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

Authors: Do Thi Tuyen; Nguyen Tien Cuong; Nguyen Sy le Thanh; Nguyen Thi Thao; Le Thanh Hoang; Nguyen Thi Hien Trang; Nguyen Thi Trung; Dao Thi Mai Anh
Journal: BioMed Research International
 (2021)

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## Abstract

The study focuses on engineering of recombinantAspergillus niger to produce highly active xylanase. The xylanase G2 encoding gene originating from Aspergillus oryzae VTCC-F187 was cloned, amplified, and inserted into the pAN7.1GluA vector with specific primers possessing BamHI. 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 purified by His-tag column. The molecular mass of the purified xylanase G2, as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), was 21 kDa with a specific activity of 1025 IU/mg towards 0.5% (w/v) of birchwood xylan. The optimal temperature and pH were 55°C and pH 6.5, 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 Hg2+ and partially by Zn2+, Fe2+, and Ag+, while it was slightly stimulated by K+ and EDTA.


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## Body

## 1. Introduction

Xylanase is a class of enzymes produced by microorganisms to breakdown xylans, the most abundant hemicellulose-polysaccharides 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-D-xylanase (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 positive 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 health-promoting 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 fromE. 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 Km of 5.1 mg/mL and a Vmax 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 Km and Vmax 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 inPichia 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 pAnGluA 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.


## 2. Materials and Methods

### 2.1. Chemicals and Reagents

Restriction enzymes, Taq DNA polymerase, and T4 ligase were purchased from Fermentas, Thermo Fisher Scientific Inc. (Waltham, USA). Kit ProBond™ Nickel-chelating resin was from Invitrogen Corp. (Carlsbad, CA, USA). 3,5,5-Dinitrosalisilic acid (DNS), birchwood xylan, and SDS were from Sigma-Aldrich Co. (St. Luis, USA). Peptone, yeast extract, Tween 80, and Tween 20 were from BioBasic Inc. (Ontario, Canada). Triton X-100 was from Merck (Darmstadt, Germany). All other chemicals were of analytical grade unless otherwise stated.


### 2.2. Vectors, Strains, and Culture Conditions

Aspergillus oryzae VTCC-F187 and A. niger VTCC-017 strains were provided by Vietnam Type Culture Collection (VTCC). They are used as the source for the xylanase gene (xlnG2).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.The recombinantE. coli was inoculated in Luria-Bertani medium (LB) containing (g/L) bacto tryptone 10, yeast extract 5, NaCl 10, and at a pH of 7–7.5. Next, LB agar contained additional 20 agar and 100 μg ampicillin.Yeast peptone glucose medium (YPG) contains (g/L) yeast extract 10, and peptone 20 supplemented with glucose 10. Yeast peptone dextrose (YPD) medium (g/L) contains yeast extract 10, peptone 20, dextrose 20, and agar 20 for plates only. Minimal medium (AMM) (g/L) contains NaNO3 0.6, KCl 0.52, KH2PO4 1.52, MgSO4.7H2O 0.52, sucrose 2, and 1 ml MNS. Complete media (CM) contain 10 mL solution A (Ca(NO3)2.4H2O 10%), 10 mL solution B (KH2PO4 2 g/L, MgSO4.7H2O 2.5 g/L, NaCl 1.5 g/L, and pH 5.3), yeast-casein 0.2 g/L, glucose 1 g/L, 1 mL MNS, and 900 mL H2O. TB3 medium (g/L) contains yeast extract 3, casamino-acid 3, glucose 10, and sucrose 20. All medium was from Merck (Darmstadt, Germany).


### 2.3. Total RNA Extraction

Total RNA fromA. 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].


### 2.4. PCR Amplification

Based on the nucleotide sequence ofxlnG2 available in GenBank, two oligonucleotides xlnG2F (5′-CG GGATCCATGGTGTCCTTCTCCTCCATCC-3′) and xlnG2R (5′-CG GGATCCTCAGTGGTGGTGGTGGTGGTGATAAACAGTGATAGCAGA-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 first-strand cDNA product was used to amplify xlnG2 using two primers xlnG2F and xlnG2R.The PCR mixture contained 2.5μL of 10 times PCR buffer, 2 μL of 2.5 mM dNTP, 1.5 μL of 25 mM MgCl2, 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.


### 2.5. Analysis of Genomic DNA

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


### 2.6. Transformation of Xylanase Gene inA. 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 (100μm) 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 CaCl2 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).


### 2.7. DNA Isolation fromA. niger

A square of 0.5 cm2 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.


### 2.8. Southern Blot

After treatment with restricted enzymes, the DNA samples isolated fromA. 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 pre-wet 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) 2 times, 5 min each time at room temperature (25°C) with 20 mL of W1 solution; (2) 2 times, 15 min each time at 68°C with 20 mL of W2 solution; (3) 30 min in WP solution and B2 solution at room temperature (25°C). The antibody was mounted on the membrane for 30 min (5 μL antibody was mixed in 50 mL B2 solution). The membrane was washed 3 times, 20 min each time with WP solution at room temperature (25°C), then washed for 5 min in B3 solution. 500 μL substrate was spread onto the nylon plastic sheet, then covered the membrane with DNA sample on the substrate for 5 min at room temperature (25°C); the results were visualized by fluoghraphy.


### 2.9. Gene Expression

For the expression ofxylanase G2 in recombinant A. niger VTCC-F017, 0.5 mL of an overnight culture was inoculated into 50 mL YP medium supplemented with 1% (w/v) glucose in a 1 L-flask bottle and grown at 30°C with agitation at 200 rpm. The culture supernatant was collected periodically to detect xlnG2 activity during 120 h incubation.


### 2.10. Purification of Recombinant xlnG2

The culture supernatant containing xlnG2 was harvested from 50 mL culture by centrifugation at 5000 rpm and 4°C for 10 min. After that, 8 mL of culture supernatant was applied to a ProBond™ Ni2+ affinity chromatography column containing 2 mL resin, which had been equilibrated with native binding buffer (250 mM NaH2PO4, 2.5 M NaCl, and pH 8.0) and incubated for 60 min at room temperature (25°C) with gentle handshaking for several times. The column was washed 4 times with 8 mL of native wash buffer (250 mM NaH2PO4, 2.5 M NaCl, 20 mM imidazole, and pH 8.0). The binding protein was eluted with 1 mL of a native elution buffer containing 250 mM imidazole (250 mM NaH2PO4, 2.5 M NaCl, 300 mM imidazole, and pH 8.0). The purified enzyme was used for characterization.


### 2.11. Determination of Xylanase Activity

Xylanase activity was determined by measuring the concentration of reducing sugars produced by enzymatic hydrolysis of birchwood xylan. A reaction mixture of 100μL 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.


### 2.12. 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.


### 2.15. Effect of Metal Ions

We assessed the influences of metal ions on xylanase activity by incubating enzyme with 10 mM metal ions (Mg2+, Fe2+, Cu2+, Ca2+, Ag+, Ni2+, K+, Hg2+, and Zn2+) and ethylenediaminetetraacetic acid (EDTA) at room temperature (25°C) for 2 h. The residual activity of the enzyme was observed by standard assay procedure.


### 2.16. 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.


## 2.1. Chemicals and Reagents

Restriction enzymes, Taq DNA polymerase, and T4 ligase were purchased from Fermentas, Thermo Fisher Scientific Inc. (Waltham, USA). Kit ProBond™ Nickel-chelating resin was from Invitrogen Corp. (Carlsbad, CA, USA). 3,5,5-Dinitrosalisilic acid (DNS), birchwood xylan, and SDS were from Sigma-Aldrich Co. (St. Luis, USA). Peptone, yeast extract, Tween 80, and Tween 20 were from BioBasic Inc. (Ontario, Canada). Triton X-100 was from Merck (Darmstadt, Germany). All other chemicals were of analytical grade unless otherwise stated.


## 2.2. Vectors, Strains, and Culture Conditions

Aspergillus oryzae VTCC-F187 and A. niger VTCC-017 strains were provided by Vietnam Type Culture Collection (VTCC). They are used as the source for the xylanase gene (xlnG2).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.The recombinantE. coli was inoculated in Luria-Bertani medium (LB) containing (g/L) bacto tryptone 10, yeast extract 5, NaCl 10, and at a pH of 7–7.5. Next, LB agar contained additional 20 agar and 100 μg ampicillin.Yeast peptone glucose medium (YPG) contains (g/L) yeast extract 10, and peptone 20 supplemented with glucose 10. Yeast peptone dextrose (YPD) medium (g/L) contains yeast extract 10, peptone 20, dextrose 20, and agar 20 for plates only. Minimal medium (AMM) (g/L) contains NaNO3 0.6, KCl 0.52, KH2PO4 1.52, MgSO4.7H2O 0.52, sucrose 2, and 1 ml MNS. Complete media (CM) contain 10 mL solution A (Ca(NO3)2.4H2O 10%), 10 mL solution B (KH2PO4 2 g/L, MgSO4.7H2O 2.5 g/L, NaCl 1.5 g/L, and pH 5.3), yeast-casein 0.2 g/L, glucose 1 g/L, 1 mL MNS, and 900 mL H2O. TB3 medium (g/L) contains yeast extract 3, casamino-acid 3, glucose 10, and sucrose 20. All medium was from Merck (Darmstadt, Germany).


## 2.3. Total RNA Extraction

Total RNA fromA. 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].


## 2.4. PCR Amplification

Based on the nucleotide sequence ofxlnG2 available in GenBank, two oligonucleotides xlnG2F (5′-CG GGATCCATGGTGTCCTTCTCCTCCATCC-3′) and xlnG2R (5′-CG GGATCCTCAGTGGTGGTGGTGGTGGTGATAAACAGTGATAGCAGA-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 first-strand cDNA product was used to amplify xlnG2 using two primers xlnG2F and xlnG2R.The PCR mixture contained 2.5μL of 10 times PCR buffer, 2 μL of 2.5 mM dNTP, 1.5 μL of 25 mM MgCl2, 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.


## 2.5. Analysis of Genomic DNA

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


## 2.6. Transformation of Xylanase Gene inA. 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 (100μm) 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 CaCl2 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).


## 2.7. DNA Isolation fromA. niger

A square of 0.5 cm2 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.


## 2.8. Southern Blot

After treatment with restricted enzymes, the DNA samples isolated fromA. 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 pre-wet 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) 2 times, 5 min each time at room temperature (25°C) with 20 mL of W1 solution; (2) 2 times, 15 min each time at 68°C with 20 mL of W2 solution; (3) 30 min in WP solution and B2 solution at room temperature (25°C). The antibody was mounted on the membrane for 30 min (5 μL antibody was mixed in 50 mL B2 solution). The membrane was washed 3 times, 20 min each time with WP solution at room temperature (25°C), then washed for 5 min in B3 solution. 500 μL substrate was spread onto the nylon plastic sheet, then covered the membrane with DNA sample on the substrate for 5 min at room temperature (25°C); the results were visualized by fluoghraphy.


## 2.9. Gene Expression

For the expression ofxylanase G2 in recombinant A. niger VTCC-F017, 0.5 mL of an overnight culture was inoculated into 50 mL YP medium supplemented with 1% (w/v) glucose in a 1 L-flask bottle and grown at 30°C with agitation at 200 rpm. The culture supernatant was collected periodically to detect xlnG2 activity during 120 h incubation.


## 2.10. Purification of Recombinant xlnG2

The culture supernatant containing xlnG2 was harvested from 50 mL culture by centrifugation at 5000 rpm and 4°C for 10 min. After that, 8 mL of culture supernatant was applied to a ProBond™ Ni2+ affinity chromatography column containing 2 mL resin, which had been equilibrated with native binding buffer (250 mM NaH2PO4, 2.5 M NaCl, and pH 8.0) and incubated for 60 min at room temperature (25°C) with gentle handshaking for several times. The column was washed 4 times with 8 mL of native wash buffer (250 mM NaH2PO4, 2.5 M NaCl, 20 mM imidazole, and pH 8.0). The binding protein was eluted with 1 mL of a native elution buffer containing 250 mM imidazole (250 mM NaH2PO4, 2.5 M NaCl, 300 mM imidazole, and pH 8.0). The purified enzyme was used for characterization.


## 2.11. Determination of Xylanase Activity

Xylanase activity was determined by measuring the concentration of reducing sugars produced by enzymatic hydrolysis of birchwood xylan. A reaction mixture of 100μL 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.


## 2.12. 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.


## 2.15. Effect of Metal Ions

We assessed the influences of metal ions on xylanase activity by incubating enzyme with 10 mM metal ions (Mg2+, Fe2+, Cu2+, Ca2+, Ag+, Ni2+, K+, Hg2+, and Zn2+) and ethylenediaminetetraacetic acid (EDTA) at room temperature (25°C) for 2 h. The residual activity of the enzyme was observed by standard assay procedure.


## 2.16. 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.


## 3. Results and Discussion

### 3.1. Cloning Xylanase Gene

The mRNA total fromA. 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 intoE. 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.


### 3.2. Xylanase Gene Sequence Analysis

After nucleotide sequencing, the putative sequence of xylanase G2 gene in GeneBank and amino acid sequences were compared using the DNAstar software. The gene sequence of xylanase G2 ofA. oryzae VTCC-F187 (Ao_Xln_07) had a high similarity to the sequences of xylanase genes of A. oryzae Ao_Xln_41 gene (99.3%) in GenBank (Figure 1(a)). Accordingly, the predicted amino acid sequence of A. oryzae VTCC-F187 xylanase had a high homology to Ao_Xln_41 (99.9%) (Figure 1(b)). The xlnG2 (699 bp, 232 aa) from A. oryzae VTCC-F187 belongs to the glycosyl hydrolase family 11, which was completely similar to the xlnG2 (767 bp, 232 aa) (glycosyl hydrolase family 11) [14], A. oryzae xlnG1 (725 bp, 189 aa) [21], xlnC (636 bp, 211 aa) [22], xlnA (glycosyl hydrolase family 10) [16], and xlnD (957 bp, 318 aa) [23] from A. niger strains (from GenBank). The phylogenetic tree of nucleotide and amino acids are not much different; this can be explained by a multitude of codons encoding for an amino acid. The nucleotide sequence of gene xylanase G2 of Aspergillus oryzae VTCC-F187 had been registered on GenBank with the code of EU848307.Figure 1
Phylogenetic tree of the (a) xylanase G2 gene family and (b) xylanase G2 protein family. Ao_Xln_41:Aspergillus oryzae AB044941; Ao_Xln_54: Aspergillus oryzae AP007154; Ao_Xln_64: Aspergillus oryzae AP007164; Ao_Xln_74: Aspergillus oryzae AP007174; Ao_Xln_76: Aspergillus oryzae AB066176; Ao_Xln_85: Aspergillus oryzae AB003085.
(a)(b)


### 3.3. Design of Plasmid to Express the Xylanase Gene inAspergillus sp.

The xylanase gene was amplificated from pJxlnG2 by PCR reaction with specific primers containingBamHI 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 fromA. 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 (Supplement3C). Recombinant plasmids carrying the gene encoding xlnG2 were denoted by pANxlnG2.


### 3.4. Expression of Xylanase G2 Gene inAspergillus niger VCCT-F017

Recombinant plasmid pANxlnG2 was cut by restriction enzymeEheI 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 enzymeXhoI 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)).Figure 2
(a) Southern blot analysis of the recombinantxlnG2 in A. niger (M: marker; 1-6: recombinant strains; 7: wild-type strain; 8: plasmid pANxlnG2); (b) Xylanase activity (1: wild-type strain; 2 and 3: xylanase xlnG2 of recombinant A. niger VTCC-F017).
(a)(b)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 recombinantA. niger VTCC-017 strains had higher xylanase activity than the wild-type strains (Figure 2(b)).


### 3.5. Xylanase Purification

Xylanase from crude extract obtained after 120 h of culture was purified through the Ni2+ 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 [24–27]. 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.Figure 3
Electrophoresis of the xylanase G2: 1: extracellular extract of wild-type strain; 2: extracellular extract of recombinant strain; 3-6: purified xylanase G2; M: marker.Table 1
Purification steps of recombinant xylanase G2.
Purification stepTotal activity (U)Total protein (mg)Specific activity (U/mg)Purification factorYield (%)Culture supernatant922.1±8.61.84±0.05501.1±7.81100Ni2+ProBond™ resin column485.1±13.20.44±0.0021102.5±12.52.252.6


### 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 (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)).Figure 4
(a) Temperatures and (b) pH optimum of recombinant xylanase (the relative activity (%) was expressed as a percentage of optimal activity, and 100% xylanase activity was1085±36.8 U/mg at (a) 55°C and 852.3±41.3 U/mg at (b) pH 7).
(a)(b)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 (Figure4(b)).The optimal temperature and pH for family 11Aspergillus 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 (Figure4(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)).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 was921.9±15.02 U/mg and 1097.2±76.3 U/mg for temperature and pH stability, respectively).
(a)(b)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 (Figure5(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% (Table2).Table 2
Effect of organic solvents on the recombinant xylanase G2 activity.
Organic solventsResidual activity (%)aMeOH84.6±0.4EtOH62.4±4.2IsOH66.5±1.7Act72.0±3.1n-BtOH40.8±2.7aRelative activity was expressed as a percentage of the control reaction without any additive (100% xlnG2 activity was 927.21±38.3 U/mg).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%.Previous studies also showed the high retained activity of xylanase fromA. awamori VTCC-F312 63-86% [31] and xlnA from Pichia pastoris GS115 [16]. However, methanol and ethanol reduced significantly the activity of xylanase from A. niger C3486 [32] and completely inhibited the enzymatic activity from Termitomyces sp. [33].


#### 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 (Table3). 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 of SDS strongly inhibited the enzyme with residual activity of 20% comparing to the initial activity [34].Table 3
Effect of detergents on the recombinant xylanase G2 activity.
DetergentsResidual activity (%)aTriton X-100103±5.6Tween 80117±0.7Tween 2073±2.8SDS49±6.9aRelative activity was expressed as a percentage of the control reaction without any additive (100% xlnG2 activity was 900.12±38.3 U/mg).


### 3.7. Effect of Metal Ions

The effect of various metal ions and EDTA on the purified xylanase activity was investigated. After 2 h of incubation at the concentration of 10 mM, only Hg2+ completely inhibited enzyme activity while Mg2+, Ca2+, and Ni2+ slightly reduced the enzyme activity. The presence of Fe2+, Zn2+, and Ag+ partially reduced enzyme activity by 56, 57, and 57%, respectively (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]. Hg2+ 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 Hg2+ 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 Zn2+ and Hg2+) may also occur due to the formation of a complex with the reactive groups of the enzyme, such as SH, CONH2, NH2, COOH, and PO4 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].Table 4
Effect of metal ions and EDTA on the recombinant xylanase G2 activity.
Additive (10 mM)Residual activity (%)aMg2+96±1.1Fe2+44±5.8Cu2+53±7.7Ca2+99.6±13.5Ag+43±9.1Ni2+96±3.1K+101±0.1EDTA106±10.4Zn2+43±5.3Hg2+0±0.0aRelative activity was expressed as a percentage of the control reaction without any additive (100% xlnG2 activity was 881.0±65.4 U/mg).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.Some xylanases require Ca2+ 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, Mg2+ and Ca2+ 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.


## 3.1. Cloning Xylanase Gene

The mRNA total fromA. 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 intoE. 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.


## 3.2. Xylanase Gene Sequence Analysis

After nucleotide sequencing, the putative sequence of xylanase G2 gene in GeneBank and amino acid sequences were compared using the DNAstar software. The gene sequence of xylanase G2 ofA. oryzae VTCC-F187 (Ao_Xln_07) had a high similarity to the sequences of xylanase genes of A. oryzae Ao_Xln_41 gene (99.3%) in GenBank (Figure 1(a)). Accordingly, the predicted amino acid sequence of A. oryzae VTCC-F187 xylanase had a high homology to Ao_Xln_41 (99.9%) (Figure 1(b)). The xlnG2 (699 bp, 232 aa) from A. oryzae VTCC-F187 belongs to the glycosyl hydrolase family 11, which was completely similar to the xlnG2 (767 bp, 232 aa) (glycosyl hydrolase family 11) [14], A. oryzae xlnG1 (725 bp, 189 aa) [21], xlnC (636 bp, 211 aa) [22], xlnA (glycosyl hydrolase family 10) [16], and xlnD (957 bp, 318 aa) [23] from A. niger strains (from GenBank). The phylogenetic tree of nucleotide and amino acids are not much different; this can be explained by a multitude of codons encoding for an amino acid. The nucleotide sequence of gene xylanase G2 of Aspergillus oryzae VTCC-F187 had been registered on GenBank with the code of EU848307.Figure 1
Phylogenetic tree of the (a) xylanase G2 gene family and (b) xylanase G2 protein family. Ao_Xln_41:Aspergillus oryzae AB044941; Ao_Xln_54: Aspergillus oryzae AP007154; Ao_Xln_64: Aspergillus oryzae AP007164; Ao_Xln_74: Aspergillus oryzae AP007174; Ao_Xln_76: Aspergillus oryzae AB066176; Ao_Xln_85: Aspergillus oryzae AB003085.
(a)(b)


## 3.3. Design of Plasmid to Express the Xylanase Gene inAspergillus sp.

The xylanase gene was amplificated from pJxlnG2 by PCR reaction with specific primers containingBamHI 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 fromA. 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 (Supplement3C). Recombinant plasmids carrying the gene encoding xlnG2 were denoted by pANxlnG2.


## 3.4. Expression of Xylanase G2 Gene inAspergillus niger VCCT-F017

Recombinant plasmid pANxlnG2 was cut by restriction enzymeEheI 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 enzymeXhoI 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)).Figure 2
(a) Southern blot analysis of the recombinantxlnG2 in A. niger (M: marker; 1-6: recombinant strains; 7: wild-type strain; 8: plasmid pANxlnG2); (b) Xylanase activity (1: wild-type strain; 2 and 3: xylanase xlnG2 of recombinant A. niger VTCC-F017).
(a)(b)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 recombinantA. niger VTCC-017 strains had higher xylanase activity than the wild-type strains (Figure 2(b)).


## 3.5. Xylanase Purification

Xylanase from crude extract obtained after 120 h of culture was purified through the Ni2+ 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 [24–27]. 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.Figure 3
Electrophoresis of the xylanase G2: 1: extracellular extract of wild-type strain; 2: extracellular extract of recombinant strain; 3-6: purified xylanase G2; M: marker.Table 1
Purification steps of recombinant xylanase G2.
Purification stepTotal activity (U)Total protein (mg)Specific activity (U/mg)Purification factorYield (%)Culture supernatant922.1±8.61.84±0.05501.1±7.81100Ni2+ProBond™ resin column485.1±13.20.44±0.0021102.5±12.52.252.6


## 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 (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)).Figure 4
(a) Temperatures and (b) pH optimum of recombinant xylanase (the relative activity (%) was expressed as a percentage of optimal activity, and 100% xylanase activity was1085±36.8 U/mg at (a) 55°C and 852.3±41.3 U/mg at (b) pH 7).
(a)(b)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 (Figure4(b)).The optimal temperature and pH for family 11Aspergillus 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 (Figure4(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)).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 was921.9±15.02 U/mg and 1097.2±76.3 U/mg for temperature and pH stability, respectively).
(a)(b)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 (Figure5(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% (Table2).Table 2
Effect of organic solvents on the recombinant xylanase G2 activity.
Organic solventsResidual activity (%)aMeOH84.6±0.4EtOH62.4±4.2IsOH66.5±1.7Act72.0±3.1n-BtOH40.8±2.7aRelative activity was expressed as a percentage of the control reaction without any additive (100% xlnG2 activity was 927.21±38.3 U/mg).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%.Previous studies also showed the high retained activity of xylanase fromA. awamori VTCC-F312 63-86% [31] and xlnA from Pichia pastoris GS115 [16]. However, methanol and ethanol reduced significantly the activity of xylanase from A. niger C3486 [32] and completely inhibited the enzymatic activity from Termitomyces sp. [33].


### 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 (Table3). 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 of SDS strongly inhibited the enzyme with residual activity of 20% comparing to the initial activity [34].Table 3
Effect of detergents on the recombinant xylanase G2 activity.
DetergentsResidual activity (%)aTriton X-100103±5.6Tween 80117±0.7Tween 2073±2.8SDS49±6.9aRelative activity was expressed as a percentage of the control reaction without any additive (100% xlnG2 activity was 900.12±38.3 U/mg).


## 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 (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)).Figure 4
(a) Temperatures and (b) pH optimum of recombinant xylanase (the relative activity (%) was expressed as a percentage of optimal activity, and 100% xylanase activity was1085±36.8 U/mg at (a) 55°C and 852.3±41.3 U/mg at (b) pH 7).
(a)(b)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 (Figure4(b)).The optimal temperature and pH for family 11Aspergillus 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 (Figure4(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)).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 was921.9±15.02 U/mg and 1097.2±76.3 U/mg for temperature and pH stability, respectively).
(a)(b)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 (Figure5(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% (Table2).Table 2
Effect of organic solvents on the recombinant xylanase G2 activity.
Organic solventsResidual activity (%)aMeOH84.6±0.4EtOH62.4±4.2IsOH66.5±1.7Act72.0±3.1n-BtOH40.8±2.7aRelative activity was expressed as a percentage of the control reaction without any additive (100% xlnG2 activity was 927.21±38.3 U/mg).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%.Previous studies also showed the high retained activity of xylanase fromA. awamori VTCC-F312 63-86% [31] and xlnA from Pichia pastoris GS115 [16]. However, methanol and ethanol reduced significantly the activity of xylanase from A. niger C3486 [32] and completely inhibited the enzymatic activity from Termitomyces sp. [33].


## 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 (Table3). 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 of SDS strongly inhibited the enzyme with residual activity of 20% comparing to the initial activity [34].Table 3
Effect of detergents on the recombinant xylanase G2 activity.
DetergentsResidual activity (%)aTriton X-100103±5.6Tween 80117±0.7Tween 2073±2.8SDS49±6.9aRelative activity was expressed as a percentage of the control reaction without any additive (100% xlnG2 activity was 900.12±38.3 U/mg).


## 3.7. Effect of Metal Ions

The effect of various metal ions and EDTA on the purified xylanase activity was investigated. After 2 h of incubation at the concentration of 10 mM, only Hg2+ completely inhibited enzyme activity while Mg2+, Ca2+, and Ni2+ slightly reduced the enzyme activity. The presence of Fe2+, Zn2+, and Ag+ partially reduced enzyme activity by 56, 57, and 57%, respectively (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]. Hg2+ 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 Hg2+ 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 Zn2+ and Hg2+) may also occur due to the formation of a complex with the reactive groups of the enzyme, such as SH, CONH2, NH2, COOH, and PO4 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].Table 4
Effect of metal ions and EDTA on the recombinant xylanase G2 activity.
Additive (10 mM)Residual activity (%)aMg2+96±1.1Fe2+44±5.8Cu2+53±7.7Ca2+99.6±13.5Ag+43±9.1Ni2+96±3.1K+101±0.1EDTA106±10.4Zn2+43±5.3Hg2+0±0.0aRelative activity was expressed as a percentage of the control reaction without any additive (100% xlnG2 activity was 881.0±65.4 U/mg).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.Some xylanases require Ca2+ 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, Mg2+ and Ca2+ 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.


## 4. Conclusion

In this study, cloning of the gene encoding xylanase G2 fromA. 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 Hg2+, and organic solvents. Tween 80 increased, and Triton X-100 had no effect on xylanase activity.



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*Source: 8840038-2021-03-12.xml*