Secondary substrate binding strongly affects activity and
binding affinity of Bacillus subtilis and Aspergillus niger
GH11 xylanases
Sven Cuyvers, Emmie Dornez, Mohammad N. Rezaei, Annick Pollet, Jan A. Delcour and
Christophe M. Courtin
Laboratory of Food Chemistry and Biochemistry & Leuven Food Science and Nutrition Research Centre (LFoRCe), Katholieke Universiteit
Leuven, Belgium
Introduction
Glycoside hydrolases can possess noncatalytic polysac-
charide binding sites that facilitate attack on the natu-
ral substrate. Most of these sites belong to separate
domains, referred to as carbohydrate-binding modules
(CBMs), linked to the catalytic domain through flexi-
ble linker regions. Elaborate research has clarified the
functional relevance of these CBMs [1–4]. CBMs are
considered to target the enzyme towards specific cell
wall regions and to keep it in proximity of the sub-
strate. In some cases, distortion of the substrate struc-
ture by the CBMs is considered to facilitate hydrolysis
[5]. CBMs can also be involved in binding the bacterial
cell wall, thereby anchoring the attached enzyme onto
the bacterial surface [6,7].
Despite the clear advantage of having CBMs, some
glycoside hydrolases consist of a catalytic domain only
[8]. Studies investigating the structure of carbohydrate-
active enzymes have revealed the presence of other
substrate binding regions situated on the surface of the
structural unit that contains the catalytic site, rather
than on an auxiliary domain [9]. These substrate bind-
ing sites are located at a certain distance from the
active site and are called secondary binding sites

on their natural substrates.
Abbreviations
AX, arabinoxylan; AzU, unit of enzyme activity on Azo-wheat AX; CBM, carbohydrate-binding module; GH, glycoside hydrolase family;
OSX, insoluble xylan from oat spelts; SBS, secondary binding site; X
6,
xylohexaose; X
6
U, unit of enzyme activity on xylohexaose;
XAN, Aspergillus niger xylanase; XBS, Bacillus subtilis xylanase; XyU, unit of enzyme activity on Xylazyme AX.
1098 FEBS Journal 278 (2011) 1098–1111 ª 2011 The Authors Journal compilation ª 2011 FEBS
reported in enzymes belonging to glycoside hydrolase
family (GH) 8, 10, 11, 13, 14, 15, 16 and 77 [9–20].
The widespread occurrence of these binding sites indi-
cates that incorporation of a SBS provides an evolu-
tionary benefit for these enzymes. However, the
function of these SBS in many enzymes remains to be
unraveled. To date, most work aiming to understand
the role of SBS has been performed on starch degrad-
ing enzymes. Human salivary a-amylase contains sev-
eral SBS. Mutational analysis demonstrated that these
SBS residues are important for the activity on starch
and that they play a role in the binding of the enzyme
to bacteria of the oral cavity [15]. Nielsen et al. [9]
concluded that the two SBS in barley a-amylase each
have a distinct binding specificity, although they both
play a role in substrate targeting. In two single domain
glucoamylases from Saccharomycopsis fibuligera, a SBS
was also found to enhance binding to starch granules
[18].
In xylanases (EC 3.2.1.8), the existence of a SBS was

LIGPLOT [37] based on Vandermarliere et al. [12]. Amino acid residues that form direct protein–ligand
interactions with their main chain only are indicated by an asterisk.
S. Cuyvers et al. Secondary substrate binding in GH11 xylanases
FEBS Journal 278 (2011) 1098–1111 ª 2011 The Authors Journal compilation ª 2011 FEBS 1099
investigation of whether the SBS has a similar func-
tionality in different GH11 xylanases and hence
whether the occurrence of SBS is a more general strat-
egy of GH11 xylanases to compensate for their lack of
CBMs.
Results and Discussion
Genetic engineering of the SBS
Residues of the SBS of both XBS and XAN involved
in substrate interaction were selected based on the
crystal structures of XBS and XAN soaked with xylo-
tetraose and xylopentaose, respectively [12] (Fig. 1),
and were subjected to genetic engineering using site-
directed mutagenesis. Amino acid residues reported to
potentially play a role in secondary substrate binding
were mutated to Ala aiming to investigate their impor-
tance. Several mutations were also combined to assess
the importance of the SBS as a whole for the biochem-
ical properties of XBS and XAN. In an attempt to
increase substrate binding affinity of the SBS, aromatic
residues were introduced at certain places to create
extra or stronger hydrophobic stacking interactions or
residues were replaced to create new hydrogen bonds.
Screening procedure
To examine the impact of different mutations on the
functionality of the SBS in XBS and XAN, a screening
method on nonpurified enzyme samples was developed.

lysates containing (mutant) XBS and on P. pastoris
expression media containing (mutant) XAN. The
results of the screening for XBS and XAN with a
modified SBS are shown in Table 1.
XBS
The results of the screening clearly show that modifica-
tion of the SBS of XBS leads to a lower relative effi-
ciency towards the water-unextractable Xylazyme AX
compared to that towards X
6
. These results are in
agreement with the observations of Ludwiczek et al.
[13] on GH11 B. circulans xylanase, which has a SBS
equivalent to that of XBS. Replacement of residues
considered to play a role in secondary binding with
Ala leads to a lower screening ratio for all enzymes.
For the G56A-T183A-W185A mutant, a large drop in
the screening ratio is seen, resulting in a ratio that is
only half the ratio obtained for the wild-type XBS.
The results shown in Table 1 also demonstrate the
importance of the hydrophobic stacking interaction
that Trp185 makes with bound substrate. Hydrogen
bonds with other residues also appear to be of major
importance. Mutation of residues Thr183, Asn181 and
Gly56 to Ala leads to a large drop in the screening
ratio. A smaller effect is also observed upon mutation
of Asn54 and Asn141. These results correspond well
with the results obtained in the previous study by Van-
dermarliere et al. [12] that reported residues important
for secondary substrate binding. The results obtained

because Asp32 makes only one hydrogen bond with
surface bound substrate through a main chain amine
group that is not abolished by the D32A mutation
(Fig. 1). The acidic side chain is 3.7 A
˚
away from a
hydroxyl group of the bound substrate and this dis-
tance is too far to form a relevant hydrogen bond [12].
The introduction of amino acid residues to create new
or stronger hydrophobic stacking of hydrogen bonds
with substrate in the SBS has led to screening ratios
similar to that of the wild-type XAN for most
enzymes. Subtle changes in the hydrogen bonding
appear to have no (or only a very minor) effect on the
functionality of the SBS. In some cases, the introduc-
tion of aromatic residues even lowered the screening
ratio, as was seen for some of the XBS mutants.
Activity measurements
After the screening procedure, a smaller set of enzymes
was selected for purification and further biochemical
characterization. The activity of these enzymes was
determined on X
6
and two chromophoric polymeric
substrates: the water-unextractable Xylazyme AX and
the water-extractable Azo-wheat AX. Table 2 lists
these results along with temperature and pH optima of
the enzymes. Most mutations lead to a lower tempera-
ture optimum, whereas little or no change is observed
in the pH optimum. The lowered temperature opti-

. Screening ratios are expressed relative
to the ratio of the wild-type enzyme (100%) and were calculated
based on two independent activity measurements on X
6
and two
independent activity measurements on Xylazyme AX on the same
unpurified enzyme sample, with each independent assay compris-
ing three replicates. Data are shown as the mean ± SD.
Screening
ratio (%)
XBS
Wild-type 100 ± 13
N54A 86 ± 19
G56A 70 ± 15*
N141A 81 ± 13
N181A 70 ± 21
T183A 63 ± 13*
W185A 62 ± 12*
N54A-G56A 68 ± 13*
N54A-T183A 65 ± 16*
N181A-T183A 66 ± 23
G56A-T183A-W185A 54 ± 14*
N54F 83 ± 10
N54W 76 ± 17
N141Q 80 ± 26
N54W-N141Q 79 ± 21
XAN
Wild-type 100 ± 13
D16A 92 ± 6
Y29A 82 ± 3

XBS solubilizes water-unextractable AX and OSX.
Especially in the case of OSX, the solubilization by the
enzymes is greatly hampered upon modification of the
SBS. At the same enzyme concentration, the wild-type
XBS solubilized the most. The maximal attainable sol-
ubilization of these substrates by different mutants was
also measured, although no clear differences were
observed (results not shown). This indicates that the
SBS influences the rate of hydrolysis, most likely by
enhancing substrate recognition, rather than affecting
the real catalytic potential and substrate specificity of
the enzyme.
XAN
By contrast to results on XBS, the activity on X
6
is
affected by a number of the mutations made in the
SBS of XAN. A much lower activity is observed espe-
cially for those enzymes containing the E31A mutation
display. This single mutation leads to an activity on X
6
that is only half that of the wild-type XAN. However,
a loss of activity on X
6
in the set of purified XAN
mutants does not appear to be correlated with a weak-
ening of the SBS. For example, Y29A does not display
a lower activity on X
6
, whereas it is regarded as one of

U = 1.02 · 10
)10
M, 1 XyU = 7.72 · 10
)10
M and
1 AzU = 9.65 · 10
)10
M for the activity on X
6
, Xylazyme AX and Azo-wheat AX, respectively (data are shown as the mean ± SD). K
d
values
are expressed in mgÆmL
)1
and are apparent K
d
values in many cases as a result of substrate concentration limitations in the test (data are
shown as the mean ± SE from the fit on a single curve). The reported temperature and pH ranges indicate the intervals in which the
observed activity was at least 95% of the maximal activity of the enzyme.
Activity (%) on Affinity (K
d
) towards Optimal conditions
Xylohexaose
Xylazyme
AX
Azo-wheat
AX
Water-
unextractable AX OSX Temperature pH
XBS

zyme AX over X
6
, however, do confirm the results of
the screening. Solubilization experiments with the nat-
ural substrates OSX and water-unextractable AX also
clearly demonstrate that the solubilizing capacity is
strongly decreased upon modification of SBS residues.
The observed drops in solubilizing capacity are espe-
cially spectacular on OSX.
Comparison of XBS and XAN
For XBS, it is clear that the residues involved in sec-
ondary substrate binding play no (or a very minor)
role in the hydrolysis of oligomeric substrates. The
SBS is located too far from the active site to influence
the binding and catalysis of these substrates in the
active site. The same statement is probably true for
most residues in the SBS of XAN, although, in this
case, some mutants (especially those containing the
E31A mutation) display a strong decrease in activity
on X
6
. The reason for this is not clear. Possibly, these
mutations provoke subtle positional changes of resi-
dues located in the active site. The results of activity
measurements on purified enzymes, as presented in the
present study, support the results already obtained by
screening and thereby indicate that the screening pro-
cedure can indeed provide a valuable tool for the ini-
tial selection of mutant enzymes. The SBS in XBS and
XAN mainly function to increase the efficiency of the

XBS G56A-T183A-W185A
XBS N54W
XBS N54W-N141Q
LEGEND
XAN wild-type
XAN Y29A
XAN E31A
XAN Y29A-E31A
XAN D16A-Y29A-E31A
A
WU-AX solubilisation
(% of WU-AX)
Enzyme concentration (×10
–9
M)
0
10
20
30
40
0 10203040
C
D
Enzyme concentration (×10
–8
M)
0
10
20
30

to the hydrolysis of these substrates. Whether the
observed differences in substrate selectivity are relevant
in applications remains to be explored. Strikingly, the
W185A mutation in XBS has already been character-
ized with regard to its effect on substrate selectivity.
However, Moers et al. [24] reported a drop in the sub-
strate selectivity factor for the W185A mutant. One
possible explanation for this discrepancy could be the
use of a His-tagged protein in their study. The C-ter-
minal location of this His-tag suggests a likely interfer-
ence with the functionality of the SBS because three
important residues for secondary binding are located
near the C-terminus.
Binding affinity towards insoluble polymers
As outlined above, the effect of the SBS on activity
towards different substrates was studied. Obviously,
substrate binding is closely linked to activity. There-
fore, the binding of the different XBS and XAN
mutants towards water-unextractable AX and OSX
was assessed by constructing binding curves, as shown
for several mutants in Fig. 3. Table 2 gives the overall
dissociation constants (K
d
) derived from these curves.
XBS
Weakening of the SBS of XBS clearly increases K
d
val-
ues and therefore lower affinities towards both water-
unextractable AX and OSX. The differences on OSX

values, similar to the combined N54W-N141Q mutant,
which has a K
d
of 5.3 mgÆmL
)1
. By contrast, on OSX,
all mutants give higher K
d
values than the wild-type
enzyme. It is difficult to pinpoint the reason for the
higher affinity of these mutants towards water-unex-
tractable AX. One possibility is that the attempt to
enhance substrate binding was successful for water-
unextractable AX, although a higher affinity is
not necessarily correlated with a higher activity.
Table 3. Substrate selectivity factors of B. subtilis and A. niger
xylanases with a modified secondary binding site. The substrate
selectivity factor is calculated as the ratio of activity on Xylazyme
AX over the activity on Azo-wheat AX, with both activities calcu-
lated based on the values obtained from five independent trials,
each comprising three replicates. All values are expressed relative
to the wild-type enzyme (1.00). Data are shown as the mean ± SD.
Substrate
selectivity factor
XBS
Wild-type 1.00 ± 0.10
N181A 1.05 ± 0.10
T183A 1.07 ± 0.08
W185A 1.31 ± 0.10*
G56A-T183A-W185A 1.57 ± 0.09*

site. Binding experiments have shown that N54W,
N141Q and N54W-N141Q also display higher affinity
towards some other polysaccharides such as cellulose
and barley b-glucan than the wild-type XBS (results
not shown). This might indicate that these mutations
create a ‘sticky patch’ causing aspecific binding to all
kinds of substrates, rather than enhancing the specific
binding of xylan substrates in a correct orientation to
help provide the catalytic site with substrate for
hydrolysis.
XAN
Affinity towards both water-unextractable AX and
OSX is decreased upon modification of the SBS of
XAN. The wild-type XAN has lower K
d
values than
mutants that weaken the SBS, as well as mutants
aimed at creating a SBS with increased substrate bind-
ing affinity. The K
d
of wild-type XAN is 24 mgÆmL
)1
for water-unextractable AX and 3.8 mgÆmL
)1
for
OSX. The Y29A mutation increases the K
d
values to
93 mgÆmL
)1

100
0 10203040
Enzyme bound to substrate (%)
OSX (mg·mL)
LEGEND
Wild-type XAN
XAN Y29A
XAN E31A
XAN Y29A-E31A
XAN D16A-Y29A-E31A
LEGEND
Wild-type XBS
XBS W185A
XBS G56A-T183A-W185A
XBS N54W
XBS N54W-N141Q
A
0
20
40
60
80
100
0 10203040
Enzyme bound to substrate (%)
OSX (mg·mL)
B
D
WU-AX (mg·mL)
0

function of the SBS is not merely to bring the enzyme
into contact with insoluble substrates, as is often pro-
posed as one of the functions of CBMs [1,2]. The SBS
possibly has a more pronounced role, such as assisting
catalysis by leading the substrate into the active site.
Relevance of the present findings
Many glycoside hydrolases contain one or more CBMs
that are considered to function as an aid to target sub-
strates and to keep enzymes in proximity with their
substrate [1,2]. The discovery of a SBS in two single
domain xylanases from B. subtilis and A. niger led to
the suggestion that these structures compensate for the
lack of CBMs in these enzymes [12]. In the B. circulans
xylanase, it was found that the SBS assists the active
site by binding larger substrates cooperatively, thereby
facilitating their hydrolysis [13]. In the present study,
the demonstrated effects of modification in the SBS of
XBS and XAN on binding affinities, as well as on
activity, indicate that these sites are of significant
importance for the enzymes. Previously, the deletion of
CBMs from (or fusion of CBMs to) xylanases was
shown to lead to lower activities in the absence of the
CBM comparable to those seen for the elimination of
the SBS in the present study [26–28]. In most studies,
however, the presence of CBMs is correlated with a
higher activity on insoluble substrate, whereas the
activity on soluble substrate is often unaffected [26,27].
However, in the present study, the presence of a SBS
gives rise to higher activities on all tested polymeric
substrates (i.e. both water-extractable and water-

several xylanases of other Bacillus species. Further-
more, it is possible that other GH11 xylanases also
contain undiscovered SBS in different regions of the
enzyme. The concept of a SBS in GH11 xylanases
demonstrated in the present study is possibly also valid
for other single domain xylanases. As noted in the
Introduction, the presence of a SBS has also been sug-
gested in GH8 and GH10 xylanases [10,11]. Future
work on these enzymes will aim to clarify whether the
SBS has the same functional relevance for these
enzymes.
Conclusions
Screening of a large set of XBS and XAN with a mod-
ified SBS clearly established that the SBS raises the rel-
ative activity of single domain xylanases on polymeric
versus oligomeric substrate. Activity measurements on
purified enzymes confirmed these findings. For XBS,
the activity on X
6
is independent of the strength of
the SBS; for XAN, this is probably also the case,
although, for a few mutations, the overall activity was
seriously decreased. Although the differences are small,
the efficiency of the SBS in XBS and XAN appears to
be higher for water-extractable substrate, probably
because it is less rigid or more accessible than water-
unextractable substrate, which is tightly associated
with other components in the cell wall matrix.
Secondary substrate binding in GH11 xylanases S. Cuyvers et al.
1106 FEBS Journal 278 (2011) 1098–1111 ª 2011 The Authors Journal compilation ª 2011 FEBS

15 min at 4 °C. After centrifugation (1500 g for 10 min),
the residue was boiled for 30 min in 20 mL water per milli-
gram of starting material. After a new centrifugation step
(11000 g for 30 min), the remaining soluble components
were removed from the residue by shaking in 20 mL of
water per milligram of starting material for 15 min at room
temperature. After centrifugation (11000 g for 30 min), the
insoluble fraction was lyophilized.
Site-directed mutagenesis
Expression plasmid pEXP5-CT-xyna was used for heterolo-
gous expression of XBS (UniProtKB P18429) in E. coli
[31]. For the heterologous expression of XAN (differing in
three amino acids from UniProtKB P55329, namely K50N,
E57D and M167V) in P. pastoris , the pPicZaC-exlA plas-
mid was used [32]. In both plasmids, a stop codon was
incorporated after the last nucleotide encoding for the C-
terminal amino acid of the native protein (Trp185 and
Ser184 for XBS and XAN, respectively). All mutants were
constructed using a QuikChange site-directed mutagenesis
kit (Stratagene, La Jolla, CA, USA). The template DNA
and oligonucleotide primers used are shown in Table S1.
E. coli TOP10 cells (Invitrogen, Groningen, the Nether-
lands) were transformed with the modified plasmids (3 lL)
by heat shock (30 s at 42 °C). Success of mutagenesis was
verified by sequence analysis (Genetic Service Facility, VIB,
Wilrijk, Belgium).
Recombinant expression
XBS
E. coli * BL21 (DE3) pLysS cells transformed with pEXP5-
CT-xynA (or mutant constructs) were used to express XBS

XBS
XBS and its mutant variants were purified from the E. coli
cell lysates with cation exchange chromatography, as previ-
ously described by Pollet et al. [33]. To remove the last con-
taminating proteins, an additional gel filtration step was
performed on a Sephacryl S-100 column (GE Healthcare,
Uppsala, Sweden) with sodium acetate buffer (250 mm,
pH 5.0) as elution buffer.
XAN
XAN and its mutant variants were purified with anion
exchange chromatography, as previously described by Van-
S. Cuyvers et al. Secondary substrate binding in GH11 xylanases
FEBS Journal 278 (2011) 1098–1111 ª 2011 The Authors Journal compilation ª 2011 FEBS 1107
dermarliere et al. [12]. Xylanase containing fractions were
pooled and dialyzed against a sodium acetate buffer
(200 mm, pH 4.0).
XBS and XAN
All purified XBS, XAN and mutant variants were free
from protein impurities as verified by SDS ⁄ PAGE and
silver staining was performed on a PhastSystem Unit
(GE Healthcare) (Fig. S2).
Protein quantification
XBS
The protein concentration of purified XBS samples was
determined by measurement of E
280
with a Nanodrop-1000
spectrophotometer (Thermo Fisher Scientific, Waltham,
MA, USA) using molar extinction coefficients calculated
with protparam software (http://expasy.org/tools/protpa-

McIlvaine buffer at pH 4.0. Both buffers contained
0.50 mgÆmL
)1
BSA. The incubation time in all assays was
60 min and the pre-incubation and incubation temperature
was 40 °C. The stability of the different purified enzymes in
these measurement conditions was tested to avoid errone-
ous conclusions with respect to differences in activity
(Fig. S1). The substrates used in these measurements were
X
6
, Xylazyme AX, Azo-wheat AX, water-unextractable AX
and OSX. X
6
is a soluble, linear, oligomeric substrate.
Xylazyme AX and Azo-wheat AX are polymeric chromo-
phoric AX that are water-unextractable and water-
extractable, respectively. Water-unextractable AX is a
substrate isolated from wheat flour. Its water-unextractabil-
ity is mainly a result of ferulic acid cross-links between AX
molecules and interactions with other cell wall components.
OSX has a low degree of substitution and is considered to
be insoluble as a result of partial alignment of unsubstitut-
ed regions [35]. The water-unextractable AX used in the
present study had an average arabinose ⁄ xylose ratio of 0.60
and the average arabinose ⁄ xylose ratio of the OSX was
0.08, as assessed by hydrolysis of the materials and analysis
of the noncellulosic monosaccharide content by GC follow-
ing derivatization, as described previously [36]. For mea-
surements on X

over 30 min was used for elution (1.0 mLÆmin
)1
). A stan-
dard solution containing xylooligosaccharides (xylose to
X
6
) and rhamnose was used to identify and quantify the
hydrolysis products. One unit of enzyme activity on X
6
(X
6
U) corresponds to the enzyme concentration needed
for the formation of 1.0 lm xylotriose from excess X
6
under the conditions of the assay.
Secondary substrate binding in GH11 xylanases S. Cuyvers et al.
1108 FEBS Journal 278 (2011) 1098–1111 ª 2011 The Authors Journal compilation ª 2011 FEBS
Activity on Xylazyme AX
After 10 min of pre-incubation, a Xylazyme AX tablet was
added to 1.0 mL of an appropriate enzyme dilution. The
reaction was terminated by the addition of 10.0 mL of Tris
solution (1.0% w ⁄ v), vigorous vortex-mixing and immediate
filtration. E
590
of the filtrate was measured. One unit of
enzyme activity on Xylazyme AX (XyU) corresponds to the
enzyme concentration required to obtain E
590
= 1.0 after
subtraction of the control value (no enzyme) under the con-

ren, Ger-
many). To avoid alkaline solubilization of water-
unextractable AX, the reaction with water-unextractable
AX was terminated by first separating the nonsolubilized
from the solubilized material by filtration and by then
immediately mixing the filtrate with 230 lL of 4.00 m
NaOH. The monosaccharide content in the filtrate was ana-
lyzed by GC following hydrolysis and derivatization, as
previously described by Gebruers et al. [36].
Temperature and pH optima
The optimal temperature for enzyme activity under the con-
ditions of the activity measurement assays was determined
by measuring activity on Xylazyme AX, under the condi-
tions described above, using different incubation tempera-
tures, in the range 30–60 °C at temperature intervals of
5 °C. The optimal pH for enzyme activity was determined
by measuring activity on Xylazyme AX, under the condi-
tions described above, in McIlvaine buffers with different
pH, in the pH range 4.5–7.5 for XBS and 2.5–6.5 for XAN
at intervals of 0.5 pH.
Binding affinity towards insoluble polymers
To study the binding affinity of enzymes towards insoluble
substrates, 1.0 mL of enzyme solution (91.4 · 10
)10
m),
diluted in the same buffer as that used for activity mea-
surements, was incubated for 10 min on ice with different
substrate concentrations. The 14 concentrations used var-
ied in the range 0–40.0 mg for water-unextractable AX
and 0–30.0 mg for OSX. The mixtures were then centri-

Brussels, Belgium) is gratefully acknowledged for the
postdoctoral fellowship of E. Dornez. This study is
part of the Methusalem programme ‘Food for the
Future’ at the Katholieke Universiteit Leuven.
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Supporting information
The following supplementary material is available:
Fig. S1. Enzyme stability under the conditions of the
activity assays.
Fig. S2. Purity of XBS, XAN and their mutant vari-
ants as verified by SDS ⁄ PAGE and silver staining.
Table S1. Summary of template DNA and oligonu-
cleotide primers used in site-directed mutagenesis for
the genetic engineering of XBS and XAN.
This supplementary material can be found in the
online version of this article.
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from supporting information (other than missing files)
should be addressed to the authors.
S. Cuyvers et al. Secondary substrate binding in GH11 xylanases
FEBS Journal 278 (2011) 1098–1111 ª 2011 The Authors Journal compilation ª 2011 FEBS 1111