Inhibition kinetics of acetosyringone on xylanase in hydrolysis of hemicellulose.

Many phenolic compounds, derived from lignin during the pretreatment of lignocellulosic biomass, could obviously inhibit the activity of cellulolytic and hemicellulolytic enzymes. Acetosyringone (AS) is one of the phenolic compounds produced from lignin degradation. In this study, we investigated the inhibitory effects of AS on xylanase activity through kinetic experiments. The results showed that AS could obviously inhibit the activity of xylanase in a reversible and noncompetitive binding manner (up to 50% activity loss). Inhibitory kinetics and constants of xylanase on AS were conducted by the HCH-1 model (ß = 0.0090 ± 0.0009 mM-1). Furthermore, intrinsic and 8-anilino-1-naphthalenesulfonic (ANS)-binding fluorescence results showed that the tertiary structure of AS-mediated xylanase was altered. These findings provide new insights into the role of AS in xylanase activity. Our results also suggest that AS was an inhibitor of xylanase and targeting AS was a potential strategy to increase xylose production.

Sensitivity of the Bacillus subtilis Xyn A Xylanase and Its Mutants to Different Xylanase Inhibitors Determines Their Activity Profile and Functionality during Bread Making.

The importance of inhibition sensitivity for xylanase functionality in bread making was investigated using mutants of the wild-type Bacillus subtilis xylanase (XBSTAXI), sensitive to Triticum aestivum xylanase inhibitor (TAXI). XBSNI, a mutant with reduced sensitivity to TAXI, and XBSTI, a mutant sensitive to all wheat endogenous proteinaceous inhibitors (TAXI, Xylanase Inhibiting Protein and Thaumatin-like Xylanase Inhibitor) were used. The higher inhibition sensitivity of XBSTAXI and XBSTI compared to XBSNI was associated with a respective 7- and 53-fold increase in enzyme dosage required for a maximal increase in bread loaf volume. XBSTI and XBSTAXI were only active during the mixing phase and the beginning of fermentation, while XBSNI was able to hydrolyze arabinoxylan until the end of fermentation. In spite of this difference in activity profile, no differences in loaf volume were observed for the different xylanases at optimal concentrations. Dough extensional viscosity analysis suggests that increased water availability as a result of xylanase activity favors starch-starch and starch-gluten interactions and drives the improvement in bread loaf volume.

Differential inhibition of GH family 11 endo-xylanase by rice xylanase inhibitor and verification by a modified yeast two-hybrid system.

Rice xylanase inhibitor (RIXI) is a XIP-type xylanase inhibitor protein that protects rice cells from pathogenic organisms. RIXI inhibits most microbial xylanases and thus decreases their practical application. The recombinant RIXI (rePRIXI) showed evident inhibitory activities against several family 11 endo-xylanases. After interaction with rePRIXI at 50 °C for 40 min, the residual activities of reBaxA50, reBaxA, TfxA_CD214, and TfxA_CD were 55.6%, 30.3%, 30.09%, and 11.20%, respectively. Intrinsic fluorescence of reBaxA50 and TfxA_CD214 was statically quenched after interaction with rePRIXI. rePRIXI decreased hydrolysis of beechwood xylan by reBaxA50 and TfxA_CD214. Molecular dynamics simulations revealed the long loop (residues 144-153) of RIXI inserts into the catalytic cleft of family 11 xylanases. Native PAGE results revealed the formation of RIXI-xylanase complex after their interaction in the test tube. Interactions were also observed between RIXI and xylanases in living yeast cells. The results of inhibitory activity assay and modified yeast two-hybrid revealed that the inhibitory activity of RIXI on family 11 xylanase improved with the interaction strength of the RIXI-xylanase complex, indicating their positive correlation. The modified yeast two-hybrid system is relatively simple and has low cost, and its use may be extended to other studies on protein-protein interactions.

Recombinant rice xylanase-inhibiting protein inhibits GH11 endo-xylanases through competitive inhibition.

The ricexip gene, which encodes the rice xylanase-inhibiting protein (riceXIP), was recombinantly expressed in Pichia pastoris GS115 under the control of AOX1 promoter. Recombinant riceXIP (rePriceXIP) was secreted into the supernatant and purified to homogeneity with the use of Ni-affinity resin. The molecular mass of rePriceXIP was approximately 44.0 kDa. RePriceXIP significantly inhibited the activity of GH11 xylanases in a concentration-dependent manner. The optimal inhibitory activity of rePriceXIP on GH11 xylanases (TfxA_CD214, reBaxA454, and TfxA_CD526) occurred at 40 °C for 30 min. The IC50 values of rePriceXIP inhibiting TfxA_CD214, reBaxA454, and TfxA_CD526 (0.5 U) were 45, 40, and 40 nM, respectively. The Ki of rePriceXIP on TfxA_CD214 xylanase was 12.2 nM. Increasing the concentration of rePriceXIP did not change Vmax, but increased Km, thereby suggesting that the inhibition was competitive. Analysis of the fluorescence intensities revealed that the Kq values for TfxA_CD214, reBaxA454, and TfxA_CD526 exceeded 2.0 × 1010 L mol-1•s-1, implying that static quenching occurred. Circular dichroism spectroscopy demonstrated that rePriceXIP bound to xylanase, thereby changing the secondary structure and reducing the catalytic activity of xylanase. Lower levels of hydrolytes are released from beechwood xylan by xylanases in the presence of rePriceXIP.

Superior Growth Rates in Broilers Fed Wheat with Low In Vitro Feed-Xylanase Inhibition.

Grain-batch variation in xylanase-inhibitor levels may account for variations in the efficacy of feed xylanase supplementation. This would make inhibition an important quality parameter in the routine analysis of feedstuffs. Two analytical procedures for testing feedstuffs against specific xylanases were researched: the high-throughput viscosity-pressure assay (ViPr) and the extraction-free remazol-brilliant-blue-beechwood-xylan (RBBX) assay. Thirty-two wheat cultivars were analyzed for inhibition of a commercial xylanase, Ronozyme WX. Four cultivars were selected for a feeding experiment in which the growth of 1440 broilers from ages 7-33 days was monitored. The treatments resulted up to 7 % difference (day 14) in broiler weight . The cultivar choice had an effect throughout the experiment ( p < 0.05). The performance ranking of the treatments corresponded better to xylanase inhibition than to crude-protein content or nonstarch-polysaccharide content. Wheat-grain xylanase-inhibitor content is therefore a highly relevant quality parameter when broiler diets are supplemented with feed xylanase.

Recombinant rice xylanase inhibitor (RIXI) expressed in Escherichia coli and its inhibitory activity on family GH11endo-xylanases.

The rice xylanase inhibitor gene, rixi, was cloned from rice genome. The open reading frame of rixi was 915 bp and encoded 304 amino acids with the theoretical molecular mass of 33.9 kDa. The rixi was inserted into the new-type expression vector pCold TF, and was high-level expressed in Escherichia coli BL21 (DE3). SDS-PAGE and Western blot analysis revealed that the molecular weight of the recombinant rice xylanase inhibitor, namely reERIXI, was approximately 89.8 kDa. The reERIXI exhibited significant inhibitory activities against several family GH11 xylanases. After interaction with reERIXI, the residual activity of reBaxA50 and TfxA_CD214 were 59.24% and 44.41%, respectively. The optimal temperature of reERIXI inhibitory activity to reBaxA50 and TfxA_CD214 were 60 °C and 50 °C, respectively. The thermostability assay revealed that reERIXI was stable below 60 °C. reERIXI showed high inhibitory when interacting with reBaxA50 and TfxA_CD214 for 30-60 min. The intrinsic fluorescence spectroscopy of reBaxA50 and TfxA_CD214 was quenched with increasing reERIXI concentration. Circular dichroism measurement revealed that ratio of helix of reBaxA50 and ratio of beta of TfxA_CD214 significantly decreased when interacting with reERIXI. The total concentration of hydrolytic products from beechwood xylan decreased when reERIXI was added.

Molecular Cloning and Characterizations of Xylanase Inhibitor Protein from Wheat (Triticum Aestivum).

Xylanase inhibitor proteins (XIPs) were regarded to inhibit the activity of xylanases during baking and gluten-starch separation processes. To avoid the inhibition to xylanases, it is necessary to define the conditions under which the inhibition takes place. In this study, we cloned the XIP gene from 2 different variety of Triticum aestivum, that is, Zhengmai 9023 and Zhengmai 366, and investigated the properties of XIP protein expressed by Pichia pastoris. The results showed that the 2 XIP genes (xip-9023 and xip-366) were highly homologous with only 3 nucleotide differences. XIP-9023 showed the optimal inhibition pH and temperature were 7 °C and 40 °C, respectively. Inhibition of xylanase by XIP-9023 reached the maximum in 40 min. At 50% inhibition of xylanase, the molar ratio of inhibitor: xylanase was 26:1. XIP-9023 was active to various fungal xylanases tested as well as to a bacterial xylanase produced by Paenibacillus sp. isolated from cow rumen.

Improved enzyme properties upon glutaraldehyde cross-linking of alginate entrapped xylanase from Bacillus licheniformis.

Enzyme immobilization is an exciting alternative to improve the stability of enzymatic processes and economic viability in terms of reusability. In the current study, purified xylanase from B. licheniformis Alk-1 was immobilized within glutaraldehyde activated calcium alginate beads and characterized in respect of free enzyme. Immobilization increases the optimum pH and temperature of entrapped and cross-linked enzyme from pH=8.0 to 9.0 and 50-60°C. The kinetics parameter of immobilized (cross-linked) enzyme showed an increase in Km (from 4.36mg/mL to 5.38mg/mL) and decrease in Vmax (from 383 IU/mg/min to 370 IU/mg/min). Immobilization increases the optimum reaction time for xylan degradation of immobilized xylanase from 15 to 30min when compare to free form. The storage stability study suggested that the immobilized enzyme retains 80% of its original activity at 4°C after 30days compared to free enzyme (5%). Further, immobilization improved enzyme stability in presence of different additives. The immobilized (cross-linked) enzyme also exhibited adequate recycling efficiency up to five reaction cycles with 37% retention activity. The finding of this study suggests improvement of overall performance of immobilized xylanase in respect to free form and can be used to make a bioreactor for various applications such as poultry feed preparations.

Deciphering the factors defining the pH-dependence of a commercial glycoside hydrolase family 8 enzyme.

A prerequisite to the use of any enzyme in any industrial process is an understanding of its activity and stability under process conditions. Glycoside hydrolase family 8 enzymes include many important biotechnological biocatalysts yet little is known of the performance of these with respect to pH. A better understanding of this parameter and its relationship to structure and function in these enzymes will allow for an improved use of these in industry as well as an enhanced ability in their engineering and optimisation for a particular application. An in-depth analysis of the pH induced changes in activity, irreversible inactivation, conformation, stability and solubility of a commercial glycoside hydrolase family 8 xylanase was carried out with the aim of identifying the factors determining the pH dependence of this enzyme. Our study showed that different phenomena play different roles at the various pHs examined. Both reversible and irreversible processes are involved at acidic pHs, with the irreversible processes dominating and being due to protein aggregation and precipitation. At basic pHs, loss of activity is principally due to reversible processes, possibly deprotonation of an essential catalytic residue, but at higher pHs, near the pI of the protein, precipitation again dominates while structure unfolding was discerned at the higher pHs investigated. Such insights demonstrate the complexity of factors involved in the pH dependence of proteins and advances our knowledge on design principles and concepts for engineering proteins. Our results highlight the major role of protein precipitation in activity and stability losses at both low and high pHs but it is proposed that different strategies be used in tailoring the enzyme to overcome this in each case. Indeed the detailed understanding obtained here will allow for a more focused, informed and hence successful tailoring of glycoside hydrolase family 8 proteins for a specific pH and process application.

Hyperthermostable Thermotoga maritima xylanase XYN10B shows high activity at high temperatures in the presence of biomass-dissolving hydrophilic ionic liquids.

The gene of Thermotoga maritima GH10 xylanase (TmXYN10B) was synthesised to study the extreme limits of this hyperthermostable enzyme at high temperatures in the presence of biomass-dissolving hydrophilic ionic liquids (ILs). TmXYN10B expressed from Pichia pastoris showed maximal activity at 100 °C and retained 92 % of maximal activity at 105 °C in a 30-min assay. Although the temperature optimum of activity was lowered by 1-ethyl-3-methylimidazolium acetate ([EMIM]OAc), TmXYN10B retained partial activity in 15-35 % hydrophilic ILs, even at 75-90 °C. TmXYN10B retained over 80 % of its activity at 90 °C in 15 % [EMIM]OAc and 15-25 % 1-ethyl-3-methylimidazolium dimethylphosphate ([EMIM]DMP) during 22-h reactions. [EMIM]OAc may rigidify the enzyme and lower V max. However, only minor changes in kinetic parameter K m showed that competitive inhibition by [EMIM]OAc of TmXYN10B is minimal. In conclusion, when extended enzymatic reactions under extreme conditions are required, TmXYN10B shows extraordinary potential.

Biochemical and Thermodynamic Characterization of a Novel, Low Molecular Weight Xylanase from Bacillus Methylotrophicus CSB40 Isolated from Traditional Korean Food.

A low molecular weight xylanase from Bacillus strain CSB40, isolated from traditional Korean food and produced in beechwood xylan, was biochemically and thermodynamically characterized. It was purified 8.12-fold with a 15.88 % yield using DEAE sepharose fast flow, and it was determined to have a mass of ∼27 kDa via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and xylan zymography. The purified xylanase was optimally active at 50 °C and pH 6 and stable over a wide range of pH (4.5-12.5). The N-terminal amino acid sequence of xylanase was GIQQGDDGKL. The activation energy for beechwood xylan hydrolysis was 29.39 kJmol(-1) with k cat value of 927.582 × 10(2) s(-1). K m and V max were 0.080 mg/ml and 794.63 mmol min(-1) mg(-1). The analysis of other thermodynamic parameters like ∆H, ∆G, ∆S, Q10, ∆GE-S, and ∆GE-T also supported the spontaneous formation of products, greater hydrolytic efficiency, and feasibility of enzymatic reaction, which also ratifies the novelty of this xylanase. The enzyme was strongly activated by Zn(2+) and inhibited by Cu(2+). The principal hydrolyzed end-products of this xylanase are xylobiose, xylotriose, and xylotetrose, which can be used in the pharmaceutical industry and as prebiotic in food.

Novel alkali-thermostable xylanase from Thielaviopsis basicola (MTCC 1467): Purification and kinetic characterization.

A novel extracellular alkali-thermostable xylanase was purified to an apparent homogeneity from the submerged fermented culture filtrate of Thielaviopsis basicola MTCC 1467, wherein, the fungus was fed with rice straw as prime carbon source. SDS-PAGE analysis of the xylanase showcased molecular weight of ∼ 32 kDa. This extracellular protein macromolecule had maximum xylanolytic activity at pH 5.5 and 60°C, and was stable in the range of pH 5.0-10.0 for 5 days retaining >70% activity. The enzyme was stable at 30-50°C for 5h retaining >85% activity and further by retaining 70% activity at 60°C for 2h. The enzyme deactivation constants (kd) were in range of 0.41-1.3. The kinetic experiments specified that the enzyme had Km and Vmax values of 1.447 ± 0.22 mg mL(-1) and 60.04 ± 1.25 IU mL(-1), respectively, for xylan. The purified xylanase was significantly inhibited by Cu(2+) and Zn(2+) (∼ 58%), whilst Ca(2+) and Na(+) ions displayed partial inhibition (<8%) Intriguingly, the K(+) and Mn(2+) ions enhanced the activity by about ∼ 10%. Both SDS and EDTA reduced its activity by ∼ 20%.

Fusarium graminearum produces different xylanases causing host cell death that is prevented by the xylanase inhibitors XIP-I and TAXI-III in wheat.

To shed light on the role of Xylanase Inhibitors (XIs) during Fusarium graminearum infection, we first demonstrated that three out of four F. graminearum xylanases, in addition to their xylan degrading activity, have also the capacity to cause host cell death both in cell suspensions and wheat spike tissue. Subsequently, we demonstrated that TAXI-III and XIP-I prevented both the enzyme and host cell death activities of F. graminearum xylanases. In particular, we showed that the enzymatic inhibition by TAXI-III and XIP-I was competitive and only FGSG_11487 escaped inhibition. The finding that TAXI-III and XIP-I prevented cell death activity of heat inactivated xylanases and that XIP-I precluded the cell death activity of FGSG_11487 - even if XIP-I does not inhibit its enzyme activity - suggests that the catalytic and the cell death activities are separated features of these xylanases. Finally, the efficacy of TAXI-III or XIP-I to prevent host cell death caused by xylanases was confirmed in transgenic plants expressing separately these inhibitors, suggesting that the XIs could limit F. graminearum infection via direct inhibition of xylanase activity and/or by preventing host cell death.

GH11 xylanase from Emericella nidulans with low sensitivity to inhibition by ethanol and lignocellulose-derived phenolic compounds.

An endo-ß-1,4-xylanase (X22) was purified from crude extract of Emericella nidulans when cultivated on submerged fermentation using sugarcane bagasse as the carbon source. The purified protein was identified by mass spectrometry and was most active at pH and temperature intervals of 5.0-6.5 and 50-60°C, respectively. The enzyme showed half-lives of 40, 10 and 7 min at 28, 50 and 55°C, respectively, and pH 5.0. Apparent Km and Vmax values on soluble oat spelt xylan were 3.39 mg/mL and 230.8 IU/mg, respectively, while Kcat and Kcat/Km were 84.6 s(-1) and 25.0 s(-1) mg(-1) mL. Incubation with phenolic compounds showed that tannic acid and cinnamic acid had an inhibitory effect on X22 but no time-dependent deactivation. On the other hand, ferulic acid, 4-hydroxybenzoic acid, vanillin and p-coumaric acid did not show any inhibitory effect on X22 activity, although they changed X22 apparent kinetic parameters. Ethanol remarkably increased enzyme thermostability and apparent Vmax and Kcat values, even though the affinity and catalytic efficiency for xylan were lowered.

The xylanase inhibitor TAXI-III counteracts the necrotic activity of a Fusarium graminearum xylanase in vitro and in durum wheat transgenic plants.

The xylanase inhibitor TAXI-III has been proven to delay Fusarium head blight (FHB) symptoms caused by Fusarium graminearum in transgenic durum wheat plants. To elucidate the molecular mechanism underlying the capacity of the TAXI-III transgenic plants to limit FHB symptoms, we treated wheat tissues with the xylanase FGSG_03624, hitherto shown to induce cell death and hydrogen peroxide accumulation. Experiments performed on lemmas of flowering wheat spikes and wheat cell suspension cultures demonstrated that pre-incubation of xylanase FGSG_03624 with TAXI-III significantly decreased cell death. Most interestingly, a reduced cell death relative to control non-transgenic plants was also obtained by treating, with the same xylanase, lemmas of TAXI-III transgenic plants. Molecular modelling studies predicted an interaction between the TAXI-III residue H395 and residues E122 and E214 belonging to the active site of xylanase FGSG_03624. These results provide, for the first time, clear indications in vitro and in planta that a xylanase inhibitor can prevent the necrotic activity of a xylanase, and suggest that the reduced FHB symptoms on transgenic TAXI-III plants may be a result not only of the direct inhibition of xylanase activity secreted by the pathogen, but also of the capacity of TAXI-III to avoid host cell death.

Thermal behaviour and tolerance to ionic liquid [emim]OAc in GH10 xylanase from Thermoascus aurantiacus SL16W.

GH10 xylanase from Thermoascus aurantiacus strain SL16W (TasXyn10A) showed high stability and activity up to 70-75 °C. The enzyme's half-lives were 101 h, 65 h, 63 min and 6 min at 60, 70, 75 and 80 °C, respectively. The melting point (T m), as measured by DSC, was 78.5 °C, which is in line with a strong activity decrease at 75-80 °C. The biomass-dissolving ionic liquid 1-ethyl-3-methylimidazolium acetate ([emim]OAc) in 30 % concentration had a small effect on the stability of TasXyn10A; T m decreased by only 5 °C. It was also observed that [emim]OAc inhibited much less GH10 xylanase (TasXyn10A) than the studied GH11 xylanases. The K m of TasXyn10A increased 3.5-fold in 15 % [emim]OAc with xylan as the substrate, whereas the approximate level of V max was not altered. The inhibition of enzyme activity by [emim]OAc was lesser at higher substrate concentrations. Therefore, high solid concentrations in industrial conditions may mitigate the inhibition of enzyme activity by ionic liquids. Molecular docking experiments indicated that the [emim] cation has major binding sites near the catalytic residues but in lower amounts in GH10 than in GH11 xylanases. Therefore, [emim] cation likely competes with the substrate when binding to the active site. The docking results indicated why the effect is lower in GH10.

Constitutive expression of the xylanase inhibitor TAXI-III delays Fusarium head blight symptoms in durum wheat transgenic plants.

Cereals contain xylanase inhibitor (XI) proteins which inhibit microbial xylanases and are considered part of the defense mechanisms to counteract microbial pathogens. Nevertheless, in planta evidence for this role has not been reported yet. Therefore, we produced a number of transgenic plants constitutively overexpressing TAXI-III, a member of the TAXI type XI that is induced by pathogen infection. Results showed that TAXI-III endows the transgenic wheat with new inhibition capacities. We also showed that TAXI-III is correctly secreted into the apoplast and possesses the expected inhibition parameters against microbial xylanases. The new inhibition properties of the transgenic plants correlate with a significant delay of Fusarium head blight disease symptoms caused by Fusarium graminearum but do not significantly influence leaf spot symptoms caused by Bipolaris sorokiniana. We showed that this contrasting result can be due to the different capacity of TAXI-III to inhibit the xylanase activity of these two fungal pathogens. These results provide, for the first time, clear evidence in planta that XI are involved in plant defense against fungal pathogens and show the potential to manipulate TAXI-III accumulation to improve wheat resistance against F. graminearum.

A sorghum xylanase inhibitor-like protein with highly potent antifungal, antitumor and HIV-1 reverse transcriptase inhibitory activities.

A 25-kDa protein, with an N-terminal amino acid sequence homologous to that of xylanase inhibitor and designated as xylanase inbibitor-like protein (XILP) was purified from sorghum seeds. The isolation protocol consisted of affinity chromatography, ion exchange chromatography, and gel filtration. XILP inhibited mycelial growth in various phytopathogenic fungi. The antifungal activity was thermostable and pH-stable. XILP inhibited proliferation of various cancer cell lines but did not do so in human embryonic liver (WRL 68) cells. There was no mitogenic activity toward mouse splenocytes. XILP reduced the activity of HIV-1 reverse transcriptase with an IC50 of 11.1µM, but lacked inhibitory activity toward HIV-1 integrase and SARS coronavirus proteinase. In conclusion, sorghum XILP is thermostable and pH stable and exhibits potent antifungal, antiproliferative, and HIV-1 reverse transcriptase inhibitory activities.

Two ß-xylanases from Aspergillus terreus: characterization and influence of phenolic compounds on xylanase activity.

Sugarcane bagasse was used as an inexpensive alternative carbon source for production of ß-xylanases from Aspergillus terreus. The induction profile showed that the xylanase activity was detected from the 6th day of cultivation period. Two low molecular weight enzymes, named Xyl T1 and Xyl T2 were purified to apparent homogeneity by ultrafiltration, gel filtration and ion exchange chromatographies and presented molecular masses of 24.3and 23.60 kDa, as determined by SDS-PAGE, respectively. Xyl T1 showed highest activity at 50 °C and pH 6.0, while Xyl T2 was most active at 45 °C and pH 5.0. Mass spectrometry analysis of trypsin digested Xyl T1 and Xyl T2 showed two different fingerprinting spectra, indicating that they are distinct enzymes. Both enzymes were specific for xylan as substrate. Xyl T1 was inhibited in greater or lesser degree by phenolic compounds, while Xyl T2 was very resistant to the inhibitory effect of all phenolic compounds tested. The apparent km values of Xyl T2, using birchwood xylan as substrate, decreased in the presence of six phenolic compounds. Both enzymes were inhibited by N-bromosuccinimide and Hg(2+) and activated by Mn(2+). Incubation of Xyl T1 and Xyl T2 with L-cysteine increased their half-lives up to 14 and 24 h at 50 °C, respectively. Atomic force microscopy showed a bimodal size distribution of globular particles for both enzymes, indicating that Xyl T1 is larger than Xyl T2.

Enhanced thermostability of a mesophilic xylanase by N-terminal replacement designed by molecular dynamics simulation.

BACKGROUND: Xylanases have attracted much attention owing to their potential applications. The applicability of xylanases, however, was bottlenecked by their low stabilities at high temperature or extreme pH. The purpose of this work was to enhance the thermostability of a mesophilic xylanase by N-terminal replacement. RESULTS: The thermostability of AoXyn11, a mesophilic family 11 xylanase from Aspergillus oryzae, was enhanced by replacing its N-terminal segment with the corresponding one of EvXyn11(TS) , a hyperthermotolerant family 11 xylanase. A hybrid xylanase with high thermostability, NhXyn1157, was predicted by molecular dynamics (MD) simulation. An NhXyn1157-encoding gene, Nhxyn1157, was then constructed as designed theoretically, and overexpressed in Pichia pastoris. The temperature optimum of recombinant NhXyn1157 (re-NhXyn1157) was 75 °C, much higher than that of re-AoXyn11. Both xylanases were thermostable at 65 and 40 °C, respectively. Additionally, the pH optimum and stability of re-NhXyn1157 were 5.5 and at a range of 4.0-8.5. Its activity was not significantly affected by metal ions tested and EDTA, but strongly inhibited by Mn²âº and Ag⁺. CONCLUSION: This work obviously enhanced the thermostability of a mesophilic xylanase, making re-NhXyn1157 a promising candidate for industrial processes. It also provided an effective technical strategy for improving thermostabilities of other mesophilic enzymes.