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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.
264
265
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.
266
Table 1
Summary of the purication protocol of the His-tagged r-XAn11 produced by E. coli BL21.
Steps
Purication (-fold)
Yield (%)
54
15.7
12.7
7811
6248.8
5271.86
144.6
398
415.1
1
2.75
2.87
100
80
67.5
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)
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.)
268
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 )
514.88
164.75
400.25
102.25
280.28
58.07
190.87
26.81
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.
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
270
[29] G.M. Zhang, J. Huang, G.R. Huang, L.X. Ma, X.E. Zhang, Appl. Microbiol. Biotechnol. 74 (2007) 339346.
[30] M. Jayakishan, R. Mukesh Kumar, D. Gananath, M. Pramod Kumar, JBiSE 4 (2011)
562568.
[31] J. Kyte, R.F. Doolittle, J. Mol. Biol. 157 (1982) 105132.
[32] M. Hara, M. Fujinaga, T. Kuboi, J. Exp. Bot. 56 (2005) 26952703.
[33] M.M. Harding, Acta Crystallogr. Sect. D: Biol. Crystallogr. 57 (2001) 401411.
[34] H. Jun, Y. Bing, Z. Keying, D. Xuemei, C. Daiwen, Protein Exp. Purif. 67 (2009)
16.
[35] W. Liu, P. Shi, Q. Chen, P. Yang, G. Wang, Y. Wang, H. Luo, B. Yao, Appl. Biochem.
Biotechnol. 162 (2010) 112.