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International Journal of Biological Macromolecules 74 (2015) 263–270

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

 j ournal homepage: www.elsevier .com/ locate / i jb iomac

Expression of  A. niger US368 xylanase in E. coli: Purification,characterization and copper activation

Fatma Elgharbi, Hajer Ben Hlima, Ameny Farhat-Khemakhem, Dorra Ayadi-Zouari,Samir Bejar∗, Aïda Hmida-SayariLaboratoire de Microorganismeset de Biomolécules (LMB), Centre de Biotechnologie de Sfax (CBS),Université 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 2014Received in revised form10 December 2014Accepted 11 December 2014Available online 18 December 2014

Keywords:

 Aspergillus niger US368XylanaseGene cloningExpressionEscherichia coliBL21Copper

a b s t r a c t

The  XAn11 cDNA was cloned in pET-28a(+) and the recombinant plasmid was transformed inEscherichia coli. The His-tagged r-XAn11 was purified using Ni-NTA affinity and anion exchange chro-matography. The enzyme showed a specific activity of 415.1 U mg−1 and a molecular mass of 25 kDa. Ithad an optimal activity at pH 5 and 50 ◦C. It was stable in a wide range of pH and inthe presence of somedetergents and organic solvents. In the presence of 3 mM Cu2+, the relative activity of the His-tagged r-XAn11 was enhanced by 54%. This isthe first work reporting that copper is a strong activator for xylanaseactivity making this enzyme very attractive for future industrial applications. Molecular modeling sug-gests that the contact region between the catalytic site and the N-terminal His-tag fusion peptide couldbe 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 hemicellulosefraction of plant cell walls and accounts for 20–30% of their totaldry mass. It is covalently and noncovalently attached to cellu-lose, lignin, pectin and other polysaccharides to maintain cell wallintegrity [1,2]. Xylan thus belongs to the main food source of  farm animals and also represents a major component of the rawmaterial for many industrial processes ranging from baking topaper production [3]. It consists of a backbone of -1,4-linkedxylopyranose residues, usually with branches of -1,3-linked l-arabinose and -1,2-linked d-glucopyranose [4]. -1,4-Xylanases(EC 3.2.1.8) are the key enzymes that hydrolyze the backbonestructure of -1,4-xylans to initiate degradation of the polysaccha-ridic complex by microorganisms.Several microorganismsproducemultiple xylanases, implying a strategy for effective hydrolysisof -1,4-xylan. Each enzyme may have a specialized function inthe degradation of the polysaccharidic complex. These specializedfunctions of individual xylanases may be useful for applications inthe 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).

Xylanases have been classified into two families, F and G, basedonhydrophobicclusteranalysisandsequencehomology [6,7]. Fam-ilies F and G correspond to families 10 and 11 respectively in thenumerical classification of glycosyl hydrolases [8,9]. F10 Familyare endo--1,4-xylanses with higher molecular mass than familyG11 xylanases, and presenting eight (/) barrel folds in three-dimensional (3D) structure [7,10]. G11 Family are xylanases withlower molecular masses (<30kDa) [7,9] and are encoded as pre-cursors composed of signal peptide and a mature xylanase. The 3Dstructures of familyG11 xylanaseshave theoverall shape of a “righthand” [11].

During the last decades, heterologous expression is becomingone of the main tools forthe production of industrial enzymes [12].Many xylanase genes have been isolated, cloned and expressed inEscherichia coli from microorganisms including fungi, bacteria, andyeasts [13–15]. Aspergillus niger  is a filamentous fungus that has been shown

to secrete large amounts of efficient xylan-degrading enzymes[3,16–19], and most of them belong to G11 family.

Heterologous expression of  A. niger xylanase was of interest forthe production of large quantities of a single xylanolytic enzyme.The E. coli BL21 due to its clear genetic background, simple opera-tion, short growthperiod andhigh expression became an attractiveexpressionsystem of foreign proteins,includingthose of eukaryoticorigin.

http://dx.doi.org/10.1016/j.ijbiomac.2014.12.0050141-8130/© 2014 Elsevier B.V. All rights reserved.

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Previous study conducted in our laboratory described a newlyisolated A. niger strain US368 producing an endo-xylanase XAn11with a molecular masse of about 26kDa and a maximal xylanaseactivity at pH 5 and 55◦C [18].

In this study, we describe the molecular cloning and expressionof the A. niger US368 XAn11gene inE. coli as well as the purificationand biochemical characterization of the recombinant xylanase. Wealso described the differential behavior of the His-tagged r-XAn11toward 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 iso-lated in our laboratory [18].

The pCR ®-Blunt vector (Invitrogen, USA) andE. coli Top10 (Invi-trogen, USA) were used for subcloning.

E. coli BL21 ((DE3) pLysS) was used to express recombinantprotein using the expression vector pET-28a(+) (Novagen, Merck,America).

 2.2. Cloning of XAn11 cDNA

Exon I (without thesignalpeptide) of  XAn11gene was amplifiedwith PCR primers XylF (CCG GAA TTC CGG GCT CCG GAG CCT GTTCTG G) and ASR2 (AGG TGA TAG ACT TAG AGG AGC CAG TGG TCCAGC C), in a first round. The ASR2 primer has 10 base pair over-lapping region from 5 end of exon II. Similarly, Exon II of  XAn11gene was amplified with primers ASF2 (CTC CTC TAA GTC TAT CACCTA CTC TGC CCA ATA C) and XylR (TTG CGG CCG CAA TTA AGTGGA GAT CGT GAC ACT GGC), the forward primer ASF2 has 10 basepair overlapping region from 3 end of exon I. Polymerase chainreactions (PCR) were carried out in a Gene Amp® PCR System 2700(Applied Biosystems). The amplification reaction mixture (50l)wascomposedof  pfuDNApolymeraseamplificationbuffer(1×finalconcentration), 10pmol of each primer, 0.2 mM of dNTPs, 300 ng

of DNA template (recombinant plasmid p XAn11 [18]) and 2 U of  pfu enzyme (Thermo Scientific, USA). The cycling parameters were94 ◦C for5 min followed by 30 cycles at 94◦C for 30s, 50 ◦C for 45sand 72◦C for 60s, with a final extension of 72◦C for 10min.

Both amplified exon I and exon II were purified and mixed in1:1 molar ratio. These fragments were joined by the second roundof PCR with no primers. The cycling parameters were 15 cycles at94 ◦C for 30s, 60 ◦C for 60s and 72 ◦C for 90s.

In the third round of PCR, the fused product was amplified withprimers XylF and XylR carrying flanking restriction sites (EcoRIandNot I respectively) to facilitate further cloning into pET-28a(+) vec-tor. The cycling parameters were 30 cycles at 94◦C for 30s, 50 ◦Cfor60sand72◦C for 90s, witha finalextensionof 72◦C for 10min.

The fusion product was purified and cloned into pCR ®-Blunt

vector, the resulting plasmid was transformed into E. coli Top10.The transformants were screened on LB agar supplemented with50g ml−1 of kanamycin and the recombinant plasmid wasdigested with EcoRI and Not I and cloned into pET-28a(+) vectorpredigested with the same restriction enzymes.

Digestion of DNA with restriction endonucleases, separation of fragments by agarose gel electrophoresis, ligation of DNA frag-ments, transformation of E. coliwith plasmidic DNA and extractionof recombinant DNA were all performed according to standardmethods described by Sambrook et al. [20].

 2.3. Expression and purification 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 with50g ml−1 of kanamycin and incubated at 37◦C for 24h. A singlecolony was isolated and inoculated, into 5ml of Luria–Bertani(LB) medium and cultured overnight at 37◦C on a rotary shaker at250 rpm. After that, 1 ml of overnight culture was inoculated into100ml of fresh medium and incubated at 37◦C on a rotary shakerat 250 rpm until the  A600nm reached 0.6–0.8. Then, isopropyl-d-1-thiogalactopyranoside (IPTG) was added into the culture brothto a final concentration of 0.1 mM. The culture was subsequentlyincubated for another 24h at 20 ◦C and centrifuged at 7000rpmfor 10min. The supernatant was decanted and the wet weight of the cell pellets was determined. The cell pellets were crushed withthe same weight of powder aluminia (aluminum oxide Type A-5,Sigma-Aldrich Co., St. Louis, MO, USA)usingapestleandamortarat4 ◦C for 30min in the presence of 100 mM PMSF (phenylmethane-sulfonyl fluoride; Sigma–Aldrich Co., St. Louis, MO, USA) preparedin isopropyl alcohol. The mixture was suspended in 1× phosphatebuffer (Amersham Pharmacia Biotech, USA) and cell debris wasremoved by centrifugation at 9500rpm for 30min. The clearsupernatant containing the His-tagged r-XAn11 was adjusted to10mM imidazole and loaded on 1 ml HisTrap Chelating Ni-affinitycolumn (Amersham Pharmacia Biotech, USA) equilibrated with 1×phosphate buffer (PB, 10mM imidazole). The adsorbed proteins

were eluted using a linear gradient of imidazole (50–200mM)and recombinant enzyme (His-tagged r-XAn11) was eluted with100mM imidazole. The fractions containing the xylanase activitywere pooled, dialyzed, concentrated and the pH was adjustedto 7. The dialyzed enzyme solution was further purified by fastperformance liquid chromatography (FPLC), using a UNO Q-12(15mm×68mm) anion exchange column pre-equilibrated with25mM phosphate buffer (pH 7). The proteins were eluted at a flowrate of 5ml/min by using linear NaCl gradient ranged from 0 to1 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 R-250 (Bio-Rad) staining.

Protein concentration was determined using Bio-Rad proteinassay kit, based on the method of Bradford using bovine serumalbumin as the standard [22].

 2.4. Assay of xylanolytic activity

The His-tagged r-XAn11 activity was measured at 50 ◦C and pH5. 0.5ml of the enzyme solution, diluted in citrate buffer (0.1M,pH 5), was incubated for 20 min with 0.5ml of 1% soluble birch-wood xylan (Sigma–Aldrich Co., St. Louis, MO, USA). The amountof reducing sugars released was determined by the dinitrosalicylicacid (DNS) method [23], using d-xylose as standard.

One unit of xylanase activity was defined 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 severalmetallic ions (Co2+, Zn2+, Mg2+, Ca2+, Cu2+, Fe2+ and Mn2+) orEDTAat5 mM.

 2.6. Effects of pH and temperature on recombinant xylanase

activity and stability

The effectof pH on His-tagged r-XAn11 activity wasdeterminedat 50 ◦C using the following buffer solutions at 100mM. Buffersused were citrate buffer (pH 3–5.5), phosphate buffer (pH 5.5–8),

Tris–HCl buffer (pH 8–9) and glycine–NaOH buffer (9–10). The

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stability of -glucanase was investigated over a pH range of 3–10in 100mM buffers. Aliquots were taken after 1 h of incubation at37 ◦C. The residual activity was determined, after centrifugation,under standard assay conditions.

The optimum temperature for xylanase activity was deter-mined by carrying out the enzyme assay at different temperatures(40–70 ◦C) at pH 5. The thermal stability of the enzyme was deter-mined by incubating the pure enzyme at different temperatures(45, 50 and 55 ◦C) at pH 5. Aliquots were drawn at regular timeintervals and immediately cooled in ice-cold water. The residualactivity was determined, aftercentrifugation,under standard assayconditions.

 2.7. Effect of additives on XAn11 stability

Solvents and detergents were incubated with the pure enzymefor 1 h at 40 ◦C. The final concentration of additives in the reac-tion mixture was 1%. The xylanase activity was measured underthe standard test conditions.

 2.8. Enzyme specificity

Recombinant xylanase activity was determined on various sub-strates (birchwood xylan, beechwood xylan, arabinoxylan, oatspeltxylan, avicel and CM-cellulose). The reaction was carried out in100mM citrate buffer (pH 5) containing 1% of each substrate at50 ◦C for 20 min in the presence of 3mM Cu2+.

 2.9. Kinetics parameters

Xylanase assays were performed using different substrates(birchwood xylan, beechwood xylan, arabinoxylan and oatspeltxylan), at various concentrations ranging from 0 to 2mg ml−1.Assays were performed for 5 min at 50 ◦C in the presence of 3mMCu2+ usinga50g ml−1 enzymeconcentrationintheassaymixture.The maximum velocity (V max) and the Michaelis–Menten constant(K m) values were calculated from a Lineweaver and Burk plot usingthe hyper-32 program.

 2.10. Polysaccharide-binding properties

The polysaccharide-binding capacity of the recombinantxylanase was determined by the method described by Tenkanenet al. [24]. 30g of purified xylanase was incubated with differ-ent concentrations of avicel, arabinoxylan or birchwood xylan in50mM citrate buffer (pH 5) and 3mM of Cu2+, a t 4 ◦C for 1 h withslow shaking. Unbound enzyme was determined by measuringresidual activity in the supernatant.

 2.11. Homologymodeling 

The3D structural modelsof theHis-taggedr-XAn11were gener-ated using the SWISS-MODEL (http://www.expasy.org/swissmod/)and the crystal structure of Xyn1 from A. niger (PDB accession code1UKR) as template with which the His-tagged r-XAn11 possess93.48% sequence identity. The automated comparative proteinstructure homology modeling server, phyre 2 (Protein Homol-ogy/analogY Recognition Engine V.2) was used to analyze thetagged model structures and construct illustrative figures. ThePyMol molecular Graphics System (DeLano Scientific, San Carlos,CA, http://www.pymol.org.) was used to visualize the constructed

model structures and generate graphical figures.

3. Results and discussion

 3.1. cDNA cloning 

The entire XAn11 coding region was already cloned andsequenced [18]. The sequence analysis showed two exons and oneintron of 51 pb. In order to express this gene, a PCR fragment of 582 pb carrying the open reading frame was generated by delet-ing the unique intron and the N-terminal signal sequence usingthe sequence-specific PCR primers as described in Section 2. ThePCR product was cloned into pCR ®-Blunt vector and the result-ing plasmid was transformed into E. coli Top10. The recombinantplasmid was digested with EcoRI and Not I and directly ligated inpET-28a(+) expression vector, in frame with N-terminal His-tagspeptide sequence, under the control of strong bacteriophage T7promoter. Ligation products were transformed intoE. coli BL21 andthe transformants were screened on LB agar supplemented with50g ml−1 of kanamycin. Recombinant plasmids were checkedby digestion followed by sequence analysis of the inserted frag-ment (data not shown). Sequencingdataproved thatthe expressionplasmid was correctly constructed. The predicted mature peptideconsisted of 229amino acids including 36 extra amino acids due tothe presence of the His-tag fusion peptide.

 3.2. Expression of XAn11 in E. coli

The recombinant plasmid pET28a- XAn11was transformed intoE. coli BL21. Then, one single colony was cultured and inducedby IPTG. The xylanase activity assay of the resulting cell crudeextract showed a maximum specific activity of 144.6U mg−1

(78.11Uml−1) which washigher than that of recombinant xylanasefrom  A. niger XZ-3S [15] Glaciecola mesophila KMM 241 [25] andStreptomyces thermocyaneoviolaceus [26].

When the cell extract of the recombinant xylanase was ana-lyzed by SDS-PAGE a clear band with molecular weight of about25kDa(Fig. 1, lane2) wasmonitored, whereas, no band of thesamesize was observed in the extract from the uninduced strain (Fig. 1,lane 1).

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-taggedr-XAn11 without induction; lane 2, crude cell extracts of His-tagged r-XAn11 withIPTG induction; lane 3, His-tagged r-XAn11 purified by Ni-NTA affinity chromatog-raphy; lane 4, pooled fraction from the FPLC-UNO Q-12 anion exchange column.

60g of protein was loadedin each laneof the SDS–PAGE gel.

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 Table 1

Summary of thepurification protocol of theHis-tagged r-XAn11 produced by E. coli BL21.

Steps Total protein (mg) Total activity (U) Specific activity (U mg−1) Purification (-fold) Yield (%)

Crude cell extracts 54 7811 144.6 1 100Ni-NTA resin 15.7 6248.8 398 2.75 80FPLC Mono-Q chromatograph 12.7 5271.86 415.1 2.87 67.5

 3.3. Purification of recombinant XAn11

The cell crude extract was loaded into a Ni-NTA affinity chro-matography column. The fractions containing the xylanase activitywerepooled,dialyzedandconcentrated.Then,thedialyzedenzymesolution was further purified by FPLC using a UNO Q-12 anionexchange column. Fraction containing xylanase activity was ana-lyzed by SDS-PAGE indicating a clear band with molecular weightabout 25kDa (Fig. 1, lane 4). As mentioned by previous study, themature XAn11 secreted by A. niger US368 has a molecular weightof 26kDa [18]. However, the expressed recombinant His-tagged r-XAn11 in this study has a different molecular weight than that of native enzyme (25 vs. 26kDa) caused by the absence of glycosyla-tion and the addition of N-terminal fusion peptide (His-tag), whichis commonly used for the protein purification.

Protein and enzyme assay demonstrated that the recombinantenzyme was purified 2.87 fold with 67.5% recovery (Table 1). Thepurified enzyme, having a specific activity of 415.1U mg−1, wasused to determine its biochemical properties.

 3.4. Effects of metal ions

The effect of bivalent metallic ions on purified His-tagged r-XAn11 activity was carried out at concentration of 5mM usingbirchwood xylan as substrate under standard conditions (Table 2).The effects of bivalent metallic ions on His-tagged r-XAn11 arealmost the same as the  A. niger US368 XAn11. Indeed, in vari-ous metal ions assayed, Co2+ and Mn2+ reduced enzyme activityby 84.3% and 54.2%, respectively. The His-tagged r-XAn11 activity

was not significantly affected by EDTA and Fe2+. Slight activationof His-tagged r-XAn11 (up to 10%) was observed in the presenceof Zn2+ and Mg2+. However, in the presence of 5mM Ca2+ orCu2 +,which were inhibitors for the native xylanase, the enzyme activityincreased by 25% and 40% respectively compared to the control.

The activity of His-tagged r-XAn11 was investigated at differentconcentrations of Cu2+ or Ca2+ (from 1 to 8mM) using birchwoodxylan as substrate. The results showed that the activity of His-tagged r-XAn11 reach a maximum to be 154.2% or 130%, whenit was measured in the presence of 3mM of Cu2+ or Ca2+ respec-tively. In previous reports, copper significantly inhibited xylanasesactivity of S. cellulosum [27], Geobacillus thermoleovorans [28] andPlectosphaerella cucumerina [29] and slightly inhibited the activityof 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 5mM of metal ions and EDTA on the His-tagged r-XAn11 activity. Therelative activities were measured at optimum of pH and temperature and enzymeactivities without metal ions were taken as 100%.

Metal ions (5mM) Relative activity (%)

None 100EDTA 102.5±1.05Mg2+ 111.5±0.5Cu2+ 140.3±0.8Zn2+ 111.2±0.7Fe2+ 100±0.5Co2+ 84.3±0.6Ca2+ 125.1±0.5Mn2+ 54.2±0.7

the first work reporting thata xylanase was activated with a rate of 

54%in thepresenceof copper. Activation of theHis-tagged r-XAn11by copper may greatly facilitate the application of the enzyme inimproving the efficiency of xylan degradation for biofuel ethanolproduction [29].

The different inhibition/stimulation effects on His-tagged r-XAn11 by various metals compared to the native enzyme mightbe related to interaction of metals with critical amino acid residuesin the N-terminal His-tag fusion peptide (36 residues). It is widelyknown that the knowledge of hydrophobic regions is of great helpin defining the environment required for metal binding [30]. Inthis context and in order to investigate the possible creation of anion binding site after the N-terminal His-tag fusion peptide intro-duction, the hydrophobicity plot of the His-tagged r-XAn11 wasdetermined (Fig. 2a) according to the method of Kyte and Doolit-

tle [31]. Findings showed that the amino acids sequence had animportant hydrophobic inclination (−2.4) in the N-terminal regionsuggesting its possible implication in a novel metal binding site. Inaddition, the His-rich sequence has been characterized as a metalbinding site in many metal-binding proteins especially with Cu2+,Ca2+ and Zn2+ ions [32].

In order to correlate the structural features responsible for thepossible ion interaction, the 3D model of the His-tagged r-XAn11wasgenerated using thecrystal structureof Xyn1 from A.niger (PDBaccession code 1UKR) as template with 93.48% sequence identity.The inspection of the generated model showed that the introducedN-terminal His-tag fusion peptide is tightly linked to the enzymecore and that it forms a kind of hindrance at the end of the activesite (Fig. 2b) compared to the non tagged XAn11 (Fig. 2c). A care-

ful examination of the contact region between the catalytic cavityand the N-terminal His-tag fusion peptide showed the appearanceof a pocket that could be the cause of the observed changes in theenzyme properties(Fig. 2b). To further consolidate this hypothesis,the amino acids forming this region were checked as well as theirspatial distribution and coordinations. It is noticed that the cop-per ion can describe most commonly a square planar geometry (4coordinations), a square pyramidal geometry (5 coordinations) andrarely trigonal bipyramidal (5 coordinations) or octahedral geome-try exhibiting Jahn–Teller distortion (6 coordinations) [33]. In fact,distancesbetween copper and donoratoms in four-coordinateCu2+

or basal plane must be in the range of 1.97–2 A and consequentlythe 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 (3.4–4.4 A)between these residues showed in Fig. 2d, the trigonal bipyrami-dal geometry is the most probable and the apical ligand could be awater molecule which lies further from the copper than the otherfour ligands. It should be noted that for this type of arrangementmore variability in length is allowed and probably the ion bindingwould create a slight displacement of donors to meet the appropri-ate distances.

Interestingly, it was demonstrated that calcium ion could notestablish four coordinated geometry but it can adopt trigonalbipyramidal geometry in few cases. This could be the case for theHis-tagged r-XAn11 leading to a possible activation of the enzymebyCa2+. However, thecopper bind more easilyto theprobableionicsite than calcium ion since the latter has a larger ionic radius.

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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) andthe 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 andthe enzyme core. The probable residues implicated in ion binding are shown in yellow and distances (Å) between them are shown as dotted yellow lines. (e)Close up viewshowing the catalytic residues in pink color and their proximity to theprobable ion binding site especially the Glu 215. (For interpretation of the references to color in thisfigure legend, thereader is referred to the web version of thearticle.)

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 3.5. OptimumpH, temperature and stability

The effects of pH and temperature on the activity of the puri-fied His-tagged r-XAN11 are shown in Fig. 3a and b respectively.Enzymeassaysrevealedthatthehighestactivityoftherecombinantxylanase was at 50 ◦C and in the pH range of 4.5–5. The opti-mum temperature and pH for the His-tagged r-XAn11 are almostthe same as the A. niger US368 XAn11 [18] or other recombinantxylanases [25].

The thermostability of the enzyme is shown in Fig. 3c. Therecombinant enzyme had similar thermal stability in the presenceof3mMCu2+, compared with the native enzyme and the half-livesrecorded for the purified enzyme at 45 ◦C, 50 ◦C and 55 ◦C were300 min, 210 min, and 50min, respectively. In the absence of Cu2+,the enzyme was noted to be unstable since it was inactivated after30min of incubation at 50◦C and 5 min at 55 ◦C (Fig. 3c). A care-ful inspection of the residues that could coordinates the metalion 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 donorresidues and consequently theneighbor environmentincludingthecatalytic cavity. Stabilization of the active site would contribute tomore rigidity of the enzyme especially at high temperature. Fur-

thermore,the residuesaddedin theN-terminalsequence extensionwould 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 3–10(Fig. 3d) with maximum activity at the range of 4.5–5 and at 50◦Csuggesting that it may be a good candidate in various applicationssuch as in the animal feed industry since the pH and temperatureof the livestock digestive tracts are approximately 4.8 and 40◦C,respectively [34].

 3.6. Substrate specificity polysaccharide-binding properties and

kinetic parameters

The hydrolytic activity of the purified enzyme on various sub-

strates was determined in the presence of 3 mM Cu2+. The highestactivity was observed with the birchwood xylan (850.3U mg−1)followed by the beechwood xylan (788.5Umg−1). The enzymewas less active on oatspelt xylan (450.3U mg−1) and arabinoxy-lan(324.2Umg−1). No activitywas detected forthe enzyme againstavicel (3%)and CM-cellulose (2%).The specific activityof His-taggedr-XAn11onbirchwoodxylan(1%)inthepresenceof3mM Cu2+ wasfound to be 850.3Umg−1, two fold higher than that measured inthe absence of copper and almost the same as the native enzymeof  A. niger US368 XAn11 [18]. The observed decrease in the specificactivity, in absence of copper, compared to the non tagged enzymecould be due to the N-terminal His-tag fusion peptide that formsa kind of obstruction at the end of the active site. The ion bindingcould restore the conformation of the catalytic pocket allowing the

appropriate substrate fixation and consequently could increase thespecific activity of the tagged enzyme.

The polysaccharide-binding capacity of the purified His-taggedr-XAN11 was determined by incubating the enzyme with avicel,arabinoxylanor birchwoodxylan. As shownin Fig.4, the His-taggedr-XAn11 could not bind to avicel. In contrast, the enzyme showedability to bind to birchwood xylan and arabinoxylan, since about71% and 79% enzyme activity still remained in the supernatantrespectively.

In this study theHis-tagged r-XAn11 exhibiteda high activity onxylan, but no activity was detected for cellulosic substrates, prov-ing the absence of cellulose-binding sites in the structure of theenzyme. These results were confirmed by polysaccharide-bindinganalysis. When incubating the His-tagged r-XAn11 with avicel,

more than 98% unbound enzyme was detected in the supernatant.

Fig. 3. Characterization of the purified His-tagged r-XAn11 enzyme. (a) Effect of pH on the activity of the purified His-tagged r-XAn11. The relative activities weredetermined according to the standard assay with buffers solutions at 100mM asfollow: citrate buffer(pH 3–5.5)and phosphatebuffer (pH5.5–7). (b)Effect of tem-perature on the activity of the purified His-tagged r-XAn11. The relative activitieswere determined according to thestandard assay at pH 5 andtemperatures varyingfrom 40to 70◦C. (c) Thermostability of the purified His-tagged r-XAn11 at pH 5.0and temperatures of 45 ◦C inpresence of3 mM ofCu2+(), 45◦C in the absence of Cu2+ () , 50◦C in presence of 3 m M of Cu2+ () , 50◦C in the absence of Cu2+ (),55 ◦C in presence of 3mM of C u2+ (), and 55 ◦C in the absence of Cu2+ (). Theenzymewithout incubation wasdefined as 100% relative activity.(d) pH stability of the purified His-tagged r-XAn11. The relative activities were determinedaccordingto the standard assayat pH values ranging from 3 to 10. Aliquots were taken after1 h ofincubation at 37◦C and immediately assayedfor residual xylanase activity in

standard assay conditions.

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 Table 3

Kinetic parameters of purified His-tagged r-XAn11 on different substrates.

Substrate V max (Uml−1) K m (mg ml−1) K cat (s−1) K cat/K m (ml mg−1 s−1)

Birchwood 823.8 ± 7.215 1.837 ± 0.215 514.88 280.28Oalt spelt xylan 263.6 ± 3.852 2.837 ± 0.127 164.75 58.07Beechwood 640.4 ± 4.512 2.097 ± 0.185 400.25 190.87Arabinoxylan 163.6 ± 6.315 3.814 ± 0.228 102.25 26.81

Fig. 4. Effect of differentconcentrations of avicel, birchwoodxylan and arabinoxy-lan on the binding ability of the recombinant xylanase XAn11: purified xylanase(30g) was incubated with 6 to 24mgml−1 avicel (), arabinoxylan () or birch-wood xylan () in100mM citrate buffer(pH5) at4 ◦C for1 h. Therelativeactivitieswere determined according to the standard assayat pH 5 and 50◦C.

Previous study showed that the cellulose-binding domains aremore common in family 10 xylanases than in family 11 xylanases[34].

The Michaelis-Menten constants were determined for differentsubstrates (Table 3) and the K m  and K cat  were 1.837 mgml−1 and514.88s−1 for birchwood xylan, and 2.097mg ml−1 and 400.25s−1

for beechwood xylan, respectively.

 3.7. Effect of additives on His-tagged r-XAn11 stability

Theenzymewas pre-incubated at 40 ◦Cfor1hinthepresenceof several detergents or organic solvents and theresidual activity wasdetermined at pH 5 and 50◦C (Table 4). Findings showed that theeffects of additives on His-tagged r-XAn11 stability are almost thesame as the  A. niger US368 XAn11 [18]. Since, the enzyme retainsonly 15% of its initial activity upon treatment with 1% SDS. Signif-icant inhibition of xylanase activity by SDS was also recorded inPenicillium 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 thepurified enzymewith 1% of TritonX-100and

 Table 4

His-tagged r-XAn11 stability in thepresenceof some additives.

Additive (1%) Relative activity (%)

None 100

Detergents

SDS 15±2.3Tween 80 121.43±1.2Triton X-100 117.82±2.45

Organic solvents

Ethanol 130.2±1.57Isopropanol 122.64±2.23Butanol 112.03±1.81Methanol 104.8±2.2Glycerol 102.12±2.4Acetone 98.36±1.02Chloroform 95.45±1.32

Tween 80, respectively. Also, the His-tagged r-XAN11 presenteda high stability in various organic solvents such as acetone andchloroform, since it retained more than 95% of its initial activityupon incubation in these solvents. The His-tagged r-XAN11 wasalso stable in the presence of 1% of ethanol and the activityreached130%. High stabilityto organic solvents, at relatively high tempera-ture and at a broad range of pH indicated that His-tagged r-XAn11has great potential for industrial applications such as a biocata-lyst in food industry, bioconversion processes and pharmaceuticalindustry.

4. Conclusion

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

to organic solvents and metallic ions,at relativelyhigh temperatureand at a broad range of pH. More interestingly, the relative activityof the His-tagged r-XAn11 was enhanced by 54%, in the presence of 3 mM Cu2+. Our findings indicatethatthe His-taggedr-XAn11couldbe considered as a potentialstrong candidatefor future applicationparticularly in the animal feed industry, prebleaching of Kraft pulpin paper industries and degradation for biofuel ethanol production.

Futureresearch will be focused on development of a more effec-tive expression system for this xylanase.

 Acknowledgements

This work was funded by the Tunisian Ministry of Higher Edu-cation and Scientific Research and Technology (contract program

LMB-CBS, grant no. RL02CBS01).

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