Báo cáo khoa học: Cloning, expression and characterization of a family-74 xyloglucanase from Thermobifida fusca pptx

Cloning, expression and characterization of a family-74
xyloglucanase from
Thermobifida fusca
Diana C. Irwin, Mark Cheng*, Bosong Xiang†, Jocelyn K. C. Rose‡ and David B. Wilson
Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA
Thermobifida fusca xyloglucan-specific endo-b-1,4-gluca-
nase (Xeg)74 and the Xeg74 catalytic domain (CD) were
cloned, expressed in Escherichia coli, purified and charac-
terized. This enzyme has a glycohydrolase family-74 CD that
is a specific xyloglucanase followed by a family-2 carbo-
hydrate binding module at the C terminus. The Michaelis
constant (K
m
) and maximal rate (V
max
) values for hydrolysis
of tamarind seed xyloglucan (tamXG) are 2.4 l
M
and
966 lmol xyloglucan oligosaccharides (XGOs) min
)1
Ælmol
protein
)1
. More than 75% of the activity was retained after a
16-h incubation at temperatures up to 60 °C. The enzyme
was most active at pH 6.0–9.4. NMR analysis showed that
its catalytic mechanism is inverting. The oligosaccharide
products from hydrolysis of tamXG were determined by MS
analysis. Cel9B, an active carboxymethylcellulose (CMC)ase
from T. fusca, was also found to have activity on xyloglucan

(XG) at 49 lmolÆmin
)1
Ælmol protein
)1
, but it could not
hydrolyze XG units containing galactose. An XG/cellulose
composite was prepared by growing Gluconacetobacter
xylinus on glucose with tamXG in the medium. Although a
mixture of purified cellulases was unable to degrade this
material, the composite material was fully hydrolyzed when
Xeg74 was added. T. fusca was not able to grow on tamXG,
but Xeg74 was found in the culture supernatant at the same
level as was found in cultures grown on Solka Floc. The
function of this enzyme appears to be to break down the
XG surrounding cellulose fibrils found in biomass so that
T. fusca can utilize the cellulose as a carbon source.
Keywords: xyloglucanase; cellulase; inverting; regulation;
plant cell walls.
Converting plant biomass into ethanol for use as fuel has
been a long-term goal of scientists studying cellulases and
related glycosyl hydrolases. Much progress has been made
in identifying, cloning, expressing and characterizing cellu-
lases from both aerobic and anerobic bacteria and from
fungi. Hundreds of such enzymes have been identified and a
list of glycohydrolase families can be found at http://
afmb.cnrs-mrs.fr/CAZY/. Lynd et al. [1] have written a
comprehensive review of the current information on pos-
sible future strategies for biomass hydrolysis. A natural
biomass substrate, such as corn fiber [2], is structurally
complex and many other enzymes besides cellulases are

needed for efficient degradation of the polysaccharides to
monosaccharides.
The load-bearing structure of primary plant cell walls
comprises a network of cellulose fibrils complexed through
noncovalent associations with hemicelluloses such as xylo-
glucan (XG) and arabinoxylan (AXG) [3]. An additional
network consists of pectic polysaccharides as well as other,
less abundant, wall components, including structural pro-
teins, proteoglycans and hydrophobic compounds [4].
Cellulose microfibrils are composed of noncovalently, but
tightly associated, linear chains of b-1,4-linked
D
-gluco-
pyranosyl residues. XG is the predominant hemicellulose
in dicotyledon type I cell walls and has a b-1,4-linked
glucopyranosyl backbone of repeating cellotetraose units
with a-
D
-xylosyl residues attached to C6 of two or more of
the first three residues. In addition, some of the xylosyl
residues are substituted to form oligomeric side-chains
containing galactosyl, arabinose or fucosyl residues [3]. The
nomenclature for XG subunits is given in Fig. 1 [5]. XG
is proposed to form a monolayer coating the surface of
cellulose microfibrils and to penetrate the cellulose in
amorphous areas [6]. Experiments using differential extrac-
tion of etiolated pea stems with a family-12 xyloglucanase,
KOH and a cellulase, suggested that some of the XG is
entrapped within or between cellulose microfibrils [3].
Individual XG polymers are also thought to cross-link

adjacent microfibrils, forming a complex three-dimensional
lattice [4,6], underscoring the potentially important struc-
tural role of XG.
Thermobifida fusca YX is a thermophilic actinomycete
that was originally isolated by Dexter Bellamy [7]. It grows
well at 50 °C in minimal medium with carbohydrate
Correspondence to D. B. Wilson, 458 Biotechnology Building,
Cornell University, Ithaca, New York, USA.
Fax: 1607 255 6249, Tel.: 1607 255 5706,
E-mail:
Abbreviations: AXG, arabinoxylan; BMCC, bacterial microcrystalline
cellulose (Cellulon
TM
); CBM, cellulose-binding module; CD, catalytic
domain; CMC, carboxymethylcellulose; GBG, bacterial cellulose;
GBX, bacterial cellulose/xyloglucan composite; PAHBAH,
p-hydroxybenzoic acid hydrazide; SC, phosphoric acid swollen cellu-
lose; tamXG, tamarind seed xyloglucan; TFSF, Thermobifida fusca
crude supernatant enzymes; Xeg, xyloglucan-specific endo-b-1,4-
glucanase; XG, xyloglucan; XGO, xyloglucan oligosaccharide.
Present addresses: *Beth Israel Hospital, New York City, NY, USA.
US Plant, Soil and Nutrition Laboratory, Tower Road., Ithaca,
NY 14853, USA.
àDepartment of Plant Biology, Cornell University, Ithaca, New York,
USA.
(Received 3 April 2003, revised 19 May 2003, accepted 30 May 2003)
Eur. J. Biochem. 270, 3083–3091 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03695.x
polymers such as cellulose, starch, xylan, or mannan as the
sole carbon source and it is thought to be an important
organism in the degradation of biomass. Six T. fusca

cellulase genes and a xylanase gene have been cloned,
expressed and characterized [8–11]. The genome of this
organism has been sequenced ( />microbial/tfus/) and includes many genes coding for a
variety of glycosyl hydrolases. One such gene codes for a
glycosyl family-74 hydrolase with a typical bacterial
family-2 cellulose-binding module (CBM) at the C terminus.
In the present study we show that this enzyme is a
xyloglucanase and investigate its properties and its possible
role in the degradation of plant biomass.
Materials and methods
Protein production and purification
Clone KPR17269, containing the gene for T. fusca xylo-
glucan-specific endo-b-1,4-glucanase (Xeg)74, was obtained
from Stephanie Stilwagen Malfatti (DOE Genome Institute,
Walnut Creek, CA, USA). A PCR product of the gene was
made using the forward primer, 5¢-CGTCCACTCC
GC
GGCCGCCCCCGCCTC, which creates a NotI site (under-
lined) near the predicted mature N-terminal codon (in
italics), and the reverse primer, 5¢-CCCTCGTGCG
CTCG
AGGTACCAGGGCTTTGC, which has an XhoI site
after the C-terminal codon. Plasmid pD1164 was created
by ligating gel-purified fragments of pET26B+ (Novagen)
digested with NdeIandXhoI, a NdeI–NotI fragment
containing the T. fusca Cel6A signal peptide and the above
PCR fragment digested with NotIandXhoI. This ligation
mixture was transformed into Escherichia coli DH5 alpha,
and plasmid minipreps (Qiagen) were used to identify a
transformant containing the desired plasmid which was

then transformed into E. coli BL21-DE3 (Novagen) and
also into BL21 (DE3)-RP codon plus (Stratagene). An
Xeg74 catalytic domain (CD) expression plasmid was
created by ligating the 1.2-kb NotI–SphI fragment of
pD1164, the 5.4-kb SpeI–NotI fragment of pD1164 and a
1.0-kb SphI–SpeI PCR fragment, which created a stop
codon after the amino acid sequence GDLDG (mature
amino acid 736). The ligation mix was transformed into
BL21 gold (DE3) (Stratagene) and the correct strain
(pMC1, D1212) was identified, as described above. The
portion of each plasmid created by PCR was sequenced and
no unwanted mutations were detected.
D1170 (Xeg74) or D1212 (Xeg74CD) were cultured,
from frozen stocks, overnight at 30 °C in Luria–Bertani
(LB) broth containing 0.5% glucose and 60 lgÆmL
)1
kanamycin. Thirty milliliters of culture was used to ino-
culate 1 L of M9 containing 0.5% glucose and 60 lgÆmL
)1
kanamycin. These cultures were grown to a D
600
of 0.9,
isopropyl thio-b-
D
-galactoside was added to 0.8 m
M
,and
the cultures were allowed to grow overnight at 30 °C. The
supernatant was clarified by centrifugation, NH
2

SO
4
was
addedto1.2
M
, and the supernatant was further clarified by
depth microfiltration using a CUNO Beta Pure polyolefin
#46368-02L cartridge. This material was loaded onto a
phenyl sepharose CL-4B (Sigma) column (10 mLÆL
)1
of
supernatant) and the column was washed with two volumes
of 0.8
M
NH
2
SO
4
+10m
M
NaCl + 5 m
M
Kpi, pH 6,
followed by three volumes of 0.4
M
NH
2
SO
4
+5m

M
NaCl + 5 m
M
Kpi, pH 6, and eluted with 5 m
M
Kpi,
pH 6. The purest fractions, as determined by SDS/PAGE,
were combined and loaded on a Q-Sepharose Fast Flow
(Pharmacia Biotech) column (2 mg of proteinÆmL
)1
of
column). The protein was eluted with a gradient (20·
column volume) of 0–0.7
M
NaCl in 10 m
M
Bis/Tris,
pH 5.6, + 10% glycerol. The purest fractions were com-
bined and concentrated in a stirred cell using a PTGC
10 000 MWCO ultrafiltration membrane (Millipore). The
proteins were stored at )70 °Cin5m
M
NaOAc, pH 5.5,
containing 10% glycerol. The final yield of purified protein
was 22 mgÆL
)1
Xeg74 and 14 mgÆL
)1
Xeg74CD. T. fusca
crude cellulase was prepared by concentration of T. fusca

culture supernatant after growth on Solka Floc powdered
cellulose (James River Corporation, Berlin, New Hamp-
shire, USA), as previously described [2].
Cel9B was expressed in the supernatant of a Strepto-
myces lividans clone, pSHE1, and purified as previously
described [12]. The molecular masses of the purified proteins
were determined using a Bruker Biflex III MALDI-TOF
spectrometer instrument at the Cornell University Bio-
resources Center.
Assay methods
Tamarind seed xyloglucan (arabinose/galactose/xylose/glu-
cose; 3 : 16 : 36 : 45) (tamXG) was obtained commercially
(Megazyme, Ireland) or extracted as described previously
[13]; XG-bean was isolated from the media of bean
(Phaseolus vulgaris) suspension cell cultures, as described
previously [14,15]. Except where stated otherwise, xylo-
glucanase activity was assayed in 500 lL microcentrifuge
tubes by incubating 200 lL of samples containing
2–2.5 mgÆmL
)1
tamXG, 0.05-
M
NaH
2
PO
4
/K
2
HPO
4

(pH 7.5) buffer, and enzyme for 30 min at the desired
temperature. Twenty-microliter samples (in triplicate) were
removed and added to 1.5 mL of p-hydroxybenzoic acid
hydrazide (PAHBAH) reagent [16] and boiled for 6 min
according to the published procedure. The absorbance (A)
at 410 nm was read and a reference curve was prepared
using glucose (0–25 lg).
Fig. 1. Example of a xyloglucan (XG) oligosaccharide structure
(XXLG) and nomenclature [5]. XXXG, XXLG, XLXG, and XLLG
are known subunits of tamarind seed xyloglucan (tamXG) [15]. XGs
from other sources vary in composition and the XG from some
dicotyledonous plants has a-
L
-fucose (1 fi 2) added to some of the
galactose residues, while solanaceous plants produce more complex
arabinoxyloglucans (AXG) [23,26,28,34]. In addition, residues may
contain either one or two O-acetyl groups on C-2, C-3 or C-6.
3084 D. C. Irwin et al. (Eur. J. Biochem. 270) Ó FEBS 2003
The pH optimum assays used buffers prepared by mixing
30-m
M
solutions of citric acid, NaH
2
PO
4
, boric acid, and
barbital to obtain the desired pH. The final concentration of
buffer in the assays was 15 m
M
. The temperature stability of

the enzyme was tested by incubation of 2.7 l
M
Xeg74 for
16 h at 0–75 °C followed by performing the XG activity
assay at 50 °C.
Kinetic constants were determined with substrate con-
centrations from 2 to 60 lgÆmL
)1
and 0.375 pmolÆmL
)1
enzyme. The bicinchoninate assay [17] was used to quantify
the reducing sugars produced. Michaelis constant (K
m
)and
maximal rate (V
max
) values were calculated from a plot of
the initial reaction rates vs. substrate concentration using
Kaleidagraph (
SYNERGY
Software) to fit the data to the
Michaelis-Menten equation.
All reducing sugar assays included a glucose standard
curve. The average molecular mass of the XG oligosaccha-
rides (XGOs) was calculated from the manufacturer’s data
and was found to be 1293 Da. This value agreed with the
MS analysis of the tamXG digestion products. Exhaustive
digestion of tamXG by Xeg74 created a set of XGOs from
known amounts of substrate (0–1500 lg), and the approxi-
mate relationship between the glucose standard curve and

the nmoles of XGOs produced was determined for both the
PAHBAH and the bicinchoninic acid reducing sugar
methods. For the PAHBAH method, the nmol of
XGOs ¼ nmol of glucose equivalents ·0.481; for the
bicinchoninic acid method, the nmol of XGOs ¼ nmol of
glucose equivalents ·0.686.
NMR experiments were performed at the Chemistry and
Chemical Biology NMR facility, Cornell University, by the
method of Pauly et al. [18]. TamXG (5 mgÆmL
)1
)was
addedto10m
M
NaCl in D
2
Oat80°C and stirred
overnight. Xeg74 was prepared for NMR experiments by
resuspending and concentrating it six times with 10-m
M
NaCl in D
2
O using a Centricon 30 (Millipore). Three-
hundred micrograms of Xeg74 was added to 0.7 mL of
tamXG in an NMR tube and spectra were taken over a 100-
min time course.
Samples were prepared for MS by reacting 0.4 nmol
Xeg74 or Cel9B with 5 mg of tamXG in 1 mL of 5 m
M
NaOAc (pH 5.5) buffer containing 0.02% NaN
3

,at50°C
for 48 h on a rotator. Positive-mode electrospray MS was
performed on a Bruker Esquire LC ion-trap mass spectro-
meter.
TLC of hydrolysis products was performed using What-
man LK5D 150-A silica gel thin-layer plates with two
ascents of the solvent, ethyl acetate/water/MeOH
(40 : 15 : 20). Plates were stained (100 mL of acetic acid,
1 mL of p-anisaldehyde, 2 mL of concentrated sulfuric
acid) and then heated for 1 h at 95 °C, as described by
Chirco & Brown [19] and Jung et al. [12]. Glucose and
xylose oligomer standards were obtained from Seikagaku
America or Sigma.
Preparation of GBG, GBX and tomato cell walls
Bacterial cellulose (GBG) and bacterial cellulose/xyloglucan
composite (GBX) were produced from Gluconaceto-
bacter xylinus (formerly Acetobacter xylinus) ATCC 53524
according to a procedure published previously [6]. The
resultant gel-like material was harvested, rinsed six times on
afilterwithdH
2
O and stored at 4 °C in 0.04% NaN
3
.
Culture reducing sugars were removed by rotating a
solution of the coarsely chopped GBG or GBX in 0.05-
M
NaKPi, pH 7.4, for at least 2 h at 50 °C followed by further
washing with 0.04% NaN
3

. Pieces of the gels were rinsed
with water, blotted on filter paper and chopped finely with a
razor blade. The dry mass of the material was approxi-
mately 1% of the blotted gel and overdigestion of GBX with
Xeg74 gave an XG content of 16%. In order to equalize the
amount of cellulose to be digested, 42 mg of GBG or 50 mg
of GBX were weighed into 0.5-mL screw-cap microfuge
tubes. Buffer (0.05-
M
NaKPi, pH 7.4) and enzymes were
added to achive a final volume of 0.5 mL and the microfuge
tubes were then incubated on a rotator for 16 h at 50 °C.
Reducing sugars in the supernatant were measured by the
PAHBAH method, as described above, using glucose as a
standard. Each data point represents the average of three
separate digestions and the PAHBAH measurements for
each digestion were run in triplicate.
Tomato cell walls were isolated as follows [14,20]: 50 g of
outer pericarp tissue from green tomatoes was finely diced,
frozen in liquid nitrogen and ground into a fine powder.
This material was boiled in 500 mL of 95% EtOH for
40 min, filtered on Miracloth
TM
(Calbiochem), resuspended
in boiling EtOH, and refiltered. The solid material was
resuspended in 500 mL of CHCl
3
/MeOH (1 : 1), filtered on
a glass frit and washed with 500 mL of acetone. These steps
inactivate endogenous cell-wall enzymes and extract low-

molecular-mass solutes. To remove starch, the solid mater-
ial was resuspended in 15 mL of dimethylsulfoxide/H
2
O
(9 : 1) and stirred for 1 h, centrifuged and washed three
times with dimethylsulfoxide/H
2
O (9 : 1). The solid mater-
ial was resuspended in trans-1,2-diaminocyclohexane-
N,N,N¢,N¢-tetraacetic acid (CDTA)/bicarb buffer (50 m
M
CDTA, 50 m
M
Na
2
CO
3
) containing 20-m
M
NaBH
4
(pH 6.5) to a final volume of 125 mL and stirred overnight.
(The CDTA chelates Ca
2+
, facilitating the release of pectin,
and the NaBH
4
removes reducing groups.) This material
was filtered on Miracloth
TM

(Calbiochem), rinsed several
times with CDTA/bicarb buffer, stirred overnight at room
temperature, homogenized, filtered again, and washed with
CDTA/bicarb buffer until the filtrate was clear. The solid
material was resuspended and dialyzed against 0.01
M
Tris
(pH 7.0) containing 0.02% NaN
3
,at4°C. Assays were
carried out, as for GBG and GBX, using 100 lL
(6.8 mgÆmL
)1
dry mass) of the tomato cell wall preparation
as substrate in a total volume of 500 lL. The tomato XG
was from Bree Urbanovicz and prepared by extraction of
tomato cell walls with 1
M
KOHfor1handthenwith4
M
KOH at room temperature with stirring overnight. The
supernatants were filtered through nylon mesh and neut-
ralized, on ice, to pH 7.0 with glacial acetic acid. XG was
precipitated from the 4
M
extraction with two volumes of
ethanol, cooled on ice, centrifuged, washed three times with
cold ethanol, resuspended in water and then freeze dried.
Western blots
Culture supernatants were analyzed for the presence of

Xeg74 by separation on SDS/polyacrylamide gels [21]
followed by transfer to Immobilon-P poly(vinylidene diflu-
oride) membranes (Millipore). Rabbit polyclonal antisera
Ó FEBS 2003 Thermobifida fusca xyloglucanase Xeg74 (Eur. J. Biochem. 270) 3085
raised against recombinant Xeg74 was used as the first
antibody and goat anti-rabbit Ig alkaline phosphatase
conjugate (Bio-Rad) was used as the second antibody.
Immunodetection was performed using nitro-blue tetra-
zolium and 5-bromo-4-chloro-3-indolyl phosphate, accord-
ing to the Bio-Rad protocol.
The GenBank accession number for the T. fusca genome
is NZ_AAAQ00000000. The file containing the Xeg74 gene
is NZ_AAAQ01000018 and the gene is Tfus0318, CDS
39974.42751. The protein identification is ZP_00056977.
The accession numbers for T. fusca cellulases are: Cel5A,
Q01786; Cel6A, P26222; Cel6B, Q60029; Cel9A, P26221;
Cel9B, Q08166; and Cel48A, AAD39947. T. fusca YX
(BAA-629) and T. fusca ER1 (BAA-630) have been depo-
sited in the American Type Culture Collection.
Results
Cloning and purification
The gene for Xeg74 was cloned into pET26b using the
T. fusca Cel6A signal sequence (MRMSPRPLRALL
GAAAAALVSAAALAFPSQAA) in place of its native
signal sequence. Cel6A, Cel6Acd, and Cel48A have been
expressed and secreted successfully by E. coli [11,22] using
this signal sequence and it has a convenient NotIsiteatits
C terminus, which allows cloning in-frame with alanine as
the first amino acid of the mature protein. An alignment
of the amino acid sequences of 11 family-74 catalytic

domains shows that their amino acid identity to T. fusca
Xeg74 ranges from 29 to 63% with the alignment starting
at GYTWR. Xeg74 was predicted by SignalP (http://
www.cbs.dtu.dk/services/SignalP-2.0/#submission) to have
the mature N-terminal sequence, APASATTGYTWR,
with the start of the conserved sequence at amino acid 8.
The catalytic domain was produced from the same vector
after inserting a stop codon after amino acid 736 ending
with VGDLDG. This C-terminal sequence agrees well
with that of other family-74 catalytic domains and, in the
native protein, is followed by a linker region, (737)
PPPQPTEEP…, which is similar to the linker region in
T. fusca Cel9A [12]. Of the expressed protein, about 90%
was secreted to the culture supernatant and about 10%
was in the shock fluid, as determined by SDS gels (data
not shown). The molecular mass of purified Xeg74CD, as
determined by MS, was 79 443 Da (expected mass
79 480 Da). However, the mass spectrum of Xeg74
showed a broad set of peaks with mass values from
96 200 to 94 718 (expected mass 94 705 Da), indicating
that the signal peptide was being cut in different places,
resulting in the addition of up to 17 extra amino acids at
the N terminus of the protein. It is not clear why the
Xeg74CD signal peptide cuts cleanly and Xeg74 does not,
as both genes are cloned in the same way except for the
presence of the linker and the family-2 CBM at the C
terminus of XEG74. The molecular masses of Cel9A-68
(CD + family3c CBM) and Cel48ACD, both cloned with
the Cel6A signal peptide, were also determined by MS
and the peaks also had several small shoulders at higher

molecular masses although they were much sharper than
the peak for Xeg74. Possibly the more complicated
domain structure of Xeg74 (a catalytic domain, a linker,
and a CBM) results in nonspecific cleavage of the signal
sequence.
Characterization
Xeg74 had very low activity on swollen cellulose (SC) or
carboxymethyl cellulose, and assays using tamXG, barley
b-glucan, Avicel, locust bean gum (galactomannan), soluble
starch, xylan, pectin, or corn fiber as substrates showed
that Xeg74 had significant activity only on tamXG. The
products of Xeg74 hydrolysis of tamXG were analyzed by
TLC (Fig. 2A). Digestion of tamXG was complete within
1.5 h and produced three main bands with no carbohydrate
remaining at the origin. In contrast, Xeg74 digestion of SC
and carboxymethylcellulose (CMC), after overnight incu-
bation, produced only very faint bands of cellotriose (G3),
cellotetraose (G4) and cellopentaose (G5). Each of the six
purified T. fusca cellulases was tested for activity on tamXG
and only Cel9B was active. However, it produced only two
of the three product bands and there was a large quantity of
undigested material remaining at the origin (Fig. 2B).
Xeg74 had very limited activity on G4, G5 and G6 and
only faint product bands were produced after overnight
digestion (Fig. 2C). Xeg74 was active on both bean and
tomato XG and inactive on boiled barley b-glucan
(Fig. 2D,E).
The molecular masses of the digestion products of
tamXG by Xeg74 and Cel9B were determined by MS
(Fig. 3). Using the known composition of tamXG [15,23],

the possible products were determined, as shown in the
figure. Xeg74 cleaved the backbone of XG to give products
decorated with two or three xylose units and further
decoratedwithuptotwogalactosemolecules.XXGGwas
not reported by York et al. [15] to be a component of
tamXG, but there is a peak at mass 953.8, which appears to
be XXGG in Fig. 3A. Cel9B only cleaved a portion of the
tamXG (Fig. 2B, lane 5) and did not produce any products
containing galactose, XXGG appears to represent the
major product of hydrolysis by this enzyme and this
suggests that Cel9B prefers to cleave where there are two
undecorated glucoses, although it can also cleave slowly to
produce XXXG.
The K
m
wasdeterminedtobe3.2and3.9lgÆmL
)1
for
Xeg74 and Xeg74CD, respectively. This corresponds to 2.4
and 3.0 l
M
using a molecular mass of 1293 for the average
XGO hydrolyzable unit. The V
max
of Xeg74 was 966 lmol
XGOÆmin
)1
Ælmol
)1
protein, while that of Xeg74CD was

somewhat higher, at 1257 XGOÆmin
)1
Ælmol
)1
protein. The
specific activities were also determined using a similar assay
to that used for cellulases [8] with a substrate concentration
of 2.5 mgÆmL
)1
and increasing amounts of enzyme. The
data shows a linear relationship up to 50% digestion and the
specific activities were 578 and 875 lmol XGOÆmin
)1
Æ
lmol
)1
enzyme for Xeg74 and Xeg74CD, respectively.
The extra amino acids left on the N terminus from the signal
peptide may have caused the whole protein to have lower
specific activity than the CD, or the smaller size of the CD
may enable it to fit into the xyloglucan/cellulose matrix
more easily, increasing the availability of the insoluble
xyloglucan substrate to the enzyme. The specific activity of
Cel9B was also measured in this way and was found to be
46 lmol XGOÆmin
)1
Ælmol enzyme at 15% digestion.
3086 D. C. Irwin et al. (Eur. J. Biochem. 270) Ó FEBS 2003
However, the maximum digestion obtained was about
30% of the total substrate and the curve was not linear.

In contrast, Cel9B has activities of 5410 and 363 lmol
cellobioseÆmin
)1
Ælmol
)1
, respectively, on CMC and SC
while Xeg74 activity on CMC and SC was 1.1 and 1.9 lmol
cellobioseÆmin
)1
Ælmol
)1
, respectively.
Xeg74 retained more than 60% activity at pH 6.0–9.4
and 90% activity at pH 6.9–8.6 (Fig. 4A). When assayed
with 0.025
M
(pH 7.5) NaH
2
PO
4
/K
2
HPO
4
, Hepes, Tris/
HCl, or NaOAc buffers, the enzyme had 100%, 83%, 73%,
and 92% activity, respectively. Temperature stability tests
showed that the enzyme retained full activity after 16 h of
incubation at 55 °C and 76% activity after incubation at
60 °C (Fig. 4B). Thirty-minute activity assays showed that

the activity increased with temperature up to 76 °C
(Fig. 4B).
Enzymatic hydrolysis of b-glycosidic bonds occurs by
two general mechanisms giving rise to either retention or
inversion of the anomeric configuration [24]. Figure 5
shows the [
1
H]-NMR spectra of tamXG after reaction with
Xeg74for0,5,20and100min.Thea-anomeric proton
appears at 5 min, indicating an inverting enzyme, and after
20 min the b-anomeric proton appears as the result of
mutarotation. The NMR assignments relied on the work of
Pauly et al. [18] for the family-12 Aspergillus aculeatus
xyloglucanase, a retaining enzyme, in which the b-anomeric
proton appeared first followed by the subsequent appear-
ance of the a-anomeric proton.
Concentrated supernatants from T. fusca cultures
grown on Solka Floc, xylan, or corn fiber, all had activity
on tamXG and revealed the same product pattern on
TLC as observed for Xeg74 (Fig. 2B). The xylan-grown
supernatant produced four additional brown-colored
bands of lower molecular mass; although the composition
of these bands is not known, the brown color indicates
that they contain xylose. A Western blot with polyclonal
rabbit antiserum prepared against purified Xeg74 showed
that the level of this protein was highest in the super-
natants of the Solka Floc-, xylan-, or corn fiber-grown
cultures and was present at low levels when T. fusca was
grown on glucose, cellobiose, xylose, or bacterial micro-
crystalline cellulose (BMCC) (Fig. 6). T. fusca was not

able to grow with tamarind, tomato, or bean XG as the
sole carbon source. When glucose was added to the
culture, growth resumed, ruling out the presence of an
inhibitor. Although attempts to culture T. fusca on
tamXG produced little or no growth, high levels of
Xeg74 were induced (Fig. 6, lane 11) and TLC analysis of
the culture supernatant showed that the XG had been
degraded to the expected products (data not shown). The
Xeg74 antiserum also reacted with T. fusca Cel6A, but
not with the Cel6A catalytic domain (Fig. 6, lanes 9 and
10), which indicates that the CBM of Cel6A cross-reacts
with the Xeg74 antiserum. The family-2 CBMs of all six
cellulases show 33–52% amino acid identity with the
Xeg74 CBM (43% identical to Cel6A CBM). Presumably
these two CBMs have antigenic epitopes in common while
the others do not. This cross-reaction is useful for
comparing the differences in induction of the two
enzymes. Cel6A is induced by growth on cellobiose,
Solka Floc, BMCC, and corn fiber, while Xeg74 is present
in all of the supernatants but is found at a higher level in
cultures grown on XG, xylan, Solka Floc, and corn fiber
(Fig. 6). The low level of Xeg74 found in BMCC cultures
Fig. 2. TLC analyses of reaction products (A–E). All reactions were run at 50 °C for 16 h except for lane 2, which was incubated for 1.5 h. The
enzymes and substrates used are noted on the figure under each lane using the following abbreviations: 74, xyloglucan-specific endo-b-1,4-glucanase
(Xeg)74; and 9B, Cel9B. CF, XY, and SF indicate hydrolysis by enzymes in concentrated crude supernatants from cultures of Thermobifida fusca
ER1 grown on corn fiber, xylan, or Solka Floc, respectively. Abbreviations: tom, tomato; tam, tamarind; and G6, cellohexose. Standards for each
TLC analysis were glucose, cellobiose, cellotriose, cellotetraose, cellopentaose (G1–G5) and xylose and xylobiose (X1–X2).
Ó FEBS 2003 Thermobifida fusca xyloglucanase Xeg74 (Eur. J. Biochem. 270) 3087
shows that it is not the cellulose in Solka Floc which is
inducing the higher level of Xeg74 but some minor

component.
Role of Xeg74
G. xylinus synthesizes cellulose I chains that extrude parallel
to the bacterial wall and which coalesce into bundles of
highly crystalline microfibrils [25]. When G. xylinus was
grown in the presence of glucose and tamXG, 38% of the
added XG was incorporated into the cellulose pellicle and
microscopic analysis showed the formation of cross-linkages
between the ribbons [6]. The level of incorporated XG was
similar to that found in primary cell walls and much of the
XG was thought to be intimately bound to the surface or
woven into the cellulose fibers [6]. The role of Xeg74 in plant
biomass degradation was studied using the GBX composite
produced by G. xylinus when grown on glucose plus
tamXG and, as a control, GBG was prepared by growth
on glucose. A mixture of purified T. fusca cellulases (cel
mix) consisted of Cel5A (an endocellulase), Cel6A (a
nonreducing end-directed exocellulase), Cel9A (a processive
endocellulase), and Cel48A (a reducing end-directed
exocellulase). Under the assay conditions, Xeg74 enzyme
alone (0.05 nmol, 4.7 lg) produced 58 lg of reducing sugar
from GBX and 6.9 lg from GBG. The cel mix alone was not
able to degrade GBX to any appreciable extent; however,
when Xeg74 was added, the activity was very similar to that
of concentrated T. fusca crude supernatant enzymes (TFSF)
(Fig. 7A). The reactions of the cel mix and the cel
mix + Xeg74 on GBG were very similar to that of TFSF
on GBG (data not shown). XG protects the cellulose
microfibrils from degradation by cellulases. Xeg74CD was
foundtohave 86% of the activity of whole Xeg74 when

combined with the cel mix on GBX, implying that the CBM
contributes to the degradation, but is not essential.
Tomato cell walls that had been processed to inactivate
endogenous enzyme activity and remove low-molecular-
mass solutes and starch, were used as a substrate in a similar
assay. Figure 7B shows that the amount of reducing sugar
produced by the cel mix, plus and minus Xeg74, was about
the same. However, TFSF was able to produce four times
as much reducing sugar as the cel mix. Analysis of the
hydrolysate by TLC showed glucose and cellobiose to be the
Fig. 3. MS analysis of the products of tamarind seed xyloglucan (tam-
XG) hydrolysis by xyloglucan-specific endo-b-1,4-glucanase (Xeg)74
(A) and Cel9B (B). The theoretical mass values are given in parentheses
for possible products.
Fig. 4. Temperature and pH optima for Xeg74 activity. (A) Percentage
xyloglucanase activity at various pH values. (B) Effect of temperature
on the activity and stability of xyloglucan-specific endo-b-1,4-gluca-
nase (Xeg)74. Stability was tested by incubating xyloglucan-specific
endo-b-1,4-glucanase (Xeg)74 at the indicated temperatures for 16 h
followedbydilutionanda30-minassayat50°C.
3088 D. C. Irwin et al. (Eur. J. Biochem. 270) Ó FEBS 2003
dominant products, with only faint bands at the higher
molecular massess expected for XGOs (Fig. 2D, lane 19).
The products from Xeg74 hydrolysis of the 4
M
KOH-
extracted tomato XG are shown in Fig. 2E, lane 22. XGO
bands are produced, but much of the substrate remains
undegraded, at the origin. An activity assay measuring
reducing sugars showed that a maximum of  26% of this

tomato XG extract could be degraded by Xeg74. This may
reflect the presence of small amounts of polysaccharides
other than XG in the extract and/or variability in the
structure of tomato XG. For example, the AXG of
solanaceous plants, such as tomato, is characterized by
the presence of arabinose-containing side-chains, such as
b-ara-(1fi 3)-a-
L
-ara-(1fi 2)-a-
D
-xyl [26]. It is possible that
theseresiduesdonotfitintheactivesiteofXeg74.
Discussion
In this study, results were obtained which showed that XG
protects the cellulose in the cellulose/xyloglucan composite
and that the activity of Xeg74 with a synergistic mixture of
cellulases can easily degrade this material. However, tomato
cell walls have a much more complex structure, and
additional enzymes in the TFSF crude mixture are essential
for tomato cell-wall digestion. Similar results were seen in
studies by Vincken et al. [27,28], which showed good
synergistic activity on water-unextractable solids from
apples (about 24% xyloglucan and 33% cellulose) when
Trichoderma viride Endo IV (a family-12 CMCase/xyloglu-
canase) was added to a mixture of EXOIII and EndoI. The
amount of cellobiose released was twice as high as the
amounts released by EXOIII and EndoI alone; however,
even the most effective combination was not able to
solubilize all of the cellulose.
The results of the NMR experiment show that Xeg74 and

presumably all family-74 enzymes catalyze hydrolysis with
inversion of the anomeric configuration. This is in contrast
to the family-12 A. aculeatus xyloglucanase, which is a
retaining enzyme [18]. The Xeg74 gene does not have an
upstream 14-bp DNA-binding site for the regulatory
protein, CelR, which is found in the six T. fusca cellulase
genes. This is consistant with our observation that Xeg74
and Cel6A are regulated differently. It is interesting that
T. fusca produces Xeg74 during incubation with tamXG,
even though it cannot grow on the XGOs released from XG
hydrolysis. The amount of enzyme produced in a nongrow-
ing culture appears to be equal to or even a little higher than
the amount produced in growing cultures, using either
Solka Floc or corn fiber as the sole carbon source. Another
example of an enzyme produced in a nongrowing culture is
a polyester-degrading extracellular hydrolase from T. fusca
DSM43793 [29], which is induced in cultures containing
Ecoflex
TM
(BASF AG, Germany), a random co-polyester of
1,4-butanediol, terephthalic acid and adipic acid, as the sole
carbon source. It is curious that T. fusca grows on xylose
and yet does not metabolize the a-
D
-xylose in the XGOs
produced by Xeg74. There are open-reading frames in the
T. fusca genome which are homologous to a-xylosidases;
however, neither extracts nor concentrated supernatants
Fig. 6. Western blot of an SDS/polyacrylamide gel using rabbit
polyclonal antiserum against xyloglucan-specific endo-b-1,4-glucanase

(Xeg)74. Supernatants (10 lL) from cultures of Thermobifida fusca
grown on glucose (lane 1), cellobiose (lane 2), xylan (lane 3), Solka Floc
(lane 4), and corn fiber (lane 5); lane 6, Benchmark molecular mass
standards; lane 7, Xeg74 (0.05 lg); lane 8, Xeg74CD (0.05 lg); lane 9,
Cel6ACD (0.5 lg); lane 10, Cel6A (0.05 lg); supernatants (10 lL)
from cultures of T. fusca grownontamXG,nogrowthwasapparent
(lane 11), bacterial microcrystalline cellulose (BMCC) (lane 12), Solka
Floc (lane 13).
Fig. 5. [
1
H] NMR spectra of tamarind seed xyloglucan (tamXG) reac-
ted with xyloglucan-specific endo-b-1,4-glucanase (Xeg)74 at 50 °C. The
a-anomeric proton appears rapidly, indicating an inverting enzyme,
while the b-anomeric proton appears later as a result of mutarotation.
Ó FEBS 2003 Thermobifida fusca xyloglucanase Xeg74 (Eur. J. Biochem. 270) 3089
from T. fusca cultures grown on Solka Floc, xylan, or corn
fiber had measurable activity on p-nitro-phenol a-
D
-xylose
[2].
The first family-74 enzyme to be reported was an
A. aculeatus enzyme (FIII avicelase), which produces cello-
triose and cellobiitol from reduced cellopentaose, clearly
showing that it releases cellobiose from the reducing end
[30]. However, the enzyme was not tested on XG and the
degree of hydrolysis was only about 0.7%. Thermotoga
maritima Cel74 is most active on barley b-glucan, which has
a mixed-linkage b-1,3/4 glucose backbone and only 23% as
much activity on tamXG [31]. A. niger EglC [32] resembles
Xeg74 in having the highest activity on tamXG with about

5% as much activity on CMC or b-glucan and it has a
C-terminal family-1 fungal CBM. EglC is regulated by
XlnR, a transcriptional activator which binds to the DNA
sequence, GGCTAA. We did not find a protein homolog-
ous to XlnR in the T. fusca genome, nor did we find
GGCTAA upstream of the Xeg74 start codon. The
Geotrichum sp. M128 family-74 enzyme is an exoglucanase,
which attacks the reducing end of XG, releasing GG, XG,
or LG [33]. This protein is unique in having four regions of
amino acids (235–251; 310–318; 361–372; 398–413) that are
not found in the other family-74 proteins. These may form
loops that make the active site a tunnel rather than an open
cleft, leading to exoglucanase rather than endoglucanase
activity. The nature and presence of a CBM varies among
the family-74 proteins: four of 13 family-74 genes code for a
family-2 CBM; three have a family-1 fungal CBM; one has
a family-3 CBM; and five have no CBM. The low K
m
of
T. fusca Xeg74 shows that the binding of the catalytic site to
the substrate is very tight and perhaps this makes the CBM
useful but not essential for hydrolytic activity. Overall, the
characterized family-74 enzymes show a variety of substrate
specificities with most having a strong preference for a
cellulosic backbone substituted with xylose (and further
decorated) side-chains. The structure of the Xeg74 active
cleft should provide interesting insights into its mechanism
of action and efforts are underway to solve the structure of
Xeg74CD.
In summary, Xeg74 is produced at low levels on all

substrates, is induced at higher levels by growth on Solka
Floc, corn fiber and xylan, and is also highly induced by XG
in the media, although additional growth does not take
place. The purpose of the enzyme seems to be to degrade
XG surrounding cellulose microfibrils to allow the organism
access to a substrate which it can hydrolyze and metabolize
efficiently. This function is consistent with the presence of a
family-2 CBM on the enzyme. Xeg74 may also function to
help T. fusca grow on other plant cell-wall polymers, such
as xylan or mannan, which may be protected by xyloglucan.
Acknowledgements
This work was supported by DE-FG02-01ER63150, from the US
Department of Energy.
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Ó FEBS 2003 Thermobifida fusca xyloglucanase Xeg74 (Eur. J. Biochem. 270) 3091