Cloning, Expression, and Characterization of Xylanase G2 from Aspergillus oryzae VTCC-F187 in Aspergillus niger VTCC-F017

The study focuses on engineering of recombinant Aspergillus niger to produce highly active xylanase. The xylanase G2 encoding gene originating from Aspergillus oryzae VTCC-F187 was cloned, ampli ﬁ ed, and inserted into the pAN7.1GluA vector with speci ﬁ c primers possessing Bam HI. The recombinant plasmid was introduced into Aspergillus niger VTCC-F017 by chemical methods. The recombinant strain was checked by polymerase chain reaction method and Southern blot. Next, the recombinant protein was expressed and puri ﬁ ed by His-tag column. The molecular mass of the puri ﬁ ed xylanase G2, as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), was 21kDa with a speci ﬁ c activity of 1025IU/mg towards 0.5% (w/v) of birchwood xylan. The optimal temperature and pH were 55 ° C and pH6.5, respectively. The enzyme was stable in a temperature ranges 25 – 40 ° C and a pH ranges 5 – 7. The presence of Tween 80 enhanced xylanase activity. Triton X-100, however, had no impact on the function of the enzyme. The xylanase activity was reduced by Tween 20, SDS, and organic solvents. The enzyme was completely inhibited by Hg 2+ and partially by Zn 2+ , Fe 2+ , and Ag + , while it was slightly stimulated by K + and EDTA.


Introduction
Xylanase is a class of enzymes produced by microorganisms to breakdown xylans, the most abundant hemicellulosepolysaccharides found in plant cell walls. The complete hydrolysis of xylan requires the synergistic action of several xylanases of different functions including endo-β-1,4-Dxylanase (EC 3.2.1.8). Endoxylanases have a wide variety of biotechnological applications. In the pulp industry, they are used as a pre-bleaching agent which facilitates subsequent chemical bleaching, reduces the toxic chemical demand (especially organochlorine compounds), and improves the brightness of the pulp [1]. Xylanases are also used in the clarification of fruit juice, wine, beer, and forming xylitol in the confectionery industry [2]. Besides, the enzymes hydrolyze food containing xylan and, at the same time, help to reduce the viscosity in the digestive system followed by many posi-tive effects such as improved food absorption, improved microorganism populations of the intestine in the advantageous direction, and reduced digestion disorder [3,4].
On another hand, arabinoxylan oligosaccharides, the main products of xylanase-catalyzed hydrolysis reaction, are widely known as prebiotic carbohydrates with promising healthpromoting properties by improving the gut microbes [5] and enhancing the quality of immunotherapy of cancer patients [6]. An alternative form of arabinoxylan with ferulic acid has been drawn interests in the pharmaceutical industry which is presented as antioxidant and anticancer components [7].
Various microorganisms such as bacteria, yeast, and filamentous fungi have been reported with the ability to produce xylanase. Among them, fungi are capable of synthesizing highly active xylanases [8]. Owing to different applications, the purifications and biochemical characterizations of xylanase from various Aspergillus strains such as A. awamori, A. ficuum, A. giganteus, A. niger, and A. sydowii have been described in previous lectures [2,9,10].
In order to produce highly active enzymes, many recombinant microorganisms were created. The recombinant xylanase from E. coli, Bacillus subtilis, and Pichia pastoris were purified and characterized. The gene encoding xylanase from B. pumilus was overexpressed in E. coli and B. subtilis. These B. subtilis clones produced 2.7-3.0 folds higher than those of B. pumilus [11]. Aureobasidium pullulans Y-2311-1 xynA had an open reading frame encoding a polypeptide of 221 amino acids containing a putative 34 amino acids. Purified xylanase had the highest activity at pH 4.8 and 54°C. This enzyme was stable at 55°C and pH 4.5 [12]. Two family xylanases xynG2 and xynF3 were overexpressed and purified from A. oryzae [13,14]. The structural part of xynG2 was found to be 767 bp. The nucleotide sequence of cDNA amplified by reverse transcription polymerase chain reaction (RT-PCR) showed that the open reading frame of xynG2 was interrupted by a single intron which was 71 bp and encoded 232 amino acids. The purified xynG2 showed optimal activity at pH 6.0 and 58°C. It had a K m of 5.1 mg/mL and a V max of 123 μmol/min/mg when birchwood xylan was used as a substrate. The structural part of xynF3 was found to be 1468 bp. The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynF3 was interrupted by ten short introns, and encoded 323 amino acids. The purified xynF3 showed an optimal activity at pH 5.0 and 58°C [14]. The xlnR gene encoded a polypeptide of 875 amino acids was cloned and overexpressed in A. niger with goxC promoter [15]. Xylanase B from Aspergillus cf. niger BCC14405 was purified and characterized. It was found to have a molecular mass of 21 kDa, an optimal pH of 5.0, and a temperature of 55°C. When xylanase was tested using xylan from birchwood, it showed K m and V max values of 8.9 mg/mL and 11,100 U/mg, respectively [9].
In our previous study, a GH 10 enzyme, xylanase A, was expressed in Pichia pastoris GS115. The gene encoding this xylanase A has been cloned from A. niger DSM1957. The maximal activity of this recombinant xylanase (808.5 U/mg protein) was reached after 144 h of fermentation with shaking (200 rpm) at 30°C in YP medium (induced by methanol 0.5%) [16]. In this study, the gene encoding xylanase G2 from A. oryzae VTCC-F187 has been cloned and analyzed in pAn-GluA vector. This gene was used to engineer recombinant protein in A. niger with the aim of producing highly active xylanase to produce arabinoxylan as functional foods. The enzyme was purified and characterized to determine its potential in this regard. Escherichia coli DH5α (F-, ø80dlacZDM15, D(lacZYA-argF)U169, deoR, recA1, endA1, hsdR17(rk-, mk+), phoA, supE44, k-, thi-1, gyrA96, and relA1) and pJET1.2/blunt vector (Fermentas, Thermo Fisher Scientific Inc., Waltham, USA) were used for DNA manipulations and amplification. pAN7.1GluA vector was from Department of Genetic and Plant Pathology, University of Hamburg, Germany.

Total RNA Extraction.
Total RNA from A. oryzae VTCC-F187 was extracted from 2 g of mycelia using Trizol Reagent Kit (Invitrogen Corp., Carlsbad, CA, USA). The protocol was previously described in detail [17].

PCR Amplification.
Based on the nucleotide sequence of xlnG2 available in GenBank, two oligonucleotides xlnG2F (5 ′ -CG GGATCCATGGTGTCCTTCTCCTCCATCC-3 ′ ) and xlnG2R (5 ′ -CG GGATCCTCAGTGGTGGTGGTGGTGGT GATAAACAGTGATAGCAGA-3 ′ ) were designed as primers to amplify the xlnG2 from A. oryzae VTCC-F187 with the introduction of the underlined EcoRI and XbaI restriction sites at 5 ′ of forward and reverse primer, respectively. The firststrand cDNA product was used to amplify xlnG2 using two primers xlnG2F and xlnG2R.
The PCR mixture contained 2.5 μL of 10 times PCR buffer, 2 μL of 2.5 mM dNTP, 1.5 μL of 25 mM MgCl 2 , 2 μL of cDNA (50 ng) from control RT reaction, 0.5 μL of 5 unit Taq polymerase, and 1 μL of each primer (10 pmol), supplemented with 14.5 μL of distilluted water to a final volume of 25 μL. The thermocycler conditions were as follows: 94°C/3 min; 35 cycles of 95°C/45 s, 54°C/1 min, and 72°C/1 min; 72°C/10 min. PCR products were inserted into the pJET1.2/blunt vector resulting in pJxlnG2 and transformed into the E. coli DH5α. The recombinant plasmid was confirmed by restriction enzyme analysis and DNA sequencing. 2.6. Transformation of Xylanase Gene in A. niger. Approximately 30000 spores were bred in 50 mL YPD medium at 150 rpm, 28°C overnight; then, the whole culture was ground with 150 mL YPD medium (3 times, 10 s/time). Next, 50 mL of the above mixture was added to 150 mL YPD medium and shaken overnight at 150 rpm, 28°C in a 500 mL-flask bottle. The entire pre-culture was filtered by a Wilson-sieve filter funnel (100 μm) and washed several times with deionized water. The weight of the hyphae mixture was determined after drying with sterilized blotting paper. The enzyme solution was mixed with the above hyphae mixture (20 mL with 1-1.5 g), in a 100 mL-flask bottle at 75 rpm, 28°C. Next, the collected cells were filtered through the 100 μm and 40 μm filter funnel Wilson-sieve, respectively. The dregs of the cell were dissolved carefully in 12 mL 0.7 M NaCl pH 5.6, then centrifuged it again and reconstituted. The number of cells was checked with a microscope. 50 μg plasmid-DNA and 1 mL of precooled PTC (40% PEG 4000 and 60% STC (20% sucrose, 10 mM Tris-HCl, pH 8.0, and CaCl 2 50 mM)) were added and mixed well. The mixture was kept at 4°C for 25 min and shaken every 2 min and incubated at 80 rpm, 28°C overnight. Later, the cells were mixed with AMM medium added 50 μl hygromycin, then poured into 2 plates, and kept at 28°C overnight. Each plate was coated with 10 mL of 1.2% agar containing 50 μL of hygromycin. The plates were checked every day until colonies became visible. The colonies were picked and transferred to a selective agar plate of CM containing 900 μg/L hygromycin (w/v).

DNA Isolation from A. niger.
A square of 0.5 cm 2 agar plates were cut and cultured in 50 mL of YPG liquid medium, at 200 rpm, 28°C for 3-4 days. Hyphae were collected by filtering through a filter funnel and rushed in porcelain pestle with liquid nitrogen, then transferred into 2 mL eppendorf. 600 μL lysis buffer was added to each eppendorf tube and placed them at 65°C from 40 min to 1 h. After that, all eppendorf tubes above were centrifuged 13000 rpm, 10 min at 4°C. The upper phase was transferred to another 1.5 mL eppendorf tube. Chloroform : isoamyl alcohol solution (24 : 1; v/v) was added with the equivalent volume and shaken well, then centrifuged the mixture 13000 rpm at 4°C for 15 min. The upper phase was transferred to a new 1.5 mL eppendorf tube, and the DNA was precipitated by adding 750 μL of isopropanol, shaking well, leaving it in the refrigerator for 30 min at 4°C and centrifuging 13000 rpm at 4°C for 20 min. Then the residue was collected and washed with 500 μL cold alcohol (70%), dried, and precipitated in TE.

Southern Blot.
After treatment with restricted enzymes, the DNA samples isolated from A. niger were checked by electrophoresis on an agarose gel. Next, the gel was placed in a plastic box and washed 2 times with distilled water, then soaked in denaturation solution, and shaken for 30 min at room temperature (25°C). After that, the gel was washed with distilled water and soaked in neutralization solution, shaken for 30 min at room temperature (25°C), and then balanced in 20 × SSC for at least 10 min. After had being removed from the balanced solution, the gel was placed on top of a sheet of Whatman 3MM blotting paper which had been already prewet with 20 × SCC solution. Air bubbles between the two layers should be avoided. The nylon membrane was placed on top of the gel, and 4-5 sheets of Whatman 3MM blotting paper were put on the nylon membrane. A 10 cm stack of absorbent paper and a heavy object weighing about 200-500 g were placed on top, then leaving them overnight in order to transfer DNA onto the nylon membrane. Before fixing, the film was washed by 2 × SSC buffer, then placed on the 3MM Whatman plate (placing the face containing DNA on top). The membrane was laid under UV light for 1-3 min, washed with 2 × SSC, then put it back on the 3MM Whatman plate, and allow it to dry, shaken overnight at 68°C. Next, the solution covering the membrane was removed, then poured the probe mixture, and incubated overnight at 68°C. Accordingly, the membranes were washed in three steps: (1)  sugars produced by enzymatic hydrolysis of birchwood xylan. A reaction mixture of 100 μL of the crude or purified xylanase containing 11 μg for each reaction was incubated with 400 μL of 0.5% (w/v) birchwood xylan in 20 mM potassium phosphate buffer pH 6.5 at 55°C for 5 min. To arrest the reducing sugar released in the reaction mixture, 1.25 mL of DNS was added. The reducing sugars were determined by measuring the absorbance at 540 nm [18]. D-xylose was used as a standard. One unit (U) of xylanase activity was defined as the amount of enzyme that released 1 mol of xylose per minute under the standard assay conditions.

SDS-PAGE Electrophoresis and Protein Concentrations.
The relative molecular weight and purity of the xylanase G2 enzyme were analyzed through 12.5% (w/v) SDS polyacrylamide gel electrophoresis [19]. Proteins were visualized by staining with 0.1% (w/v) Coomassie Brilliant Blue R-250. Protein concentrations were estimated by the method of Bradford with bovine serum albumin as a standard [20].
2.13. Optimal Temperature and Thermostability. We determined the optimal temperatures by carrying out the experiments at different temperatures at 37, 40, 45, 50, 55, 60, 65, and 70°C. To determine the thermostability, the purified enzyme was incubated 0-24 h at different temperatures from 25 to 50°C. The residual activity of the enzyme was observed by standard assay procedure.
2.14. Optimal pH and pH Stability. We determined the optimal pH by carrying out experiments at different pH 3.5-8 (acetate: 3.5-5; phosphate: 6-8). To determine the pH stability, the enzyme was incubated in 10 mM buffers with different pH 3.5-8 (acetate: 3.5-5; phosphate: 6-8) at 4°C for 2, 8, and 24 h. The residual activity of the enzyme was observed by standard assay procedure.

Effect of Metal Ions.
We assessed the influences of metal ions on xylanase activity by incubating enzyme with 10 mM metal ions (Mg 2+ , Fe 2+ , Cu 2+ , Ca 2+ , Ag + , Ni 2+ , K + , Hg 2+ , and Zn 2+ ) and ethylenediaminetetraacetic acid (EDTA) at room temperature (25°C) for 2 h. The residual activity of the enzyme was observed by standard assay procedure.

Organic Solvent Tolerant.
We assessed the effects of organic solvents on xylanase activity by incubating the enzyme with organic solvents (methanol, ethanol, isopropanol, acetone, and n-butanol) at the final concentration 30% (v/v) at 30°C, for 1 h. The residual activity of the enzyme was observed by standard assay procedure.
2.17. Effect of Detergents. The enzyme was incubated with 2% Tween 20, Tween 80, Triton X-100, and SDS (v/v) at 30°C, for 1 h. The residual activity of the enzyme was observed by standard assay procedure. All measurements were carried out in triplicate with the resulting values being the mean of the cumulative data obtained.

Results and Discussion
3.1. Cloning Xylanase Gene. The mRNA total from A. oryzae VTCC-F187 was extracted and purified to be used as a template to create cDNA with Fementas kit (Supplement 1A). The xylanase genes were then amplified with xlnG2F/xlnG2R primer pairs. PCR product of size 699 bp was observed (Supplement 1B).
These PCR amplification products were inserted to the vector pJET1.2 blunt to be transformed into E. coli DH5α. The recombinant colonies were collected; recombinant plasmid was extracted and then identified by electrophoresis. The plasmid contained in the lower band was selected to be used as the template for PCR reaction using primer xlnG2F/xlnG2R. The PCR product had a size of 699 bp, which corresponded to the size of the xlnG2 (Supplement 1C). These results suggested that the xlnG2 was successfully cloned in to pJET1.2 and transformed into E. coli DH5α. Recombinant plasmids carrying the gene encoding xlnG2 were denoted by pJxlnG2.

Design of Plasmid to Express the Xylanase Gene in
Aspergillus sp. The xylanase gene was amplificated from pJxlnG2 by PCR reaction with specific primers containing BamHI site and His coding sequence (Supplement 2A). PCR product was attached to the pGEMT vector. The adhesive product is transformed on a medium containing X-gal and ampicillin. White colonies were selected to separate plasmid and examine the presence of the xylanase gene (Supplement 2B).
To design the vector expressing xylanase gene, the plasmid pAN7.1GluA with a strong promotor GPDA from A. nidulan and a BamHI site was used. Plasmids containing the xylanase gene were cut by BamHI (Supplement 3A), purified, and tested by electrophoresis on agarose gel (Supplement 3B). Vector and the xylanase genes were ligated together by the T4-ligate. The adhesive product was transferred into E.coli DH5α.
The colonies were randomly selected, separated plasmid, and examined the presence and direction of the gene by primers in which the forward primer was designed in the promoter GpdA, and the reverse primer was used to amplify the xylanase gene. The PCR product had a size of about 1200 bp (Supplement 3C). Recombinant plasmids carrying the gene encoding xlnG2 were denoted by pANxlnG2.

Expression of Xylanase G2 Gene in Aspergillus niger
VCCT-F017. Recombinant plasmid pANxlnG2 was cut by restriction enzyme EheI and was inserted into the genome at the location of the gene encoding GluA. DNA from recombinant fungal strains was amplified by PCR reaction with primers in which forward primer was from promoter GpdAF and reverse primer was the primer in the gene coding xylanase G2 (Supplement 4).
For further examination, the DNA from the recombinant strain was cut with the restriction enzyme XhoI and examined with Southern blot with the probe of the xylanase G2 gene. According to calculations, when cutting the recombinant DNA with XhoI, the segment containing xlnG2 would be 2700 bp (Figure 2(a)).
After checking the recombinant DNA strain carrying the gene coding xylanase G2, the recombinant and wild-type strains were cultured in the YPD medium. After 120 h of incubation, the recombinant A. niger VTCC-017 strains had higher xylanase activity than the wild-type strains (Figure 2(b)).

Xylanase
Purification. Xylanase from crude extract obtained after 120 h of culture was purified through the Ni 2+ affinity column. The purified xylanase G2 showed a single protein band of about 21 kDa on SDS-PAGE (Figure 3, lanes 3-6) and revealed a specific activity of 1102:5 ± 12:5 U/mg protein. The yield of purification was 52.6%, and the purification factor was 2.2 (Table 1). Family 11 xylanases from A. oryzae strain overexpressed in A. oryzae showed a molecular mass of 21 kDa [14]. However, this enzyme from A. niger strains expressed in P. pastoris showed a molecular mass of <21 kDa [24][25][26][27]. On the other hand, family 10 endo-1,4-β-xylanases expressed in P. pastoris from A. usamii E001 [28] and A. terreus BCC129 [29] showed a molecular mass of >32 kDa. These results were obvious evidence of the diversity in Aspergillus xylanase production by different organisms.
3.6. Characterization of Recombinant Xylanase 3.6.1. Optimization of Temperature and pH. When the reaction temperature increased from 37 to 55°C, the xylanase activity also increased gradually from 56 to 100%. The highest activity of the enzyme was observed at 55°C

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(1085 ± 36:8 IU/mg). When the reaction temperature continued to rise above 55°C, the hydrolysis activity tended to decrease. The optimal temperature for the recombinant xylanase was 55°C (Figure 4(a)).
At pH 4.0, the relative activity of xylanase is 38%. When the reaction pH increases to 6.5, the activity reached the highest point (1031 IU/ml). The residual activity decreased steadily to 50% at pH 9. So the recombinant xylanase activity was optimized in a neutral environment (Figure 4(b)).
The optimal temperature and pH for family 11 Aspergillus xylanases were observed at 50-60°C and pH 5-6 [13,28,29] while these of xylanase G2 from A. oryzae KBN616 were observed at 58°C and pH 6, respectively [14]. The optimal temperature and pH of a recombinant enzyme, purified from A. niger XZ-3S, which has a good stability in alkaline conditions, were 40°C and 5.0, respectively [30].
3.6.2. Temperature and pH Stability. Enzyme activity at temperatures of 25, 37, and 40°C was not much different (Figure 4(a)), and the overall trend was downward over time. The higher the temperature, the more rapid the enzyme activity loss. After 5 h of incubation at 25 and 40°C, enzyme activity decreased to 86 and 92%, respectively, compared to the initial activity. After 24 h of incubation at 50°C, activity plunged to 53% compared to the initial activity ( Figure 5(a)).
Medium pH affects the stability of the enzyme, so it is important to select a suitable pH buffer for enzyme preservation. Xylanase from recombinant strains was relatively stable at pH 4.0-8.0. After 24 h of incubation, the activity remains 78.4% and 75.0%, respectively ( Figure 5(b)). Therefore, this enzyme retained its function in both weak alkali and acidic environment. This characteristic of GH11 family xylanase was reported in previous study when the xylanase G2 from A. oryzae KBN616 preserved its activity at pH 4.0-8.0 after incubated for 12 h at 25°C [14], 3.6.3. Effect of Organic Solvents. The organic solvent resistance of an enzyme is important to its catalytic capabilities. There are more and more advantages of employing enzymes as catalysts in organic solvents have been reported. Therefore, the search for organic solvent tolerant enzymes has been an extensive area of research. In our study, the effects of several widely used organic solvents such as methanol, ethanol, isopropanol, acetone, and n-butanol on the purified recombinant xylanase G2 were assessed. The purified recombinant xylanase G2 showed a high tolerance to nearly all the tested organic solvents with the residual activity of 62.4-84.6% ( Table 2).
The highest tolerance was observed in presence of methanol. The xylanase activity was remained 84.6%, followed by acetone (reached 72.0%). The lowest was the solvent ethanol; the remaining xylanase activity was only 62.4%.
3.6.4. Effect of Detergents. Detergents such as Tween 20 and SDS affect xylanase activity. The addition of 2% (v/v) of Tween 20 and SDS reduced the xylanase activity by 27% and 51%, respectively, after 1 h of treatment. Particularly, Tween 80 slightly increased the activity of xylanase 17% while Triton X-100 had no effect on the function of xylanase (Table 3). Our research was also coincident with Hmida-Sayari et al. 's study that the addition of Tween 80 increased the XAn11 A. niger xylanase activity by 33%, and an addition    (Table 4). On the other hand, K + and EDTA slightly stimulated xylanase activity. The inhibition or stimulation of enzyme activity may be due to metal ions interacted with SH or carboxyl groups which led to an altered conformation of protein subsequent inactivation [35]. Hg 2+ is a strong inhibitor of most xylanases, including those produced by Aspergillus sp., such as Aspergillus giganteus [36], Aspergillus awamori [37], and Aspergillus niger [38]. The inhibition of xylanase by Hg 2+ has been reported as related to the presence of histidine and tryptophan residues, which oxidize the indole ring, thereby inhibiting the enzyme activity. In addition, the inhibition by heavy metal ions (such as Zn 2+ and Hg 2+ ) may also occur due to the formation of a complex with the reactive groups of the enzyme, such as SH, CONH 2 , NH 2 , COOH, and PO 4 or the catalysis of the cysteine thiol group auto-oxidation, which leads to the formation of intra-and intermolecular disulfide bonds or to the formation of sulfenic acid [39]. However, in our study, the addition of chelating agents, EDTA, did not affect xylanase activity. These data suggest that sulfhydryl groups are not related to the active site of the purified xylanase. Therefore, histidine and tryptophan might play an important role in the active site of the purified xylanase. Further structural studies are needed to confirm this hypotheses. Relative activity was expressed as a percentage of the control reaction without any additive (100% xlnG2 activity was 900:12 ± 38:3 U/mg). Hg 2+ 0 ± 0:0 a Relative activity was expressed as a percentage of the control reaction without any additive (100% xlnG2 activity was 881:0 ± 65:4 U/mg).   Figure 5: (a) Temperature and (b) pH stability of recombinant xylanase (the relative activity (%) was expressed as a percentage of the control reaction at time zero, and 100% xylanase activity was 921:9 ± 15:02 U/mg and 1097:2 ± 76:3 U/mg for temperature and pH stability, respectively).

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Some xylanases require Ca 2+ and Mg 2+ for their stability and activities, such as xylanase from A. Niger SCTCC 400264 [22], A. niger US368 [34], and A. niger BCC14405 [9]. However, in our study, Mg 2+ and Ca 2+ had no significantly effects on xylanase activity. These results suggested that these metal ions do not play important role in the activation and protection of the active site of the purified enzyme.

Conclusion
In this study, cloning of the gene encoding xylanase G2 from A. oryzae was performed successfully. The expression of recombinant xylanase G2 in A. niger was successful. The xylanase G2 activity reached 1025 IU/mg. Xylanase G2 displayed its optimal activity at 55°C, pH 6.5. This enzyme was stable at the temperatures ranging from 25 to 40°C, pH 5-7. Metal ions like K + and EDTA slightly increased xylanase activity. On other hand, the purified recombinant xylanase was inhibited by Tween 20, SDS, heavy metal ions such as Ag + and Hg 2+ , and organic solvents. Tween 80 increased, and Triton X-100 had no effect on xylanase activity.

Data Availability
The nucleotide sequence is determined by the Sanger method, on the ABI PRISM 3100 Avant Genetic Analyzer. Nucleotide sequence analysis is performed by the DNAstar software.

Conflicts of Interest
The authors declare that they have no competing interests.