their sur- vival and propagation in colder climates with a shorter growing season (Gilbertson, 1980). On the other hand, there are numerous examples of wood-rotting fungi that have a geographic distri- bution not corresponding to their most suitable hosts (Gilbertson, 1980).

In contrast, white-rot fungi predominantly de- grade wood from deciduous trees (angiosperms). In a survey of a total of 65 central European wood-destroying basidiomycetes, four were re- ported to only attack coniferous wood, whereas 34 were attacking angiosperms exclusively. 27 of the fungi attack both (Rypacek, 1977). Table 2 presents a overview of white-rot fungi whose ligninolytic system has been investigated. Where known, their preferred host is indicated, and the dominance of hard-wood substrates is evident.

In general, wood rotting fungi have broad host ranges and can only be designated soft- or hard- wood degraders. Growth on principal monoses of soft- or hardwood hemicellulose correlates with the specialization of white-rot and brown-rot fungi. It was therefore suggested that the ability of hemicellulose degradation may determine preference for coniferous or deciduous substrates (Rypacek, 1977).

3. Oxidative enzymes in lignin biodegradation

The extracellular oxidative enzymes ligninase, manganese peroxidase and lactase may be de- fined as phenoloxidases. Both ligninase (EC No. 1.11.1.14 Diarylpropane: oxygen, hydrogen-per-

oxide oxidoreductase) and manganese peroxidase (EC No. 1.11.1.13 Mn(I1): hydrogen-peroxide oxi- doreductase) belong to the class of peroxidases and oxidize their substrates by two consecutive one-electron oxidation steps with intermediate cation radical formation. Due to its high redox potential, the preferred substrates for LIP are nonphenolic methoxyl-substituted lignin subunits, whereas MnP acts exclusively as an phenoloxi- dase on phenolic substrates using Mn2+/Mn3+ as an intermediate redox couple. Oxidation of phe- nolic substrates by ligninase leads to their poly- merization (Higuchi, 1986; Odier et al., 1988). Ring-cleavage of aromatic rings is a key step of lignin mineralization. Non-phenolic syringyl and biphenyl model compounds are oxidized by lignin peroxidase and subsequent ring cleavage. In con- trast, oxidation of the corresponding phenolic compounds by ligninase did not yield ring-opened products (Hattori and Higuchi, 1991a,b). Alkoxyl groups activate aromatic rings towards oxidation by ligninase, partly explaining why syringyl lignin is easier to degrade than guaiacyl lignin (Eriksson et al., 1990). Lactase (EC No. 1.10.3.2. (be- nzenediol: 0, oxidoreductase) is a true phenolox- idase with broad substrate specificity. It oxidizes phenols and phenolic lignin substructures by one-electron abstraction with formation of radi- cals that can repolymerize or lead to depolymer- ization (Higuchi, 1989). Demethylation reactions of terminal phenolic units catalyzed by lactase may therefore be of importance for native lignin degradation, and the utilization of lactase or lac- case-producing white-rot fungi for biopulping has become a focus of interest recently (Reid and Paice, 1994; Messner and Srebotnik, 1994). In view of these findings it seems likely that struc- tural and chemical differences in the very inho- mogeneous lignin substrate should lead to a spe- cialization in the respective degrading microor- ganisms, particularly the oxidative enzymes ex- pressed by each organism. Table 2 gives an overview of the white-rot fungi investigated and the oxidative enzyme activities as published in the literature. They are classified in Table 2 into five main groups:

I. White-rot fungi expressing Lip, MnP and lactase. This group contains the best known

P

Table

2

Whit

e-r

ot fu

ngi,

their

oxi

dati

ve enzy

mes a

nd their

pre

ferr

ed h

ost

s

Mic

roorg

anis

m

Enzy

mes

Ref.

H

ost

R

em

ark

s

UP

Mn

P

Lact

ase

Ph

en

ol-

A

tyl

alc

oh

ol

oxi

dase

oxi

dase

act

ivity

(EC

1.1

1.1

.14)

(EC

1.1

i.i.1

.3)

(15c

I

10.3

.2)

(EC

1.

1.3.

7)

x (l

IttI

e)

x x x x x x

Malt

seva

et al.,

19Y 1:

deci

du

ou

s tr

ees

class

ific

ati

on

whit

e-r

ot

Golo

vleva

tl

al.,

1993

fungus unce

rtain

x x x

I C

onalu

s LY

rsic

olo

r x

x x

Fahra

eus a

nd

Rein

ham

mar.

1967.

Dodso

n e

t al.,

1987;

Johanss

on a

nd

Nym

an, 1

987;

Wald

ner,

1987:

Wald

ner et al.,

1988

Gle

nn e

t al.,

1983;

Tie

n a

nd K

irk,

1983:

Leis

ola

et al.,

1987

Pere

z and Jc

ffri

cs.

1990:

Riit

tim

ann c

t al..

19

92~

Hata

kka e

t al..

1987:

Nik

u-P

aavo

la e

t al..

1988

Sonnia

et al.,

19%

; W

aldn

rr,

19X

7:

Wal

dnrl

- rt

al

,

198X

;

San

nia

et

al.,

1991

Fu

ku

rum

i.

LY8

7,

Bou

rbon

nais

and

Paic

e, 1

989;

Boyl

e e

t al.,

1992

Riit

tim

ann e

t al..

1992a,b

: Lo

bos

ct a

l.. l

r)4

none

Petr

osk

i et al.,

1980;

P&

it

and G

old

, 1991

Leath

am

and

Sta

hm

ann, 1

981:

Leath

am

. 1986:

Forr

ente

r et al.,

1990

deci

duous t

rees

deci

duous t

ree\

rare

ly c

om

ters

deci

duous l

reen

nd

deci

duous t

rccb

littl

e ia

ccasr

pro

duct

ion

\ 2

(Andcr

ct

al..

1980:

5

Eri

ksso

n c

l al,

1983)

$

%

5

e

R

%

pre

sum

ably

whit

e-r

ot

bir

ch, a

spen, s

pru

ce, w

heat,

barl

ey,

gra

pe s

traw

deci

duous t

rees

Rig

idop

oru

s iig

nosu

s n

on

e

x G

eig

er

et al.,

1986a,b

; H

evea bra

silie

nsi

s

Galli

ano e

t al.,

1991

(ru

bb

er

tre

e)

Wald

ner,

1987;

deci

duous t

rees

Wald

ner

et al.,

1988

Dill

and K

raepelin

, 1988

na

Hiit

term

ann e

t al.,

1989

beech

Vare

s et al.,

1993

na

Bis

was-

Haw

kes e

t al.,

1987; d

eci

duous t

rees

syn. D

aed

ale

op

sis

conf

rago

sa

* in

dir

ect

dete

ctio

n

111 C

ori

olu

s pru

inosu

m x

Gan

od

en

na

x

ap

pla

natu

m

Ou

dem

an

siella

x

rad

icata

Phle

bia

och

ra-

x

ceofu

loa

Phle

bia

wem

ello

ws

x

x

x x

syn. M

eru

lius t

rem

el~

osu

s x x x

Hiit

term

ann e

t al.,

1989

Hiit

term

ann e

t al.,

1989

Hiit

term

ann e

t al.,

1989

x H

iitte

rmann e

t al.,

1989

x H

iitte

rmann e

t al.,

1989

x H

iitte

rmann e

t al.,

1989

x H

iitte

rmann e

t al.,

1989

x W

ald

ner,

1987;

Hiit

term

ann e

t al.,

1989;

Muheim

et al.,

1990;

Muheim

, 1991;

Hilt

term

ann e

t al.,

1989

Hiit

term

ann e

t al.,

1989

Hiit

term

ann e

t al.,

1989

and c

onifers

na

na

Ple

uro

tus fl

ori

da

x

Poly

port

~s pla

ten

sis

x

pre

sum

ably

whit

e-r

ot

fungus

deci

duous t

rees

na

deci

duous t

rees

(beech

)

deci

duous t

rees

(beech

) deci

duous t

rees

Poly

poru

s brw

nalis

x

Poly

poru

s pin

situ

s x

Ust

ulin

a deu

sta

x

syn. H

yp

oxylo

n deu

stu

m

Xyla

ria poly

morp

ha x

IV

Bje

rkan

dera

ad

&a

x

none

deci

duous t

rees

(cherr

y)

deci

duous t

rees

and c

onifers

deci

duous t

rees

(beech

, ald

er)

Daed

ale

op

sis

x

con

frag

osa

Phallu

s im

pu

dic

us

x

not w

hit

e-r

ot fu

ngus

Poly

poru

s oari

us

x

V

Fam

es l

ign

osu

s n

on

q

none

none

Kir

k, 1

987; W

ald

ner,

1987; n

a

Wald

ner

et al.,

1988

Tra

mete

s cin

gu

lata

n

on

e

none

none

K

irk,

1987; W

ald

ner,

1987; n

a

Wald

ner

et al.,

1988

Phelii

nus n

oxiu

s x

Geig

er et al.,

1986a,b

H

eoea bra

silie

nsi

s (r

ubber t

ree)

Phelli

nus w

eir

ii x

Must

ranta

, 1987

conifers

Ple

uro

tw e

ryn

gii

x

Guill

en e

t al.,

1990

na

pre

sum

ably

whit

e-r

ot

fungus

syn. I

non

otu

s weir

ii cl

ass

ific

ati

on w

hit

e-r

ot

fungus

unce

rtain

sy

n. C

ori

olu

s u

ers

icolo

r Poly

stic

rus u

ers

icolo

r x

Farm

er

et al.,

1960

na

For

exp

lanati

ons s

ee te

xt.

x =

enzy

me act

ivity

report

ed; n

one =

does

not

pro

duce

enzy

me; b

lank

= n

o r

eport

s on e

nzy

me act

ivit

y; n

a =

info

rmati

on n

ot

ava

ilable

.

6 U. Tuor et al. /Journal of Biotechnology 41 (1995) I-1 7

white-rot fungi Coriotus L:ersicolor, Phane- rochaete chrysosporium and Phlebia radiata. P. chrysosporium is listed within this group since lactase production was reported (Ander et al., 1980; Eriksson et al., 1983). However, this fun- gus is generally considered not to produce lac- case. All of them usually colonize deciduous trees, only Phlebia radiata occasionally degrades conifers. II. White-rot fungi simultaneously producing both types of phenoloxidases MnP and lactase, but reportedly not secreting detectable levels of lignin peroxidase. Nevertheless, these fungi are strong lignin degraders. Dichomitus squalens and the edible fungus Lentinulu edodes belong to this group. III. White-rot fungi with LIP and one of either phenoloxidases. Lactase is the predominant phenoloxidase produced, only in the case of Coriolus pruinosum was MnP production re- ported. These fungi grow on hardwood. As an exception, only Phlebia tremellosus also de- grades coniferous wood. IV. Four white-rot fungi secrete LIP without phenoloxidases. Again with one exception they are hardwood degraders. V. The last group probably consists of fungi which are incompletely characterized. Notably Fames lignosus and Trametes cingulata are white-rot degraders, but neither of the oxidative enzymes was detected. Furthermore, references to aryl alcohol oxi-

dase and more general reports on phenoloxidase activity are given in Table 2. Apparently, white-rot fungi achieve lignin biodegradation with several different combinations of peroxidases and oxi- dases. Lignin degradation by fungi that mainly produce LiP most likely runs along different pathways and involves different redox mediators as opposed to fungi predominantly secreting MnP and laccases. The fungal species investigated may also express gene products differently in agitated or stationary cultures than during growth on solid substrates in natural environments. Contrary to liquid culture, P. chrysosporium produces MnP as major peroxidase when grown on wood (Datta et al., 1991). C. subvermispora and P. brevispora tested negative for LIP production in agitated

culture, whereas the use of southern hybridiza- tion technique with a cDNA probe from P. chrysosporium revealed the presence of LiP genes in both fungi (Riittimann et al., 1992a,b). In the case of P. brevispora, LIP production in liquid culture was reported earlier (Perez and Jeffries, 1990).

Rather than pointing to a particular set of enzymes responsible for lignin breakdown, the data of Table 2 suggest that neither ligninase, manganese peroxidase nor lactase is essential for white-rot wood decay. Work with a ligninase- negative mutant of P. chrysosporium showed no obligatory relationship between ligninase expres- sion and lignin degradation. This LiP mutant produced MnP, but no detectable amounts of Lip, and was still able to mineralize lignin, albeit at a lower rate than the wild type (Boominathan et al., 19901. Lignin degradation by other wild-type white-rot and brown-rot fungi which do not se- crete lignin peroxidase was observed (Buswell and Odier, 1987; Bourbonnais and Paice, 1989). C. subvermisporu, known not to produce Lip, does degrade nonphenolic synthetic lignin and model compounds when grown on its natural substrate wood, but does not do so in liquid culture (Srebotnik et al., 1994). In cell free sys- tems, LiP does not mineralize lignin (Kirk and Farrell, 1987; Hammel et al., 1993). Haemmerli et al. (1986) observed polymerization of different lignin preparations when incubated with purified ligninase. However, partial depolymerization of synthetic, radiolabeled lignin was achieved using ligninase (Hammel and Moen, 1991; Hammel et al., 1993) or MnP (Wariishi et al., 1991), provided that low hydrogen peroxide concentrations are maintained. Depolymerization by MnP alone is limited to phenolic lignin preparations and can be stopped completely by chemical blocking of phenolic functions (Hammel et al., 1993). Com- plete mineralization requires cellular uptake of depolymerization products and further intracellu- lar catabolism.

3.1. Crystal structure of lignin peroxidase and man- ganese peroxidase

Communication$ on the tertiary structure of ligninase at 2.6 A resolution (Edwards et al.,

II Tuor et L /Journal of Biotechnology 41 (1995) l-17 7

19931, 2.5 A resolution (Piontek et al., 1993) and 343 amino acids with three ordered sugar moi- 2.0 A (~oulos et al., 1993) have appeared rz- eties and one heme molecule. A view of the cently. The chainfold of the enzyme comprises crystallographically determined structure is given

Fig. 2. A view of the crystallographically determined structure of lignin peroxidase (Piontek et al., 1993). a-Helices are shown in blue, while p-strands are drawn as yellow arrows. The catalytic heme molecule together with the proximal and distal histidine are shown as ball and stick models, as well as three glycosylation sites and the eight cysteine residues forming four disulfide bridges. Two calcium ions are represented as enlarged, violet spheres. The plot was generated using the program MOLSCRIPT.

x U. Tuor et ul. /Journal of Biotechnology 41 (1995) I - 17

in Fig. 2. Noteworthy features are the mostly helical fold with separate domains on either side of the heme. LIP strikingly resembles cytochrome c peroxidase (CCP) with a similar spatial arrange- ment of 11 helical segments, despite the fact that the amino acid sequence identity of the two en- zymes is below 20% (Piontek et al., 1993: Poulos et al., 1993). LiP contains four disulfide bridges formed by eight cysteine residues which are ab- sent in CCP (Piontek et al., 1993; Poulos et al.. 1993).

Comparisons of the active centers of LiP and CCP show differences which in part account for the reactivity differences observed. The smaller heme pocket in LiP is separated from the solvent and probably provides the binding site for small aromatic molecules, e.g., veratryl alcohol. In ad- dition, the active site of LIP seems to be stabi- lized by two structural calcium ions which have not been observed in CCP (Poulos et al., 1993). The enzyme contains one N-acetylglycosamine at Asn-257 and two mannose residues, supposedly originating from an 0-glycosylation site (Piontek et al.. 1993: Poulos et al.. 1993).

The porphyrin system consists0 of one pentaco- ordinate iron placed 0.15-0.27 A above the sad- dle-shaped heme plane. It coordinates with a distal and a proximal histidine and a distal argi- nine. Unique features of LIP like a low pH opti- mum near 3.0 (Tien et al., 1986) and the capacity to oxidize nonphenolic substrates with high redox potential (Schoemaker. 1990; Gold et al., 1989; Kirk and Farrell, 1987) can now be explained on a molecular level. pH-dependent protonation of carboxylates which are present in the heme pocket favour substrate binding and electron transfer (Poulos et al., 1993). Weaker ligation of the ligni- nase heme iron with the proximal histidine re- sults in a higher electron deficit and thus higher redox potential than in CCP (Piontek et al., 1993). Since LIP and MnP exhibit good sequence ho- mology, LIP electron density maps were also ap- plied for the refinement of crystallographic data obtained from MnP crystals. Preliminary crystal- lographic high-resolution data has been pre- sented, but a detailed structural description of MnP and its binding site is not available as yet (Sundaramoorthy et al., 1994).

3.2. Enzyme multiplicity

Ligninolytic activity in P. chrysosporium and other white-rot fungi is associated with multiple isoenzymes. At least 21 heme peroxidases are produced in liquid cultures of P. chrysosporium (Leisola et al., 1987; Tien, 1987). The physical and kinetic characteristics of the isozymes are very similar, but differences in stability, quantity and catalytic properties have been described (Farrell et al., 1989; Giumoff et al., 1990). They may be the consequence of three different struc- tural genes or post-translational modifications. Coriolus versicolor (Morohoshi, 1991), Panus tigrinus (Maltseva et al., 1991), Rigidoporus ligno- sus (Geiger et al., 1986b), and Ceriporiopsis sub- cermispora (Lobes et al., 1994) secrete lactase isoenzymes. The significance of the multiplicity of ligninolytic enzymes to lignin degradation re- mains to be explained; it was suggested that they possess different importance in the delignifica- tion process, or that they may act synergistically (Farrell et al., 1989). The isozymes may also re- flect the inhomogeneity of the substrate and in- termediate products at different stages of wood decay and among plant species.

4. Importance of ecological conditions

All white-rot basidiomycetes degrade the three wood polymers cellulose, hemicellulose and lignin, albeit at different rates and extent. Based on these criteria white-rot fungi have been divided into classes of simultaneous degraders of lignin along with wood polysaccharides or selective lignin degraders (Eriksson et al., 1990; Blanchette, 1991). Classification according to these groups proved to be difficult since many white-rot fungi show both types of decay while acting on the same substrate, or they attack lignin and polysac- charides sequentially (Eriksson et al., 1990). Envi- ronmental conditions such as temperature, hu- midity, microclimate, nitrogen content of the sub- strate and compartimentalization may also govern the selectivity of lignin biodegradation in vivo. The gaseous regimes within the wooden substrate influence enzyme activies. At the hyphal level,

U. Tuor et al. /Journal of Biotechnology 41 (1995) 1 -I 7 9

elevated carbon dioxide levels (> lo-20%) with concomitant depressed oxygen levels ( < 1%) were measured (Thacker and Good, 1952), suggesting that in vivo oxidative lignin degradation may oc- cur under microaerobic conditions. P. chrysospo- rium efficiently degraded dioxane/HCl-extracted straw lignin even under an atmosphere of 30% CO, and 10% 0, (Leisola et al., 1984). There is experimental evidence that a pure oxygen atmo- sphere is toxic to the fungus (Leisola et al., 1984; Reid and Seifert, 1980).

A special case of selective lignin removal in hardwoods is caused by Ganoderma applanatum in evergreen rainforests of southern Chile and known as palo podrido. Comparing palo podrido with lignin degradation by G. applanatum in tem- perate climate it was concluded that low nitrogen concentration, high carbon dioxide/low oxygen partial pressure, high humidity and low tempera- ture are the most important environmental fac- tors for selective lignin degradation (Dill and Kraepelin, 1988). Palo podrido is a result of the rainforest climate, whereas in Europe this type of wood decay is very atypical. It will only appear if a similar microclimate can build up in a secluded compartment of a tree or stem. The same report states that the substrate of palo podrido contains lower amounts of nitrogen of 0.037-0.073% dry weight as opposed to amounts > 0.07% in Euro- pean tree species. Under these conditions micro- bial cellulose consumption is inhibited. Since ap- proximatly 50% of the total nitrogen in wood is bound to lignin (Dill et al., 19841, lignin degrada- tion with simultaneous nitrogen utilization would result in mycelial growth.

Lignin degradation occurs at low pH. Its opti- mum differs between species. P. chrysosporium degrades lignin best at pH 4.0 with decreasing activity towards lower pH values (Kirk et al., 1978). In another study, lignin degradation by P. chrysosporium and P. sajor-caju increased with decreasing pH down to pH 3.0 (Boyle et al., 1992). Accordingly, the enzymes purifed from P. chrysosporium have their activity optimum at pH 3.0 (Lip; Tien et al., 1986) and pH 4.5 (MnP; Glenn and Gold, 1985). The pH optimum of lactase from Phellinus noxius is at pH 4.6 (Geiger et al., 1986b). The pH of the natural substrate

varies between 4 and 6 for both hardwoods and softwoods (Gray, 1958) and corresponds to the growth optimum for most wood rotting fungi. Furthermore, fungi are able to maintain a low pH environment at the hyphal level by secreting a polysaccharide slime layer (Buchala and Leisola, 1987).

5. Importance of hemicellulose degradation and succession of microbial populations

The twisted arrangement of cellulose, hemicel- lulose and lignin was described in a model by Kerr and Goring (1975). Lignin is chemically bound to the hemicellulose moiety in the S2 layer, forming a matrix encrusting the cellulose microfibrils. Xylans and lignin are closely associ- ated and covalently bound in wood. Lignin- carbohydrate complexes (LCCs) have been iso- lated from wood meal extracts after removal of lignin or in the lignin fraction directly, and they have been recently reviewed (Koshijima et al., 1989; Jeffries, 1990).

Lignin-carbohydrate bonds are formed by ester or ether bonds, linking the cu-position of the lignin phenylpropane unit to either carboxyl or free hydroxyl groups of hemicelluloses, respec- tively. They occur in low frequency. The sugar- lignin linkage of a pine wood sample consists of benzyl ether bonds predominantly at C-6 in hex- oses and mostly at C-3 and to a lesser extent at C-2 in pentoses (Koshijima et al., 1989). o-Xylose and o-mannose are the major LCC components. Evidence for glycosylic lignin-carbohydrate bonds was not reported.

5.1. Accessability of the substrate

Lignin is a high-molecular, hydrophobic poly- mer and thus not soluble in aqueous solution, raising the question about its accessability by extracellular oxidative enzymes and cofactors. The situation is complicated by the fact that lignin does not serve as a sole growth substrate for white-rot fungi (Ander and Eriksson, 1975; Kirk et al., 1976). It was therefore suggested that the purpose of lignin removal is to expose cellulose

10 U. Tuor et al. /Journal of Biotechnology 41 (1995) l-1 7

and hemicellulose fibers for consumption and further fungal growth (Kirk and Fenn, 1982). On the other hand, the hemicelluloses also physically restrict the access of enzymes to lignin. The close association of lignin and hemicelluloses suggests that the primary attack of the wood cell wall requires enzymatic degradation of the hemicellu- loses prior to lignin degradation. The covalent bonds of hemicelluloses to lignin may be hydro- lyzed, resulting in lignin-carbohydrate complexes of lower molecular weight capable of diffusing from the matrix. Alternatively, the hemicellulases could strip their substrates off the cell wall, ren- dering the residual lignin exposed to attack by LiP or MnP. Sound wood cells cannot be invaded by wood decay enzymes (Srebotnik et al., 1988; Blanchette et al., 1989). Hydrolyzation and de- polymerization of the hemicellulose component would render lignin accessible for LiP and MnP.

5.2. Degradation of hemicellulose

Hemicellulose degradation could happen in two ways: either the wood-rotting fungi secrete hemicellulases, or the fungal lignin degradation is preceeded by hemicellulolytic activity of bacterial consortia. Endo-xylanases, @xylosidases and endo-mannanases have been purified from a large number of fungi and bacteria, and they are pro- duced by most wood-degrading fungi (for an overview see Eriksson et al., 1990). At an early stage of wood inoculation, P. chrysosporium ex- presses xylanases which require close contact with their substrates as shown by immunolabeling ex- periments (Joseleau and Ruel, 1992). Xylanases and hemicellulases together with peroxidases ap- pear during incipient stages of decay in the hy- phal cell wall of P. chrysosporium and subse- quently move towards the secondary cell wall (Blanchette et al., 1989). Immunolabeling showed that xylanases do not diffuse into degraded wood cell walls, whereas LiP and MnP penetrate de- graded sections of the cell wall. MnP exhibited a pronounced preference for the highly lignified middle lamella and cell corners (Joseleau and Ruel, 1992). Although white-rot fungi are investi- gated for their ability to selectively remove lignin, one should not overlook their ability to degrade

hemicelluloses during the process. Hemicellu- lases, primarily xylanases and mannanases of fun- gal origin, are currently used in the treatment of pulp on a technical scale as a biological pretreat- ment for delignification in paper manufacture (Buchert et al., 1992; Viikari et al., 1992,1994).

5.3. Bacterial wood biodegradation

Bacteria also attack softwood and hardwood cell walls. They have been described as primary wood colonizers (Liese and Greaves, 1975) and may inhibit or synergistically promote growth of wood degrading fungi. Cell wall decay by mixed bacterial cultures in natural environments as well as in vitro was classified in degradation by ero- sion, cavitation and tunneling (Daniel et al., 1987). Fungal lignin degradation results in the forma- tion of low molecular weight, mostly aromatic carboxylic acids, which may be further metabo- lized by bacteria. Bacterial cultures mineralize intermediates from model compound degradation by white-rot fungi with little concomitant label incorporation into bacterial biomass Wicuna et al., 1993).

6. The role of cr-carbonyl groups in lignin degra- dation

Structural analysis of spruce lignin determined a frequency of 15-30 free phenolic hydroxyl groups and 20 carbonyl groups per 100 phenyl- propane units (Table 1; Sarkanen and Ludwig, 1971). Frequent occurrence of phenolic, C,-oxo- substituted substructures in native lignin and in partially degraded lignin can therefore be as- sumed. Furthermore, formation of a-carbonyl groups is prominent during lignin biodegradation (Kirk and Chang, 1975; Chen and Chang, 1985) and during metabolism of a dimeric P-0-Cmodel compound (Fenn and Kirk, 1984). Destroying chi- ral centers by C,-oxidation not alone at C,, but also via enolization at C,, was suggested to be important in decreasing the steric complexity of the polymer (Shimada, 1980).

Enhanced depolymerization of spruce lignins in ligninolytic cultures of P. chrysosporium was

U. Tuor et al. /Journal of Biotechnology 41 (1995) 1-I 7 11

observed after selective chemical C,-oxidation, whereas C,-oxidation actually retarded degrada- tion of an alkylated P-0-4-model compound (Fenn and Kirk, 1984). In a more recent study with dimeric P-0-Cmodel compounds, homoge- neous MnP oxidized a phenolic C,-carbonyl model compound, leading to alkyl-phenyl cleav- age and C,-C, cleavage (Tuor et al., 1992). Based on these results, the earlier observation could be explained by oxidative C,-C, cleavage and alkyl-phenyl cleavage in the lignin polymer which was initiated by MnP. The findings strongly suggest that the MnP-system can directly attack phenolic lignin substructures with cr-carbonyl groups, leading to oxidative degradation of the lignin polymer via C,-C, cleavage or alkyl- phenyl cleavage. The free diffusion of the Mn/organic acid complex would favour such a depolymerization pathway. The observed degradative pathway for the C,-carbonyl com- pound by the MnP system suggests an important, hitherto unreported pathway for the degradation of polymeric lignin.

7. Hypothetical schemes for Iignin biodegradation

It is evident that mineralization of the complex lignin substrate by a microorganism requires two key processes: breakdown of the polymer and ring cleavage of the aromatic nuclei. Model com- pound studies in vivo and with ligninolytic en- zymes yielded a wealth of knowledge on oxidative cleavage reactions promoting these degradative reactions (reviewed in: Higuchi, 1986; Kirk and Farrell, 1987; Gold et al., 1989; Schoemaker, 1990; Umezawa, 1988). Analysis of degraded lignin preparations and lignin model compounds sug- gested that lignin biodegradation is an oxidative process. This view was confirmed with the discov- ery of Iignin peroxidase and manganese peroxi- dase and the elucidation of the mechanistic ac- tion of these enzymes. However, we must be extremely careful in extrapolating the already complex results obtained from model compound degradation studies to lignin biodegradation. Ini- tial expectations that complete in vitro lignin mineralization could be achieved by LiP and/or

MnP alone have not been fulfilled. There is grow- ing experimental evidence that reductive pro- cesses play a pivotal roIe in lignin biodegradation and are also part of the ligninolytic system. A range of monomeric and dimeric aromatic alde- hydes and acids are reduced to the corresponding alcohols in ligninolytic cultures of P. chrysospo- rium, and the responsible enzymes have been purified and characterized (Ander et al., 1980; Enoki et al., 1981; Leisola and Fiechter, 1985; Muheim et al., 1990). In addition, the enzymes cellobiose:quinone oxidoreductase (Westermark and Eriksson, 1975) and NADH:quinone oxidore- ductase (Constam et al., 1991; Muheim, 1991), which are involved in quinone reduction, have been isolated and purified from the same fungus.

Hypothetical schemes of lignin biodegradation taking these conversions into account have been published by Eriksson et al. (19901, Schoemaker (19901, and Schoemaker et al. (1991). The degra- dation scheme by the latter authors emphasizes that lignin degradation by P. chrysosporium must involve both oxidative and reductive conversions. Oxidative fragmentation and cleavage reactions are required for chemical breakdown of the poly- mer, whereas extracellular and intracellular stages of degradation are distinguished: given the size and the insoluble nature of lignin, its breakdown must take place outside the cell. This is followed by cellular uptake of the soluble, monomeric and small oligomeric fragments for further processing. In general, H,O,-requiring oxidations occur ex- tracellularly and intracellularly, whereas the NAD(P)H-dependent reductions take place in the cell. Lignin depolymerization therefore can be considered as an oxidative process, whereas the metabolism of lignin fragments involves a combi- nation of oxidations and reductions. The com- partmentalization of oxidative and reductive con- versions calls for a powerful transport mechanism through the cell wall. Evidence for this was pre- sented with the rapid removal of quinone reduc- tase substrates in liquid culture of P. chrysospo- rium, and the observation of subsequent secretion of the reduction products by the cells shortly after (Tuor et al., 1993).

The cleavage of phenylpropane side-chains in the course of the degradation yields C,, C, and

C, fragments, which can be considered as sub- strates for the enzyme glyoxal oxidase and thus supply extracellular hydrogen peroxide. Aromatic aldehydes formed by C,,-C, cleavage in the de- polymerization process can be reduced to the corresponding benzyl alcohols by the enzyme aryl alcohol dehydrogenase. This makes these com- pounds susceptible to attack by the peroxidases of the ligninolytic system, generating quinones and ring-opened products.

The importance of C,-oxidation in the primary attack of the lignin polymer and subsequent poly- mer degradation was already outlined. Its role in the depolymerization of the polymer is shown in Fig. 3 (Tuor, 1992). The initial steps of lignin degradation by P. chrysosporiunz seem to involve a combination of oxidative conversions catalyzed by MnP and LIP, possibly in conjunction with the action of Mn ions, veratryl alcohol. or other compounds, as redox mediators. This primary at- tack would lead to C,,-oxidation in the lignin

polymer or to C,-C, cleavage and alkyl-phenyl cleavage under release of small lignin fragments (Fig. 3). The C,-carbonyl groups aIready present or formed in the process above are linked to both phenolic and non-phenolic aromatic rings. For further degradation, the latter have to undergo demethylation to yield phenols or can be sepa- rated from the polymer by oxidation of an adja- cent B-ring by LIP, followed by Cp-ether bond cleavage (Kirk et al., 1986).

Phenolic lignin substructures with a C,- carbonyl function, both native or stemming from oxidation of phenolic substructures by MnP or oxidation of non-phenolic structures by LIP and subsequent demethylation, could in contrast be attacked by MnP. This process would lead to the liberation of lignin fragments from the polymer via C,-C, cleavage and alkyl-phenyl cleavage. Importantly, these results suggest that no further enzymes capable of cleaving C,-0x0 substructures need be contemplated. Since MnP production by

Ihi/ 1%0-4-cleavage

f

phenolic lr-c=O q

C,,-Clc-cleavage

=;)alkyl-phenyl- cleavage

11-I/(+0-4-cleavage

Fig. 3. Hypothetical scheme leading to oxidative depolymerization of the lignin polymer under the action of LiP and MnP. possibly involving mediation by veratryl alcohol or Mn. respectively. = . potentially depolymerizative reaction pathways: + . lignin-alter- ing reactions without structural breakdown.

U. Tuor et al. /Journal of Biotechnology 41 (1995) l-l 7 13

P. chrysosporium reaches its maximum about 2 d before LiP production (Leisola, 1988), it is con- ceivable that the oxidation of C,-carbonyl residues by MnP is key to the primary attack of the lignin polymer.

Direct degradation of the non-phenolic lignin substructures can occur only under the action of LiP via C,-C, cleavage or alkyl-phenyl cleavage according to Fig. 3. Recent results with cultures of C. subvermispora (Table 21, however, suggest that an efficient in vivo depolymerization path- way of nonphenolic lignin exists that does not depend on the secretion of Lip. The presence of the natural substrate wood triggered the forma- tion of C,-C, cleavage products, which were not detected in liquid medium, and subsequent min- eralization by an hitherto unknown mechanism (Srebotnik et al., 1994).

8. Concluding remarks

The ubiquitous presence of the substrate wood has always been a trigger for the investigation of lignin biodegradation. During the last decade the focus of applied research moved from the utiliza- tion of a single purified enzyme preparation to the selective use of different enymes or whole organisms. The main fields of application are biobleaching and biopulping in paper manufac- ture and biodegradation of xenobiotics, especially chlorinated phenols and dioxins. Remarkably, fungal hemicelluloses could be faster developed into application on an industrial scale than the lignin degrading enzymes. Despite the progress in our understanding of catalytic function and mechanism of the latter enzymes, the knowledge of their attack on the polymeric lignin in the natural substrate is too poor for being applied today.

More recent research work indicates that white-rot fungi produce a great diversity of en- zyme combinations to accomplish a very compli- cated task generally termed lignin degradation. Environmental conditions like temperature, mois- ture, as well as pH and nitrogen and oxygen levels at the location of enzyme action, are gov- erning factors. The growing knowledge will make

it possible to exploit metabolic pathways specifi- cally and allow access to the tremendous biotech- nological potential of white-rot fungi.

Acknowledgements

The authors thank Dr. 0. Petrini (Institute of Microbiology, Swiss Federal Institute of Technol- ogy, Zurich) for his competent advice in taxon- omy.

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