A termite fungus and basic xylanase produced thereby and use thereof

Abstract

The present application provides a termite fungus and the produced alkaline xylanase and application. The screened termite fungus Isoptericola sp. WL6 belongs to alkaliphilic extremophiles, can be cultured under high alkaline conditions (pH 9.0), has the characteristics of effectively inhibiting the pollution of miscellaneous bacteria and being conducive to large-scale fermentation production. The alkaline xylanase protein produced by the termite fungus Isoptericola sp. WL6 has a molecular weight of about 43 kD, and the enzymatic property research shows that the optimal reaction temperature is 50 DEG C, the optimal reaction pH is about 8, especially between pH 6.5-10.5, the relative enzyme activity of the enzyme is all above 80%, which shows that the enzyme has good pH stability, strong stress resistance characteristics of both acid and alkali, meets the basic requirements of industrial enzymes such as papermaking and washing, and has good industrial application prospect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a termite fungus and the alkaline xylanase it produces and its applications. Background Technology

[0002] Xylan is the most abundant hemicellulose in nature, found in almost all plant tissues. It is a complex molecule mainly present in the cell walls of plant cells. The backbone of this polymer consists of D-xylanose residues linked by β-1,4 glycosidic bonds. Various short groups are attached to the side chains, with substituents on the side chains present as residues such as L-Arabinose, FerulcAcid, AcetyL, Coumaricacid, and Glucuronicacid. Therefore, different xylans exhibit significant differences in structure and chemical composition. Due to its structural complexity, the complete conversion into monosaccharides requires the synergistic action of multiple hydrolases with different functions and mechanisms of action.

[0003] Xylanases are a collective term for a series of enzymes that can hydrolyze xylan into oligosaccharides or monosaccharides, including endo-1,4-D-xylanase (EC.3.2.1.8), β-D-xylosidase (EC.3.2.1.37), α-L-arabinofuranosidase (EC.3.2.1.55), α-D-glucuronidase (EC.3.2.1.139), and acetylxylanase (EC.3.2.1.139), etc. In a narrow sense, xylanase refers specifically to endo-β-1,4-D xylanase, which catalyzes the hydrolysis of β-1,4-glycosidic bonds within the xylan backbone. It cleaves these bonds to generate xylo-oligosaccharides or xylooligosaccharides with side chains, thereby reducing the degree of polymerization of xylan and hydrolyzing it into xylooligosaccharides. Therefore, endo-xylanase is also known as a key enzyme in xylan hydrolysis.

[0004] The industrial use of xylanases still faces several challenges: low stability, poor substrate specificity, and a narrow activity range, limiting their widespread application. Most reported GH10 xylanases cannot tolerate temperatures exceeding 50°C. Studying extremophiles has proven to be an effective strategy for discovering novel enzymes with higher stability. Furthermore, to enable large-scale production of screened xylanases, genetic engineering is typically used to achieve anomalous source expression, exploring more efficient expression systems to ultimately achieve large-scale fermentation production to meet industrial application requirements. In addition, exploring the relationship between enzyme structure and function from a molecular evolutionary perspective using high-throughput equipment and computer software is also a recent research hotspot. Rational design and directed evolution methods have been widely used to improve xylanase performance, and significant progress has been made in related research both domestically and internationally. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and to provide a termite fungus, Isoptericolasp.WL6, which can produce an alkaline xylanase.

[0006] Another object of the present invention is to provide an alkaline xylanase produced by the termite fungus Isoptericola sp. WL6. This enzyme has excellent enzymatic properties, including a wide pH adaptability range and resistance to heat, alkalis, metal ions, and surfactants, making it suitable for industries such as papermaking and detergents.

[0007] Another object of the present invention is to provide the application of the above-mentioned termite fungus and the alkaline xylanase it produces.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A termite fungus, Isoptericola sp.WL6, is a strain of alkali-producing xylanase that was screened from soil.

[0010] The termite fungus, with accession number CCTCC NO:M 20221888, was deposited on December 7, 2022, at the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China.

[0011] Colony morphology characteristics: Colonies on LB solid medium have relatively neat edges, are pale yellow in color, and have a smooth and glossy surface.

[0012] The selective medium for this strain was formulated as follows: xylan 8.0 g / L, KNO3 1.0 g / L, MgSO4·7H2O 0.5 g / L, NaCl 15 g / L, KH2PO4 1.5 g / L, agar 15–20 g / L, pH 9.0.

[0013] The alkaline xylanase produced by the termite fungus Isoptericola sp. WL6 is prepared according to the following method:

[0014] (1) Preparation of crude enzyme solution

[0015] The termite fungus Isoptericola sp. WL6 was liquid fermented in a fermentation medium at 37°C and 200 rpm for 7 days. After fermentation, it was centrifuged at 4°C and 12,000 rpm for 25 min. The supernatant was collected, filtered through a 0.45 μm filter membrane, and dialyzed to obtain crude enzyme solution, which was used for ion exchange chromatography.

[0016] (2) Isolation and purification of alkaline xylanase

[0017] (a) Packing of the chromatography column

[0018] Pour 100 mL of well-stirred anion exchange chromatography medium, SP-Sepharose Fast Flow chromatography gel, into a 2.6 cm × 30 cm chromatography column. Then, install the column into the GE Healthcare Rapid Protein Chromatography System. Rinse the column with deionized water at a flow rate of 2 mL / min for 15 min, followed by equilibration with 5–6 column volumes of equilibration buffer. Once the A280 UV absorption peak baseline has stabilized, seal the column head to complete the column packing.

[0019] (b) Anion exchange chromatography of endoxylanase

[0020] The packed chromatography column was installed into the GE Healthcare Rapid Protein Chromatography System. Protein separation and purification were performed using a pH 6.5 tris-HCl solution as the equilibration buffer and a pH 6.5 tris-HCl solution containing 1M NaCl as the elution buffer. Before loading, the column was equilibrated with 5–6 column volumes of equilibration buffer. After the A280 baseline was stabilized, 5 mL of fermentation broth filtered through a 0.22 nm microporous membrane was loaded. Washing continued with equilibration buffer until the breakthrough peak appeared. Once the A280 baseline stabilized, the column was switched to elution buffer, and gradient elution was performed by washing the column with different salt concentrations. The eluent at different salt concentrations under UV absorption peaks was collected in 2 mL EP tubes, and xylanase activity was measured in each tube using the DNS method.

[0021] (c) Desalting treatment of crude enzyme solution

[0022] The collected crude enzyme solution was desalted using a desalting column. The desalting column was then installed into a GE Healthcare rapid protein chromatography system. Equilibration was performed using 5–6 column volumes of equilibration buffer. After the ion concentration baseline stabilized, 10 mL of the crude enzyme solution, filtered through a 0.22 nm microporous membrane, was loaded onto the system. Elution continued with equilibration buffer until fluctuations in the A280 baseline were observed. The A280 eluent was collected in a 20 mL centrifuge tube. Sample loading was stopped when the ion concentration baseline increased, yielding the crude basic xylanase solution. The crude enzyme solution was analyzed by SDS-PAGE protein electrophoresis and stored at 4°C for subsequent gel filtration chromatography.

[0023] (d) Gel filtration chromatography using alkaline xylanase

[0024] After ultrafiltration concentration in a 10 kDa ultrafiltration tube, the crude enzyme solution was further separated using a pre-packed Superdex 200 column. The buffer system consisted of deionized water at pH 8.0. After stabilizing the A280 baseline by washing with 5–6 column volumes of buffer, 500 μL of the concentrated enzyme solution was loaded onto the plate. The elution peak was collected using an EP tube. The elution solution, i.e., the pure enzyme solution, was collected for enzyme activity detection and protein electrophoresis analysis, and stored for subsequent enzymatic property studies.

[0025] The alkaline xylanase has a protein molecular weight of approximately 43 kD. Enzymatic characterization studies show that its optimal reaction temperature is 50°C and its optimal reaction pH is around 8. In particular, the relative enzyme activity is above 80% between pH 6.5 and 10.5, indicating that the enzyme has good pH stability and strong resistance to both acid and alkali. It meets the basic requirements for enzymes used in industries such as papermaking and detergents, and has good prospects for industrial applications.

[0026] The complete open reading frame (ORF) of the XynWL6 xylanase gene was amplified from the genomic DNA of the termite fungus Isoptericola sp. WL6, with a length of 1443 bp. BLAST analysis of the amino acid coding sequence of this enzyme gene in the NCBI database revealed that this conserved sequence shares 77% similarity with the xylanase Isoptericola halotoleransWP171782838.1, which belongs to the same xylanase family 10. Based on the molecular weight, isoelectric point, and enzyme activity characteristics of the enzyme protein, it can be preliminarily inferred that the gene encoding this amino acid sequence is a novel xylanase gene.

[0027] The application of the termite fungus Isoptericola sp. WL6 and its produced alkaline xylanase in industries such as papermaking and washing.

[0028] The present invention has the following advantages and effects compared with the prior art:

[0029] (1) The termite fungus Isoptericola sp.WL6 screened in this invention is an alkaliphilic extremophile that can be cultured under high alkaline conditions (pH 9.0), which can effectively inhibit contamination by other bacteria and is conducive to large-scale fermentation production.

[0030] (2) The basic xylanase protein produced by the termite fungus Isoptericola sp. WL6 of the present invention has a molecular weight of about 43kD. Enzymatic characterization studies show that its optimal reaction temperature is 50℃ and the optimal reaction pH is about 8. In particular, the relative enzyme activity of the enzyme is above 80% between pH 6.5 and 10.5, indicating that the enzyme has good pH stability and strong resistance to both acid and alkali. It meets the basic requirements of enzymes used in papermaking and washing industries and has good prospects for industrial application. Attached Figure Description

[0031] Figure 1 These are photographs of the morphology of Isoptericola sp. WL6 in solid culture medium and the clear zone of xylan hydrolysis.

[0032] Figure 2 This is an electrophoretic banding diagram of the 16S rDNA of Isoptericola sp. WL6.

[0033] Figure 3 These are anion exchange spectra of the elution peaks of the Isoptericola sp. WL6 fermentation broth; (A) is the DNS enzyme activity detection spectrum of each elution peak, and (B) is a photograph of the sample with obvious color change in elution peak F3.

[0034] Figure 4 This is the electrophoretic pattern of XynWL6 anion exchange separation and purification; where M: protein marker; 1-7: protein patterns corresponding to the elution peaks of each protein.

[0035] Figure 5 This is the gel filtration chromatography exchange pattern of XynWL6 protein; where (A) is the electrophoresis pattern after gel filtration chromatography purification (B), M: protein marker; 1: crude enzyme solution protein pattern; 1-4: enzyme activity peaks from gel filtration chromatography.

[0036] Figure 6 The graph shows the results of the optimal reaction temperature of XynWL6 (A) and the stability of xylanase temperature (B).

[0037] Figure 7 This is a graph showing the optimal reaction pH (A) of XynWL6 and the pH stability of xylanase (B).

[0038] Figure 8This is a graph showing the effects of metal ions and surfactants on enzyme activity.

[0039] Figure 9 This is a graph showing the effect of laundry detergent on enzyme activity.

[0040] Figure 10 This is a TLC analysis of the xylan hydrolysis products by alkaline xylanase; where: M: xylo-oligosaccharide label; S1: hydrolysis product of XynWL6 reacting with substrate for 12 hours; S2: hydrolysis product of XynWL6 reacting with substrate for 24 hours; S3: hydrolysis product of XynWL6 reacting with substrate for 48 hours; N: xylan substrate.

[0041] Figure 11 This is an electrophoresis image of the Isoptericola sp. WL6 genome.

[0042] Figure 12 This is an electrophoresis image of TouchDown PCR amplification of the conserved sequence of the XynWL6 xylanase gene; where: M: molecular weight marker; 1-2: conserved sequence of the XynWL6 gene; 3: negative control.

[0043] Figure 13 This is an electrophoresis diagram of the upstream gene of the conserved XynWL6 sequence cloned by FPNI; where: M: molecular weight marker; 1-8: results of second-round PCR amplification with different nested primers.

[0044] Figure 14 This is an electrophoresis image of the cloning of downstream genes of the conserved XynWL6 sequence by FPNI-PCR, where: M: molecular weight marker; 1-8: results of the third round of PCR amplification with different nested primers.

[0045] Figure 15 This is a diagram showing the alignment results of the XynWL6 xylanase gene coding sequence with NCBI. Detailed Implementation

[0046] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0047] Unless otherwise specified in the following implementation plan, the test conditions are generally as per standard test conditions or the test conditions recommended by the reagent company. Unless otherwise specified, all materials and reagents used are commercially available.

[0048] Example 1: Screening of target strains

[0049] (1) Enrichment culture: Weigh 1g of silt soil sample, mix it thoroughly in 100mL of sterile water to make a bacterial suspension, then add 2mL of the bacterial suspension to 50mL of sterile enrichment culture medium, and place it in a shaker at 37℃ and 200rpm for 48h of enrichment culture.

[0050] Enrichment medium: xylan 8.0g, peptone 10g, NaCl 15g, KH2PO4 1.5g, Na2HPO4·12H2O 9.0g, MgSO4·7H2O 2.0g, adjusted to 1L ddH2O, pH 9.0.

[0051] (2) Initial screening on plates: The enriched bacterial culture was serially diluted 10-fold, and samples were taken from each of the 10-fold dilutions. -3 10 -5 10 -7 0.5 mL of bacterial suspension was spread on alkaline selective medium plates containing xylan and incubated at 37°C for 48 h. Single colonies with large clear zones were screened repeatedly until a single colony was obtained. This single colony was then inoculated onto slant medium and preserved as the original strain.

[0052] Selective culture medium: xylan 8.0g, KNO3 1.0g, MgSO4·7H2O 0.5g, NaCl 15g, KH2PO4 1.5g, agar 15-20g, dissolved in 1L ddH2O, pH 9.0.

[0053] Slant culture medium: glucose 10g / L, peptone 5g / L, yeast extract 5g / L, KH2PO4 1g / L, MgCl2 0.2g / L, NaCl 50g / L, Na2CO3 10g / L, agar 15-20g / L, pH 9.0.

[0054] (3) The above steps screened a strain with alkaline xylanase activity, with a liquid fermentation enzyme activity of 65 U / mL. The morphology of this strain in LB solid medium is as follows: Figure 1 As shown, the colony edges are relatively neat, the color is pale yellow, and the surface is smooth and glossy. Using universal primers (27F and 1492R) and the bacterial genome as a template, the 16S rDNA sequence was amplified, producing a band of approximately 1500 bp, as shown. Figure 2 As shown, the band was recovered and analyzed by 16S rDNA sequencing. BLAST comparison was performed on NCBI, and the fungus was preliminarily identified as a termite fungus, named Isoptericola sp.WL6, with accession number CCTCCNO:M 20221888. It was deposited on December 7, 2022, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China.

[0055] LB solid medium: 10g tryptone, 5g yeast extract, 10g NaCl, add 15-20g agar to the solid medium, bring the volume to 1L, and autoclave at 121℃ for 20min.

[0056] Compared with current industrial enzyme strains, the termite fungus Isoptericola sp. WL6 is an alkaliphilic extremophile that can be cultured under highly alkaline conditions (pH 9.0). It has the characteristics of effectively inhibiting contamination by other microorganisms and is conducive to large-scale fermentation production.

[0057] The 16S rDNA sequence of the termite fungus Isoptericola sp. WL6 is shown in SEQ ID No: 1, and its length is 1440 bp.

[0058] Example 2

[0059] (I) Experimental Materials

[0060] 1. Species: Isoptericola sp.WL6, a termite fungus obtained in Example 1.

[0061] 2. Culture medium and buffer solution

[0062] (1) Fermentation medium: 6g wheat bran, 1g peptone, 0.7g K2HPO4 dissolved in 100mL Gly-NaOH buffer, pH 9.0.

[0063] (2) 1% xylan solution: Dissolve 1g of xylan in 0.05M Gly-NaOH buffer solution at pH 9.0, and bring the volume up to 100mL. Shake well before use.

[0064] (3) Gly-NaOH buffer (0.05M): 3.8g glycine, 0.35g sodium hydroxide, dissolved in 1L ddH2O, pH 9.0.

[0065] (4) 3,5-Dinitrosalicylic acid (DNS) solution: Accurately weigh 7.5g of DNS, 14.0g of sodium hydroxide (added slowly), 216g of potassium sodium tartrate, and 6.0g of sodium metabisulfite. Stir well, add 5.6mL of phenol (toxic, wear a mask) that has been melted in a 60℃ water bath beforehand, dissolve completely, add deionized water to make up to 1L, store in a brown bottle, and use after one week at room temperature away from light.

[0066] (5) Protein separation and purification buffer:

[0067] 1. Tris-HCl buffer A (equilibration buffer): 20 mM Tris, adjusted to pH 8.0 with HCl.

[0068] 2. Tris-HCl buffer B (elution buffer): 20mM Tris, 1M NaCl, pH adjusted to 8.0 with HCl.

[0069] 3. Main reagents: Protein markers were purchased from Sangon Biotech (Shanghai) Co., Ltd. Xylan was produced by Megazyme. The anion exchange chromatography medium DEAE Sepharose Fast Flow was produced by GE. 10kDa protein ultrafiltration tubes were purchased from Millipore. All other reagents were domestically produced analytical grade.

[0070] 4. Major Instruments: Thermo high-speed refrigerated centrifuge, Bio-Rad electrophoresis apparatus, GE Healthcare rapid protein chromatography system, Thermo ultra-micro UV spectrophotometer, Thermo low-temperature incubator, Eppendorf large-capacity high-speed refrigerated centrifuge, shaking incubator, pressure steam sterilizer, Millipore ultrapure water system, gel imaging system, spectrophotometer, electronic balance, water bath, electric thermostatic incubator, stirrer, microwave oven, forced-air drying oven, Eppendorf pipette, BioTek microplate reader.

[0071] (II) Experimental Methods

[0072] 1. Preparation of crude enzyme solution

[0073] The strain Isoptericola sp. WL6 obtained from plate screening was cultured in a fermentation medium at 37℃ and 200 rpm for 7 days. The culture medium was then centrifuged at 12000 rpm at 4℃ for 25 min, and the supernatant was collected. The supernatant was filtered through a 0.45 μm filter membrane, and the filtrate was poured into a 10000 kDa ultrafiltration concentration bag. An appropriate amount of PEG20000 powder was evenly covered on the outside of the ultrafiltration concentration bag. After standing at 4℃ for 24 h, the concentrated fermentation broth inside the bag was collected to obtain the crude enzyme solution. Its xylanase activity was measured and used for subsequent chromatography experiments.

[0074] 2. Method for determining xylanase activity

[0075] Using a 1% beechwood xylan solution prepared with Gly-NaOH buffer (pH 9.0) as the substrate, 1 mL of the solution was added to 40 μL of the appropriately diluted crude enzyme solution. The mixture was stirred and reacted at 45°C for 20 min. Then, 1 mL of DNS (3,5-dinitrosalicylic acid) solution was quickly added, and the mixture was incubated in a boiling water bath for 10 min. After cooling in an ice bath, the reducing sugar content was measured at 540 nm, and the results were subtracted from the blank test values. The amount of enzyme required to produce 1 μmol of xylose per minute from the substrate under the above conditions was defined as one unit of enzyme activity, expressed in U / mL.

[0076] 3. Plotting the xylose standard curve

[0077] Weigh 1g of anhydrous xylose and dilute to 100mL to prepare a 1% standard xylose solution. Then, take 0.25mL, 0.5mL, 0.75mL, 1mL, 1.25mL, and 1.5mL of the prepared 1% standard xylose solution and dilute again to 10mL to obtain xylose concentrations of 0.25mg / mL, 0.5mg / mL, 0.75mg / mL, 1mg / mL, 1.25mg / mL, and 1.5mg / mL, respectively. Add 1mL of each of the above xylose solutions of different concentrations to different test tubes (with another tube containing 1mL of deionized water as a control). Add 1mL of DNS solution to each test tube, develop the color in a boiling water bath for 10min, cool in an ice bath, and measure the absorbance at 540nm. Plot a xylose standard curve with the mean of three repeated experiments on the ordinate and the corresponding xylose concentration of the standard xylose solution on the abscissa. The xylose concentration of the experimental group after the enzyme activity reaction was calculated based on the xylose standard curve, and then the enzyme activity units (U / mL) were calculated according to the standard enzyme activity calculation formula.

[0078] Xylan-DNS enzyme activity calculation formula:

[0079]

[0080] In the formula: 6.66 represents the number of μmol of xylose in 1 mg. (1000 / 150.13=6.66)

[0081] 4. Isolation and purification of alkaline xylanase

[0082] (a) Packing of the chromatography column

[0083] Pour 100 mL of well-stirred Q-Sepharose Fast Flow anion exchange chromatography medium into a 2.6 cm × 30 cm chromatography column, then install the column into the GE Healthcare Rapid Protein Chromatography System. Rinse the column with deionized water at a flow rate of 2 mL / min for 15 min, then equilibrate with 5–6 column volumes of equilibration buffer. Once the A280 UV absorption peak baseline has stabilized, seal the column head to complete the column packing.

[0084] (b) Anion exchange chromatography of endoxylanase

[0085] The packed chromatography column was installed into the GE Healthcare Rapid Protein Chromatography System. Protein separation and purification were performed using a pH 6.5 tris-HCl solution as the equilibration buffer and a pH 6.5 tris-HCl solution containing 1M NaCl as the elution buffer. Before loading, the column was equilibrated with 5–6 column volumes of equilibration buffer. After the A280 baseline was stabilized, 5 mL of fermentation broth filtered through a 0.22 nm microporous membrane was loaded. Washing continued with equilibration buffer until the breakthrough peak appeared. Once the A280 baseline stabilized, the column was switched to elution buffer, and gradient elution was performed by washing the column with different salt concentrations. The eluent at different salt concentrations under UV absorption peaks was collected in 2 mL EP tubes, and xylanase activity was measured in each tube using the DNS method.

[0086] (c) Desalting treatment of crude enzyme solution

[0087] The collected crude enzyme solution was desalted using a desalting column. The desalting column was then installed into a GE Healthcare rapid protein chromatography system. Equilibration was performed using 5–6 column volumes of equilibration buffer. After the ion concentration baseline stabilized, 10 mL of the crude enzyme solution, filtered through a 0.22 nm microporous membrane, was loaded onto the system. Elution continued with equilibration buffer until fluctuations in the A280 baseline were observed. The A280 eluent was collected in a 20 mL centrifuge tube. Sample loading was stopped when the ion concentration baseline increased, yielding the crude basic xylanase solution. The crude enzyme solution was analyzed by SDS-PAGE protein electrophoresis and stored at 4°C for subsequent gel filtration chromatography.

[0088] (d) Gel filtration chromatography using alkaline xylanase

[0089] After ultrafiltration concentration in a 10 kDa ultrafiltration tube, the crude enzyme solution was further separated using a pre-packed Superdex 200 column. The buffer system consisted of deionized water at pH 8.0. After stabilizing the A280 baseline by washing with 5–6 column volumes of buffer, 500 μL of the concentrated enzyme solution was loaded onto the plate. The elution peak was collected using an EP tube. The elution solution, i.e., the pure enzyme solution, was collected for enzyme activity detection and protein electrophoresis analysis, and stored for subsequent enzymatic property studies.

[0090] (e) SDS-PAGE protein electrophoresis analysis

[0091] (1) Preparation and formulation of reagents

[0092] The reagents required for preparing SDS-PAGE gels were prepared using Sangon Biotech's denaturing gel preparation kit.

[0093] Coomassie Brilliant Blue Staining Solution: Dissolve 2.5g of Coomassie Brilliant Blue R-250 in 0.5L of methanol, 0.1L of glacial acetic acid, and deionized water to a final volume of 1L. Mix thoroughly, filter, and store.

[0094] Decolorizing solution: 50 mL ethanol, 100 mL acetic acid, and deionized water to a final volume of 1 L.

[0095] (2) Preparation of SDS-PAGE gel:

[0096] a. Prepare a 15% separating adhesive according to the table below. Add the prepared separating adhesive between the fixed glass plates and wait for it to solidify.

[0097] Table 1. Preparation of SDS-PAGE gel separating gel

[0098]

[0099]

[0100] b. After the separating gel solidifies, prepare the stacking gel according to the formula in the table below. Add the prepared stacking gel between the fixed glass plates, insert the comb, and wait for it to solidify. After complete polymerization, start SDS-PAGE gel electrophoresis.

[0101] Table 2. Preparation of SDS-PAGE gel stacking gel

[0102]

[0103] (3) Steps of electrophoresis:

[0104] a. Sample preparation: Add 5× Loading buffer to an appropriate amount of sample, boil in water for 5 min, then centrifuge at 12000 rpm for 10 min, and collect the supernatant for later use.

[0105] b. Add freshly prepared electrophoresis buffer to the inner and outer tanks of the electrophoresis tank.

[0106] c. Sample loading: Add 20 μL of sample supernatant to the well, followed by 5 μL of pre-stained protein marker as a standard for determining protein molecular weight.

[0107] d. Electrophoresis: Turn on the power supply and connect the positive and negative electrodes correctly. The voltage is 120V. Electrophoresis is stopped after about 50 minutes, when the bromophenol blue indicator has descended to the bottom of the gel.

[0108] e. Staining: Carefully remove the gel from the glass plate, add an appropriate amount of Coomassie Brilliant Blue staining solution, and stain on a shaking table for 20-30 minutes.

[0109] f. Destaining: Discard the staining solution, rinse once or twice with deionized water, then add destaining solution, changing the destaining solution several times until the protein bands are clearly visible.

[0110] (III) Experimental Results

[0111] 1. Anion exchange chromatography

[0112] The Q-Sepharose Fast Flow column was equilibrated with 3–5 column volumes of pH 6.5 Tris-HCl buffer. After the A280 baseline was stabilized by the equilibration buffer, an appropriate amount of crude enzyme solution filtered through a 0.22 μm filter was loaded onto the column. Washing continued with pH 6.5 Tris-HCl buffer until the breakthrough peak appeared. After the A280 baseline stabilized again, a salt gradient elution was performed using pH 6.5 Tris-HCl buffer containing NaCl. Elution peaks at different salt concentrations were collected, and all collected peak solutions were analyzed for xylanase activity. Ion exchange profiles are shown below. Figure 3 As shown in the figure. The peak solution showing enzyme activity was concentrated and desalted using a 10 kDa ultrafiltration tube to obtain a pure enzyme solution of alkaline xylanase. An appropriate amount of the concentrated pure enzyme solution was analyzed by SDS-PAGE protein electrophoresis, and the results are shown in the figure. Figure 4 In lane 2, a band of approximately 43 kDa can be clearly seen.

[0113] 2. Gel filtration chromatography

[0114] The enzyme solution, which had undergone anion exchange chromatography, was separated again using a pre-packed Superdex 75 column. The buffer system was a Tris-HCl buffer at pH 8.0. After rinsing the A280 baseline to a stable state with 3–5 volumes of buffer, an appropriate amount of concentrated enzyme solution was loaded onto the sample, followed by elution with one volume of buffer. The elution peak was collected for enzyme activity detection and protein electrophoresis analysis. The protein electrophoresis results after gel filtration are shown below. Figure 5 As shown in lane 3, a clear band of the target protein (molecular weight approximately 43 kDa) is visible, essentially achieving the purpose of separation and purification.

[0115] Example 3: Enzymatic Properties Study of Alkaline Xylanase

[0116] (I) Experimental Methods

[0117] 1. Optimal reaction temperature and temperature stability

[0118] Take 40 μL of diluted pure enzyme solution and react it at different temperatures (30℃~80℃) for 15 min. Determine the enzyme activity using the DNS method to determine the optimal reaction temperature. To investigate temperature stability, the pure enzyme solution was appropriately diluted and incubated at 5℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, and 70℃ for 1 h, followed by reaction at 45℃ for 15 min, and the enzyme activity was measured.

[0119] 2. Optimal reaction pH and pH stability

[0120] 20 mL of diluted pure enzyme solution was reacted at different pH values ​​(3.0–12.0) for 20 min, and the enzyme activity was determined by the DNS method to determine the optimal reaction pH. To study pH stability, an appropriate amount of diluted pure enzyme solution was incubated at different pH values ​​for 1 h, followed by reaction at pH 8.0 for 20 min, and the enzyme activity was then measured.

[0121] 3. Effects of metal ions and surfactants on enzyme activity

[0122] A certain volume of metal ion solution was mixed with an appropriate amount of pure enzyme solution to achieve a final concentration of 10 mM. The following metal ions (final concentration 5 mM) were then added to the enzyme-catalyzed reaction system: K... + Na + Ca 2+ Li + Ni 2+ Cu 2+ Mg 2+ Fe 2+ Mn 2+ 、Xn 2+ The effects of the metal chelating agent EDTA (0.05%) and the surfactant SDS (0.05%) on enzyme activity were investigated. Enzyme activity was measured under optimal pH and temperature conditions, with an enzymatic reaction under the same conditions but without the addition of metal ions and chemical reagents serving as a control. Enzyme activity was measured after 60 min of reaction.

[0123] 4. The effect of laundry detergent on enzyme activity

[0124] Commercially available laundry detergent solutions were prepared to final concentrations of 0.5%, 1%, 1.5%, 2%, and 5%, respectively. Enzymatic reactions were carried out in the commercially available laundry detergent solutions at 37°C for 30 minutes, and then the enzyme activity was measured.

[0125] 5. Analysis of xylanase hydrolysis products

[0126] The hydrolysis products of xylanase were analyzed by thin-layer chromatography (TLC). The preparation method of the hydrolysis product samples is as follows:

[0127] Add 20 μL of purified enzyme solution to 1 mL of 1% beech xylan solution substrate and mix well. React at 50 °C and pH 8.0 for 12 h, 24 h, and 48 h respectively, and then stop the reaction by boiling in a water bath for 10 min. Then, centrifuge the above terminated reaction solution at 10000 r / min for 5 min and collect the supernatant for thin-layer chromatography analysis.

[0128] Thin-layer chromatography (TLC) experimental procedures:

[0129] (1) Prepare the developing solvent (ethyl acetate: methanol: water: glacial acetic acid (V:V:V) = 1:3:1:0.25) and the color developing agent (acetone: diphenylamine: aniline: phosphoric acid (V:V:V) = 50:1:1:5). The color developing agents must be added in order, otherwise they will be difficult to dissolve.

[0130] (2) Pour the developing solvent into the chromatography tank in advance to saturate the gas in the chromatography tank. The depth of the developing solvent should not exceed the spotting point.

[0131] (3) Place the unspotted silica gel plate in a 110℃ oven and activate it for 20 minutes. Then, take out the silica gel plate and cool it to room temperature. Draw a line with a pencil 1.5cm away from the bottom of the silica gel plate and mark the location of the spotting point.

[0132] (4) Use a capillary tube to pick up the sample and spot it. The spotting volume is 1 μL. Each spotting must be repeated only after the previous spotting sample is completely dry. After the spotting is completed, place the silica gel plate in the chromatography tank and start the development.

[0133] (5) After the development is completed, place the silica gel plate at room temperature to dry. After drying, put it in the color developer for a few seconds and then immediately take it out and place it in a 90℃ oven to heat for 30 minutes to develop the color.

[0134] (II) Experimental Results

[0135] 1. Optimal reaction temperature and temperature stability

[0136] Depend on Figure 6 A shows that the optimal reaction temperature for this alkaline xylanase is 50℃, and the relative enzyme activity is greater than 60% between 35-50℃. Temperature stability results are as follows... Figure 6 B. The enzyme is more stable below 50℃, but its activity gradually decreases above 50℃.

[0137] 2. Optimal reaction pH and pH stability

[0138] Depend on Figure 7As shown in Figure A, the optimal reaction pH for this enzyme is around 8, indicating it is a typical alkaline xylanase. Appropriately diluted pure enzyme solutions were incubated at different pH values ​​for 1 hour, followed by reaction at pH 9.0 for 20 minutes to determine enzyme activity. The pH stability results are as follows... Figure 7 B. As shown in the figure, the relative enzyme activity of this enzyme is above 80% between pH 6.5 and 10.5, indicating that the enzyme has good pH stability and strong stress resistance to both acid and alkali. It meets the basic requirements for enzymes used in industries such as papermaking and washing, and has good prospects for industrial application.

[0139] 3. Effects of metal ions and surfactants on enzyme activity

[0140] Take 1 mL of reaction substrate, add 20 μL of metal ions (final concentration 10 mmol / L) and 100 μL of enzyme solution, and measure enzyme activity to investigate the effect of metal ions on the activity of recombinant endoxylanase XynWL6. Figure 8 It can be seen from Li 2+ Ag + and Ca 2+ It has virtually no effect on enzyme activity; Mg 2+ and Ni + It has a certain degree of promoting effect on enzyme activity; Mn 3+ EDTA and SDS have an inhibitory effect on enzyme activity. + Cu 2+ and Ag + It has minimal impact on enzyme activity. This demonstrates that the enzyme possesses excellent resistance to various metal ions and surfactants.

[0141] 4. The effect of laundry detergent on enzyme activity

[0142] Add a certain amount of commercially available laundry detergent to an appropriate amount of pure enzyme solution to make the final reaction concentration 2%, 4%, 6%, 8%, and 10%, incubate at 37°C for 30 minutes, and then add 1 ml of reaction substrate to determine the enzyme activity. Figure 9 It is evident that XynWL6 can maintain more than 50% activity in laundry detergent at concentrations of 2% to 4%, demonstrating its good application value in the detergent and other industries.

[0143] 5. Analysis of xylanase hydrolysis products

[0144] Analysis results as follows Figure 10 As shown, S1 to S3 are the results of XynWL6 xylanase reacting with the substrate at 50℃ and pH 8.0 for 12h, 24h, and 48h, respectively. It can be seen that the main hydrolysis products of beech xylan are xylobiose and xylotriose, etc., and almost no xylose is produced, which is consistent with the enzymatic characteristics of endoxylanase.

[0145] Example 4

[0146] (I) Experimental Materials

[0147] The termite fungus Isoptericola sp.WL6 obtained in Example 1.

[0148] (II) Experimental Methods

[0149] 1. Genome extraction

[0150] Genomic DNA was extracted using a bioengineering bacteria genomic DNA rapid extraction kit, following these steps:

[0151] (1) Take 1 mL of overnight bacterial culture and add it to a 1.5 mL centrifuge tube. Centrifuge at 8,000 rpm for 1 min at room temperature, discard the supernatant, and collect the bacterial cells. Add 180 μL of lysozyme solution (prepare a 20 mg / mL lysozyme solution by adding the corresponding amount of lysozyme to an Enzymaticlysis Buffer before use) to resuspend the bacterial culture. Incubate at 37°C for 30–60 min, then add 400 μL of Buffer Digestion and vortex to mix. Incubate at 65°C for 1 h until the cells are completely lysed.

[0152] (2) Add 200 μL BufferPB, mix thoroughly by inverting, and place in a -20℃ refrigerator for 5 min.

[0153] (3) Centrifuge at 10,000 rpm for 5 min at room temperature, and transfer the supernatant (500-550 μL) to a new 1.5 mL centrifuge tube.

[0154] (4) Add an equal volume of isopropanol, invert 5-8 times to mix thoroughly, and let stand at room temperature for 2-3 minutes. Centrifuge at 10,000 rpm for 5 minutes at room temperature and discard the supernatant.

[0155] (5) Add 1 mL of 75% ethanol, rinse by inversion for 1-3 min, centrifuge at 10000 rpm for 2 min, discard the supernatant, and repeat the steps once.

[0156] (6) Open the lid and invert at room temperature for 5-10 minutes until the residual ethanol has completely evaporated. Dissolve the obtained DNA in 50-100 μL of TE Buffer. The extracted DNA can be used immediately for the next experiment or stored at -20℃.

[0157] 2. Cloning of conserved gene sequences and flanking sequences

[0158] 2.1 Cloning of the conserved sequence of the xylanase gene (Touch Down PCR)

[0159] (1) Design of conserved sequence primers

[0160] Primers were designed using PrimerPremier 5.0 software. First, all G10 xylanase genes belonging to the same genus as *Isoptericola* sp. were searched and downloaded from the Cazy database. Then, DNAMAN software was used to perform multiple sequence alignment of the xylanase genes in this genus to obtain the conserved sequences of the G10 xylanases. Based on the alignment results, upstream primers (CTCTGGAAGCCNAYNCMRTSNA) and downstream primers (GACTGGGAYGTNGTNAAYGA) were designed to clone unknown G10 family xylanase genes in *Isoptericola* sp.

[0161] (2) Touch Down PCR Amplification Reaction

[0162] Prepare the reaction solution (total volume 25 μL) according to the following system using a PCR amplification kit:

[0163] 20 μL of 10×PCR mixture, 2 μL each of upstream primer, downstream primer and genomic DNA, and deionized water to bring the total volume to 25 μL.

[0164] The amplification reaction procedure is as follows:

[0165] Pre-denaturation at 94℃ for 5 min; denaturation at 94℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 30 s, followed by a 1℃ decrease in annealing temperature for each subsequent cycle, for a total of 12 cycles (annealing temperature decreasing from 55℃ to 43℃); denaturation at 94℃ for 30 s, annealing at 43℃ for 30 s, extension at 72℃ for 30 s, for 28 cycles, with a final extension at 72℃ for 7 min; storage at 4℃. After amplification, the PCR products were detected by 1% agarose gel electrophoresis.

[0166] (3) Recovery of Touch Down PCR Products

[0167] The PCR product recovery used the Steady Pure PCR DNA Purification Kit manufactured by AG Company. The main recovery steps are as follows:

[0168] a. Under blue light, cut the agarose gel containing the target DNA band. Blot the surface of the gel dry with a paper towel. At this stage, try to remove as much gel as possible that does not contain the target DNA to reduce the gel volume and improve DNA recovery. Then place the gel in an EP tube. After cutting the gel, add gel dissolving buffer MB to the gel block. Add 3-5 times the volume of buffer MB per 100 mg of gel.

[0169] b. Dissolve the gel at 37℃ for 5–10 minutes. During the dissolution process, remove the centrifuge tube and invert it every 2 minutes to mix thoroughly and ensure complete dissolution. After the gel has dissolved, allow the solution to return to room temperature.

[0170] c. Transfer the above solution to a DNA Fragment Mini Column, let it stand at room temperature for 1 min, then centrifuge at 12,000 rpm for 1 min at room temperature and discard the filtrate.

[0171] d. Add 750uL of Buffer WB to the Mini Column, centrifuge at 12,000rpm for 1 minute at room temperature, and discard the filtrate.

[0172] e. Repeat step (4) once.

[0173] f. Place the Mini Column onto a new 2 mL CoLLectionTube and centrifuge at 12,000 rpm for 2 minutes at room temperature.

[0174] g. Place the Mini Column onto a new 1.5 mL centrifuge tube, add 50 μL of Elution Buffer or sterile water to the center of the Mini Column membrane, and let it stand at room temperature for 1 minute.

[0175] h. Elute DNA by centrifuging at 12,000 rpm for 2 minutes at room temperature.

[0176] 2.2 Preparation of E. coli DH5α competent cells

[0177] (1) Take the laboratory-preserved bacterial strain DH5α and streak it on an LB plate. Once a single colony grows, inoculate it into 10 mL of liquid culture medium and incubate overnight at 37°C and 200 rpm on a shaker.

[0178] (2) Take 0.5 mL of the bacterial culture from the overnight shaker and add it to 50 mL of LB liquid medium. Incubate at 37°C and 200 rpm. When the OD600nm is between 0.3 and 0.6, collect the bacterial culture and place it on ice.

[0179] (3) Centrifuge at 5600 rpm for 10 min at 4℃, remove the supernatant, add 10 mL of ice-cooled 0.1 mol / L CaCl2, and carefully suspend the bacterial cells with a pipette tip. Place on ice.

[0180] (4) Repeat step 3;

[0181] (5) Add 1.5 mL of 0.1 mol / L CaCl2 and 0.5 mL of 60% glycerol to gently suspend the bacterial cells, dispense 100 μL into 1.5 mL centrifuge tubes, and store at -80 °C for later use.

[0182] 2.3 TA Cloning of Touchdown PCR Products

[0183] (1) Mix 4 μL of the recovered PCR product with 1 μL of the cloning vector pMD-19T and 5 μL of Solution I, and ligate in a water bath at 16°C for 3 h.

[0184] (2) Take 100 μL of competent cell suspension from the -80℃ freezer and thaw it in ice.

[0185] (3) Add the ligated product to competent cells and mix carefully. Place on ice for half an hour.

[0186] (4) Heat shock in a 42℃ water bath for 90s, then quickly place on ice to cool for 15-30min.

[0187] (5) Add 1 mL of LB liquid medium (containing Amp) and incubate at 37°C and 250 rpm for 1 h on a constant temperature shaker. (6) Centrifuge the culture medium at 6000 rpm for 5 min, remove part of the supernatant and mix well. Take 100 μL and spread it on a plate containing Amp, and incubate overnight at 37°C.

[0188] (7) Pick the bacteria with a sterilized pipette tip, place them in 1 mL of LB liquid medium containing Amp, shake overnight at 37°C and 250 rpm, and send them for sequencing. Add glycerol and store at -80°C.

[0189] 3. Cloning of upstream and downstream genes of the conserved sequence of the xylanase gene

[0190] 3.1 Cloning of upstream genes

[0191] To obtain the complete gene sequence of xylanase, a gene walking approach was used to amplify unknown sequences upstream and downstream of a known sequence, and the resulting splices yielded the complete gene. Primer Premier 5.0 software was used to design three nested primers (USP) based on known conserved sequences, which, along with universal nested degenerate primers (FP), were used for three rounds of FPNI-PCR reactions.

[0192] Table 3. FP primers and universal primers used in FPNI-PCR

[0193]

[0194]

[0195] Table 4 Nested primers in FPNI-PCR

[0196]

[0197] FPNI-PCR first round reaction:

[0198] Using eight highly degenerate fusion degenerate primers with the bacterial genome as a template, PCR reactions were performed with the gene-specific primer USP1. The obtained PCR products were used as templates for the second round of PCR reactions.

[0199] Table 5 Amplification program for the first round of FPNI-PCR reaction

[0200]

[0201] FPNI-PCR second round reaction:

[0202] The product from the first round of PCR was diluted 100-fold and used as a template for the second round of PCR. The second round of PCR was then performed using FSP1 and USP2 primers.

[0203] Table 6. Amplification program for the second round of FPNI-PCR reaction.

[0204]

[0205] FPNI-PCR third round reaction:

[0206] The third round of PCR reaction solution was prepared by diluting the product of the second round reaction by 100 times and using it as a template. Similarly, the FSP1 primer was replaced with the FSP2 primer and the USP2 primer was replaced with the USP3 primer. After the reaction was completed, an appropriate amount of the reaction product was taken for detection by 1% agarose gel electrophoresis.

[0207] Table 7 Amplification program for the third round of FPNI-PCR reaction

[0208]

[0209] 3.2 Cloning of downstream genes

[0210] Replace USP1, USP2, and USP3 with DSP1, DSP2, and DSP3 respectively, and clone the downstream gene using the same method as for upstream gene cloning in FPNI-PCR. After the reaction, the third round of PCR products were detected by 1% agarose gel electrophoresis. The ligation, transformation, and sequencing steps for the FNPI-PCR products were the same as those for the TouchDown PCR products.

[0211] 3.3. Assembly of the full-length xylanase gene sequence

[0212] Based on the sequencing results of the upstream and downstream genes of the conserved sequence obtained by FNPI-PCR cloning, the sequences were submitted to the NCBI gene database for BLASTn alignment to match known upstream and downstream gene fragments of xylanase in the database. Subsequently, the upstream, conserved, and downstream gene fragments obtained from sequencing were spliced ​​to obtain the full-length xylanase gene sequence. Using the genome of Isoptericolasp.WL6 as a template, conventional PCR was performed using upstream primer F1: 5′-ATGCGTCCGCACCCGG-3′ and downstream primer R1: 5′-AGTCAACCGGTGGTACAC-3′. The PCR products were recovered and sent for bioengineering sequencing to obtain the full-length xylanase gene.

[0213] (III) Experimental Results

[0214] 1. Genome extraction of Isoptericola sp. WL6

[0215] The genome of Isoptericola sp. WL6 was extracted using a Genray bacterial genome extraction kit, and then detected by 1% agarose gel electrophoresis. The results are as follows: Figure 11 As shown, the genome size is over 10kb, allowing for further experiments.

[0216] 2. Cloning results of the conserved sequence of the xylanase gene

[0217] 2.1 Products obtained by cloning conserved sequences using Touch Down PCR

[0218] Conserved sequences of known xylanase families 10 were compared with those in the Cazy database. Upstream and downstream primers were designed based on the alignment results. Touch-down PCR was performed using these primers, amplifying the conserved sequence of the XynWL6 xylanase gene. After agarose gel electrophoresis, the following results were obtained: Figure 12 The bands in lanes 1 and 2 shown are approximately 240 bp. These bands conform to the conserved sequence size of the 10 family xylanase gene in the reference alignment. The bands were subsequently gel-cleaved and recovered. Sequencing of the recovered product yielded a 232 bp sequence, the nucleotide sequence of which is shown in SEQ ID No: 2.

[0219] The coding sequence was compared with the NCBI database using BLASTx. It was found that the conserved sequence had 88% similarity to the xylanase endo-1,4-beta-xylanase [Isoptericola haiaiotoierans] gene fragment, which is also a xylanase in the GH10 family. It is preliminarily inferred that the gene containing the conserved sequence of the cloned XynWL6 belongs to the xylanase gene of the GH10 family.

[0220] 3. Cloning of upstream genes with conserved sequences using FNPI-PCR

[0221] Upstream gene amplification of the conserved sequence of the XynWL6 xylanase gene was performed using three nested specific primers (DSP) and eight fusion degenerate primers. The electrophoresis image of the FNPI-PCR amplification products is shown below. Figure 13 As shown in the figure, the results indicate that after three rounds of staggered thermal asymmetric PCR, several specific bands of approximately 1000 bp were selected, gel-extracted, and sequenced. The sequencing results were then compared with the BLASTx database of NCBI to obtain the upstream unknown target fragment.

[0222] 4. Using FNPI-PCR to clone downstream genes of conserved sequences

[0223] The downstream gene fragment was obtained in the same way as above. The upstream gene and the conserved downstream gene sequence were spliced ​​together to form a complete gene sequence. The electrophoresis image of the FNPI-PCR amplification product is shown below. Figure 14 As shown.

[0224] 5. XynWL6 whole genome sequence assembly

[0225] After sequencing the unknown upstream and downstream sequences amplified by FNNI-PCR, BLAST alignment was performed to find the corresponding ORF fragments, which were then spliced ​​with the conserved sequences amplified by TouchDown-PCR to obtain a gene with a length of 1443bp, starting with ATG and ending with TGA, as shown in SEQ ID No: 3.

[0226] After translating the gene sequence into an amino acid sequence, the sequence is shown in SEQ ID No: 4. This amino acid sequence was then aligned (BLASTx) in the NCBI database. The alignment result is as follows: Figure 15 As shown, the conserved sequence was found to have 77% similarity to the xylanase Isoptericola halotolerans WP171782838.1, which is also a member of the 10 family. It is preliminarily inferred that the gene encoding this amino acid sequence is a new xylanase (a new xylanase protein) in the 10 family.

[0227] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A type of termite fungus ( Isoptericola ), characterized by: Name is Isoptericola sp.WL6, accession number CCTCC NO:M 20221888, was deposited on December 7, 2022, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China.

2. A method for preparing alkaline xylanase, characterized in that: The alkaline xylanase was prepared according to the following method: (1) Preparation of crude enzyme solution The termite fungus described in claim 1 Isoptericola sp.WL6 was subjected to liquid fermentation in fermentation medium. The supernatant was collected by centrifugation, filtered, and dialyzed to obtain crude enzyme solution, which was used for ion exchange chromatography. (2) Isolation and purification of alkaline xylanase (a) Packing of the chromatography column Pour 100 mL of well-stirred anion exchange chromatography medium Q-Sepharose Fast FLow chromatography gel into a Φ2.6 cm × 30 cm chromatography column. Then, install the chromatography column into the GE Healthcare Rapid Protein Chromatography System. Rinse the chromatography column with deionized water at a flow rate of 2 mL / min for 15 min. Then, equilibrate with 5 to 6 column volumes of equilibration buffer. After the A280 UV absorption peak baseline stabilizes, seal the column head to complete the column packing. (b) Anion exchange chromatography of endoxylanase The packed chromatography column was installed into the GE Healthcare rapid protein chromatography system. Protein separation and purification were performed using a tris-HCl solution at pH 6.5 as the equilibration buffer and a tris-HCl solution at pH 6.5 containing 1M NaCl as the elution buffer. Before loading, the column was equilibrated with 5–6 column volumes of equilibration buffer. After the A280 baseline was stabilized, 5 mL of fermentation broth filtered through a 0.22 nm microporous membrane was loaded. Washing continued with equilibration buffer until the breakthrough peak appeared. Once the A280 baseline stabilized, the column was switched to elution buffer, and gradient elution was performed by washing the column with different salt concentrations. The eluent at different salt concentrations under UV absorption peaks was collected in 2 mL EP tubes, and xylanase activity was detected in each tube using the DNS method. (c) Desalting treatment of crude enzyme solution The collected crude enzyme solution was desalted using a desalting column. The desalting column was then installed into a GE Healthcare rapid protein chromatography system. Equilibration was performed using 5–6 column volumes of equilibration buffer. After the ion concentration baseline was stabilized by the equilibration buffer, 10 mL of the crude enzyme solution filtered through a 0.22 nm microporous membrane was loaded onto the system. Elution continued with equilibration buffer until fluctuations appeared in the A280 baseline. The A280 eluent was collected in a 20 mL centrifuge tube. Sample loading was stopped when the ion concentration baseline increased, yielding the crude basic xylanase solution. The crude enzyme solution was analyzed by SDS-PAGE protein electrophoresis and stored at 4°C for subsequent gel filtration chromatography. (d) Gel filtration chromatography of alkaline xylanase After ultrafiltration concentration of the crude enzyme solution in a 10 kDa ultrafiltration tube, the crude enzyme solution was separated again using a pre-packed Superdex 200 column. The buffer system was deionized water at pH 8.

0. After stabilizing the A280 baseline by washing with 5-6 column volumes of buffer, 500 μL of concentrated enzyme solution was loaded onto the sample. When the A280 elution peak appeared, it was collected using an EP tube. The elution peak solution, i.e., the pure enzyme solution, was collected for enzyme activity detection and protein electrophoresis analysis, and stored for subsequent enzymatic property studies.

3. The preparation method according to claim 2, characterized in that: The conditions for liquid fermentation described in step (1) are fermentation at 37°C and 200 rpm for 7 days.

4. The preparation method according to claim 2, characterized in that: The centrifugation conditions described in step (1) are: high-speed refrigerated centrifugation at 4°C and 12,000 rpm for 25 min.

5. The method for preparing alkaline xylanase according to claim 2, characterized in that: The filtration described in step (1) is filtration through a 0.45μm filter membrane.

6. The application of the termite fungus described in claim 1 in the papermaking and washing industries.

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