Purification, characterization and partial amino acid sequences of thermo-alkali-stable and mercury ion-tolerant xylanase from Thermomyces dupontii KKU–CLD–E2–3

We investigated the properties of the low molecular weight thermo-alkali-stable and mercury ion-tolerant xylanase production from Thermomyces dupontii KKU-CLD-E2-3. The xylanase was purified to homogeneity by ammonium sulfate, Sephadex G–100 and DEAE–cellulose column chromatography which resulted 27.92-fold purification specific activity of 56.19 U/mg protein and a recovery yield of 2.01%. The purified xylanase showed a molecular weight of 25 kDa by SDS–PAGE and the partial peptide sequence showed maximum sequence homology to the endo-1,4-β-xylanase. The optimum temperature and pH for its activity were 80 °C and pH 9.0, respectively. Furthermore, the purified xylanase can maintain more than 75% of the original activity in pH range of 7.0–10.0 after incubation at 4 °C for 24 h, and can still maintain more than 70% of original activity after incubating at 70 °C for 90 min. Our purified xylanase was activated by Cu2+ and Hg2+ up to 277% and 235% of initial activity, respectively but inhibited by Co2+, Ag+ and SDS at a concentration of 5 mM. The Km and Vmax values of beechwood xylan were 3.38 mg/mL and 625 µmol/min/mg, respectively. Furthermore, our xylanase had activity specifically to xylan-containing substrates and hydrolyzed beechwood xylan, and the end products mainly were xylotetraose and xylobiose. The results suggested that our purified xylanase has potential to use for pulp bleaching in the pulp and paper industry.

Xylanase purification. The purification of crude xylanase was carried out with three steps consisted of ammonium sulfate precipitation, gel filtration and ion exchange chromatography by following the method described by Seemakram et al. 16 . The crude enzyme was precipitated by ammonium sulphate ((NH 4 ) 2 SO 4 ) at concentration of 20-80% for overnight at 4 ºC. The precipitated protein was separated by centrifugation at 6000 rpm for 15 min and dissolve in 0.05 M Tris-HCl, pH 9.0, then precipitated protein was dialyzed against the same previously used buffer. Thereafter, 1.0 mL of protein sample was purified by gel filtration with Sephadex G-100 column (100 cm × 1.0 cm) and equilibrated with 0.05 M Tris-HCl buffer (pH 9.0). The protein sample was eluted by 0.05 M Tris-HCl buffer pH 9.0 at a flow rate of 0.25 mL/min, and 2 mL fractions were collected. Then, all active fractions were pooled and applied to final of purification step by ion exchange chromatography with a DEAE-Cellulose column (20 cm × 1.5 cm) equilibrated with the same previously used buffer at a flow rate of 0.5 mL/min. The bound proteins were eluted with a NaCl gradient (2 M) in the same buffer at a flow rate of 0.5 mL/min, and 2 mL fractions were collected. The highly active fractions were gathered and stored at − 40 °C for further characterization.

Scientific Reports
| (2020) 10:21663 | https://doi.org/10.1038/s41598-020-78670-y www.nature.com/scientificreports/ Protein identification by LC-MS/MS and data analysis. The protein was loaded in 14% acrylamide gel, and then visualized by Coomassie brilliant blue R-250. The specific band of target purified xylanase was cut by new clean scalpel blade and transferred into a microcentrifuge tube. The purified xylanase was analyzed for amino acid identification by LC-MS/MS system model API 2000 MDS SCIEX. The amino acid sequences were used for analyzing by the MASCOT program (www.matri xscie nce.com) and a database of annotated comparative protein structure models by the Protein Modeling 101 (https ://www.prote inmod elpor tal.org) with the initial searching parameters; Enzyme: Trypsin; carbamidomethylation (C) as fixed modification, and oxidation (HW) and oxidation (M) as variable modification; peptide mass tolerance of 0.5 Da and fragment mass tolerance of 0.5 Da; a peptide charge state of + 1, + 2, + 3. The proteins were identified through the service of the Research Instrument Center, Khon Kaen University, Thailand.
Effect of pH on the activity and stability of xylanase. Investigation for determining the optimum pH of purified xylanase activity was determined in a pH ranging from 3.0 to 12.0 at 80 °C and performed by following the method described by Seemakram et al. 16 . We used various buffers including 0.1 M McIlvaine (pH 3.0-8.0), 0.05 M Tris-HCl (pH 8.0-9.0), 0.05 M Glycine-NaOH (pH 9.0-10.0), and 0.05 M Na 2 HPO 4 -NaOH (pH 10.0-12.0). The pH stability of purified xylanase was pre-incubated in these buffers at 4 °C for 24 h. The residual activity was measured under the standard assay conditions.
Effect of temperature on the activity and stability of xylanase. The optimum temperature for xylanase activity was determined at various temperatures (50-90 °C) in an optimum buffer and performed by following the method described by Seemakram et al. 16 . The thermal stability of purified xylanase in the optimum buffer was incubated at different temperatures of 50-90 °C for 90 min. After cooling, the residual xylanase activity was measured according to the standard assay conditions. Determination for substrate specificity and kinetic parameters. The purified xylanase was measured substrate specificity using 1% (w/v) of xylan consist of Beechwood xylan, Birchwood xylan, Oat spelt xylan, Carboxymethyl cellulose (CMC), Cellulose powder and Avicel, respectively 13 . The relative activity of enzyme for each substrate were analyzed at optimum temperature for 15 min and measured according to the standard assay conditions with slightly modification described by Seemakram et al. 15 . The kinetic experiments were performed at different concentrations of each substrate ranged from 2.5-30 mg/mL 14  Hydrolysis products of beechwood xylan. The hydrolysis products from purified xylanase were determined by incubating the beechwood xylan with purified enzyme under the optimum conditions assay for 12 h. The hydrolyzed products were measured at different time intervals using thin layer chromatography (TLC) on silica gel plates. Xylose and xylooligosaccharide were used as standards. The products on TLC plates were developed using methanol: glacial acetic acid: H 2 O (6 : 7 : 2, v/v/v) as the mobile phase which modified from the method described by Li et al. 21 . The hydrolysis products were detected by spraying with methanol/sulfuric acid mixture (95 : 5, v/v), followed by heating at 100 °C for 15 min.

Results
Purification of xylanase. Purification of crude xylanase was achieved by classical method including ammonium sulfate precipitation, Sephadex G-100 gel filtration and DEAE-cellulose ion-exchange column chromatography. The crude xylanase, having a total activity of 5102.22 U and specific activity of 2.01 U/mg protein, was precipitated using ammonium sulfate to 30% saturation, further purified by Sephadex G-100 gel filtration and DEAE-cellulose ion-exchange column chromatography. After three steps of purification, the specific activity of the crude enzyme increased from 2.01 to 56.19 U/mg protein with a recovery yield of 2.01% and the 27.92-fold apparent homogeneity (Table 1). In addition, the purified xylanase from T. dupontii KKU-CLD-E2-3 was subjected to determination for molecular weight and homogeneous by SDS-PAGE. The purified xylanase appeared as a single protein band with a molecular mass of approximately 25 kDa (Fig. 1).

Protein identification by LC-MS/MS and data analysis.
The sample from T. dupontii KKU-CLD-E2-3 was analyzed with amino acid identification of purified analyze by LC-MS/MS. The obtained peptide sequences were used for analyzing by the MASCOT search (Table 2), which amino acid similar to those fungi previously reported (Table 3). This strain was related to members of the description endo-1,4-β-xylanase, where it showed maximum sequence identities of 90% and similarity of 1.452 at resolution 1.55 Å with the endo-1,4-βxylanase from Thermomyces lanuginosus (Accession number gi|3915307).    Table 3. Identification of purified xylanase from Thermomyces dupontii KKU-CLD-E2-3.

NCBInr ID Nominal mass (Mr) Sequencing of generated peptide by MS/MS Protein identification Organism
This work 2851 dupontii KKU-CLD-E2-3 was active at pH 9.0 (Fig. 2a), a higher pH than those described in previous reports for other fungi ( Table 4). The purified endo-1,4-β-xylanase retained over 75% of the original activity in pH range of 7.0-10.0 after incubation at 4 °C for 24 h (Fig. 2b).

Substrate specificity and kinetic constants.
The substrate specificity of the purified endo-1,4β-xylanase was determined on several substrates ( Table 5). The results showed that the highest activity was obtained in beechwood xylan, followed by birchwood xylan, and oat spelt xylan, respectively. In contrast, the xylanase did not have an activity towards Avicel, CMC and cellulose powder. The K m and V max of endo-1,4-βxylanase was determined by beechwood xylan as a substrate. In this case, the K m and V max values of the purified endo-1,4-β-xylanase were 3.38 mg/mL and 625 µmol/min/mg, respectively.
Effect of metal ions and reagents on xylanase activity. The effect of different metal ions and reagents on the activity of the purified xylanase from T. dupontii KKU-CLD-E2-3 is summarized in Table 6. The addition of metal ions and nonmetal reagents at a concentration of 1 mM showed increased xylanase activity, except for Ag + and SDS. The presence of Cu 2+ , Mg 2+ , Fe 2+ , Hg 2+ , Zn 2+ , Mn 2+ and EDTA at a concentration of 5 mM increased xylanase activity, especially Cu 2+ and Hg 2+ that increased the xylanase activity up to 277% and 235%, respectively. On the other hand, Co 2+ , Ag + and SDS at a concentration of 5 mM showed inhibition of enzyme activity. In addition, xylanase activity was significantly activated by Cu 2+ and Mg 2+ at a concentration of 10 mM up to 248% and 259% of activity, respectively. 1,4-β-xylanase was studied using thin layer chromatography (TLC) (Fig. 3). The result revealed that the purified endo-1,4-β-xylanase liberated mainly xylotriose and xylotetraose from beechwood xylan.  Table 5. Substrate specificity of the purified xylanase from Thermomyces dupontii KKU-CLD-E2-3.

Discussion
Among xylanase-producing thermophilic fungi, there are only a few reports on the properties of xylanase from Thermomyces dupontii. In this research, the thermo-alkali-stable and metal ions-tolerant xylanase from T. dupontii KKU-CLD-E2-3 was purified and characterized. The purification fold of xylanase purification in this study was higher than some previous studies using other thermophilic fungi. For instance, the observations of Kumar and Shukla 22 showed that the xylanase from Thermomyces lanuginosus VAPS24 was purified 5.92-fold using acetone precipitation followed by ultrafiltration. According to Yang et al. 23 , the purified xylanase from Aspergillus fumigatus Yang FC2-2 was purified to 25.5-fold and apparent homogeneity of 13.9% recovery yield. Maalej et al. 24 reported that the purified xylanase from Talaromyces thermophilus was purified 23-fold using diethylaminoethyl cellulose anion exchange chromatography, P-100 gel filtration, and Mono Q chromatography. Chanwicha et al. 13 also reported purification of xylanase to 14.5-fold with 2.3% recovery from Thermoascus aurantiacus var. evisporus KKU-PN-I2-1. The purified xylanase from T. dupontii KKU-CLD-E2-3 was appeared as a single protein band on SDS-PAGE gel with a molecular mass of approximately 25 kDa, which was in agreement with the value of 25 kDa xylanase from T. thermophiles 24 13 . Then, this protein band was identified against genome sequence assembly v1.0 database (MASCOT search) and the Protein Modeling 101 in order to obtain peptide sequences. The bands excised from the gel were analyzed by mass spectrometry and were compared to protein sequences available in the NCBI database revealed. The protein sequence matches with Endo-1,4-β-xylanase of Thermomyces spp. at 90% identity (Accession number: gi|3,915,307) 25 . In addition, these results showed that identities were similar to protein from Thermomyces lanuginosus (Accession number:gi|335,371,365) at 89% identity 26 . The relatively high yield of enzyme and the coverage of peptides from mass-spec was so sparse which the protein sequence coverage of 31% indicated that identity or extensive homology from mass-spectrometry has reliability. Because of the individual ions scores > 54 of indicating identity have the significance at p < 0.05 of mascot score histogram. Besides, the peptide sequence of xylanase has the glycans attached in the fragment after cutting with the specific enzyme 27 that attached to the amide group of an asparagine residue within the consensus peptide sequence 28 , resulting in the observed ion (m/z) to high value has changed it is not matches the coverage of another peptide sequence in the database. The results of the experiments in Table 5 confirmed that this enzyme was xylanase.
According to previous literature, the purified xylanase from Humicola grisea var. thermoidea was most active at pH of 4.5-6.5 29 . Maalej et al. 24 reported that the optimum pH of the purified xylanase from T. thermophilus was 7.0. The optimum pH of xylanase from Thermomyces lanuginosus THKU-49 was 6.0 30 . Furthermore, Li et al. 31 found that the hybrid xylanase enzyme (TXynFM) from Talaromyces thermophilus F1208 had optimum activity at pH 6.5. Also, Ping et al. 32 reported that the purified xylanase from Thermoascus aurantiacus M-2 was most active at pH 5.0. In addition, the highest xylanase activity from Myceliophthora heterothallica F.2.1.4 was observed at pH 6.0 33 . Our results showed that the purified xylanase from T. dupontii KKU-CLD-E2-3 was most active at pH 9.0, which was higher pH than those reported for other thermophilic fungi. Because this fungus www.nature.com/scientificreports/ could be grown in alkaline pH conditions resulted in the enzyme production by this fungus had an optimal pH higher than the others fungi which similar to that from the report from Chanwicha et al. 13 . The purified xylanase from strain KKU-CLD-E2-3 remained more than 75% of the original activity in pH range 7.0-10.0 after incubation at 4 °C for 24 h. This was in contrast to Vafiadi et al. 34 who found that the hybrid xylanase from Sporotrichum thermophile was stable in a pH range of 4.0-8.0. The purified xylanase from T. lanuginosus THKU-49 was stable at pH 3.5-8.0 30 . In addition, Amo et al. 35 found that the purified xylanase from M. heterothallica F.2.1.4 was stable in pH range of 4.5-9.5.
In terms of optimum temperature of enzyme, the optimum temperature of the purified enzyme from strain KKU-CLD-E2-3 was found to be 80 °C. Interestingly, the optimum temperature of this xylanase was higher than those from the other xylanase-producing thermophilic fungi. Li et al. 5 reported that xylanase from T. lanuginosus CBS 288.54 was optimally active at 70-75 °C, while Maalej et al. 24 reported the optimum temperature of the purified xylanase from T. thermophiles to be 75 °C. Khucharoenphaisan et al. 30 found that the optimum pH of xylanase from Thermomyces lanuginosus THKU-49 was 70 °C. Also, Chanwicha et al. 13 reported that the purified xylanase from T. aurantiacus var. levisporus KKU-PN-I2-1 was optimally active at 60 °C. The purified xylanase from the thermophilic fungus M. thermophila BF1-7 was 50 °C 14 . In addition, the maximum activity of purified xylanase from T. aurantiacus M-2 was determined at 75 °C 32 . As for the thermo-stability of enzyme from our strain KKU-CLD-E2-3, xylanase activity was retained more than 70% of the original activity after heating at 70 °C for 90 min. This result was similar to values for the xylanase from other strains of thermophilic fungi 13,30,32 .
The highest hydrolytic activity of purified xylanase was exhibited toward beechwood xylan, followed by birchwood xylan and oat spelt xylan, respectively. Our results indicated that substrate specificity of xylanase depends on type of xylan substrates. The purified xylanase in this study did not act towards Avicel, carboxymethyl cellulose and cellulose powder. These results indicated that the purified xylanase from T. dupontii KKU-CLD-E2-3 was a true xylanase and belongs to the glycosylic family G11. The xylanases of family G11 have no activity on cellulose and are the low-molecular-weight enzymes 24 . The K m and V max values of the purified xylanase in this study were 3.38 mg/mL and 625.00 µmol/min/mg, respectively. This study then provided better value of both K m and V max in comparison to the value previously reported from T. thermophilus (K m 22.51 mol/L and V max 1.235 µmol/min/mg) 24 , T. aurantiacus var. levisporus KKU-PN-I2-1 (K m 40.9 mol/L and V max 6.2 µmol/min/mg) 13 , M. thermophila BF1-7 (K m 9.67 mol/L and V max 5.38 µmol/min/mg) 14 , and T. aurantiacus M-2 (K m 4.81 mol/L and V max 467.2 µmol/min/mg) 32 .
The purified xylanase activity in this study was stimulated up to 277% and 235%, respectively by Cu 2+ and Hg 2+ but inhibited by Co 2+ , Ag + and SDS at a concentration of 5 mM. In addition, xylanase activity was significantly activated by Cu 2+ and Mg 2+ at a concentration of 10 mM up to 248% and 259%, respectively. According to Boonrung et al. 14 , the purified xylanase from M. thermophila BF1-7 was activated by Cu 2+ , Mg 2+ and Ag + at 1 mM. The observations of Chanwicha et al. 13 showed that the xylanase from T. aurantiacus var. levisporus KKU-PN-I2-1 was stimulated by Cu 2+ and Mg 2+ but was inhibited by Ag + at 1 mM. Maalej et al. 24 reported that the purified xylanase from T. thermophilus was stimulated by Ag + but was inhibited by Mg 2+ at 10 mM. Also, Kumar and Shukla 22 found that the xylanase from T. lanuginosus VAPS24 was strongly inhibited by Cu 2+ at a concentration of 10 mM. While the Hg 2+ has previously been reported to completely inhibit the activity of xylanase 13,14,22,24,35,36 it should be noted that our purified xylanase showed good stability in the presence of Hg 2+ . The active effect of Hg 2+ ions is possibly due to its binding to the free SH group of thiol acid, the interaction of carboxyl group and/or the imidazole group of amino acid 37 , which the thiol groups are essential for enzyme activity, because thiol groups are one of the main targets of heavy metals. However, HgCl 2 is a known inhibitor of thermitase through its binding to the free SH group 38 . Besides, the effects of metal ions and reagent to enzyme inhibition such as Co 2+ and Ag 2+ are mixed inhibitors, which do not bind to the active site, but to another region of the enzyme, and thus to the formation of complex with the reactive groups of the enzyme, resulted in reducing of the enzyme availability function 39 . In addition, the enzyme activity is inhibited by SDS, which interferes in hydrophobic regions of the enzyme, resulted in alternate of its three-dimensional structure indicating that cause enzyme denaturation 40 .
The purified xylanase from T. dupontii KKU-CLD-E2-3 hydrolyzed beechwood xylan to yield mainly xylotetraose, xylotriose and xylose as end products. This result indicated that the purified xylanase of this fungus was endo-xylanase. These data were in agreement with the observations of Li et al. 29 which found that the xylanase from Talaromyces thermophilus F1208 mainly liberated xylotriose, xylotetraose and xylopentaose from beechwood xylan as substrates. The purified xylanase from Aspergillus carneus M34 mainly liberated xylotriose and xylotetraose from beechwood xylan 41 .

Conclusions
In this study, we firstly reported the amino acid sequence of the low-molecular-weight thermo-alkali-stable xylanase of T. dupontii KKU-CLD-E2-3. Until now, no previous studies have been reported on the mercury ion (Hg 2+ ) tolerant xylanase from thermophilic fungi. Interestingly, our purified xylanase was resistant to HgCl 2 (Hg 2+ ). In addition, these enzyme characteristics suggested the high potential in the pulp and paper industry. Since the optimum pH and the temperature of purified xylanase activity are relatively similar to the pH of the initial pulp and the temperature of bleaching activity in the factory processes. The enzyme was used in bleaching processes without pH and temperature alteration in the standard bleaching process of the factory production line. In addition, purified xylanase had high specific activity and low molecular weight, which ease to access into the fiber wall structure and can efficiently hydrolyze xylan in pulp bleaching without adverted effect on the structure of cellulose in pulp fiber, resulting in an improvement of pulp and paper quality. Therefore, this enzyme can be used in place of chemicals in bleaching process, which more environmentally-friendly than the chemical