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International Journal of Biological Macromolecules 74 (2015) 263270

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules


journal homepage: www.elsevier.com/locate/ijbiomac

Expression of A. niger US368 xylanase in E. coli: Purication,


characterization and copper activation
Fatma Elgharbi, Hajer Ben Hlima, Ameny Farhat-Khemakhem, Dorra Ayadi-Zouari,
Samir Bejar , Ada Hmida-Sayari
Laboratoire de Microorganismes et de Biomolcules (LMB), Centre de Biotechnologie de Sfax (CBS), Universit de Sfax, Route de Sidi Mansour Km 6,
BP 1177 3018 Sfax, Tunisia

a r t i c l e

i n f o

Article history:
Received 22 July 2014
Received in revised form
10 December 2014
Accepted 11 December 2014
Available online 18 December 2014
Keywords:
Aspergillus niger US368
Xylanase
Gene cloning
Expression
Escherichia coli BL21
Copper

a b s t r a c t
The XAn11 cDNA was cloned in pET-28a(+) and the recombinant plasmid was transformed in
Escherichia coli. The His-tagged r-XAn11 was puried using Ni-NTA afnity and anion exchange chromatography. The enzyme showed a specic activity of 415.1 U mg1 and a molecular mass of 25 kDa. It
had an optimal activity at pH 5 and 50 C. It was stable in a wide range of pH and in the presence of some
detergents and organic solvents. In the presence of 3 mM Cu2+ , the relative activity of the His-tagged rXAn11 was enhanced by 54%. This is the rst work reporting that copper is a strong activator for xylanase
activity making this enzyme very attractive for future industrial applications. Molecular modeling suggests that the contact region between the catalytic site and the N-terminal His-tag fusion peptide could
be responsible for the different behavior of the native and recombinant enzyme toward copper.
2014 Elsevier B.V. All rights reserved.

1. Introduction
Xylan is one of the major components of the hemicellulose
fraction of plant cell walls and accounts for 2030% of their total
dry mass. It is covalently and noncovalently attached to cellulose, lignin, pectin and other polysaccharides to maintain cell wall
integrity [1,2]. Xylan thus belongs to the main food source of
farm animals and also represents a major component of the raw
material for many industrial processes ranging from baking to
paper production [3]. It consists of a backbone of -1,4-linked
xylopyranose residues, usually with branches of -1,3-linked larabinose and -1,2-linked d-glucopyranose [4]. -1,4-Xylanases
(EC 3.2.1.8) are the key enzymes that hydrolyze the backbone
structure of -1,4-xylans to initiate degradation of the polysaccharidic complex by microorganisms. Several microorganisms produce
multiple xylanases, implying a strategy for effective hydrolysis
of -1,4-xylan. Each enzyme may have a specialized function in
the degradation of the polysaccharidic complex. These specialized
functions of individual xylanases may be useful for applications in
the food, feed, and paper industries [5].

Corresponding author. Tel.: +216 74 440 451; fax: +216 74 440 451.
E-mail address: samir.bejar@cbs.rnrt.tn (S. Bejar).
http://dx.doi.org/10.1016/j.ijbiomac.2014.12.005
0141-8130/ 2014 Elsevier B.V. All rights reserved.

Xylanases have been classied into two families, F and G, based


on hydrophobic cluster analysis and sequence homology [6,7]. Families F and G correspond to families 10 and 11 respectively in the
numerical classication of glycosyl hydrolases [8,9]. F10 Family
are endo--1,4-xylanses with higher molecular mass than family
G11 xylanases, and presenting eight (/) barrel folds in threedimensional (3D) structure [7,10]. G11 Family are xylanases with
lower molecular masses (<30 kDa) [7,9] and are encoded as precursors composed of signal peptide and a mature xylanase. The 3D
structures of family G11 xylanases have the overall shape of a right
hand [11].
During the last decades, heterologous expression is becoming
one of the main tools for the production of industrial enzymes [12].
Many xylanase genes have been isolated, cloned and expressed in
Escherichia coli from microorganisms including fungi, bacteria, and
yeasts [1315].
Aspergillus niger is a lamentous fungus that has been shown
to secrete large amounts of efcient xylan-degrading enzymes
[3,1619], and most of them belong to G11 family.
Heterologous expression of A. niger xylanase was of interest for
the production of large quantities of a single xylanolytic enzyme.
The E. coli BL21 due to its clear genetic background, simple operation, short growth period and high expression became an attractive
expression system of foreign proteins, including those of eukaryotic
origin.

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F. Elgharbi et al. / International Journal of Biological Macromolecules 74 (2015) 263270

Previous study conducted in our laboratory described a newly


isolated A. niger strain US368 producing an endo-xylanase XAn11
with a molecular masse of about 26 kDa and a maximal xylanase
activity at pH 5 and 55 C [18].
In this study, we describe the molecular cloning and expression
of the A. niger US368 XAn11 gene in E. coli as well as the purication
and biochemical characterization of the recombinant xylanase. We
also described the differential behavior of the His-tagged r-XAn11
toward the copper ion comparing to the native enzyme.
2. Materials and methods
2.1. Microbial strains and plasmids
The strain A. niger US368 used in this study was previously isolated in our laboratory [18].
The pCR -Blunt vector (Invitrogen, USA) and E. coli Top10 (Invitrogen, USA) were used for subcloning.
E. coli BL21 ((DE3) pLysS) was used to express recombinant
protein using the expression vector pET-28a(+) (Novagen, Merck,
America).
2.2. Cloning of XAn11 cDNA
Exon I (without the signal peptide) of XAn11 gene was amplied
with PCR primers XylF (CCG GAA TTC CGG GCT CCG GAG CCT GTT
CTG G) and ASR2 (AGG TGA TAG ACT TAG AGG AGC CAG TGG TCC
AGC C), in a rst round. The ASR2 primer has 10 base pair overlapping region from 5 end of exon II. Similarly, Exon II of XAn11
gene was amplied with primers ASF2 (CTC CTC TAA GTC TAT CAC
CTA CTC TGC CCA ATA C) and XylR (TTG CGG CCG CAA TTA AGT
GGA GAT CGT GAC ACT GGC), the forward primer ASF2 has 10 base
pair overlapping region from 3 end of exon I. Polymerase chain
reactions (PCR) were carried out in a Gene Amp PCR System 2700
(Applied Biosystems). The amplication reaction mixture (50 l)
was composed of pfu DNA polymerase amplication buffer (1 nal
concentration), 10 pmol of each primer, 0.2 mM of dNTPs, 300 ng
of DNA template (recombinant plasmid pXAn11 [18]) and 2 U of
pfu enzyme (Thermo Scientic, USA). The cycling parameters were
94 C for 5 min followed by 30 cycles at 94 C for 30 s, 50 C for 45 s
and 72 C for 60 s, with a nal extension of 72 C for 10 min.
Both amplied exon I and exon II were puried and mixed in
1:1 molar ratio. These fragments were joined by the second round
of PCR with no primers. The cycling parameters were 15 cycles at
94 C for 30 s, 60 C for 60 s and 72 C for 90 s.
In the third round of PCR, the fused product was amplied with
primers XylF and XylR carrying anking restriction sites (EcoRI and
NotI respectively) to facilitate further cloning into pET-28a(+) vector. The cycling parameters were 30 cycles at 94 C for 30 s, 50 C
for 60 s and 72 C for 90 s, with a nal extension of 72 C for 10 min.
The fusion product was puried and cloned into pCR -Blunt
vector, the resulting plasmid was transformed into E. coli Top10.
The transformants were screened on LB agar supplemented with
50 g ml1 of kanamycin and the recombinant plasmid was
digested with EcoRI and NotI and cloned into pET-28a(+) vector
predigested with the same restriction enzymes.
Digestion of DNA with restriction endonucleases, separation of
fragments by agarose gel electrophoresis, ligation of DNA fragments, transformation of E. coli with plasmidic DNA and extraction
of recombinant DNA were all performed according to standard
methods described by Sambrook et al. [20].
2.3. Expression and purication of recombinant xylanase
The resulting plasmid pET-28a-XAn11 was transformed
into E. coli BL21 using the plasmid pET-28a(+) as control. The

transformants were screened on LB agar supplemented with


50 g ml1 of kanamycin and incubated at 37 C for 24 h. A single
colony was isolated and inoculated, into 5 ml of LuriaBertani
(LB) medium and cultured overnight at 37 C on a rotary shaker at
250 rpm. After that, 1 ml of overnight culture was inoculated into
100 ml of fresh medium and incubated at 37 C on a rotary shaker
at 250 rpm until the A600 nm reached 0.60.8. Then, isopropyl -d1-thiogalactopyranoside (IPTG) was added into the culture broth
to a nal concentration of 0.1 mM. The culture was subsequently
incubated for another 24 h at 20 C and centrifuged at 7000 rpm
for 10 min. The supernatant was decanted and the wet weight of
the cell pellets was determined. The cell pellets were crushed with
the same weight of powder aluminia (aluminum oxide Type A-5,
Sigma-Aldrich Co., St. Louis, MO, USA) using a pestle and a mortar at
4 C for 30 min in the presence of 100 mM PMSF (phenylmethanesulfonyl uoride; SigmaAldrich Co., St. Louis, MO, USA) prepared
in isopropyl alcohol. The mixture was suspended in 1 phosphate
buffer (Amersham Pharmacia Biotech, USA) and cell debris was
removed by centrifugation at 9500 rpm for 30 min. The clear
supernatant containing the His-tagged r-XAn11 was adjusted to
10 mM imidazole and loaded on 1 ml HisTrap Chelating Ni-afnity
column (Amersham Pharmacia Biotech, USA) equilibrated with 1
phosphate buffer (PB, 10 mM imidazole). The adsorbed proteins
were eluted using a linear gradient of imidazole (50200 mM)
and recombinant enzyme (His-tagged r-XAn11) was eluted with
100 mM imidazole. The fractions containing the xylanase activity
were pooled, dialyzed, concentrated and the pH was adjusted
to 7. The dialyzed enzyme solution was further puried by fast
performance liquid chromatography (FPLC), using a UNO Q-12
(15 mm 68 mm) anion exchange column pre-equilibrated with
25 mM phosphate buffer (pH 7). The proteins were eluted at a ow
rate of 5 ml/min by using linear NaCl gradient ranged from 0 to
1 M in the same buffer.
SDS-PAGE was performed using a 5% stacking gel and 10%
resolving gel under reducing conditions as described by Laemmli
[21]. Protein bands were visualized by Coomassie brilliant blue R250 (Bio-Rad) staining.
Protein concentration was determined using Bio-Rad protein
assay kit, based on the method of Bradford using bovine serum
albumin as the standard [22].
2.4. Assay of xylanolytic activity
The His-tagged r-XAn11 activity was measured at 50 C and pH
5. 0.5 ml of the enzyme solution, diluted in citrate buffer (0.1 M,
pH 5), was incubated for 20 min with 0.5 ml of 1% soluble birchwood xylan (SigmaAldrich Co., St. Louis, MO, USA). The amount
of reducing sugars released was determined by the dinitrosalicylic
acid (DNS) method [23], using d-xylose as standard.
One unit of xylanase activity was dened as the amount of
enzyme which produces one micromole of xylose equivalents per
minute.
2.5. Effect of metal ions and EDTA on His-tagged r-XAn11 activity
Xylanase activity was measured in the presence of several
metallic ions (Co2+ , Zn2+ , Mg2+ , Ca2+ , Cu2+ , Fe2+ and Mn2+ ) or EDTA
at 5 mM.
2.6. Effects of pH and temperature on recombinant xylanase
activity and stability
The effect of pH on His-tagged r-XAn11 activity was determined
at 50 C using the following buffer solutions at 100 mM. Buffers
used were citrate buffer (pH 35.5), phosphate buffer (pH 5.58),
TrisHCl buffer (pH 89) and glycineNaOH buffer (910). The

F. Elgharbi et al. / International Journal of Biological Macromolecules 74 (2015) 263270

stability of -glucanase was investigated over a pH range of 310


in 100 mM buffers. Aliquots were taken after 1 h of incubation at
37 C. The residual activity was determined, after centrifugation,
under standard assay conditions.
The optimum temperature for xylanase activity was determined by carrying out the enzyme assay at different temperatures
(4070 C) at pH 5. The thermal stability of the enzyme was determined by incubating the pure enzyme at different temperatures
(45, 50 and 55 C) at pH 5. Aliquots were drawn at regular time
intervals and immediately cooled in ice-cold water. The residual
activity was determined, after centrifugation, under standard assay
conditions.

2.7. Effect of additives on XAn11 stability


Solvents and detergents were incubated with the pure enzyme
for 1 h at 40 C. The nal concentration of additives in the reaction mixture was 1%. The xylanase activity was measured under
the standard test conditions.

2.8. Enzyme specicity


Recombinant xylanase activity was determined on various substrates (birchwood xylan, beechwood xylan, arabinoxylan, oatspelt
xylan, avicel and CM-cellulose). The reaction was carried out in
100 mM citrate buffer (pH 5) containing 1% of each substrate at
50 C for 20 min in the presence of 3 mM Cu2+ .

2.9. Kinetics parameters


Xylanase assays were performed using different substrates
(birchwood xylan, beechwood xylan, arabinoxylan and oatspelt
xylan), at various concentrations ranging from 0 to 2 mg ml1 .
Assays were performed for 5 min at 50 C in the presence of 3 mM
Cu2+ using a 50 g ml1 enzyme concentration in the assay mixture.
The maximum velocity (Vmax ) and the MichaelisMenten constant
(Km ) values were calculated from a Lineweaver and Burk plot using
the hyper-32 program.

265

3. Results and discussion


3.1. cDNA cloning
The entire XAn11 coding region was already cloned and
sequenced [18]. The sequence analysis showed two exons and one
intron of 51 pb. In order to express this gene, a PCR fragment of
582 pb carrying the open reading frame was generated by deleting the unique intron and the N-terminal signal sequence using
the sequence-specic PCR primers as described in Section 2. The
PCR product was cloned into pCR -Blunt vector and the resulting plasmid was transformed into E. coli Top10. The recombinant
plasmid was digested with EcoRI and NotI and directly ligated in
pET-28a(+) expression vector, in frame with N-terminal His-tags
peptide sequence, under the control of strong bacteriophage T7
promoter. Ligation products were transformed into E. coli BL21 and
the transformants were screened on LB agar supplemented with
50 g ml1 of kanamycin. Recombinant plasmids were checked
by digestion followed by sequence analysis of the inserted fragment (data not shown). Sequencing data proved that the expression
plasmid was correctly constructed. The predicted mature peptide
consisted of 229 amino acids including 36 extra amino acids due to
the presence of the His-tag fusion peptide.

3.2. Expression of XAn11 in E. coli


The recombinant plasmid pET28a-XAn11 was transformed into
E. coli BL21. Then, one single colony was cultured and induced
by IPTG. The xylanase activity assay of the resulting cell crude
extract showed a maximum specic activity of 144.6 U mg1
(78.11 U ml1 ) which was higher than that of recombinant xylanase
from A. niger XZ-3S [15] Glaciecola mesophila KMM 241 [25] and
Streptomyces thermocyaneoviolaceus [26].
When the cell extract of the recombinant xylanase was analyzed by SDS-PAGE a clear band with molecular weight of about
25 kDa (Fig. 1, lane 2) was monitored, whereas, no band of the same
size was observed in the extract from the uninduced strain (Fig. 1,
lane 1).

2.10. Polysaccharide-binding properties


The polysaccharide-binding capacity of the recombinant
xylanase was determined by the method described by Tenkanen
et al. [24]. 30 g of puried xylanase was incubated with different concentrations of avicel, arabinoxylan or birchwood xylan in
50 mM citrate buffer (pH 5) and 3 mM of Cu2+ , at 4 C for 1 h with
slow shaking. Unbound enzyme was determined by measuring
residual activity in the supernatant.

2.11. Homology modeling


The 3D structural models of the His-tagged r-XAn11 were generated using the SWISS-MODEL (http://www.expasy.org/swissmod/)
and the crystal structure of Xyn1 from A. niger (PDB accession code
1UKR) as template with which the His-tagged r-XAn11 possess
93.48% sequence identity. The automated comparative protein
structure homology modeling server, phyre 2 (Protein Homology/analogY Recognition Engine V.2) was used to analyze the
tagged model structures and construct illustrative gures. The
PyMol molecular Graphics System (DeLano Scientic, San Carlos,
CA, http://www.pymol.org.) was used to visualize the constructed
model structures and generate graphical gures.

Fig. 1. 10% SDS-PAGE of the His-tagged r-XAn11. Lanes are designated as follows:
lane M, molecular mass standard proteins; lane 1, crude cell extracts of His-tagged
r-XAn11 without induction; lane 2, crude cell extracts of His-tagged r-XAn11 with
IPTG induction; lane 3, His-tagged r-XAn11 puried by Ni-NTA afnity chromatography; lane 4, pooled fraction from the FPLC-UNO Q-12 anion exchange column.
60 g of protein was loaded in each lane of the SDSPAGE gel.

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Table 1
Summary of the purication protocol of the His-tagged r-XAn11 produced by E. coli BL21.
Steps

Total protein (mg)

Total activity (U)

Specic activity (U mg1 )

Purication (-fold)

Yield (%)

Crude cell extracts


Ni-NTA resin
FPLC Mono-Q chromatograph

54
15.7
12.7

7811
6248.8
5271.86

144.6
398
415.1

1
2.75
2.87

100
80
67.5

3.3. Purication of recombinant XAn11


The cell crude extract was loaded into a Ni-NTA afnity chromatography column. The fractions containing the xylanase activity
were pooled, dialyzed and concentrated. Then, the dialyzed enzyme
solution was further puried by FPLC using a UNO Q-12 anion
exchange column. Fraction containing xylanase activity was analyzed by SDS-PAGE indicating a clear band with molecular weight
about 25 kDa (Fig. 1, lane 4). As mentioned by previous study, the
mature XAn11 secreted by A. niger US368 has a molecular weight
of 26 kDa [18]. However, the expressed recombinant His-tagged rXAn11 in this study has a different molecular weight than that of
native enzyme (25 vs. 26 kDa) caused by the absence of glycosylation and the addition of N-terminal fusion peptide (His-tag), which
is commonly used for the protein purication.
Protein and enzyme assay demonstrated that the recombinant
enzyme was puried 2.87 fold with 67.5% recovery (Table 1). The
puried enzyme, having a specic activity of 415.1 U mg1 , was
used to determine its biochemical properties.
3.4. Effects of metal ions
The effect of bivalent metallic ions on puried His-tagged rXAn11 activity was carried out at concentration of 5 mM using
birchwood xylan as substrate under standard conditions (Table 2).
The effects of bivalent metallic ions on His-tagged r-XAn11 are
almost the same as the A. niger US368 XAn11. Indeed, in various metal ions assayed, Co2+ and Mn2+ reduced enzyme activity
by 84.3% and 54.2%, respectively. The His-tagged r-XAn11 activity
was not signicantly affected by EDTA and Fe2+ . Slight activation
of His-tagged r-XAn11 (up to 10%) was observed in the presence
of Zn2+ and Mg2+ . However, in the presence of 5 mM Ca2+ or Cu2 + ,
which were inhibitors for the native xylanase, the enzyme activity
increased by 25% and 40% respectively compared to the control.
The activity of His-tagged r-XAn11 was investigated at different
concentrations of Cu2+ or Ca2+ (from 1 to 8 mM) using birchwood
xylan as substrate. The results showed that the activity of Histagged r-XAn11 reach a maximum to be 154.2% or 130%, when
it was measured in the presence of 3 mM of Cu2+ or Ca2+ respectively. In previous reports, copper signicantly inhibited xylanases
activity of S. cellulosum [27], Geobacillus thermoleovorans [28] and
Plectosphaerella cucumerina [29] and slightly inhibited the activity
of xylanase of A. niger XZ-3S which was cloned into pET28a (+) and
expressed in E. coli BL21 [15]. To the authors knowledge this is
Table 2
Effects of 5 mM of metal ions and EDTA on the His-tagged r-XAn11 activity. The
relative activities were measured at optimum of pH and temperature and enzyme
activities without metal ions were taken as 100%.
Metal ions (5 mM)

Relative activity (%)

None
EDTA
Mg2+
Cu2+
Zn2+
Fe2+
Co2+
Ca2+
Mn2+

100
102.5 1.05
111.5 0.5
140.3 0.8
111.2 0.7
100 0.5
84.3 0.6
125.1 0.5
54.2 0.7

the rst work reporting that a xylanase was activated with a rate of
54% in the presence of copper. Activation of the His-tagged r-XAn11
by copper may greatly facilitate the application of the enzyme in
improving the efciency of xylan degradation for biofuel ethanol
production [29].
The different inhibition/stimulation effects on His-tagged rXAn11 by various metals compared to the native enzyme might
be related to interaction of metals with critical amino acid residues
in the N-terminal His-tag fusion peptide (36 residues). It is widely
known that the knowledge of hydrophobic regions is of great help
in dening the environment required for metal binding [30]. In
this context and in order to investigate the possible creation of an
ion binding site after the N-terminal His-tag fusion peptide introduction, the hydrophobicity plot of the His-tagged r-XAn11 was
determined (Fig. 2a) according to the method of Kyte and Doolittle [31]. Findings showed that the amino acids sequence had an
important hydrophobic inclination (2.4) in the N-terminal region
suggesting its possible implication in a novel metal binding site. In
addition, the His-rich sequence has been characterized as a metal
binding site in many metal-binding proteins especially with Cu2+ ,
Ca2+ and Zn2+ ions [32].
In order to correlate the structural features responsible for the
possible ion interaction, the 3D model of the His-tagged r-XAn11
was generated using the crystal structure of Xyn1 from A. niger (PDB
accession code 1UKR) as template with 93.48% sequence identity.
The inspection of the generated model showed that the introduced
N-terminal His-tag fusion peptide is tightly linked to the enzyme
core and that it forms a kind of hindrance at the end of the active
site (Fig. 2b) compared to the non tagged XAn11 (Fig. 2c). A careful examination of the contact region between the catalytic cavity
and the N-terminal His-tag fusion peptide showed the appearance
of a pocket that could be the cause of the observed changes in the
enzyme properties (Fig. 2b). To further consolidate this hypothesis,
the amino acids forming this region were checked as well as their
spatial distribution and coordinations. It is noticed that the copper ion can describe most commonly a square planar geometry (4
coordinations), a square pyramidal geometry (5 coordinations) and
rarely trigonal bipyramidal (5 coordinations) or octahedral geometry exhibiting JahnTeller distortion (6 coordinations) [33]. In fact,
distances between copper and donor atoms in four-coordinate Cu2+
or basal plane must be in the range of 1.972 A and consequently
the distance between donor side chains must not exceed 3.6 A [33].
In our case, the residues likely to be implicated as donors in the
ion binding are His 9 (N2 atom), His 10 (O atom), Thr 90 (O atom)

and Asn 208 (OD1 atom). According to the distances (3.44.4 A)


between these residues showed in Fig. 2d, the trigonal bipyramidal geometry is the most probable and the apical ligand could be a
water molecule which lies further from the copper than the other
four ligands. It should be noted that for this type of arrangement
more variability in length is allowed and probably the ion binding
would create a slight displacement of donors to meet the appropriate distances.
Interestingly, it was demonstrated that calcium ion could not
establish four coordinated geometry but it can adopt trigonal
bipyramidal geometry in few cases. This could be the case for the
His-tagged r-XAn11 leading to a possible activation of the enzyme
by Ca2+ . However, the copper bind more easily to the probable ionic
site than calcium ion since the latter has a larger ionic radius.

F. Elgharbi et al. / International Journal of Biological Macromolecules 74 (2015) 263270

267

Fig. 2. (a) Hydrophilicity plot for His-tagged r-XAn11 according to Kyte and Doolittle [31]. Electrostatic potential surface representation of the His-tagged r-XAn11 (b) and
the non-tagged XAn11 (c); the probable ion site is showed as a yellow circle. (d) Close up view of the contact region between the N-terminal His-tag fusion peptide and
the enzyme core. The probable residues implicated in ion binding are shown in yellow and distances () between them are shown as dotted yellow lines. (e) Close up view
showing the catalytic residues in pink color and their proximity to the probable ion binding site especially the Glu 215. (For interpretation of the references to color in this
gure legend, the reader is referred to the web version of the article.)

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F. Elgharbi et al. / International Journal of Biological Macromolecules 74 (2015) 263270

3.5. Optimum pH, temperature and stability


The effects of pH and temperature on the activity of the puried His-tagged r-XAN11 are shown in Fig. 3a and b respectively.
Enzyme assays revealed that the highest activity of the recombinant
xylanase was at 50 C and in the pH range of 4.55. The optimum temperature and pH for the His-tagged r-XAn11 are almost
the same as the A. niger US368 XAn11 [18] or other recombinant
xylanases [25].
The thermostability of the enzyme is shown in Fig. 3c. The
recombinant enzyme had similar thermal stability in the presence
of 3 mM Cu2+ , compared with the native enzyme and the half-lives
recorded for the puried enzyme at 45 C, 50 C and 55 C were
300 min, 210 min, and 50 min, respectively. In the absence of Cu2+ ,
the enzyme was noted to be unstable since it was inactivated after
30 min of incubation at 50 C and 5 min at 55 C (Fig. 3c). A careful inspection of the residues that could coordinates the metal
ion showed that the Asn 208 is located at the N-terminal end of
the -sheet that carry the catalytic amino acid Glu 215 (Fig. 2e).
The probable attachment of a metal ion would stabilize the donor
residues and consequently the neighbor environment including the
catalytic cavity. Stabilization of the active site would contribute to
more rigidity of the enzyme especially at high temperature. Furthermore, the residues added in the N-terminal sequence extension
would create a kind of protection by stabilizing the right-hand sandwich structure of the enzyme.
The His-tagged r-XAn11 was stable over the range of pH 310
(Fig. 3d) with maximum activity at the range of 4.55 and at 50 C
suggesting that it may be a good candidate in various applications
such as in the animal feed industry since the pH and temperature
of the livestock digestive tracts are approximately 4.8 and 40 C,
respectively [34].
3.6. Substrate specicity polysaccharide-binding properties and
kinetic parameters
The hydrolytic activity of the puried enzyme on various substrates was determined in the presence of 3 mM Cu2+ . The highest
activity was observed with the birchwood xylan (850.3 U mg1 )
followed by the beechwood xylan (788.5 U mg1 ). The enzyme
was less active on oatspelt xylan (450.3 U mg1 ) and arabinoxylan (324.2 U mg1 ). No activity was detected for the enzyme against
avicel (3%) and CM-cellulose (2%). The specic activity of His-tagged
r-XAn11 on birchwood xylan (1%) in the presence of 3 mM Cu2+ was
found to be 850.3 U mg1 , two fold higher than that measured in
the absence of copper and almost the same as the native enzyme
of A. niger US368 XAn11 [18]. The observed decrease in the specic
activity, in absence of copper, compared to the non tagged enzyme
could be due to the N-terminal His-tag fusion peptide that forms
a kind of obstruction at the end of the active site. The ion binding
could restore the conformation of the catalytic pocket allowing the
appropriate substrate xation and consequently could increase the
specic activity of the tagged enzyme.
The polysaccharide-binding capacity of the puried His-tagged
r-XAN11 was determined by incubating the enzyme with avicel,
arabinoxylan or birchwood xylan. As shown in Fig. 4, the His-tagged
r-XAn11 could not bind to avicel. In contrast, the enzyme showed
ability to bind to birchwood xylan and arabinoxylan, since about
71% and 79% enzyme activity still remained in the supernatant
respectively.
In this study the His-tagged r-XAn11 exhibited a high activity on
xylan, but no activity was detected for cellulosic substrates, proving the absence of cellulose-binding sites in the structure of the
enzyme. These results were conrmed by polysaccharide-binding
analysis. When incubating the His-tagged r-XAn11 with avicel,
more than 98% unbound enzyme was detected in the supernatant.

Fig. 3. Characterization of the puried His-tagged r-XAn11 enzyme. (a) Effect of


pH on the activity of the puried His-tagged r-XAn11. The relative activities were
determined according to the standard assay with buffers solutions at 100 mM as
follow: citrate buffer (pH 35.5) and phosphate buffer (pH 5.57). (b) Effect of temperature on the activity of the puried His-tagged r-XAn11. The relative activities
were determined according to the standard assay at pH 5 and temperatures varying
from 40 to 70 C. (c) Thermostability of the puried His-tagged r-XAn11 at pH 5.0
and temperatures of 45 C in presence of 3 mM of Cu2+ (), 45 C in the absence of
Cu2+ (), 50 C in presence of 3 mM of Cu2+ (), 50 C in the absence of Cu2+ (),
55 C in presence of 3 mM of Cu2+ (), and 55 C in the absence of Cu2+ (). The
enzyme without incubation was dened as 100% relative activity. (d) pH stability of
the puried His-tagged r-XAn11. The relative activities were determined according
to the standard assay at pH values ranging from 3 to 10. Aliquots were taken after
1 h of incubation at 37 C and immediately assayed for residual xylanase activity in
standard assay conditions.

F. Elgharbi et al. / International Journal of Biological Macromolecules 74 (2015) 263270

269

Table 3
Kinetic parameters of puried His-tagged r-XAn11 on different substrates.
Substrate

Vmax (U ml1 )

Birchwood
Oalt spelt xylan
Beechwood
Arabinoxylan

823.8
263.6
640.4
163.6

7.215
3.852
4.512
6.315

Km (mg ml1 )
1.837
2.837
2.097
3.814

0.215
0.127
0.185
0.228

Kcat (s1 )

Kcat /Km (ml mg1 s1 )

514.88
164.75
400.25
102.25

280.28
58.07
190.87
26.81

Tween 80, respectively. Also, the His-tagged r-XAN11 presented


a high stability in various organic solvents such as acetone and
chloroform, since it retained more than 95% of its initial activity
upon incubation in these solvents. The His-tagged r-XAN11 was
also stable in the presence of 1% of ethanol and the activity reached
130%. High stability to organic solvents, at relatively high temperature and at a broad range of pH indicated that His-tagged r-XAn11
has great potential for industrial applications such as a biocatalyst in food industry, bioconversion processes and pharmaceutical
industry.
4. Conclusion

Fig. 4. Effect of different concentrations of avicel, birchwood xylan and arabinoxylan on the binding ability of the recombinant xylanase XAn11: puried xylanase
(30 g) was incubated with 6 to 24 mg ml1 avicel (), arabinoxylan () or birchwood xylan () in 100 mM citrate buffer (pH 5) at 4 C for 1 h. The relative activities
were determined according to the standard assay at pH 5 and 50 C.

Previous study showed that the cellulose-binding domains are


more common in family 10 xylanases than in family 11 xylanases
[34].
The Michaelis-Menten constants were determined for different
substrates (Table 3) and the Km and Kcat were 1.837 mg ml1 and
514.88 s1 for birchwood xylan, and 2.097 mg ml1 and 400.25 s1
for beechwood xylan, respectively.
3.7. Effect of additives on His-tagged r-XAn11 stability
The enzyme was pre-incubated at 40 C for 1 h in the presence of
several detergents or organic solvents and the residual activity was
determined at pH 5 and 50 C (Table 4). Findings showed that the
effects of additives on His-tagged r-XAn11 stability are almost the
same as the A. niger US368 XAn11 [18]. Since, the enzyme retains
only 15% of its initial activity upon treatment with 1% SDS. Significant inhibition of xylanase activity by SDS was also recorded in
Penicillium sp. CGMCC1669 [35] and G. mesophila KMM 241 [25].
The His-tagged r-XAN11 activity was increased by 17.8% or 21.43%
after incubation of the puried enzyme with 1% of Triton X-100 and
Table 4
His-tagged r-XAn11 stability in the presence of some additives.
Additive (1%)

Relative activity (%)

None

100

Detergents
SDS
Tween 80
Triton X-100

15 2.3
121.43 1.2
117.82 2.45

Organic solvents
Ethanol
Isopropanol
Butanol
Methanol
Glycerol
Acetone
Chloroform

130.2 1.57
122.64 2.23
112.03 1.81
104.8 2.2
102.12 2.4
98.36 1.02
95.45 1.32

In this study, the His-tagged r-XAn11 exhibited a high stability


to organic solvents and metallic ions, at relatively high temperature
and at a broad range of pH. More interestingly, the relative activity
of the His-tagged r-XAn11 was enhanced by 54%, in the presence of
3 mM Cu2+ . Our ndings indicate that the His-tagged r-XAn11 could
be considered as a potential strong candidate for future application
particularly in the animal feed industry, prebleaching of Kraft pulp
in paper industries and degradation for biofuel ethanol production.
Future research will be focused on development of a more effective expression system for this xylanase.
Acknowledgements
This work was funded by the Tunisian Ministry of Higher Education and Scientic Research and Technology (contract program
LMB-CBS, grant no. RL02CBS01).
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