Purification and characterization of a high-thermostable β-xylanase from newly isolated Thermomyces lanuginosus THKU-49

Highly thermostable β-xylanase produced by newly isolated Thermomyces lanuginosus THKU-49 strain was purified in a four-step procedure involving ammonium sulfate precipitation and subsequent separation on a DEAE-Sepharose fast flow column, hydroxylapatite column, and Sephadex G-100 column, respectively. The enzyme purified to homogeneity had a specific activity of 552 U/mg protein and a molecular weight of 24.9 kDa. The optimal temperature of the purified xylanase was 70°C, and it was stable at temperatures up to 60°C at pH 6.0; the optimal pH was 5.0–7.0, and it was stable in the pH range 3.5–8.0 at 4°C. Xylanase activity was inhibited by Mn²⁺, Sn²⁺, and ethylenediaminetetraacetic acid. The xylanase showed a high activity towards soluble oat spelt xylan, but it exhibited low activity towards insoluble oat spelt xylan; no activity was found to carboxymethylcellulose, avicel, filter paper, locust bean gum, cassava starch, and p-nitrophenyl β-d-xylopyranoside. The apparent K _m value of the xylanase on soluble oat spelt xylan and insoluble oat spelt xylan was 7.3 ± 0.236 and 60.2 ± 6.788 mg/ml, respectively. Thin-layer chromatography analysis showed that the xylanase hydrolyzed oat spelt xylan to yield mainly xylobiose and xylose as end products, but that it could not release xylose from the substrate xylobiose, suggesting that it is an endo-xylanase.     

Purification and characterization of a high molecular weight endoxylanase from the solid-state culture of an alkali-tolerant Aspergillus fumigatus MKU1

Summary A high molecular weight endoxylanase (XylF2) from the solid state culture of Aspergillus fumigatus MKU1 was purified to homogeneity by a combination of tube gel electrophoresis and electroelution methods. The purity was demonstrated by SDS-PAGE and the molecular mass of the XylF2 was found to be 66 kDa. The optimal pH and temperature for activity were 5.0 and 90 °C, respectively. The apparent K _m and V _max values of XylF2 with oat spelt xylan as substrate were 1.6 mg/ml and 3.25 mmol/min/mg protein respectively. The enzyme showed high activity towards oat spelt xylan while negligible activity was observed on carboxymethylcellulose. The activity of XylF2 was strongly inhibited by Hg²⁺, Ni²⁺, Zn²⁺, SDS and N-bromosuccinimide and stimulated by l-cysteine and iodoacetamide. The hydrolysis of oat spelt xylan by XylF2 released only xylo-oligosaccharides.     

Identification and characterization of a novel xylanase derived from a rice straw degrading enrichment culture

A metagenomic library containing ca. 3.06 × 10⁸ bp insert DNA was constructed from a rice straw degrading enrichment culture. A xylanase gene, umxyn10A, was cloned by screening the library for xylanase activity. The encoded enzyme Umxyn10A showed 58% identity and 73% similarity with a xylanase from Thermobifida fusca YX. Sequence analyses showed that Umxyn10A contained a glycosyl hydrolase family 10 catalytic domain. The gene was expressed in Escherichia coli, and the recombinant enzyme was purified and characterized biochemically. Recombinant Umxyn10A was highly active toward xylan. However, the purified enzyme could slightly hydrolyze β-1,3/4-glucan and β-1,3/6-glucan. Umxyn10A displayed maximal activity toward oat spelt xylan at a high temperature (75°C) and weak acidity (pH 6.5). The K _m and V _max of Umxyn10A toward oat spelt xylan were 3.2 mg ml⁻¹ and 0.22 mmol min⁻¹ mg⁻¹ and were 2.7 mg ml⁻¹ and 1.0 mmol min⁻¹ mg⁻¹ against birchwood xylan, respectively. Metal ions did not appear to be required for the catalytic activity of this enzyme. The enzyme Umxyn10A could efficiently hydrolyze birchwood xylan to release xylobiose as the major product and a negligible amount of xylose. The xylanase identified in this work may have potential application in producing xylobiose from xylan.     

Purification and characterization of endoxylanase Xln-1 from <Emphasis Type="Italic">Aspergillus niger</Emphasis> B03

An extracellular endoxylanase was isolated from the xylanolytic complex of Aspergillus niger B03. The enzyme was purified to a homogenous form using consecutive ultrafiltration and anion exchange chromatography. The endoxylanase was a monomer protein with a molecular weight of 33,000 Da determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 34,000 Da determined by gel filtration. The optimal pH and temperature values for the enzyme action were 6.0 and 60°C, respectively. Endoxylanase was stable at 40°C, pH 7.0 for 210 min. The thermal stability of the enzyme was significantly increased in the presence of glycerol and sorbitol. The enzyme activity was inhibited by Cu²⁺, Fe²⁺, Fe³⁺, and Ag¹⁺, and it was activated by Mn²⁺. The substrate specificity and kinetic parameters of the enzyme were determined with different types of xylans. Endoxylanase displayed maximum activity in the case of oat spelt xylan, with an apparent K _m value of 8.19 mg/ml. The substrate specificity and the product profile of the enzyme suggested it to be an endoxylanase.     

Molecular and biochemical characterization of a new alkaline active multidomain xylanase from alkaline wastewater sludge

A xylanase gene, xyn-b39, coding for a multidomain glycoside hydrolase (GH) family 10 protein was cloned from the genomic DNA of the alkaline wastewater sludge of a paper mill. Its deduced amino acid sequence of 1,481 residues included two carbohydrate-binding modules (CBM) of family CBM_4_9, one catalytic domain of GH 10, one family 9 CBM and three S-layer homology (SLH) domains. xyn-b39 was expressed heterologously in Escherichia coli, and the recombinant enzyme was purified and characterized. Xyn-b39 exhibited maximum activity at pH 7.0 and 60 °C, and remained highly active under alkaline conditions (more than 80 % activity at pH 9.0 and 40 % activity at pH 10.0). The enzyme was thermostable at 55 °C, retaining more than 90 % of the initial activity after 2 h pre-incubation. Xyn-b39 had wide substrate specificity and hydrolyzed soluble substrates (birchwood xylan, beechwood xylan, oat spelt xylan, wheat arabinoxylan) and insoluble substrates (oat spelt xylan and wheat arabinoxylan). Hydrolysis product analysis indicated that Xyn-b39 was an endo-type xylanase. The K _m and V _max values of Xyn-b39 for birchwood xylan were 1.01 mg/mL and 73.53 U/min/mg, respectively. At the charge of 10 U/g reed pulp for 1 h, Xyn-b39 significantly reduced the Kappa number (P < 0.05) with low consumption of chlorine dioxide alone.     

Characterization of a thermostable and alkaline xylanase from Bacillus sp. and its bleaching impact on wheat straw pulp

Delignification efficacy of xylanases to facilitate the consequent chemical bleaching of Kraft pulps has been studied widely. In this work, an alkaline and thermally stable cellulase-less xylanase, derived from a xylanolytic Bacillus subtilis, has been purified by a combination of gel filtration and Q-Sepharose chromatography to its homogeneity. Molecular weight of the purified xylanase was 61 kDa by SDS–PAGE. The purified enzyme revealed an optimum assay temperature and pH of 60°C and 8.0, respectively. Xylanase was active in the pH range of 6.0–9.0 and stable up to 70°C. Divalent ions like Ca²⁺, Mg²⁺ and Zn²⁺ enhanced xylanase activity, whereas Hg²⁺, Fe²⁺, and Cu²⁺ were inhibitory to xylanase at 2 mM concentration. It showed K _m and V _max values of 9.5 mg/ml and 53.6 μmol/ml/min, respectively, using birchwood xylan as a substrate. Xylanase exhibited higher values of turn over number (K _cat) and catalytic efficiency (K _cat/K _m) with birchwood xylan than oat spelt xylan. Bleach-boosting enzyme activity at 30 U/g dry pulp displayed the optimum bio-delignification of Kraft pulp resulting in 26.5% reduction in kappa number and 18.5% ISO induction in brightness at 55°C after 3 h treatment. The same treatment improved the pulp properties including tensile strength and burst index, demonstrating its potential application in pre-bleaching of Kraft pulp.     

Characterization of Xyn10A, a highly active xylanase from the human gut bacterium Bacteroides xylanisolvens XB1A

A xylanase gene xyn10A was isolated from the human gut bacterium Bacteroides xylanisolvens XB1A and the gene product was characterized. Xyn10A is a 40-kDa xylanase composed of a glycoside hydrolase family 10 catalytic domain with a signal peptide. A recombinant His-tagged Xyn10A was produced in Escherichia coli and purified. It was active on oat spelt and birchwood xylans and on wheat arabinoxylans. It cleaved xylotetraose, xylopentaose, and xylohexaose but not xylobiose, clearly indicating that Xyn10A is a xylanase. Surprisingly, it showed a low activity against carboxymethylcellulose but no activity at all against aryl-cellobioside and cellooligosaccharides. The enzyme exhibited K _m and V _max of 1.6 mg ml⁻¹ and 118 μmol min⁻¹ mg⁻¹ on oat spelt xylan, and its optimal temperature and pH for activity were 37°C and pH 6.0, respectively. Its catalytic properties (k _cat/K _m = 3,300 ml mg⁻¹ min⁻¹) suggested that Xyn10A is one of the most active GH10 xylanase described to date. Phylogenetic analyses showed that Xyn10A was closely related to other GH10 xylanases from human Bacteroides. The xyn10A gene was expressed in B. xylanisolvens XB1A cultured with glucose, xylose or xylans, and the protein was associated with the cells. Xyn10A is the first family 10 xylanase characterized from B. xylanisolvens XB1A.     

Purification,characterization and regulation of the synthesis of an Aspergillus nidulans acidic xylanase

An acidic xylanase from a culture filtrate of Aspergillus nidulans grown on oat-spelt xylan was purified to apparent homogeneity. The purified enzyme showed a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis with a molecular mass of 34,000 Da and had an isoelectric point of approximately 3.4. The enzyme was a non-debranching endoxylanase highly specific for xylans. The xylanase showed an optimal activity at pH 6.0 and 56° C and had a Michaelis constant K_m of 0.97 mg oat-spelt xylan (soluble fraction) ml and a maximed reaction velocity (Vmax) of 1,091 mol min^–1 (mg^–1protein)^–1. Using polyclonal antibodies raised against the purified enzyme, the regulation of its synthesis has been studied. The xylanase production is repressed by glucose and induced by oat-spelt xylan, arabinoxylan, 4-O-methylglucurono-xylan, birchwood xylan and xylose.     

A novel neutral xylanase with high SDS resistance from Volvariella volvacea: characterization and its synergistic hydrolysis of wheat bran with acetyl xylan esterase

A neutral xylanase (XynII) from Volvariella volvacea was identified and characterized. Unlike other modular xylanases, it consists of only a single GH10 catalytic domain with a unique C-terminal sequence (W-R-W-F) and a phenylalanine and proline-rich motif (T-P-F-P-P-F) at N-terminus, indicating that it is a novel GH10 xylanase. XynII exhibited optimal activity at pH 7 and 60 °C and stability over a broad range of pH 4.0–10.0. XynII displayed extreme highly SDS resistance retaining 101.98, 92.99, and 69.84 % activity at the presence of 300 mM SDS on birchwood, soluble oat spelt, and beechwood xylan, respectively. It remained largely intact after 24 h of incubation with proteinase K at a protease to protein ratio of 1:50 at 37 °C. The kinetic constants K _m value towards beechwood xylan was 0.548 mg ml^?1, and the k _cat/K _m ratio, reflecting the catalytic efficiency of the enzyme, was 126.42 ml mg^?1 s^?1 at 60 °C. XynII was a true endo-acting xylanase lacking cellulase activity. It has weak activity towards xylotriose but efficiently hydrolyzed xylans and xylooligosaccharides larger than xylotriose mainly to xylobiose. Synergistic action with acetyl xylan esterase (AXEI) from V. volvacea was observed for de-starched wheat bran. The highest degree of synergy (DS 1.42) was obtained in sequential reactions with AXEI digestion preceding XynII. The high SDS resistance and intrinsic stability suggested XynII may have potential applications in various industrial processes especially for the detergent and textile industries and animal feed industries.     

Purification and characterization of a neutral endoxylanase from Aspergillus nidulans

Abstract A neutral endoxylanase from a culture filtrate of Aspergillus nidulans grown on oat spelt xylan was purified to apparent homogeneity. The purified enzyme showed a single band on SDS-PAGE with a molecular mass of 22,000 and had an isoelectric point of 6.4. The enzyme was a non-debranching endoxylanase highly specific for xylans and completely free from cellulolytic activity. The xylanase showed an optimum activity at pH 5.5 and 62°C and had a K m of 4.2 mg oat spelt xylan per ml and a V max of 710 μmol min⁻¹ (mg protein)⁻¹.     

Purification and partial characterization οf a new β-xylosidase from Humicola grisea var. thermoidea

Summary The thermophilic fungus Humicola grisea var. thermoidea produces a mycelium-associated β-xylosidase activity when grown in liquid-state cultures on media containing oat spelt xylan as the carbon source. The β-xylosidase was purified to apparent homogeneity by gel filtration and anion exchange chromatography. Its molecular weight was 37 and 50 kDa, as determined by MALDI/TOF mass spectrometry and SDS-PAGE, respectively. The purified enzyme exhibited maximum activity at 55 °C and pH 6.5. It was also active at pH 8.8, retaining 60% of its activity after 6 h of incubation at 50 °C. β-xylosidase was strongly inactivated by NBS and slightly activated by DTT and β-mercaptoethanol. The enzyme was highly specific for PNPX as the substrate. The purified β-xylosidase showed K _m and V _max values of 1.37 mM and 12.98 IU ml⁻¹, respectively.     

Production of alkaliphilic, halotolerent, thermostable cellulase free xylanase by Bacillus halodurans PPKS-2 using agro waste: single step purification and characterization

The alkaliphilic Bacillus halodurans strain PPKS-2 was shown to produce extracellular alkaliphilic, thermostable and halotolerent xylanase. The culture conditions for xylanase production were optimized with respect to pH, temperature, NaCl and inexpensive agro waste as substrates. Xylanase yield was enhanced more than four fold in the presence of 1% corn husk and 0.5% peptone or feather hydrolysate at pH 11 and 37°C. Xylanase was purified to 11.8-fold with 8.7% yield by using traditional chromatographic methods whereas the same enzyme purified to 20-fold with 72% yield by using corn husk as ligand. Its molecular mass was estimated to be 24 kDa by SDS–PAGE. The xylanase had maximal activity at pH 11 and 70°C. The enzyme was active over broad range, 0–20% sodium chloride. The enzyme was thermostable retaining 100% of the original activity at 70°C for 3 h. The apparent K _m values for oat spelt xylan and brichwood xylan were 4.1 and 4.4 mg/ml respectively. The deduced internal amino acid sequence of PPKS-2 xylanase resembled the sequence of β-1,4-endoxylanase, which is member of glycoside hydrolase family 11.     

A novel cold-active xylanase from the cellulolytic myxobacterium Sorangium cellulosum So9733-1: gene cloning, expression, and enzymatic characterization

The cellulolytic myxobacterium Sorangium cellulosum is able to efficiently degrade many kinds of polysaccharides, but none of the enzymes involved have been characterized. In this paper, a xylanase gene (xynA) was cloned from S. cellulosum So9733-1 using thermal asymmetric interlaced PCR. The gene is composed of 1,209 bp and has only 52.27% G + C content, which is much lower than that of most myxobacterial DNA reported (67–72%). Gene xynA encodes a 402 amino acid protein that contains a single catalytic domain belonging to the glycoside hydrolase family 10. The novel xylanase gene, xynA, was expressed in Escherichia coli BL21 (DE3) and the recombinant protein (r-XynA) was purified by Ni-affinity chromatography. The r-XynA had the optimum temperature of 30–35°C and exhibited 33.3% activity at 5°C and 13.7% activity at 0°C. Approximately 80% activity was lost after 20-min pre-incubation at 50°C. These results indicate that r-XynA is a cold-active xylanase with low thermostability. At 30°C, the K _m values of r-XynA on beechwood xylan, birchwood xylan, and oat spelt xylan were 25.77 ± 4.16, 26.52 ± 4.78, and 38.13 ± 5.35 mg/mL, respectively. The purified r-XynA displayed optimum activity at pH 7.0. The activity of r-XynA was enhanced by the presence of Ca²⁺. The r-XynA hydrolyzed beechwood xylan, birchwood xylan, and xylooligosaccharides (xylotriose, xylotetraose, and xylopentose) to produce primarily xylose and xylobiose. To our knowledge, this is the first report on the characterization of a xylanase from S. cellulosum.     

Heterologous expression,purification and characterization of a highly thermolabile endoxylanase from the Antarctic fungus Cladosporium sp.

Numerous endoxylanases from mesophilic fungi have been purified and characterized. However, endoxylanases from cold-adapted fungi, especially those from Antarctica, have been less studied. In this work, a cDNA from the Antarctic fungus Cladosporium sp. with similarity to endoxylanases from glycosyl hydrolase family 10, was cloned and expressed in Pichia pastoris. The pure recombinant enzyme (named XynA) showed optimal activity on xylan at 50 °C and pH 6–7. The enzyme releases xylooligosaccharides but not xylose, indicating that XynA is a classical endoxylanase. The enzyme was most active on xylans with high content of arabinose (rye arabinoylan and wheat arabinoxylan) than on xylans with low content of arabinose (oat spelts xylan, birchwood xylan and beechwood xylan). Finally, XynA showed a very low thermostability. After 20–30 min of incubation at 40 °C, the enzyme was completely inactivated, suggesting that XynA would be the most thermolabile endoxylanase described so far in filamentous fungi. This is one of the few reports describing the heterologous expression and characterization of a xylanase from a fungus isolated from Antarctica.     

A new xylanase from a Trichoderma harzianum strain

A new xylanase (XYL2) was purified from solid-state cultures of Trichoderma harzianum strain C by ultrafiltration and gel filtration. SDS-PAGE of the xylanase showed an apparent homogeneity and molecular weight of 18 kDa. It had the highest activity at pH 5.0 and 45°C and was stable at 50°C and pH 5.0 up to 4 h xylanase. XYL2 had a low K _m with insoluble oat spelt xylan as substrate. Compared to the amino acid composition of xylanases from Trichoderma spp, xylanase XYL2 presented a high content of glutamate/glutamine, phenylalanine and cysteine, and a low content of serine. Xylanase XYL2 improved the delignification and selectivity of unbleached hardwood kraft pulp. Received 02 February 1999/ Accepted in revised form 17 April 1999     

Purification and characterization of an extracellular alpha-L-arabinosidase from a novel isolate Bacillus pumilus ARA and its over-expression in Escherichia coli

The α-l-arabinosidase, AraB, was induced when Bacillus pumilus ARA was grown at 50°C in a minimal medium containing xylan. A 56-kDa protein with α-l-arabinosidase activity was purified from culture supernatant to gel electrophoretic homogeneity. The optimal activity was at pH 6.4 and 60°C over a 10-min assay. The purified enzyme was stable over a pH range of 5.2–7.6 and had a 1-h half life at 70°C. The enzyme released arabinose from oat spelt xylan. Kinetic experiments at 60°C with p-nitrophenyl α-l-arabinofuranoside as substrate gave a K _m, and V _max of 1.05 mM and 240 U per mg of protein. The NH₂-terminal amino acid sequence of the enzyme was determined, and its gene araB was subsequently cloned, sequenced, and over-expressed in Escherichia coli. The open reading frame of araB consists of a 1,479-bp fragment encoding a protein of 472 amino acids, which belonged to family 51 of the glycoside hydrolases with an identity of 67% to the protein encoded by abfB of Bacillus subtilis 168.     

Purification and Characterisation of Xylanolytic Enzymes of a Cellulase-free Thermophilic strain of Clostridium absonum CFR-702

Two endo-β-1-4-xylanases (EC 3.2.1.8), xylanase-I and xylanase-II, were purified fromClostridium absonum CFR-702 by ammonium sulphate precipitation and chromatographed on DEAE-Cellulose and phenyl-Sepharose. The enzymes in sodium dodecyl sulphate polyacrylamide gels resolved as proteins corresponding to molecular mass 150 and 95 kDa for xylanase-I and xylanase-II, respectively. The optimum pH and temperature ranges for the enzyme activities on birchwood xylan were between 6.5 and 7.5 and 75°C for xyl-I and 7.5 and 80°C for xyl-II. Xyl-I was stable up to 60°C whereas xyl-II was stable at 50°C. Both the enzymes liberated xylobiose, xylotriose and xylotetraose from birchwood xylan. Xyl-I and xyl-II with birchwood xylan had K_mvalues of 1.1 and 1.4%, and V_maxvalues of 454.54 and 363.63 μmol/min/mg protein respectively.     

Comparative characterization of deletion derivatives of the modular xylanase XynA of Thermotoga maritima

The modular Xylanase XynA from Thermotoga maritima consists of five domains (A1-A2-B-C1-C2). Two similar N-terminal domains (A1-A2-) are family 22 carbohydrate-binding modules (CBMs), followed by the catalytic domain (-B-) belonging to glycoside hydrolase family 10, and the C-terminal domains (-C1-C2), which are members of family 9 of CBMs. The gradual deletion of the non-catalytic domains resulted in deletion derivatives (XynAΔC; XynAΔA1C and XynAΔNC) with increased maximum activities (V _max) at 75°C, pH 6.2. Furthermore, these deletions led to a shift of the optimal NaCl concentration for xylan hydrolysis from 0.25 (XynA) to 0.5 M (XynAΔNC). In the presence of the family 22 CBMs, the catalytic domain retained more activity in the acidic range of the pH spectrum than without these domains. In addition to the deletion derivatives of XynA, the N-terminal domains A1 and A2 were produced recombinantly, purified, and investigated in binding studies. For soluble xylan preparations, linear β-1,4-glucans and mixed-linkage β-1,3-1,4-glucans, only the A2 domain mediated binding, not the A1 domain, in accordance with previous observations. The XynA deletion enzymes lacking the C domains displayed low affinity also to hydroxyethylcellulose and carboxymethylcellulose. With insoluble oat spelt xylan and birchwood xylan as the binding substrates, the highest affinity was observed with XynAΔC and the lowest affinity with XynAΔNC. Although the domain A1 did not bind to soluble xylan preparations, the insoluble oat spelt xylan-binding data suggest that this domain does play a role in substrate binding in that it improves the binding to insoluble xylans.     

Construction of an Aspergillus nidulans multicopy transformant for the xlnB gene and its use in purifying the minor X24 xylanase

 Using recombinant DNA techniques, an Aspergillus nidulans multicopy transformant for the gene xlnB coding for the minor X₂₄ xylanase has been constructed. When grown on glucose as sole carbon source this transformant secretes 114 U of xylanase (mg protein)^-1. In this culture condition, X₂₄ is the only xylanase secreted and the predominant protein in the culture filtrate. This strategy has been used to purify the X₂₄ enzyme to homogeneity. The purified xylanase showed a single band on sodium dodecyl sulphate/ polyacrylamide gel electrophoresis with a molecular mass of 24 kDa and had an isoelectric point of approximately 3.5. The enzyme was a non-debranching endo-1,4-β-xylan xylanohydrolase highly specific for xylans and showed optimal activity at pH 5.5 and 52°C. The X₂₄ xylanase had a Michaelis constant, K _m, of 12.43 mg oat spelt xylan ml^-1 and a V _max of 1639 μmol min^-1 (mg protein)^-1. Received: 17 May 1995/Received last revision: 25 September 1995/Accepted: 29 September 1995     

A new acidophilic endo-β-1,4-xylanase from Penicillium oxalicum: cloning,purification, and insights into the influence of metal ions on xylanase activity

A new acidophilic xylanase (XYN11A) from Penicillium oxalicum GZ-2 has been purified, identified and characterized. Synchronized fluorescence spectroscopy was used for the first time to evaluate the influence of metal ions on xylanase activity. The purified enzyme was identified by MALDI TOF/TOF mass spectrometry, and its gene (xyn11A) was identified as an open reading frame of 706 bp with a 68 bp intron. This gene encodes a mature protein of 196 residues with a predicted molecular weight of 21.3 kDa that has the 100 % identity with the putative xylanase from the P. oxalicum 114-2. The enzyme shows a structure comprising a catalytic module family 10 (GH10) and no carbohydrate-binding module family. The specific activities were 150.2, 60.2, and 72.6 U/mg for beechwood xylan, birchwood xylan, and oat spelt xylan, respectively. XYN11A exhibited optimal activity at pH 4.0 and remarkable pH stability under extremely acidic condition (pH 3). The specific activity, K _m and V _max values were 150.2 U/mg, 30.7 mg/mL, and 403.9 μmol/min/mg for beechwood xylan, respectively. XYN11A is a endo-β-1,4-xylanase since it release xylobiose and xylotriose as the main products by hydrolyzing xylans. The activity of XYN11A was enhanced 155 % by 1 mM Fe²⁺ ions, but was inhibited strongly by Fe³⁺. The reason of enhancing the xylanase activity of XYN11A with 1 mM Fe²⁺ treatment may be responsible for the change of microenvironment of tryptophan residues studied by synchronous fluorescence spectrophotometry. Inhibition of the xylanase activity by Fe³⁺ was first time demonstrated to associate tryptophan fluorescence quenching.