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Enzyme and Microbial Technology 91 (2016) 1–7

Contents lists available at ScienceDirect

Enzyme  and  Microbial  Technology

j o ur na l ho mepage: www.elsev ier .com/ locate /emt

iophysical  and  biochemical  studies  of  a  major  endoglucanase
ecreted  by  Xanthomonas  campestris  pv.  campestris.

lávio  Rodolfo  Rossetoa,  Livia  Regina  Manzinea, Mario  de  Oliveira  Netob,
gor  Polikarpova,∗

Instituto de Física de Sa˜o Carlos, Universidade de Sa˜o Paulo, Avenida Trabalhador São Carlense 400-Centro, São Carlos—SP 13560-970, Brazil
Instituto de Biociências, Universidade Estadual Paulista, Distrito de Rubião Jr. s/n, Botucatu—SP 18618-970, Brazil

 r  t  i  c  l  e  i  n  f  o

rticle history:
eceived 30 December 2015
eceived in revised form 14 May  2016
ccepted 20 May  2016
vailable online 21 May  2016

eywords:

a  b  s  t  r  a  c  t

Endoglucanases  are  the  main  cellulolytic  enzymes  secreted  by the  bacterium  Xanthomonas  campestris
pv.  campestris  (Xcc).  The  major  endoglucanase  exported  by this  bacterium  into  an external  milieu  is
an  enzyme  XccCel5A,  which  belongs  to  GH5  family  subfamily  1  and is  encoded  by the  gene  engXCA.
We  purified  XccCel5A  using  ammonium  sulfate  precipitation  followed  by size  exclusion  chromatogra-
phy  and  identified  it by  zymogram  analysis.  Circular  dichroism  and  fluorescence  spectroscopy  studies
showed  that  XccCel5A  is  stable  in a wide  pH range  and  up  to about  55 ◦C  and  denatures  at  the  higher tem-

◦

anthomonas campestris
ndoglucanases
ydrolysis
AXS

peratures.  The  optimal  conditions  for enzyme  activity  were  identified  as  T = 45 C  and  pH  =  7.0. Under  the
optimum  conditions  the catalytic  efficiency  (kcat/KM)  of the  enzyme  was  determined  as 5.16  ×  104 s−1 M−1

using  carboxymethylcellulose  (CMC)  as  a substrate.  Our  SAXS  studies  revealed  extended  tadpole-shape
molecular  assembly,  typical  for  cellulases,  and  allowed  to determine  an overall  shape  of  the enzyme  and
a relative  position  of  the  catalytic  and  cellulose  binding  domains.

©  2016 Elsevier  Inc.  All  rights  reserved.
. Introduction

Traditional sources of hydrocarbon energy such as coal, oil and
atural gas are limited and non-renewable. In this context, the
rowth of the world population coupled with increasing demand
or food and energy continuously intensifies interest in renewable
ources of energy, such as plant biomass and agro-industrial waste
nd their conversion into fuels, chemicals and renewable mate-
ials [1,2]. The biomass to cellulosic ethanol conversion normally
ncludes four major steps: biomass pre-treatment and enzymatic
ydrolysis of (hemi)cellulose fractions into simple sugars; their

ermentation to ethanol and, finally, ethanol separation and purifi-
ation, usually by distillation [3]. The classical view of cellulose
ydrolysis depictures a depolymerization process as being initi-
ted by endoglucanases reducing the degree of polymerization of
he substrate and releasing new cellulose chain ends which are

argets for exoglucanases [4,5]. Due to their tunnel-shaped active
ite, the exoglucanases have much higher processivity than typical
ungal endoglucanases, that have more open active side cleft [6,7]

∗ Corresponding author at: Universidade de São Paulo, Departamento de Física
 Ciência Interdisciplinar, Instituto de Física de São Carlos, Av. Trabalhador
ão−carlense, 400, São Carlos, SP 13560−970, Brazil.

E-mail address: ipolikarpov@ifsc.usp.br (I. Polikarpov).

ttp://dx.doi.org/10.1016/j.enzmictec.2016.05.007
141-0229/© 2016 Elsevier Inc. All rights reserved.
and their joint enzymatic action leads to a substantial synergism in
hydrolysis of crystalline cellulose substrates [8,9]. The hydrolysis
is terminated by �-glucosidases that enzymatically cleave released
cellobiose units into glucose. However, recent discoveries of lytic
polysaccharide monooxygenases (LPMOs) reveal that the canoni-
cal view of cellulose enzymatic hydrolysis might be somewhat over
simplified and incomplete [10].

Most of the bacterial endoglucanases belong to the glycoside
hydrolase (GH) families GH5, GH6, GH8, GH9 and GH12 [11,12].
Even though they hydrolyze the same cellulose substrate, they act
using differing catalytic mechanisms: “inverting” for GH6, GH8 and
GH9 family members and “retaining” for GH5 and GH12 endoglu-
canases. Bacterial endoglucanases are diverse in their molecular
structure, substrate specificity and the mode of action. Some of
the bacterial endoglucanases are processive and contain catalytic
domain rigidly bound to cellulose binding module [13–15]. Several
anaerobic cellulolytic bacteria produce multicomponent, multien-
zyme cellulosome complexes [16] whereas some other produce
cellulases that are multi-domain enzymes comprised of endo- and
exoglucanase catalytic domains linked to cellulose-binding mod-
ules, that “drill” into cellulose substrate instead of eroding it from

the surface [17]. Finally, various endoglucanases are also enzymat-
ically active against a number of hemicellulose polysaccharides,

dx.doi.org/10.1016/j.enzmictec.2016.05.007
http://www.sciencedirect.com/science/journal/01410229
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mailto:ipolikarpov@ifsc.usp.br
dx.doi.org/10.1016/j.enzmictec.2016.05.007


2  Micro

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 F.R. Rosseto et al. / Enzyme and

hich could be related to their function in degradation of complex
ignocellulosic biomass [18,19].

Xanthomonas campestris pv.  campestris (Xcc) is a plant
athogenic bacterium and its genome analysis revealed presence
f several genes encoding enzymes involved in the degradation of
lant cell walls including pectinases, lyases, xylanases and nine cel-

ulases [20]. This indicates that the bacterium is able to secrete
arious enzymes that degrade structural polysaccharides such as
ellulose and hemicellulose [21]. Xcc secretes two endoglucanases
nd the endoglucanase XccCel5A, encoded by the gene engXCA,
s the most abundant cellulase produced by the bacterium [22].
his endoglucanase belongs to GH5 family subfamily 1 [11] in the
AZy database [12]. BLASTP2.2.25+ program [23] analysis based on
he available enzyme sequence (NCBI: NP 638867.1) shows that
ccCel5A is composed by two domains: an N-terminal catalytic
omain (CD) and a carbohydrate binding type-2 module (CBM),

oined together by a Thr-Pro-rich linker peptide of about 30 amino
cid residues long.

Given importance of Xcc endoglucanases in biotechnological
pplications (such as production of xanthan gum and, potentially,
ellulosic ethanol) and also for plant pathogenicity of the bacteria,
ere we performed biophysical, biochemical and enzymatic char-
cterization of the major endoglucanase secreted by Xanthomonas
ampestris pv.  campestris and retrieved its low-resolution SAXS
odel in solution.

. Materials and methods

.1. Bacteria culture and production of endoglucanases

The strain of Xcc (ATCC 33913) was kindly provided by Prof.
haker C. Farah (IQ/USP, São Paulo, Brazil). Xcc cells, maintained
n 20% of glycerol, were transferred onto 100 �g mL−1 ampicillin-
ontaining LB medium plates and grown for 3–5 days at 30 ◦C. Then,
ells were grown for 16 h in 50 mL  of ampicillin-containing LB
edium at 30 ◦C at constant agitation of 250 rpm. As a next step,

he culture was transferred into a 500 mL  of the same medium and
ept at same conditions for further 36 h.

.2. Identification of Xcc major endoglucanase

In order to identify the protein of interest based on its enzymatic
ctivity, we performed zymogram analysis using 10% SDS-PAGE
upplied with 0.1% of carboxymethylcellulose (CMC) [24]. Briefly,
he polyacrylamide gel was separated in two parts: one containing
eat-denatured protein with reducing agent and another non-
eated protein without reducing agent. After electrophoresis at
0 ◦C, the denatured part was stained with Coomassie Brilliant Blue
-250 and the undenatured part was incubated with 2.5% (v/v)
riton X-100 solution (Sigma) for 30 min  followed by 2 washes
ith 50 mM sodium citrate pH 5.0 and kept at 50 ◦C for 15 min.

inally, this part of the gel was stained with 0.1% (w/v) Congo Red
nd destained with 1 M NaCl until the bands were revealed. To
onfirm which band corresponded to XccCel5A protein, the bands
ere cut off from the gel and digested with trypsin (Sigma) [25].

he resulting peptides were analyzed at an Electrospray Tandem
S/MS  (Q-TOF) mass spectrometer at the National Laboratory of

ynchrotron Light (LNLS, Brazil) and the program MASCOT (www.
atrixscience.com) was used for protein identification.

.3. Purification of XccCel5A
Xcc culture was centrifuged and the supernatant was  concen-
rated by tangential ultrafiltration using HollowFiber system (GE)
ith 10 kDa cutoff cartridge in 50 mM Tris–HCl pH 8.0. Twenty
ercent of ammonium sulfate was added to the extract to induce
bial Technology 91 (2016) 1–7

precipitation of contaminants. After centrifugation, 40% (w/v)
ammonium sulfate was  added to supernatant followed by cen-
trifugation at 13,000g for 20 min  to precipitate the enzyme. The
precipitate was resuspended in 50 mM Tris–HCl pH 8.0 and injected
in the molecular size exclusion column Sephadex G-25 Superfine
(GE) equilibrated in the same buffer. The protein concentration was
determined using the theoretical extinction coefficient at 280 nm
(117225 M−1 cm−1) calculated by ProtParam tool [26].

2.4. Thermal, pH and catalytic activity assays

Optimum temperature and pH experiments were performed
using carboxymethylcellulose (CMC) with degree of polymeriza-
tion of 400 as a substrate. For pH-dependence studies the pH was
varied from 2 to 10 and the temperature was maintained con-
stant at 50 ◦C; for thermal analysis the temperature was changed
from 30 to 80 ◦C in predetermined pH [27]. For these assays, we
used final concentrations of 400 nM of enzyme, 0.25% (w/v) of CMC
and 50 mM of the appropriate buffer in 100 �L volume reactions.
Kinetic parameters (kcat, Vmax and KM) against CMC were deter-
mined at 45 ◦C using 200 nM of XccCel5A in 50 mM Tris–HCl pH
7.0 by varying the concentration of low-viscosity CMC from 0.05 to
4% (w/v). The mixtures were incubated for 30 min  and the released
sugars were measured using 3,5-dinitrosalicylic acid (DNS) method
[28]. The data represent average of triplicate experiments and their
analyses were performed using SigmaPlot 10.0 program.

2.5. Circular dichroism (CD) and fluorescence spectroscopy
analyzes

Temperature and pH variation assays were independently mon-
itored by circular dichroism technique. Temperature denaturation
assays were conducted in a 20–80 ◦C range using 2.5 �M of enzyme.
Experiments in pHs 2.6, 4.0, 4.5, 6.0 (20 mM Na2HPO4/citric acid),
8.0 (20 mM  Na2HPO4/NaH2PO3) and 10.0 (20 mM glycine) were
conducted at 25 ◦C after incubation of the enzyme in these buffers
for 16 h. CD spectra were recorded on a Jasco J-720 spectral
polarimeter (Jasco, Tokyo, Japan) using 0.1 cm path length cuvette
within 195–245 nm wavelength range, 100 nm min−1 increment
step, 10 s averaging time, 1 nm bandwidth and a response time
of 0.5s. The same conditions of pH assays were used for fluores-
cence spectroscopy measurements using a K2 spectrofluorimeter
(ISS Inc Champaign, IL—USA). The samples were excited at 295 nm
and the fluorescence emission was monitored in the range from
300 to 450 nm,  at 25 ◦C. The buffer contribution was subtracted.

2.6. SAXS experiments and data analysis

SAXS experiments were performed with XccCel5A at
3.5 mg  mL−1 in 50 mM Tris–HCl pH 7.0 at the D02A-SAXS2
beamline of the Synchrotron Light National Laboratory (Campinas,
Brazil). The measurements were conducted using a monochromatic
X-ray beam with a wavelength � = 1.488 Å. The sample-to-detector
distance was  set at 1028.4 mm,  resulting in a scattering vector q
range of 0.041 Å−1 to 0.325 Å−1, where q is defined by q = 4�sin �/�
(2� is the scattering angle). All the X-ray patterns were recorded
using a two-dimensional CCD detector (MarResearch, USA). Pro-
tein samples were centrifuged at 23,500g for 15 min  at 4 ◦C to
remove aggregates. For SAXS measurements, protein samples
were introduced into a 1 mm path length cell with mica windows
at 10 ◦C. Two  successive frames of 300 s each were recorded for
each sample to monitor radiation damage and beam stability.

Buffer scattering was recorded before the sample scattering. SAXS
curves were corrected for the detector response, intensity of
the incident beam, sample absorption and buffer scattering. The
two-dimensional patterns were integrated in 2� resulting in an

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 Microbial Technology 91 (2016) 1–7 3

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Fig. 2. XccCel5A protein purification using ammonium sulfate precipitation and
size  exclusion chromatography (SEC). (A) Purification steps of XccCel5A analyzed
by  depositing 15 �L samples on SDS-PAGE (10%) and staining them with Comassie
Brilliant blue. Lane 1, molecular weight marker (kDa). Lane 2, total extracellular
proteins of Xcc. Lane 3 and 4, protein precipitation using 20 and 40% of ammonium
sulfate, respectively. Lane 5, first peak of SEC chromatography. Lane 6, second peak
F.R. Rosseto et al. / Enzyme and

veraged one-dimensional scattering curve. The radius of gyra-
ion, Rg was approximated using two different and independent

ethods: by Guinier’s equation [29] and by of the inverse Fourier
ransform method [30] as implemented in the Gnom package. The
istance distribution function p(r) also was evaluated by Gnom and
he maximum diameter of the protein Dmax was obtained. Dummy
tom models (DAMs) were calculated from the experimental SAXS
ata of XccCel5A using ab initio procedure implemented both in
ammin [31] and Gasbor packages [32]. For the two  XccCel5A main
omains (CD and CBM), tertiary structures of homologous proteins
ith highest amino acid identity were positioned to best fit the
olecular envelope. The 3D structures of �-1,4-glycanase cellulose

inding module from Cellulomonas fimi  (PDB id: 1EXG) [33] and
he catalytic domain of endoglucanase from archaeon Pyrococcus
orikoshii (PDB id 2ZUM) [34], respectively, were used, and the
elative adjustment of the structures against the experimental
cattering data was calculated by SASREF [35]. The SAXS-based
stimation of the molecular weight (MW)  was obtained using the
eb tool SAXS MoW  (www.ifsc.usp.br/∼saxs/saxsmow.html) [36].

. Results and discussion

.1. Identification and purification of the XccCel5A, the major
ndoglucanase from Xanthomonas campestris pv.  campestris

Supernatant produced by Xcc was first subjected to SDS-PAGE
nalysis. The theoretical molecular weight of XccCel5A computed
rom its amino acid sequence (NCBI: NP 638867.1) is close to
9.5 kDa, which is consistent with our experimental analyses using
f SDS-PAGE and zymogram methodologies (Fig. 1A and B). To con-
rm the identity of the endoglucanase XccCel5A, we  achieved mass
pectrometry analysis of the 49.5 kDa band. The obtained mass
pectra had an excellent coverage of protein amino acid sequence
approximately 55%) [25], allowing us to consistently conclude that
he XccCel5A was successfully identified among the other proteins
xpressed in the extracellular medium.

The first purification step of the protein was performed using
ith 20% (w/v) of ammonium sulfate for precipitation of contam-
nating proteins and xanthan gum. The endoglucanase XccCel5A
nly precipitated after the addition of 40% ammonium sulfate
Fig. 2A). Once resuspended in 50 mM Tris-HCl pH 8.0, the enzyme
as subjected to molecular size exclusion chromatography using

ig. 1. Total extracellular proteins produced by Xcc. (A) Different concentrations
f  the samples were analyzed by SDS-PAGE (10%) stained with Comassie Brillant
lue. Lane 1, molecular-weight marker (66, 45, 36, 29, 24 and 14 kDa). Lane 2, 3
nd 4, XccCel5A at 0.2 mg mL−1. The dashed box indicates the XccCel5A protein.
B)  Zymogram of extracellular proteins against CMC  substrate, showing XccCel5A
osition. Lane 5, 6 and 7, XccCel5A at 0.2 mg  mL−1.
of  SEC chromatography. (B) SEC profile of XccCel5A protein after 40% ammonium
sulfate precipitation, using Sephadex G-25 superfine column (GE) as monitored at
280  nm.

Sephadex G-25 Superfine column (GE). The enzyme eluted in two
peaks (as followed by absorption at 280 nm; Fig. 2B). The first
elution peak contained the enzyme together with contaminants;
however the second peak consisted only by pure XccCel5A. The
estimated experimental yield was 1 mg  of purified enzyme per liter
of culture.

3.2. XccCel5A pH and temperature optima

As a next characterization step, the optimum pH and tempera-
ture conditions for XccCel5A enzymatic activity against CMC were
determined. The pH dependence of XccCel5A activity (Fig. 3A)
reveals that the enzyme has highest activity in neutral pHs around
7.0. This is different from pH optimum of many fungal cellulases
that frequently have acidic pH optimum (typically between pHs 5.0
and 6.0) [9]. However such pH behavior is not unusual for bacte-
rial cellulases, many of which have highest activity at neutral pHs
[37,38]. Furthermore, the enzyme has significant activity (above
50% of its maximum) against CMC  in a wide pH range (between

4.5–9.0). A similar assay was also used to determine the optimum
temperature for catalytic activity. The temperature was varied in a
range of 30–80 ◦C while maintaining the optimum pH 7.0. Fig. 3B
shows the influence of temperature on XccCel5A activity against

http://www.ifsc.usp.br/~saxs/saxsmow.html
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4 F.R. Rosseto et al. / Enzyme and Micro

Fig. 3. Optimum pH, temperature and kinetic assays for XccCel5A activity against
CMC. (A) pH dependence of XccCel5A activity. The peak at pH 7.0 indicates the best
pH for the enzyme activity. (B) Thermal analysis of XccCel5A performed from 30 to
80 ◦C in pH 7.0 revealing the optimum temperature for XccCel5A activity (45–50 ◦C
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more recent electron microscopy studies defined T. reesei Cel7A and
pproximately). (C) CMC  concentration effect on the reaction rate for kinetic param-
ters determination of XccCel5A.

MC, revealing 45 ◦C as the optimum temperature for its enzymatic
ctivity. Moreover, these results indicate that the enzyme exhibits
ood thermal stability since its activity decreased only by 20% in the
emperature range between 30 and 60 ◦C. Furthermore, the enzyme
as active against Avicel, indicating its activity against crystalline

ellulosic substrates (results not shown).
We  also studied kinetic behavior of XccCel5A against

MC  under the optimum temperature and pH conditions.
inetics of the enzyme follows Michaelis-Menten model
nd application of Lineweaver-Burk linearization method to
he experimental data allowed the determination of kinetic
arameters KM = 4.50 ± 0.804 mg  mL−1 and kcat = 273.60 min−1

Fig. 3C). Maximum velocity (Vmax) of the enzyme is equal
o 0.05 ± 0.00186 �mol  min−1 mL−1 and its catalytic efficiency
kcat/KM) amounts to 60.8 mL  mg −1 min−1. The KM value is compa-
able with KM = 4.8 mg  mL−1 for �-1,4-endoglucanase from a novel
acterial Bacillus sp. strain CTP-09 [39] and considerably lower than
M values for Martelella mediterranea and Talaromyces emersonii
ndoglucanases, which were determined as 28.4 mg  mL−1 and

1.7 mg  mL−1, respectively, against the same substrate [40,41].
esides, XccCel5A catalytic efficiency is similar to (kcat/KM) of
6.9 mL  mg−1 min−1 and 83.7 mL  mg−1 min−1 determined for
bial Technology 91 (2016) 1–7

GH5 family endoglucanases from Martelella mediterranea and
Thermoanaerobacter tengcongesis, respectively [40,42].

3.3. Circular dichroism (CD) and fluorescence spectroscopy

Next, we analyzed XccCel5A stability against temperature using
circular dichroism (CD) technique. By altering a temperature in the
range from 20 to 90 ◦C and measuring CD signal, the denaturation
process was  found to be irreversible (data not shown), with the
Tm value of 69.2 ◦C (Fig. 4A). Furthermore, the temperature vari-
ation monitored by CD showed that the thermal stability of the
enzyme was  maintained up to approximately 55 ◦C, and the accen-
tuated plateau above 75 ◦C indicates a complete denaturation of the
enzyme. This is consistent with the results of temperature depen-
dence of XccCel5A enzymatic activity that shows decline in the
activity at temperatures above 55 ◦C, which probably reflects begin-
ning of the protein unfolding. Analysis of CD spectra for different
pHs (Fig. 4B) shows that the enzyme retains its folding similar to
the native in basic pHs values. Under extremely acidic pHs (i.e. 2.6)
there was  a considerable decrease in ellipticity values indicative of
unfolding of the macromolecule and loss of the secondary structure.

The tertiary structure of XccCel5A was  also assessed by trypto-
phan intrinsic fluorescence. The emission spectrum of tryptophan is
very sensitive to the local environment thus providing information
about protein folding at the tertiary structure level. Upon excitation
at 295 nm,  the XccCel5A fluorescence emission spectra have maxi-
mum  at the wavelength of approximately 335 nm for the most pHs
tested (Fig. 4C). However there was a shift to 328 nm in the flu-
orescence emission maximum when the protein was  exposed to
extremely acidic pHs, indicating changes of the tryptophans local
environments.

3.4. Small angle X-ray scattering studies of XccCel5A

To determine oligomeric state of XccCel5A in solution and to
retrieve its low-resolution molecular envelope, we performed its
SAXS analysis. Experimental SAXS curve and simulated scattering
curves from the rigid body (RBM) and dummy  atom model (DAM)
analysis are shown in Fig. 5A. The p(r) curve for XccCel5A is elon-
gated, defining a Dmax as 115.00 ± 0.50 Å (Fig. 5B). Using SAXS MoW
package [36] we calculated the approximate molecular weight of
the enzyme using experimental SAXS data. This analysis estimated
a molecular weight of XccCel5A as 50.9 kDa, which is almost exactly
equal to the theoretically calculated molecular weight based on the
amino acid sequence of the enzyme with a difference of only 2.8%.

The three-dimensional DAM was  generated ab initio from the
experimental SAXS data. To check the uniqueness of the model,
independent simulations starting from different initial parameters
were performed. With these models generated and superimposed
with known homologous crystallographic structures, we  confirmed
that the enzyme has an elongated tadpole-like molecular shape
with two  distinct portions accommodating, respectively, the cat-
alytic domain and the CBM with a linker region between them
(Fig. 6). Similar molecular shapes have already been reported for
other cellulases [43–49]. Structural parameters for the XccCel5A
obtained from SAXS analysis are summarized in Table 1.

The size of the XccCel5A (Dmax = 115 Å) is almost the same as
estimated from SAXS studies for T. harzianum Cel7A (Dmax = 110 Å)
[46] and its molecular model is quite similar in its shape to the
latter enzyme. Earlier reports described significantly larger molec-
ular sizes for C. fimi endoglucanase (Dmax = 210 Å), T. reesei Cel7A
(Dmax = 180 Å) and T. reesei Cel6A (Dmax = 215 Å) [43–45], although
Cel6A Dmax as 151 ± 13 Å and 134 ± 26 Å, respectively [48]. Why  the
earlier reports show considerably larger molecular sizes for cellu-
lases is no completely clear, although a small amount of aggregation



F.R. Rosseto et al. / Enzyme and Microbial Technology 91 (2016) 1–7 5

Fig. 4. Biophysical analysis of XccCel5A. (A) Transition curve of XccCel5A ther-
mal  denaturation monitoring changes in CD signal at 215 nm as a function of
temperature (20–90 ◦C). (B) CD spectra of XccCel5A at different pHs at 25 ◦C. (C)
Intrinsic fluorescence emission spectra of XccCel5A enzyme at different pHs. Emis-
sion  spectra in the range of 300–450 nm were recorded at 25 ◦C using an excitation
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Fig. 5. SAXS analysis of XccCel5A. (A) Scattering experimental curve of Xcc major
endoglucanase (©) simulated scattering intensity for the DAM (dashed line) and
simulated scattering intensity from the rigid body model composed of the homol-
ogous cellulose binding module and catalytic domain (solid line). (B) Distance
distribution function computed from the experimental SAXS data for XccCel5A
enzyme.

Table 1
Structural parameters derived from SAXS for XccCel5A.

Exp.a DAMb RBMc

Dmax (Å) 115.0 ± 0.50 116 108.8
Rg (Å) 33.98 ± 0.50 33.19 31.55
Resolution (Å) 19.31 – –
MW  (kDa) 50.9 – –

a

ing to pHs 5.3 and 4.2 are clearly different from the ones determined
avelength of 295 nm,  Buffers contributions have been subtracted. The results are
n average of three independent experiments.

resent in the SAXS samples and/or lower fluxes of X-rays avail-
ble at that time might have contributed to lesser precision of the
easurements [43–45].

Furthermore, recent small-angle neutron scattering (SANS)

tudies of T. reesei Cel7A revealed pH-dependent conformational
hanges of the shape of the enzyme with the average Dmax vary-
Exp., calculated from experimental data.
b DAM, Parameters of the dummy atom models from the DAMMIN package.
c RBM, Parameters calculated from rigid body model from bunch package.

ing between 90 Å (pHs 7.0 and 6.0), 100 Å (pH 4.2) and 110 Å (pH
5.3) [49]. The results of these SANS investigations reveal that at
the acidic pH optimum (pH 4.2), the enzyme adopts a range of
partially disordered structures, which might be beneficial for the
activity of T. reesei Cel7A. Although CD spectra show no consider-
able changes in the enzyme secondary structure content at different
pHs (from pH 4.2–7.0), p(r) functions and Kratky plots correspond-
at higher pHs, indicating chances in the conformation and/or flex-
ibility of the enzyme. In particular, changes at pH 4.2 are likely to
be attributed to more flexible state of the catalytic domain. Cal-



6 F.R. Rosseto et al. / Enzyme and Microbial Technology 91 (2016) 1–7

Fig. 6. Superposition of the XccCel5A dummy  atoms model with the 3D structure of the cellulose binding module of Cellulomonas fimi  �-1,4-glycanase (PDB id: 1EXG) [22]
a d: 2ZU
a l com
(

c
a
a
f
m
b
t
r
h
o
w
7
l
t

t
m
a
t

A

c

c

d

a

C

A

d
9

[

[

[
[

[

nd the catalytic domain of endocellulase from archaeon Pyrococcus horikoshii (PDB i
s  rigid bodies to provide the best fit against the experimental SAXS curves. The fina
top  left) around y and x axes (bottom left and right, respectively).

ulations of the net charge of the enzyme catalytic domain show
n abrupt change (from −10 to 0) at pH 4.2, close to the cat-
lytic domain calculated pI equal to 4.3, which may  be the reason
or increased flexibility of the catalytic domain at this pH. Aug-

ented flexibility of the catalytic core of T. reesei Cel7A might
e beneficial for enhancing substrate access to the enzyme active
unnel and could indicate that conformational selection plays a
ole in the enzyme function [49]. Finally, not all endoglucanases
ave extended molecular conformation in solution. Recent report
n Neurospora crassa Cel45A endoglucanase revealed a monkey-
rench molecular shape enzyme with the maximum dimension of

5 Å [50]. Clearly, more expensive low-resolution studies of the cel-
ulases in solution are needed in order to understand in more detail
heir full-length molecular shape, conformation and flexibility.

In conclusion, our studies permitted the identification, purifica-
ion, structural, biochemical, and enzymatic characterization of the

ajor endoglucanase from Xanthomonas campestris pv.  campestris
nd paved the way for further molecular and enzymatic studies of
his enzyme.

uthors’ contributions

Flávio Rodolfo Rosseto—has taken part in the planning, identifi-
ation and characterization of XccCel5A;

Livia Regina Manzine—contributed to the production and purifi-
ation of XccCel5A and drafted this paper;

Mario de Oliveira Neto—contributed to SAXS experiments and
ata analysis;

Igor Polikarpov—planned the experiments, discussed the results
nd participated in the writing of the manuscript.

onflict of interest

The authors declare that no conflict of interest exists.

cknowledgments
This work was supported by Fundaç ão de Amparo à Pesquisa
o Estado de São Paulo (FAPESP) via grant numbers 2008/56255-
, 2009/54035-4, 2010/08370-3, 2010/16542-9; Conselho Nacional

[

M) [23]. The individual domains positions and orientations were mutually adjusted
bined models were rotated by 90◦ with respect to the originally chosen orientation

de Desenvolvimento Científico e Tecnológico (CNPq) via grant
number 482166/2010-0 and Coordenaç ão de Aperfeiç oamento de
Pessoal de Nível Superior (CAPES). We also thank Laboratório
Nacional de Luz Sincrotron (LNLS) for beam time and help with
SAXS data collection.

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