Báo cáo khoa học: Swollenin, a Trichoderma reesei protein with sequence similarity to the plant expansins, exhibits disruption activity on cellulosic materials pptx

Swollenin, a
Trichoderma reesei
protein with sequence similarity to
the plant expansins, exhibits disruption activity on cellulosic materials
Markku Saloheimo
1
, Marja Paloheimo
1
, Satu Hakola
1
, Jaakko Pere
1
, Barbara Swanson
2
, Eini Nyysso¨ nen
2
,
Amit Bhatia
2,
*, Michael Ward
2
and Merja Penttila¨
1
1
VTT Biotechnology, Finland;
2
Genencor International, Inc., Palo Alto, CA, USA
Plant cell wall proteins called expansins are thought to
disrupt hydrogen bonding between cell wall polysaccha-
rides without hydrolyzing them. We describe here a novel
gene with sequence similarity to plant expansins, isolated

from the cellulolytic fungus Trichoderma reesei. The pro-
tein named swollenin has an N-terminal fungal type cel-
lulose binding domain connected by a linker region to the
expansin-like domain. The protein also contains regions
similar to mammalian fibronectin type III repeats, found
for the first time in a fungal protein. The swollenin gene is
regulated in a largely similar manner as the T. reesei cel-
lulase genes. The biological role of SWOI was studied by
disrupting the swo1 gene from T. reesei. The disruption
had no apparent effect on the growth rate on glucose or
on different cellulosic carbon sources. Non-stringent
Southern hybridization of Trichoderma genomic DNA
with swo1 showed the presence of other swollenin-like
genes, which could substitute for the loss of SWOI in the
disruptant. The swollenin gene was expressed in yeast and
Aspergillus niger var. awamori. Activity assays on cotton
fibers and filter paper were performed with concentrated
SWOI-containing yeast supernatant that disrupted the
structure of the cotton fibers without detectable formation
of reducing sugars. It also weakened filter paper as
assayed by an extensometer. The SWOI protein was
purified from A. niger var. awamori culture supernatant
andusedinanactivityassaywithValonia cellwalls.It
disrupted the structure of the cell walls without producing
detectable amounts of reducing sugars.
Keywords: cellulase; expansin; cellulose binding domain;
Trichoderma; regulation.
In the last few years, a new class of proteins called expansins
has been discovered in plants (reviewed in [1–3]). A number
of expansin genes have been identified from a wide variety of

plant species, including cucumber, Arabidopsis, rice [4] and
tomato [5]. The expansins were first implicated in loosening
the cell wall structure during plant cell growth (the acid-
growth response), and the proteins forming a distinct family
with high sequence identity and having this type of activity
are now classified as a-expansins [6]. The group 1 pollen
allergens have approximately 25% amino-acid identity with
a-expansins and have been shown to be active in an acid-
growth assay on grass cell walls. Along with their vegetative
homologues they are designated as b-expansins [6].
Expansins have been proposed to disrupt hydrogen
bonding between cellulose microfibrils or between cellulose
and other cell wall polysaccharides without having hydro-
lytic activity [7,8]. In this way they are thought to allow the
sliding of cellulose fibers and enlargement of the cell wall.
Purified cucumber expansins have been shown to catalyze
extension of isolated plant cell walls such as cucumber
hypocotyl walls when assayed using a constant load
extensometer [9]. These cucumber expansins have also been
shown to weaken filter paper without producing reducing
sugar [7]. Some of the a-expansins are functional during fruit
ripening, possibly aiding the action of hydrolytic enzymes
that degrade the cell wall polymers [5]. Experiments have
been made where expansin activity has been over expressed,
or reduced by antisense strategy in Arabidopsis thaliana.The
results suggest a role for these proteins in the control of
plant growth and morphogenesis [10].
A number of saprophytic and pathogenic fungi and
bacteria produce a wide range of enzymes designed to break
down plant biomass. These enzymes include cellulases that

break down cellulose to glucose, and hemicellulases that
degrade the different hemicelluloses to monomeric sugars.
For the degradation of the insoluble and complex plant cell
wall the microbes produce multiple enzyme forms belonging
to various enzyme categories. For example, from the fungus
Trichoderma reesei, one of the best known saprophytic
microbes, genes have been cloned that encode two exo-
acting cellulases liberating mainly cellobiose from cellulose
chain ends, five endo-acting cellulases hydrolyzing internal
linkages of cellulose chains and 10 hemicellulases represent-
ing different enzyme activities [11,12]. Most of the cellulases
and some of the hemicellulases of this fungus have a
Correspondence to M. Saloheimo, VTT Biotechnology,
PO Box 1500, 02044 VTT, Finland.
Fax: + 358 9 455 2103, Tel.: + 358 9 456 5820,
E-mail: Markku.Saloheimo@vtt.fi
Abbreviations: CBD, cellulose binding domain; CBHI, CBHII,
T. reesei cellobiohydrolases; CREI, regulatory protein involved
in catabolite repression in T. reesei;EGI,EGII,EGIV,EGV,
T. reesei endoglucanases; FnIII repeats, fibronectin III type repeats;
SWOI, T. reesei swollenin I; swo1,geneencodingSWOI;HEC,
hydroxyethylcellulose.
Enzymes: endo-1,4-glucanase (EC 3.2.1.4), cellobiohydrolase
(EC 3.2.1.91).
*Present address: EPIcyte Pharmaceutical, Inc., 5810 Nancy Ridge
Drive, Suite 150, San, Diego, CA 92121, USA.
Note: the complete swo1 sequence data has been submitted to the
EMBL database under the accession no. AJ245918.
(Received 17 May 2002, revised 3 July 2002, accepted 5 July 2002)
Eur. J. Biochem. 269, 4202–4211 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03095.x

modular structure consisting of a cellulose binding domain
(CBD) at either end of the polypeptide chain, connected to
the catalytic domain with a linker region. The role of the
CBD is to mediate binding of the enzyme to the insoluble
substrate.
In addition to plants, a protein with an endoglucanase
domain and a domain with sequence similarity to expansins
has been reported in the plant pathogen Clavibacter
michiganensis ssp. sepedonicus [13]. In this paper, we report
the discovery of a novel fungal protein having significant
sequence identity to plant expansins. Unlike plant expan-
sins, this protein has a modular structure with an
N-terminal CBD. The protein was named swollenin due
to its ability to swell cotton fibers without producing
detectable amounts of reducing sugars.
EXPERIMENTAL PROCEDURES
Strains, vectors and growth conditions
The T. reesei cDNA library in the vector pAJ401 [14] was
screened in the yeast strain H1152 (a, sso2-1, leu2-3, trp1-1,
ura3-1, sso1::HIS3, M. Aalto, unpublished results) on SC-
Ura plates with 2% galactose as the carbon source [15] at
the restrictive temperature 31 °C. The yeast strain DBY746
(a, his3D, leu2-3, ura3-52, trp1-289, Cyh
r
)wasusedfor
swollenin production. The T. reesei strain QM9414 [16] was
used in Northern studies and for detection of the swollenin
protein. For the Northern studies the strain was cultivated
in shake flasks (28 °C, 200 r.p.m.) in minimal media [17]
containing 5% glucose, 2% sorbitol, 2% cellobiose, 2%

lactose or 2% Solka floc cellulose for 3 days. Alternatively,
thestrainwasgrownon2%glycerolfor72h,followedby
addition of sophorose (1 m
M
final concentration). After
further 15 h the culture was harvested. A culture grown in a
minimal medium with 2% glycerol for 87 h was used as a
control.
Nucleic acid methods
Yeast was transformed with the LiAc method [18] or by
electroporation (Bio-Rad). Plasmid constructs were made
using standard methodology [19]. Total T. reesei RNA was
isolated as described [20]. The RNA samples (5 lg) were
treated with glyoxal and analyzed in a 1% agarose gel.
Northern blotting and hybridization were performed on a
Hybond-N nylon membrane (Amersham). T. reesei DNA
was isolated as described [21]. Stringent Southern hybrid-
ization was performed as described [19]. Nonstringent
Southern hybridization was performed in a hybridization
mixture without formamide [19] at 48 °Candthefilterwas
washed in 2 · NaCl/Cit, 0.1% SDS for 10 min at room
temperature and for 30 min at 48 °C.
Antibodies and Westerns
Swollenin antibodies were generated in rabbits by immu-
nizing with the peptide CDPNYTSSRPQERYGS (amino
acids 422–437 in the swollenin sequence). SDS gel electro-
phoresis was performed as described [22] and Western
blotting was performed on a nitrocellulose membrane
(Schleicher & Schull) and detection of the swollenin with
a secondary antibody-alkaline phosphatase conjugate

(Bio-Rad). Samples of yeast or T. reesei supernatants and
purified SWOI were denatured for Endoglycosidase H
treatment by heating 10 min at 100 °Cin0.5%SDS,1%
2-mercaptoethanol and subsequently treated with Endo H
f
(New England Biolabs) for 3 h at 37 °C in the G5 buffer
(50 m
M
Na-citrate, pH 5.5). 1000 units of EndoH
f
was used
per 20 lg of purified SWOI and per 20 lLoftheT. reesei
or concentrated yeast supernatants.
Swo1
gene disruption
The swo1 gene was disrupted from the T. reesei strain
QM9414 [16] by replacing it with a hygromycin resistance
cassette. The genomic swo1 gene was first subcloned from a
cosmid library clone into pBluescript SK
–
as a 5.5 kb
EcoRV fragment to obtain the plasmid pSH1. Most of the
swollenin-coding region was replaced from pSH1 by
digesting it with NarIandBstEII and ligating with the
hygromycin resistance cassette consisting of the Aspergillus
nidulans gpdA promoter and trpC terminator and the E. coli
hygromycin resistance gene derived from the plasmid
pBluekan (from P.J. Punt, TNO Nutrition and Food
Research, Zeist, the Netherlands). The resulting plasmid,
pSH9 was digested with EcoRV and transformed into

QM9414 as described [17], and transformants were selected
on 100 lgÆmL
)1
hygromycin and purified to uninuclear
clones by plating single spores on selective medium.
Disruptants of the swo1 gene were screened among the
transformants by Southern hybridization performed as
described [19]. Two disruptants obtained were also exam-
ined by growing them in shake flasks (28 °C, 200 r.p.m.,
5 days) in a medium with 3% whey and 1.5% complex
grain-based nitrogen source [23] and performing Western
analysis from their culture supernatants as described above.
The phenotype of the disruption was studied by plating
single spores on plates with minimal medium [17] supple-
mented with 0, 0.1 or 0.2% proteose peptone and either 2%
glucose, 2% Solka floc cellulose, 2% Avicel cellulose or 2%
complex grain-based carbon/nitrogen source [23]. An addi-
tional test was made on plates where the minimal medium
with or without peptone and without any carbon source had
been overlaid by a Whatman 1 filter paper disc. Growth of
colonies of the swo1 disruptants and the parental strain was
followed daily.
Production and characterization of the swollenin
preparations
The yeast strain DBY746 harbouring a plasmid with swo1
in the vector pAJ401 [14] or the vector alone were grown in
Chemap CMF mini 1 L or Biolafitte 14 L bioreactors. The
bioreactor medium was SC-Ura with 2% glucose as the
carbon source [15]. The yeast supernatants were concen-
trated 20 times with a Centiprep concentrator (Amicon) for

the treatments of cotton fibers and filter paper. The amount
of SWOI was estimated by comparing signals obtained in
Westerns from Endo-H-treated yeast supernatants with
signals obtained with known amounts of purified Endo-H
treated SWOI from A. niger var. awamori.
The 1.5 kb coding region of the swollenin cDNA clone
was amplified by PCR using the following primers which
were designed to add BglII and XbaI restriction endonuc-
lease sites to the 5¢ and 3¢ ends, respectively. Primer
Ó FEBS 2002 A fungal expansin homologue (Eur. J. Biochem. 269) 4203
ExAspBgl2: 5¢-CATTAGATCTCAGCAATGGCTGGT
AAGCTTATCCTC-3¢. Primer ExAspXba1:
5¢-CGACTCTAGAAGGATTAGTTCTGGCTAAAC
TGCACACC-3¢. The DNA sequence of the amplified
product was verified and the swollenin coding region was
inserted into the BglIandXbaI sites between the glaA
promoter and terminator of an Aspergillus expression vector
(pGAPT-PG) to produce pGAPT-exp. The pGAPT-PG
vector consists of pUC18 containing the A. nidulans pyrG
gene as selectable marker and a 1.1 kb fragment of the
A. niger var. awamori glaA promoter and a 0.2 kb fragment
of an A. niger glaA terminator.
The expression plasmid pGAPT-exp was transformed
into A. niger var. awamori strain dgr246 P2 as described
[24]. Transformants were selected for their ability to grow
on minimal medium lacking uridine. For swollenin protein
production the transformants were grown in liquid medium
as described [25]. Cells were removed and the culture
supernatants were equilibrated with 1
M

ammonium sul-
phate, 100 m
M
Tris pH 7. The supernantant was then
applied to a cellulose (Sigma, St. Louis, MO, USA) affinity
column and washed with 1
M
ammonium sulphate, 100 m
M
Tris pH 7 to remove unbound proteins. The purified
swolleninwaselutedasasinglepeakinwater.
The purified swollenin was tested for activity against
hydroxyethylcellulose (HEC), b-glucan, xylan and mannan.
The enzymatic activities against HEC (Fluka) and barley
b-glucan (Biocon) were determined according to IUPAC
[26] and against birch xylan (Roth) as presented [27].
Mannanase activity was assayed according to the procedure
of IUPAC (1987) but using 0.5% locust bean gum (Sigma)
as a substrate.
Action on solid substrates
Yeast supernatants. Cotton fibers were mercerized by
treating them with 25% NaOH for 15 min at 5 °Cand
washing several times with distilled water. The cotton fibers
were suspended in the concentration of 0.5 gÆmL
)1
in
50 m
M
sodium acetate, pH 5.0 containing 1/4 of the
concentrated yeast culture media from the swollenin

producing yeast and control strain. Additionally, the
purified T. reesei EG II, CBH I and cellulose binding
domain (CBD) of CBH I at a concentration of 5 lgÆmL
)1
were used as controls for the swollenin [28,29]. After
incubation for 4 h at 25 °C, the suspended fibers were
filtered off and the amount of reducing sugars released into
the filtrates was determined as described in [30]. The fibers
were rinsed once with buffer and then suspended in distilled
water with glass beads prior to sonication for one minute
using a probe tip sonicator (Vibra Cell Sonics and Materials
Inc.) The fibers were then stained and visualized by light
microscopy to determine gross effects on their structure.
For the paper strength test, Whatman no. 3 filter paper
was cut into strips measuring 7 · 2 cm. Sodium acetate
buffer (50 m
M
, pH 5) was used for all of these experiments.
The concentrated yeast samples were sometimes first
desalted by passage through a Bio-Rad Econo-Pac10 DG
column with a molecular mass cut-off of 6000 Da. After
desalting, 5 mL of the yeast samples were added to 4 mL of
buffer in 50 mL disposable conical tubes and the Whatman
strips were added. At the same time, strips were added to
buffer alone and 8
M
urea in buffer. After incubating at
room temperature for 15 min the strips were measured for
their wet tensile strength. The assay was performed by
placing each wet strip of paper between the Thwing–Albert

tensile tester (Model 5564 from Instron Corporation,
Canton, MA, USA) clamps spaced 4.5 cm apart. A 250 lb
load cell was used. Test speed was 0.1 cmÆmin
)1
,andthe
peak load was measured before breaking; it typically only
took a minute to reach the paper breaking load.
The purified swollenin. The action of the purified swollenin
preparation on plant cell wall material was followed using
Valonia cell wall fragments as substrate. Vesicles of Valonia
macrophysa were purified as described previously [31] and
cut to small pieces, 4–5 mg each (dry weight). These cell wall
fragments were suspended in 50 m
M
acetate buffer (pH 5.0)
and swollenin was added at the dosages of 10 lgÆmg
)1
and
100 lgÆmg
)1
. Treatments with the purified T. reesei CBH I
(also known as Cel7A) and EG II (also known as Cel5A)
were performed as comparison for swollenin. The samples
were incubated at +45 °C under stirring for 48 h and
thereafter examined under a stereo microscope (Leica, Wild
M10). The control sample was treated alike but omitting the
enzymes and swollenin. In addition the filtrates were
analysed for solubilized sugars by HPLC.
RESULTS
Swollenin has sequence similarity with plant expansins. The

swollenin cDNA was isolated in a screening where compo-
nents of the Trichoderma secretory pathway were searched
for by yeast complementation. The sso2 temperature-
sensitive S. cerevisiae strain was transformed with a T. reesei
cDNA expression library, and cDNAs derived from clones
able to grow at the restrictive temperature were sequenced.
One of them encoded a protein predicted to have an
N-terminal signal sequence followed by a cellulose binding
domain. A major part of the remaining sequence was found
to have sequence similarity with plant expansins in a
BLAST database search (Fig. 1C). Based on its swelling
activity on cotton fibers (see below) the protein was named
swollenin (SWOI) and the gene swo1.
The genomic copy of the swo1 cDNA was isolated from a
cosmid library, subcloned and sequenced. The gene contains
five short introns (Fig. 1A). The promoter contains a
putative TATA box 90 bp and a putative binding sequence
of the glucose repressor protein CREI [32] 117 bp upstream
from the translation start codon (data not shown).
The putative swollenin protein starts by a typical signal
sequence. It is followed by two glutamic acids. In the
T. reesei cellulases EGI (also known as Cel7B) [33] and
EGII[34]therearetwoglutamicacidsattheN-terminus
and in CBHI there is one [35], and the N-termini of these
enzymes are blocked by a pyroglutamic acid residue. By
analogy, it is suggested that the swollenin signal sequence
would be 18 amino acids in length and be cleaved before the
two glutamines in the sequence (Fig. 1b). The SWOI has
three potential N-glycosylation sites at positions 160, 336
and 406.

The swollenin cellulose binding domain (CBD) has the
typical sequence features of fungal CBDs. The amino acids
invariant in the CBDs of the T. reesei cellulases are
conserved in swollenin (Fig. 1B). In the NMR structure
solved from the CBHI CBD there are two disulphide
4204 M. Saloheimo et al. (Eur. J. Biochem. 269) Ó FEBS 2002
bridges [36]. Based on a close proximity of an additional
cysteine pair in the modelled structures of the CBHII and
EGI CBDs it has been suggested that they would have a
third disulphide bond [37]. The swollenin CBD has six
cysteines at positions conserved with those of the CBHII
(also known as Cel6A) CBD and thus it probably has three
disulphide bridges as well. The residues forming the flat
surface binding to cellulose are conserved in the swollenin
CBD with one exception (Fig. 1B). Position 8 in the
swollenin sequence has a phenylalanine while the other
cellulases have tyrosine or tryptophan. The linker region of
the swollenin is rich in serines and threonines and is
expected to be heavily O-glycosylated [38]. Without protein
structure data the length of the linker cannot be determined
unambiguously. However, the region rich in serines and
threonines in SWOI apears to be among the longest found
in T. reesei enzymes, approximately 50 amino acids. The
region of SWOI between the putative linker and the
expansin-like area shown in Fig. 1c does not match with
any sequences in databases.
The C-terminal two-thirds of the swollenin show clear
amino-acid similarity with plant expansins (Fig. 1C). The
identity between swollenin and individual a-orb-expansins
in pairwise comparisons is about 25% over an area of about

200 amino acids. The alignment of the swollenin with two
expansin sequences (Fig. 1C) suggests that two large
insertions have occurred in the swollenin gene in the
N-terminal half of its expansin-like domain. The identity
between the a-andb-expansins is 20–25% and there are five
sequence elements that are well conserved between the
expansin categories [6]. Four of the elements form the best
conserved parts between swollenin and the expansins and
Fig. 1. The basic structure of the swollenin protein (A), alignment of the swollenin CBD with the CBD sequences of T. reesei cellulases (B), Alignment
of the swollenin with two a-expansins (C) and The alignment of swollenin (SWOI) with FnIII repeat sequences (D). In (A), vertical arrows indicate the
intron positions. SS, signal sequence; CBD, cellulose binding domain, QQ, two glutamines suggested to form the N-terminus of the mature
swollenin. The numbering refers to amino-acid positions in the mature protein. In (B), the invariant amino acids are boxed and amino acids forming
the flat surface interacting with cellulose are shaded. Lines below the sequences show the proposed disulphide bridges of the SWOI CBD. (C)
Alignment of the swollenin with two a-expansins, LeEx1 of tomato (5) and CuExS2 of cucumber (4). Invariant amino acids are shown by asterisks
and conservative substitutions by dots. The regions best conserved between a-andb-expansins are underlined in CuExS2. The conserved cysteines
are shown by arrows and positions with conserved aromatic amino acids in all three sequences by + 0. The regions with homology to the FnIII
repeats in titin are in bold in SWOI. (D) The alignment of swollenin (SWOI) with FnIII repeat sequences of human titin and a consensus sequence
of the bacterial FnIII repeats (BACT). The amino acids strictly conserved between the bacterial and mammalian FnIII repeats are shown by
asterisks.
Ó FEBS 2002 A fungal expansin homologue (Eur. J. Biochem. 269) 4205
thus they are probably functionally important. The
N-terminal half of the expansins contains eight conserved
cysteines with a spacing similar to that of cysteines in the
chitin binding domain of wheat germ agglutinin [4]. Seven
of these cysteines are conserved in swollenin. Aromatic
amino acids are often important in the interaction of
enzymes and their carbohydrate substrates. In the alignment
between swollenin and expansins there are eight positions
where an aromatic amino acid is conserved (Fig. 1C).
Alignments of swollenin with individual expansins sug-

gest that it is better conserved with b-expansins than
a-expansins.
There are two short sequences in swollenin that show
relatively strong conservation with fibronectin type III
(FnIII) repeats of mammalian titin proteins (Fig. 1D).
Interestingly, such repeats have been found in prokaryotic
hydrolases such as cellulases, chitinases and amylases [39,40]
but thus far not in fungal enzymes. The amino acids
invariant between the bacterial hydrolases and mammalian
FnIII repeats are conserved in swollenin. Unlike the
continuous FnIII repeats in the bacterial enzymes, the
region with similarity to titin in swollenin is divided into two
parts about 170 amino acids apart.
Regulation of the swollenin gene
The cellulase and hemicellulase genes of Trichoderma reesei
are regulated by the carbon source [12,41] and thus it was of
interest to analyze if the swollenin gene is regulated in a
similar manner. The T. reesei strain QM 9414 was grown in
shake flasks on different carbon sources and Northern
hybridization was performed. The role of sophorose, a
strong cellulase inducer, in the swollenin gene regulation
was studied by adding it to a Trichoderma culture grown on
the neutral carbon source glycerol. The swo1 mRNA level
was undetectable in the glucose culture sample in a short
exposure (Fig. 2, lane 1), but in a long exposure a very low
level was observed (lane 9). In a late stage of a glucose
cultivation the swo1 gene was derepressed (lane 2). In
sorbitol (lane 3) and glycerol samples (lane 4) a low mRNA
level was present, and when sophorose was added to the
glycerol culture, strong induction of swo1 occurred (lane 5).

In media with lactose and cellobiose the swo1 mRNA level
was moderate and in a medium with cellulose it was at its
highest.
Production of SWOI by
T. reesei
Polyclonal antibodies against SWOI were obtained by
immunizing rabbits with a synthetic peptide designed based
on the swollenin sequence. The expected molecular mass of
the deduced SWOI protein is 49 kDa, but the antibodies
recognize in a Western a protein of approximately 75 kDa
in a T. reesei supernatant from a cellulose-based culture
(Fig. 3, lanes 1 and 3). The difference between the calculated
and observed molecular masses can not be explained by
N-glycosylation, because endoglycosidase H that removes
N-glycans changes the apparent molecular mass of SWOI
only slightly (Fig. 3, lane 4). Also the SWOI produced in
yeast and A. niger var. awamori gained a molecular mass
close to 75 kDa when they were treated with endoglycosi-
dase H (see below). This band was also absent from the
supernatants of the swo1 disruptants (Fig. 3). These data
show that the 75 kDa band is indeed derived from the
SWOI protein. The75 kDa band could not be observed in a
T. reesei culture filtrate from a culture grown on glucose
(Fig. 3, lane 2). Thus the very low basal expression detected
at the mRNA level (Fig. 2) was undetectable in the Western
analysis. As estimated from Western blotting, the produc-
tion level of SWOI in the T. reesei culture analysed was
about 1 mgÆL
)1
. This is by far less than the production levels

of the major cellulases.
Disruption of the swo1 gene
The swo1 gene was disrupted from the genome of T. reesei
by replacing it with a hygromycin resistance cassette. Two
disruptants were shown by Southern analysis to be single-
copy transformants where the gene replacement had
Fig. 2. Regulation of the swo1 gene according to the carbon source.
Northern analysis of RNA samples isolated from mycelia grown in a
medium with (1) glucose (2) glucose, sample taken after glucose
depletion (3) sorbitol (4) glycerol (5) glycerol, induced by sophorose (6)
cellobiose (7) lactose (8) Solka floc cellulose (9) glucose, 7 times longer
film exposure. The gpd1 probingservesasaloadingcontrol.
Fig. 3. Western detection of SWOI by polyclonal antibodies from cul-
ture supernatants of T. reesei and yeast. Lane 1, T. reesei culture per-
formed in a medium inducing cellulase production (1 lLofculture
medium loaded); lane 2, T. reesei culture performed in a medium with
glucose as the carbon source (15 lL; lane 3, T. reesei culture super-
natant (10 lg of total protein); lane 4, T. reesei culture supernatant
treated with endoglycosidase H (3 lg of total protein); lane 5, yeast
strain expressing swo1 cDNA (the sample corresponds to 0.5 mL of
supernatant); lane 6, yeast strain expressing swo1 cDNA, treated with
endoglycosidase H (0.2 mL of original supernatant); lane 7, yeast
strain carrying the expression vector alone (0.5 mL of original super-
natant); lanes 8, 9, swo1 disruptant T. reesei strains (15 lLofculture
supernatant); lane 10, the parental strain of the swo1 disruptants
(15 lL of culture supernatant); lane 11, Western detection of SWOI
purified from A. niger var. awamori (100 ng); lane 12, Western detec-
tion of purified SWOI, endoglycosidase H treated (30 ng); lane 13,
Coomassie-stained SWOI purified from A. niger var. awamori (3 lg).
4206 M. Saloheimo et al. (Eur. J. Biochem. 269) Ó FEBS 2002

occurred (data not shown). Western analysis of their culture
supernatants further confirmed that they do not produce the
SWOI protein (Fig. 3, lanes 8 and 9).
We attempted to demonstrate the phenotype of the swo1
disruption, its effect either on the formation of the T. reesei
cell wall and growth of the fungal mycelium or on the
degradation of cellulosic carbon sources by the fungus. This
was performed by comparing the growth rates of the
disruptants and the parental strain on plates having glucose
or different cellulosic compounds as carbon sources. The
compounds tested were two commercial celluloses, filter
paper and a complex grain-based carbon/nitrogen source
[23]. No significant differences in the growth rates could be
observed between the strains on any of the carbon sources
and thus swo1 disruption had no apparent phenotype in our
experiments.
Non-stringent hybridization of T. reesei genomic DNA
was performed with a swo1 gene fragment encoding the
expansin-like domain as a probe. Hybridization at 48 °C
revealed several other bands in addition to the ones
originating from swo1, suggesting that there are other genes
having expansin-like domains present in the T. reesei
genome in addition to swo1 (Fig. 4). The presence of these
genes could compensate the lack of swo1 in the disruptants
and thus explain the result of the disruption experiment.
Characterization of the swollenin preparations
When the swollenin cDNA was expressed in S. cerevisiae
under the PGK1 promoter in a multicopy plasmid, Western
analysis of bioreactor culture supernatants showed that a
heterogeneous high molecular mass protein reacting with

the swollenin antibodies was produced by the yeast (Fig. 3,
lane 5). In many instances it has been shown that yeast tends
to overglycosylate heterologous proteins, e.g. the T. reesei
cellulases CBHI and CBHII [42]. When the swollenin
produced in yeast was treated by endoglycosidase H to
remove N-glycans, it gained an apparent molecular mass
close to the swollenin produced by Trichoderma (Fig. 3, lane
6). The production level of SWOI in yeast was approxi-
mately 25 lgÆL
)1
as estimated from Western blotting
experiments.
Swollenin was also produced in A. niger var. awamori and
after a single step purification procedure the purified
swollenin protein was obtained for biochemical character-
ization. The SWOI expressed in this host migrated as two
relatively diffuse bands with apparent molecular masses
between 80 and 95 kDa (Fig. 3, lanes 11 and 13), and Endo-
H treatment reduced the molecular mass to the same level as
SWOI produced by T. reesei (Fig. 3, lane 12). Thus A. niger
var. awamori slightly overglycosylated SWOI. Activities of
purified swollenin against hydroxyethyl cellulose (HEC),
b-glucan, xylan and mannan were measured and the results
are shown in Table 1. Minor hydrolytic activity on
b-glucan, xylan and mannan, but not on HEC, was observed
for the purified swollenin protein expressed in A. niger.
Demonstration of the swollenin activity on solid
substrates
The swollenin expressed in yeast. The activity of the
swollenin produced in yeast towards cellulosic materials was

shown by treatments of cotton fibers and filter paper.
Cotton fibers were incubated with concentrated yeast
supernatants from bioreactor cultivations of the SWOI-
producing yeast (approximately 0.125 lgÆmL
)1
of SWOI)
and the control strain with vector alone or, as controls, with
the T. reesei cellulases EGII, CBHI (5 lgÆml
)1
)andthe
cellulose binding domain of CBHI. After the treatment the
fibers were removed from the reaction mixture by filtering,
rinsed, sonicated with glass beads, stained and analyzed by
light microscopy. Soluble reducing sugars were measured
from the reaction mixture. Treatment with the control yeast
supernatant did not change the fiber structure (Fig. 5A).
The supernatant of the yeast strain producing swollenin
caused local disruption of the fiber structure that became
visible only after sonication. This was seen as swollen areas
occurring along the fibers (Fig. 5B). CBH I caused some
light fibrillation of the fibers (Fig. 5C), whereas the
treatment with EG II resulted in damaged and rugged
outlook of the fibers accompanied by fiber cutting (Fig. 5E).
Dinandcoworkers[43]havereportedondisruptionof
cellulosic fibers by a bacterial cellulose binding domain. No
modification of fiber surface could be detected in our
experiment with the fungal (CBHI) CBD by the light
microscopical method used (Fig. 5D), suggesting that the
effect of SWOI on the fibers is not caused by its CBD. The
treatment of the cotton fibers with the yeast supernatants or

Table 1. Characteristics of the swollenin preparation purified from
A. niger var. awamori.
Protein
(mgÆmL
)1
)
a
Activity (nkatÆmL
)1
)
HEC b-glucanase Xylanase Mannanase
SWO1 0.7 0 79 7 5
a
Lowry protein.
Fig. 4. Southern hybridization of T. reesei genomic DNA with the
region encoding the expansin-like domain from the swo1 gene, performed
at stringent (68 °C) and nonstringent (48 °C) conditions. The restriction
enzymes used are indicated.
Ó FEBS 2002 A fungal expansin homologue (Eur. J. Biochem. 269) 4207
with the CBD did not release detectable amounts of
reducing sugars. In contrast, the filtrates from the CBHI-
and EGII-treated fibers contained 0.08% and 1.61%
reducing sugars of the original dry mass, respectively.
To test the effect of swollenin on paper, filter paper strips
were incubated in concentrated culture supernatants of the
swollenin-producing and control yeast strains and measured
for their wet tensile strength. The data shows how much
load each strip of paper could hold before it broke; breakage
at a lower mass indicates less tensile strength (Table 2). The
average load is only slightly decreased when broth from

yeast which does not contain the swollenin gene is used.
However, the same amount of broth from the yeast
expressing the swollenin gene results in a 15–20% decrease
in the average load compared to the control broth.
Incubation in 8
M
urea decreases the average load the
paper can bear by about 40% compared to buffer alone.
The purified swollenin. Fragments of Valonia cell walls
were used as the solid substrate for studies on the mode of
action of the purified swollenin. This algal cell wall is made
of highly crystalline cellulose with a layered structure as
shown in Fig. 6A. Fragments of the cell wall were incubated
individually with the purified cellulases, CBH I and EG II,
and swollenin and alterations in the cell wall structure were
followed microscopically. The cellulases modified the cell
wall fragments with a concomitant release of soluble sugars
(Table 3). Treatment with EG II disrupted totally the cell
wall structure resulting in a milky solution whereas with
CBH I disintegration of cell wall to fibrils was observed
(Fig. 6). The action of swollenin resembled that of CBH I,
but integrity of the cell wall was partially retained and no
soluble sugars were released.
DISCUSSION
T. reesei produces one of the most powerful mixtures of
extracellular enzymes for efficient hydrolysis of the plant
polysaccharides cellulose and hemicellulose. This fungus has
served as a model, and extensive studies on the biochem-
istry, genetics, regulation, structure-function relationships
and applications of T. reesei enzymes have been carried out

[44]. The discovery of the expansin-like protein SWOI in
Table 2. The average peak load a strip of filter paper could bear before
breakage. The filter paper strips were treated with buffer, yeast culture
supernatants or urea as indicated. The results are the average of 3 or 4
readings.
Sample Average Peak Load (g) SD
Buffer 229 10.9
Control yeast 225 10.4
Swollenin-producing yeast 186 5.0
8
M
Urea 132 16.2
Fig. 5. Light microscopy of cotton fibers treated with culture super-
natant of control yeast with the vector alone (A), supernatant of
swollenin-producing yeast (approximately 0.125 lgÆL
)1
of SWOI) (B),
and isolated T. reesei cellulases CBHI (C) and EGII (E) and the cellulose
binding domain of CBHI (D) (5 lgÆmL
)1
). The swollen areas caused by
SWOI treatment are shown by arrows. Small fibrils can be seen on the
surface of fibers treated by CBHI. EGII caused modification of the
fibers that can be seen as rugged surface outlook.
4208 M. Saloheimo et al. (Eur. J. Biochem. 269) Ó FEBS 2002
T. reesei provides new insight to the mechanism of micro-
bial lignocellulose degradation, together with the report on
an endoglucanase with an expansin-like domain in a
pathogenic bacterium [13] and the discovery of sequence
similarity of expansins with family 45 glycosyl hydrolases

(see below).
Similarly to plant expansins, filter paper was shown to be
weakened by SWOI in an extensometer assay. We also
demonstrate that the structure of mercerized cotton fibers
was changed upon swollenin treatment in a manner clearly
visible by light microscopy. It can be assumed that the
swollen areas of the cotton fibers appearing after swollenin
treatment coincide with the tilt/twist areas of cotton fibers,
where the structure of cellulose is less ordered and more
accessible for modification than in crystalline regions [45].
Both cotton and filter paper consist of relatively pure
cellulose, and therefore swollenin would appear to be able to
open the crosslinking of cellulose fibers. No reducing sugar
formation was detected in the cotton swelling test, which is
in accordance with the plant expansin results published.
Disruption activity that was detected upon treatment of
Valonia cell wall frgaments with purified SWOI is in line
with the results obtained with yeast supernatants containing
SWOI. Although the Valonia cell wall are not representative
of the higher plant cell walls, the ability of SWOI to disrupt
the Valonia cell wall without producing reducing sugars is of
special interest. Activity of this type has been reported for
the expansins. The SWOI preparate purified from A. niger
var. awamori had a slight activity towards b-glucan,
mannan and xylan, but no activity towards hydroxyethyl
cellulose. The detected enzyme activities were very low, e.g.
the specific activity of the T. reesei endoglucanases EGI or
EGII against b-glucan are thousands of nkatÆmg
)1
,whereas

the SWOI preparate had an activity of 79 nkatÆmg
)1
.At
present we can not be sure whether the activities observed in
the SWOI preparation are due to trace amounts of
contaminating A. niger var. awamori enzyme(s) or to a
weak hydrolytic activity of the SWOI protein itself.
However, the disruption ability of solid substrate structures
observed in this work is most probably not due to hydrolytic
activity of SWOI, as no reducing sugar release from the
solid substrates was detected in these activity tests.
It has been reported that expansins have limited sequence
similarity with the family 45 of glycosyl hydrolases, which
includes the T. reesei endoglucanase EGV [3,14,46]. This
similarity is in the same range in identity percentages as the
similarity in an alignment between swollenin and individual
expansins, but it is limited to a smaller area (data not
shown). The sequence conservation between EGV and
SWOI is hardly detectable and thus it is weaker than
conservation between EGV and expansins. The sequence
motif HFD forming a part of the active site of the family 45
hydrolases [47] is conserved in the expansins, and according
to the alignment in Fig. 1C would appear to be replaced by
HLD in SWOI. The degree of conservation between
swollenin and the expansin-like domain of celA from
Clavibacter michiganensis is lower than conservation
between swollenin and plant expansins (data not shown).
In a recent report the b-expansin of Phleum pratense was
shown to have proteinase activity and to have limited
sequence similarity to papain-type proteinases [48]. The

authors proposed that expansins loosen the plant cell wall
structure by cleaving cell wall proteins that crosslink
cellulose fibers together rather than by disrupting hydrogen
bonding between fibers. The regions around the three active
site residues of papain were suggested to be conserved in
both a-andb-expansins. Some amino-acid similarity
between papain and swollenin can be detected at one of
these regions (around Cys256 of swollenin) but the others
are not conserved.
An interesting feature of the T. reesei swollenin is that it
has a modular structure typical of fungal cellulases and
some hemicellulases. SWOI has an N-terminal cellulose
binding domain (CBD) that is very well conserved with
Fig. 6. Atomic force microscopy image of the structure of the Valonia
cell wall (A), and light microscopy of Valonia cell wall fragments after
treatment with buffer alone (B), SWOI (C, 10 lgÆmg
)1
), CBHI (D,
100 lgÆmg
)1
) and EGII (E, 100 lgÆmg
)1
).
Table 3. Disintegration of Valonia cell walls by the purified swollenin
and T. reesei cellulases. The treatments were performed at a consis-
tency of 0.25%, at + 25 °C for 48 h.
Treatment
Dosage
(lgÆg
)1

) Effects on cell wall
Solubilized
sugars (% of dw)
Reference – Intact 0
Swollenin 10 Partial disintegration
to fibrils
0
CBH I 10 Total disintegration
to fibrils
0.09
EG II 10 Total disintegration
to milky solution
9.2
Ó FEBS 2002 A fungal expansin homologue (Eur. J. Biochem. 269) 4209
other fungal CBDs. Thus it can be expected that its function
is to bind the SWOI protein to cellulosic compounds. An
other interesting feature, although much less clear in its
functional importance, is the sequence similarity to the
fibronectin III (FnIII) type repeats of mammalian titin
proteins (Fig. 1D). The FnIII repeats of titin form bsand-
wich domains that have been suggested to be able to unfold
and refold easily [49] and this would make the protein able
to stretch. The ability to stretch might be important for
swollenin, if its function is to allow slippage of cellulose
microfibrils in plant cell walls as suggested for expansins.
Our results suggest that swollenin is a component of the
enzyme mixture produced by the fungus which is needed for
degradation of plant biomass and not, e.g. in modifying the
Trichoderma cell wall during the growth of the fungus. The
regulation pattern of the T. reesei swo1 gene is highly

reminiscent to that of the cellulase genes of this fungus [41].
The gene is induced for instance by plant materials and
certain oligosaccharides. The swo1 gene has a low expres-
sion level on glucose, sorbitol and glycerol unlike the more
tightly repressed major cellulases [41]. This could imply to
the interesting possibility that swollenin would be among
the enzymes that, before the onset of massive cellulose
degradation, aid in liberating a soluble inducer when the
fungus is encountering the insoluble cellulosic substrate.
This soluble inducer would further induce the main
cellulolytic machinery.
According to early theories on cellulose degradation, the
cellulase system of fungi like T. reesei would comprise two
kinds of activities. It was suggested that C1 (Ôswelling
factorÕ), a nonhydrolytic component would be needed to
make the substrate more accessible to Cx, the hydrolytic
component consisting of the endo- and exo-acting enzymes
and b-glucosidases that degrade the substrate to glucose
[50]. A large number of hydrolytic enzymes have been
characterized but so far the C1 factor has remained
unsolved. The Trichoderma C1 has not been well charac-
terized, but based on gel filtration it has been reported to
have a molecular mass of 61 kDa [51], not far from 75 kDa
that was estimated by SDS gels to be the molecular mass of
SWO1. Based on the properties of swollenin shown in this
work, it provides a possible candidate for a component of
C1. Our results also point towards the existence of other
proteins with sequence similarity to SWOI in T. reesei
(Fig. 4). Thus it is possible that there exist several swollenin-
like activities as is the case with the hydrolytic enzymes,

which vary somewhat in their modes of action but all
contribute synergistically to the efficient hydrolysis of the
plant polysaccharides.
ACKNOWLEDGEMENTS
We wish to thank Riitta Nurmi and Kati Uotila for excellent technical
assistance. The work was supported by the Finnish National
Technology Agency (Tekes).
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