A beta-1, 4-endoxylanase gene bsxynA and application thereof

Abstract

The application relates to the technical field of microorganisms, in particular to a beta-1,4-endoxylanase gene BsxynA and application thereof. The beta-1,4-endoxylanase gene BsxynA is screened and identified, and its in-vitro induced expression and enzymatic properties are studied for the first time. The enzyme activity of the Bacillus safensis xylanase reaches 83.913 U / mL, and it is expected that the Bacillus safensis mutant with higher enzyme activity can be obtained through mutagenesis breeding such as physical (such as ultraviolet rays and gamma rays) or chemical (such as nitroso guanidine and diethyl sulfate) mutagenic agent treatment and directional screening. For example, ultraviolet mutagenesis can cause random mutation of the DNA of microorganisms, change the expression of genes or the structure of enzyme proteins, and possibly increase the yield or activity of xylanase. The produced BsXynA mainly promotes the hydrolysis of beechwood xylan to generate xylobiose (X2) and xylotetraose (X4), and does not generate xylose. In addition, BsXynA significantly improves the digestion performance of animal feed, can increase the xylose content in the feed, promote the nutrient absorption of animals, and reduce the production cost.

Description

Technical Field

[0001] This application relates to the field of microbial technology, specifically to a β-1,4-endoxylanase gene. BsxynA And its applications. Background Technology

[0002] Xylanase is an important industrial enzyme widely used in the food, feed, and paper industries. Xylanase hydrolyzes the glycosidic bonds of xylan, releasing xylose, thereby improving the nutritional value of feed and refining papermaking processes. Currently, xylanases are mainly derived from microorganisms, with β-1,4-endoxylanase being a key enzyme in the xylanase system. However, existing xylanases still have limitations in terms of enzyme activity, stability, and substrate specificity, restricting their widespread application in industrial production.

[0003] Heterologous expression studies of xylanase genes and in-depth investigations into the enzymatic properties of the resulting enzyme proteins can effectively address the shortcomings of natural xylanases, which are difficult to meet large-scale industrial demands due to low yields. Simultaneously, they can reduce production costs and enable xylanases to function in a wider range of environments. This not only provides solid support for the industrial application of xylanases but also promotes the development and utilization of biomass resources and sustainable agricultural development, making it a key research direction at present. Summary of the Invention

[0004] To address the aforementioned technical problems in the prior art, this application provides a β-1,4-endoxylanase gene. BsxynA Its application is specifically achieved through the following technical solutions: A β-1,4-endoxylanase gene BsxynA Its full-length nucleotide sequence is 1230 bp (GenBank assessment no. PQ816942), and its specific nucleotide sequence is as follows: Furthermore, this β-1,4-endoxylanase gene BsxynA It encodes a total of 409 amino acids, and the specific amino acid sequence is as follows: MVKERSFLHHSFNRGENGQDSLMWKKEADDRISEHRQRDLVINVTNGEKKPIAGIEVEIKQIRHEFAFGSAMNDQVLFNQQYADFFVKYFNWAVFENEAKWYANEPQRGRITYEKADAMLNFADRHQLPVRGHALFWEVEDANPSWLRSLPNHEVYEAMKNRLEHAGNHFKGRFRHWDVNNEMMHGSFFKDRFGKNIWKWMYEE TKKIDPQALLFVNDYNVISYGEHHAYKAHINELRQLGAPIEAIGVQGHFEERVDPVIVKERLDVLAELGLPIWVTEYDSVHPDPNRRADNLEALYRVAFSHP AVKGVLMWGFWAGAHWRGENAAIVNYDWSLNEAGRRYEKLLNEWTTQRIEKTDANGHVRCPAFHGTYEVRIGKESKMLKQQTIELDSNEQTPFQLDVIIPQEG Furthermore, this gene originates from *Bacillus safensis*. The *Bacillus safensis* species is *Bacillus safensis* L7 (16S rRNA-GenBank accession no. PV588283), specifically deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC AB 2025109. *Bacillus safensis* L7 has been disclosed in the journal *Enzyme and Microbial Technology* in a paper titled "Isolation, expression and characterization of a novel thermo-acidalkali-stable GH10 xylanase BsXynA from *Bacillus safensis* L7 and its potential for xylooligosaccharide production and animal feed," with the author listed as the first inventor in this application. The public can obtain the gene through the aforementioned public deposit, and the applicant guarantees to release the biological material to the public within twenty years from the application date.

[0005] The above β-1,4-endoxylanase gene BsxynA Applications in the preparation of xylanase.

[0006] Furthermore, the xylanase is a xylanase with high thermal acid / alkali stability.

[0007] A method for preparing xylanase, using the above-mentioned β-1,4-endoxylanase gene. BsxynA It is prepared through expression. Specifically, it is prepared by... BsxynA The gene was cloned into the pET32a(+) expression vector and transformed into E. coli.

[0008] A xylanase with high thermal and acid / alkali stability, achieved by employing the aforementioned β-1,4-endoxylanase gene. BsxynA The expression was prepared.

[0009] Compared with the prior art, the technical effects created by this application are reflected in: (1) This application screened and identified a novel xylanase gene, and for the first time induced its expression in vitro and studied its enzymatic properties. The xylanase activity of the described Bacillus salsa reached 83.913 U / mL, and it is expected that gene mutations can be induced by mutagenesis breeding, such as physical (e.g., ultraviolet light, gamma rays) or chemical (e.g., nitrosoguanidine, diethyl sulfate) mutagen, to selectively screen for Bacillus salsa mutants with higher enzyme activity. For example, ultraviolet mutagenesis can cause random mutations in the DNA of microorganisms, changing gene expression or enzyme protein structure, which may increase xylanase production or enhance its activity.

[0010] (2) This application provides a xylanase derived from Bacillus salsa with high heat acid / alkali stability, studies its excellent heat-resistant acid and alkali enzymatic properties, and explores its application potential in the industrial production of hydrolyzed beech xylan to prepare xylooligosaccharides and softened animal feed.

[0011] (3) The BsXynA provided in this application significantly improves the digestibility of animal feed. Studies have found that it can effectively increase the xylose content in pig feed and chicken feed, thereby improving the absorption of nutrients by animals and reducing production costs.

[0012] (4) The BsXynA provided in this application has the following beneficial effects: (1) High heat acid / alkali stability: The purified BsXynA has good stability in a wide pH range (5.0-8.0; 60°C) and at a high temperature (50°C). (2) Long shelf life: The purified BsXynA has good stability at 4°C (pH 6.0-9.0). (3) Specific substrate hydrolysis ability: BsXynA has strong hydrolysis specificity for xylan substrates and can effectively degrade specific substrates such as beech xylan, bagasse xylan, and corn cob xylan. (4) Application potential: The application research results of BsXynA in the preparation of xylooligosaccharides from hydrolyzed xylan and in the feed industry show that BsXynA mainly promotes the hydrolysis of beech xylan to generate xylobiose (X2) and xylotetraose (X4) without producing xylose. In addition, BsXynA significantly improves the digestibility of animal feed, increases the xylose content in feed, promotes nutrient absorption in animals, and reduces production costs. Attached Figure Description

[0013] Figure 1 This is the technology roadmap for the research in this application.

[0014] Figure 2 This is the BSA standard curve graph.

[0015] Figure 3 This is a standard curve graph of xylose.

[0016] Figure 4 This is a graph showing the xylanase production curve of Bacillus salsa L7.

[0017] Figure 5 It is Bacillus sarfusula L7 BsxynA PCR amplification results (a) and phylogenetic tree analysis of the encoded protein (b). M: marker.

[0018] Figure 6 This is a schematic diagram (a) of the pET32a-BsxynA recombinant expression vector and a gel electrophoresis image (b) verifying the linearization of pET32a(+). Where M: marker; 1: undigested plasmid; 2: digested plasmid.

[0019] Figure 7 This is a gel electrophoresis image of colony PCR verification of the xylanase gene. M: Marker, 1: Blank control, 2-9: Single colonies.

[0020] Figure 8 This is a gel electrophoresis image of colony PCR verification of the xylanase gene. M: Marker, 1-3: Single colonies.

[0021] Figure 9This is an SDS-polyacrylamide gel electrophoresis image. Where M: Protein Marker; 1: 40 mmol / L; 2: 60 mmol / L; 3: 80 mmol / L; 4: 100 mmol / L; 5: 150 mmol / L; 6: 200 mmol / L; 7: 250 mmol / L.

[0022] Figure 10 The graph shows the optimal reaction pH (a) of BsXynA and its stability at 60°C (b) and 4°C (c).

[0023] Figure 11 The graph shows the optimal reaction temperature of BsXynA (a) and its stability test (b).

[0024] Figure 12 The graph shows the effects of different metal ions (a) and organic solvents, SDS, and EDTA (b) on xylanase activity.

[0025] Figure 13 This is a graph showing the specific activity of xylanase under different substrate conditions.

[0026] Figure 14 This is a diagram of the dynamic parameters of BsXynA.

[0027] Figure 15 This is an analysis of the hydrolysis products of beech wood xylan.

[0028] Figure 16 This is a graph showing the changes in xylose content in animal feed. Detailed Implementation

[0029] The technical solution of this application will be further defined below with reference to specific embodiments, but the scope of protection is not limited to the description. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods, and the materials and reagents used can all be obtained commercially.

[0030] Bacillus sabolica xylanase gene BsxynA In vitro expression and enzymatic properties study 1. Introduction 1.1 Xylanase Due to the high diversity of xylan, its complete degradation requires the synergistic action of multiple xylanases, collectively known as xylanases. The xylan hydrolase system comprises a series of enzymes for degrading xylan, such as β-1,4-endoxylanase, β-xylosidase, α-L-arabinosidase, α-D-glucuronidase, acetylxylanase, and phenolic esterase. These enzymes can widely degrade xylan hemicellulose in nature. Among this enzyme system, β-1,4-endoxylanase is considered the most important hydrolytic component. This enzyme effectively cleaves the β-1,4-glycosidic bonds in the xylan chain, converting xylan into xylooligosaccharides composed of small oligosaccharides and xylobiose, as well as small amounts of xylose and arabinose.

[0031] 1.2 Research progress on in vitro expression of xylanase With the ever-increasing demand for xylanase across various industries, efficiently obtaining highly active and stable xylanases has become a research hotspot. In vitro expression technology provides a powerful tool for the large-scale production and performance optimization of xylanases. Through genetic engineering, xylanase genes can be introduced into suitable expression hosts, enabling heterologous expression of xylanases. In recent years, extensive research has been conducted on host selection and modification, gene mining and optimization, exploration of expression regulation mechanisms, and directed evolution of enzyme molecules. For example, genetic engineering of traditional expression hosts such as Aspergillus niger has increased the expression level and activity of xylanases; directed evolution technology has been used to modify the amino acid sequence of xylanases, improving their thermal stability, pH stability, and substrate specificity.

[0032] 1.3 Application of Xylanase Xylanase possesses extremely high application value and is widely used in numerous industries such as feed, papermaking, food, and pharmaceuticals. Its application is particularly prevalent in the feed industry, where it significantly improves feed utilization and has a remarkable effect on promoting animal digestion and absorption and improving growth performance. Xylanase can degrade non-starch polysaccharides in feed, especially arabinoxylan, which is difficult for animals to digest and has anti-nutritional effects. By degrading NSP, xylanase can reduce feed viscosity and optimize the physical properties of feed components. Adding xylanase to the feed of monogastric animals such as pigs and chickens can significantly improve feed digestibility and absorption. Through the rational use of xylanase and other biological enzyme preparations, feed utilization can be improved, animal digestion and absorption and growth performance can be promoted, animal intestinal health can be improved, and the range of feed ingredients can be broadened. Therefore, the application of xylanase in the feed industry has a broad prospect.

[0033] 1.4 Research Objectives and Significance 1.4.1 Research Objectives The β-1,4-endoxylanase gene of *Bacillus sabina*, a high-yield xylanase strain screened from sugarcane bagasse soil in Guangxi, was amplified and transformed into *Escherichia coli* for in vitro heterologous expression to obtain purified xylanase. Its enzymatic properties were studied to provide guidance for its application.

[0034] 1.4.2 Significance of the Research Heterologous expression studies of xylanase genes and in-depth investigations into the enzymatic properties of the enzyme proteins produced by their expression can effectively address the shortcomings of natural xylanase, which is difficult to meet the large-scale industrial demand due to its low yield. At the same time, it can reduce production costs and enable xylanase to function in a wider range of environments. This not only provides solid support for the application of xylanase in the industrial field, but also promotes the development and utilization of biomass resources and the sustainable development of agriculture.

[0035] 1.5 Research Content This study mainly covers the following: (1) Cloning and identification of highly active xylanase genes A strain of Bacillus saffron, which was previously screened and capable of degrading sugarcane bagasse xylan, was identified. Bacillus safensis The xylanase activity of strain L7, induced by enzyme-producing liquid medium, reached 83.913 U / mL, suggesting that this bacterium contains a gene encoding a highly active xylanase. The β-1,4-endoxylanase gene of this strain was retrieved using molecular biology and bioinformatics methods. BsxynA .

[0036] (2) Heterologous expression of xylanase gene Amplification, will BsxynA After ligation with the linearized pET32a(+) plasmid, the protein was transformed into competent E. coli cells, and expression was induced using isopropyl-β-D-thiogalactoside (IPTG). The protein was then bound using an affinity chromatography column containing nickel ions, and the target protein was eluted with imidazole solution to obtain purified xylanase.

[0037] (3) Study on the enzymatic properties of purified xylanase The purified xylanase was studied to determine its optimal pH value and stability under different pH conditions, as well as its optimal temperature and stability under different temperature environments. The effects of metal ions and organic solvents on xylanase activity were investigated. The substrate specificity of the enzyme was explored, i.e., the differences in its affinity and catalytic activity for different substrates. The characteristics and efficiency of the enzymatic reaction could be quantitatively described by studying the kinetic parameters.

[0038] (4) Assessment of the application potential of xylanase The addition of BsXynA to pig and chicken feed was investigated to assess its ability to increase xylose content. Higher xylose content in feed leads to higher digestibility in animals, i.e., higher feed utilization.

[0039] 1.6 Technical Approach For a detailed technical roadmap of this research, please see [link / details]. Figure 1 .

[0040] 2. Experimental Materials and Methods 2.1 Experimental Reagents The 99% xylan used in this experiment was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; tryptone, yeast extract, and agar powder were all provided by OXOID Ltd., UK; 3,5-dinitrosalicylic acid was purchased from Shanghai Maclean Biotechnology Co., Ltd.; the bacterial DNA extraction kit was a product of Tiangen Biotech Co., Ltd.; the Bradford protein concentration assay kit and nickel ion packing material were from Beyotime Biotechnology Co., Ltd.; and the ClonExpress II kit was purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0041] 2.2 Experimental Apparatus Table 1 Experimental Instruments

[0042] 2.3 Plasmids and Strains Table 2 Experimental bacterial strains and plasmids

[0043] 2.4 Preparation of solutions and culture media (1) To prepare imidazole solutions of different concentrations (total volume 1.0 L), 6.0 g disodium hydrogen phosphate, 17.5 g sodium chloride, 1.2 g Tris, and an appropriate amount of imidazole are weighed sequentially into deionized water, mixed thoroughly, and the volume is adjusted to 1.0 L. The concentration gradient of imidazole is achieved by adding 0.7 g imidazole (corresponding to 10.0 mmol / L) and adjusting the concentration proportionally. The pH of all solutions must be adjusted to 8.0 using dilute hydrochloric acid.

[0044] (2) Bradford protein chromogenic solution (total volume 1.0 L): Weigh 0.1 g Coomassie Brilliant Blue G-250, add 50.0 mL of 95% ethanol solution and 0.1 L of 85% phosphate solution, and dilute the solution to 1.0 L with deionized water.

[0045] (3) Enzyme-producing liquid culture medium (total volume 1.0 L): Dissolve 1% self-extracted sugarcane bagasse xylan, 4 g (NH4)2SO4, 4 g KH2PO4, 0.6 g MgSO4·7H2O, 0.6 g CaCl2, 1 mL Tween-80, and 100 μL of trace element mixture in 1000 mL of distilled water. Culture the bacteria at pH=8.0 and sterilize by moist heat at 121℃ for 20 minutes.

[0046] (4) DNS reagent (total volume 1.0 L): Weigh 0.5 g sodium sulfite, 20.0 g sodium hydroxide, 10.0 g 3,5-dinitrosalicylic acid, 290.0 g potassium sodium tartrate tetrahydrate, and 2.0 g redistilled chlorine. Mix thoroughly with deionized water, bring to pH at room temperature, and dilute to 1.0 L. After complete dissolution, filter to remove impurities and allow to stand at room temperature in the dark for one week before use.

[0047] (5) Trace element mixture (total volume 1.0 L): Dissolve 2.5 g ferrous sulfate heptahydrate, 0.8 g manganese sulfate monohydrate, 0.9 g zinc chloride and 1.0 g cobalt chloride in deionized water, make up to 1 L and store at room temperature.

[0048] LB medium, LA medium, 4× separating gel Tris-HCl buffer, 4× stacking gel Tris-HCl buffer, and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) buffer were all prepared according to the methods described in Zhang Ting's published paper, "Functional Identification of the Novel Transcription Factor PoxCxrC from Penicillium oxalicum and its Homologous Protein TP05746 in *Pseudomonas spp.*".

[0049] 2.5 Experimental Methods 2.5.1 Cloning and Identification of Highly Active Xylanase Genes 1. The strain with high xylanase activity, previously screened in the laboratory using xylan selection medium (and identified as having the highest homology with Bacillus safranin), was activated using LB medium. After culturing in enzyme-producing liquid medium for 48 hours, its xylanase activity was re-measured. Genomic DNA was extracted using a bacterial DNA extraction kit and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing to confirm the bacterial species.

[0050] 2. With " Bacillus safensis / pumilu Searching the NCBI database using the keywords "s" and "xylanase" revealed two previously unreported β-1,4-endoxylanase genes that met the screening criteria. Degenerate primers were designed based on their sequences, and amplification was performed using the genomic DNA of strain L7 as a template. The successfully amplified PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0051] 3. After sequencing is complete, log in to the official platform of the National Center for Biotechnology Information (NCBI). Submit the sequencing results to the GenBank database and use the database's BLAST tool for alignment analysis. Based on the alignment results, select sequences with high homology and download them. Use Mega11 software and the Neighbor-Joining method to construct a phylogenetic tree.

[0052] 2.5.2 Construction of recombinant plasmids (1) Amplification, purification and recovery of target cDNA After sequencing, accurate nucleotide sequences are obtained. Primers are designed using Primer software, and the required cDNA fragment is amplified using the L7 genomic DNA as a template. The amplified product is then purified and recovered (refer to the instructions for using a general DNA purification and recovery kit for specific operations) to obtain the target cDNA.

[0053] (2) Enzyme digestion and purification of PET32a(+) plasmid (preparation of linearized vector) Using Escherichia coli PET32a(+) as the expression vector, E. coli culture containing PET32a(+) plasmid was taken from a -80℃ freezer and inoculated into LB medium. The culture was carried out at 37℃ and 200 r / min for about 12 h. The plasmid was purified and recovered. The PET32a(+) plasmid was digested with restriction endonuclease Sac I at 37℃ (the restriction digestion system is shown in Table 3 below). After digestion, the linear DNA was purified and recovered.

[0054] Table 3 Enzyme digestion system

[0055] (3) Ligation reaction of target cDNA fragment and linearized vector Homologous recombination was used to ligate the target cDNA with the linearized plasmid. The target cDNA and linearized plasmid were placed in a system calculated to be suitable for the ligation reaction. The reaction system was then placed in a constant temperature environment precisely controlled at 37°C for 30 minutes.

[0056] 2.5.3 Transformation of recombinant plasmids into competent E. coli cells Following the instructions of the ClonExpress II kit, the ligation product was transformed into competent E. coli clones. Transformants with correct colonies were verified by PCR, inoculated into 10 ml LB medium, and cultured on a shaker for 12 h. The culture was then preserved with glycerol at -80°C. Plasmids were extracted from the remaining bacterial culture and sequenced for verification.

[0057] 2.5.4 Induction of gene expression and purification of recombinant protein The recombinant plasmid pET-32a-BsxynA was transformed into *E. coli* BL21(DE3) competent cells according to the manufacturer's instructions. Validated single-clonal transformants were inoculated and cultured in 10 mL of fresh LB medium at 37°C and 200 r / min for 12–14 hours. Subsequently, the pre-culture was inoculated into 200 mL of LB medium and cultured under the same conditions until OD (October Expiratory Time). 600 Once the pH reaches 0.6-0.8, add isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.25 mmol / L, and incubate at 16°C for 20-24 hours to induce recombinant protein expression. The recombinant protein is extracted after cell disruption by sonication and purified by affinity chromatography using BeyoGold™ His-tagged purification resin, following the provided instructions. Elute the protein using linear elution buffer (containing 40-300 mmol / L imidazole, pH 8.0). Store the purified protein at 4°C for further assays.

[10] .

[0058] 2.5.5 Protein Concentration Determination Protein concentration was determined using the Bradford Protein Assay Kit (Shanghai Beyotime Biotechnology Co., Ltd.) with bovine serum albumin as the standard. The standard curve is shown below. Figure 2 .

[0059] 2.5.6 SDS-polyacrylamide gel electrophoresis (1) Protein sample pretreatment Use a pipette to take 32 μL of the target protein sample into a clean 1.5 ml centrifuge tube, add 8 μL of SDS-PAGE protein loading buffer (5x) (Beyotime Biotechnology), mix by pipetting, and then incubate in a boiling water bath for 10 min. Remove and immediately place in ice water to cool to room temperature. Remove, wipe off moisture, and then briefly centrifuge at 12000 r / min for 30 s for later use.

[0060] (2) Preparation of SDS-polyacrylamide gel Table 4. Preparation of 12% polyacrylamide separation gel (20 mL)

[0061] Table 5. Preparation of 12% polyacrylamide concentrated gel (20 mL)

[0062] 2.5.7 Plotting the Xylose Standard Curve Prepare a xylose standard solution with a concentration of 1 mg / mL. Dilute the xylose solutions to different concentrations and add 1 mL DNS to each 2 mL centrifuge tube. Incubate in a boiling water bath for 5 min, then immediately cool in an ice water bath. After returning to room temperature, perform a brief centrifugation. Use a 96-well microplate to measure the absorbance at a wavelength of 540 nm (refer to the methods in Zhang Ting's published papers, such as "Functional Identification of the New Transcription Factor PoxCxrC from Penicillium oxalicum and its Homologous Protein TP05746 from *Pseudomonas aeruginosa*"). Plot a standard curve with the concentration of standard xylose on the x-axis and the corresponding OD value on the y-axis. A xylose standard curve needs to be prepared each time a new DNS reagent is prepared. See the details of the standard curve below. Figure 3 .

[0063] 2.5.8 Enzyme Activity Assay The activity of endo-1,4-β-xylanase (xylanase) was determined by the 3,5-dinitrosalicylic acid (DNS) method, as previously described. A simplified procedure was performed as follows: 50 µL of appropriately diluted recombinant enzyme solution was added to 450 µL of 50 mM citrate-disodium hydrogen phosphate buffer (pH 5.0) containing 10 g / L of different substrates. The enzyme-substrate mixture was incubated at 50 °C for 10 min, followed by the addition of 1 mL of DNS reagent to terminate the reaction. The amount of reducing sugar released was quantified by measuring absorbance at 540 nm and using a xylose standard curve. Xylanase activity units (U) were defined as the amount of enzyme required to produce 1 µmol xylose equivalent per minute from 10 g / L beech xylan. All assays were performed at least three times to ensure reproducibility. The specific activity of xylanase (U / mg) was calculated by dividing the enzyme activity by the detected protein concentration.

[0064] 2.5.9 Study on Enzymatic Properties (1) Determination of optimal pH and stability Using the standard enzyme activity assay method described above, the optimal pH for purifying BsXynA was determined at 50°C using the following buffers (all 50 mM): citrate-disodium hydrogen phosphate buffer (pH 2.0–5.0), disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (pH 6.0–7.0), and Tris-HCl buffer (pH 8.0–9.0). The highest enzyme activity was set to 100%. Subsequently, the recombinant enzyme was incubated at 60°C for 5 hours in different buffers with pH values ​​of 5.0, 6.0, 7.0, and 8.0, and simultaneously stored at 4°C for 14 days (pH 5.0–9.0). Samples were taken at intervals for xylanase activity assay. Freshly prepared enzyme solution (unincubated) was used as a control group, and its activity was normalized to 100%.

[0065] (2) Determination of optimal temperature and stability The optimal temperature for BsXynA, ranging from 30 to 80°C, was determined using the standard assay method described above at pH 6.0 (50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer). The highest enzyme activity was set as 100%. To assess the thermostability of BsXynA, the enzyme was incubated at 50°C, 60°C, and 70°C (pH 6.0). Samples were taken at 2, 4, 6, 8, 22, and 24 hours, and at 2, 3, 4, 5, 6, 7, 8, 9, 10, 13, 15, and 16 days of incubation, and immediately placed on ice to stop further degradation. Freshly prepared enzyme solution (unincubated) was used as a control group, and its activity was normalized to 100%. All relative enzyme activities were calculated as a percentage relative to the control group.

[0066] (3) Effect of chemical reagents on xylanase activity The effects of various chemical reagents (e.g., metal ions (Na⁺, K⁺, Cu²⁺, Ca²⁺, Ba²⁺, Mn²⁺, Zn²⁺, Mg²⁺, Co²⁺, and Fe³⁺, all derived from chlorides), organic solvents (e.g., ethanol, isopropanol, acetic acid, acetonitrile), sodium dodecyl sulfate (SDS, an anionic surfactant), and ethylenediaminetetraacetic acid (EDTA)) at concentrations of 2 mmol / L and 5 mmol / L on the activity of BsXynA were investigated. Subsequently, xylanase activity was determined according to the standard assay method described above at pH 6.0 (50 mmol / L Na₂HPO₄-NaH₂PO₄) and 60°C. Enzyme activity without the addition of any chemical reagents was defined as 100%.

[0067] (4) Substrate specificity The substrate specificity of BsXynA was assessed using the following substrates: xylan (commercial beech xylan, bagasse xylan, and corn cob xylan, initial concentration 10 g·L⁻¹) and cellulose substrates (Whatman No.1 filter paper (GE Healthcare Life Sciences, UK, Little Chalfont, 1×6 cm), Avicel, carboxymethyl cellulose (CMC, Beijing Solarbio Science & Technology Co., Ltd., 10 g·L⁻¹) and sucrose and maltose (Sangon Biotech (Shanghai) Co., Ltd., 10 g·L⁻¹). Enzyme activity was determined according to standard xylanase assays at pH 6.0 and 60°C.

[0068] (5) Dynamic parameters kinetic parameters ( V max , Km , k cat and k cat / K m The determination was achieved by incubating a fixed amount of BsXynA with different concentrations of beech xylan (0 to 10 g·L⁻¹) at 60°C and pH 6.0 for 10 minutes. The obtained data were fitted to a standard Michaelis-Menten equation model. The Michaelis constant ( K m ) and maximum reaction rate ( V max The result was obtained by fitting the Michaelis-Menten nonlinear curve using GraphPad Prism10.

[0069] 2.5.10 Application of purified enzyme preparations in feed (1) Preparation of feed ingredients Common feed ingredients such as soybean meal, corn flour, rapeseed meal, and wheat bran are selected and dried to reduce moisture content. After drying, the raw materials are pulverized using a pulverizing device to ensure uniform particle size. The pulverized raw materials are then passed through a 30-mesh sieve to ensure the particle size distribution of the feed ingredients. The pig feed formula is: 61% corn flour, 21% soybean meal, 8% wheat bran, 2% fish meal, 1% rapeseed meal, and 1% cottonseed meal. The chicken feed formula is: 55% corn flour, 32% soybean meal, 2% fish meal, 2% rapeseed meal, and 3% bone meal.

[0070] (2) Application of xylanase in feed ingredients Three treatments were designed: a control group (no enzyme added), an enzyme-added group, and a control group. 10 g of feed substrate was added to 100 mL of 50 mmol / L Na₂HPO₄-NaH₂PO₄ (pH 6.0) buffer, mixed well, and soaked for 30 min. Then, 5 mL of enzyme solution was added (buffer solution was added to the control group). The enzymatic hydrolysis temperature was 55℃, and the shaking speed was 100 r / min. The enzymatic hydrolysis time for chicken feed was 4 h, and for pig feed it was 6 h. The reaction was terminated with 5 mL of 10% trichloroacetic acid solution. Samples were taken uniformly from both the enzyme-added group and the control group, centrifuged at 4800 r / min for 2 min, and the xylose production was determined using the DNS method.

[0071] 3 Results and Analysis 3.1 Re-identification of high xylanase-producing Bacillus saffron L7 Enzyme production curves were plotted for the xylanase-producing Bacillus saffron L7 strain preserved in the laboratory. The results showed that enzyme production gradually increased after 18 h of culture, reaching its peak at 36-42 h, with an enzyme activity of 83.913 U / mL at 42 h. With prolonged culture time, the enzyme production capacity of strain L7 slowed down, and the enzyme activity began to decline. See details below. Figure 4 This indicates that Bacillus saffron L7 is a xylanase-producing strain, suggesting that the bacterium contains a gene encoding a highly active xylanase.

[0072] 3.2 L7 xylanase gene of Bacillus saffron BsxynA Cloning and Identification by" Bacillus safensis / pumilus A search of the NCBI database using the keywords "xylanase" revealed two previously unreported β-1,4-endoxylanase genes that met the screening criteria. Their sequences were retrieved, and degenerate primers were designed for amplification using the genomic DNA of strain L7 as a template. The results are as follows: Figure 5 As shown in Figure a, a band of 1000-1500 bp was obtained. Sequencing analysis of the PCR product identified its nucleotide sequence. The corresponding 409 amino acid sequence (uploaded to GenBank, ID: PQ816942) was submitted to NCBI for alignment analysis. It was found to have 99.76% sequence identity with an endonuclease-1,4-β-xylanase from Bacillus (WP_039178603.1) (query coverage 100%). Phylogenetic analysis showed that BsXynA and... B. safensis The relationship between endo-1,4-β-xylanase and ( Figure 5 b). In summary, the amplified sequence was identified as a xylanase-encoding gene and named the *Bacillus saffron* xylanase gene. BsxynA Its encoded protein is named BsXynA.

[0073] The specific nucleotide sequence is as follows: 3.3 p ET32a-BsxynA Construction of recombinant expression vectors The xylanase gene insertion site is as follows: Figure 6 As shown in a, the xylanase gene insertion site is located at... Sac The 5' end of the I restriction enzyme cleavage site carries a His tag. Restriction endonuclease is used. Sac I linearize pET32a(+), from Figure 6 The gel electrophoresis results (b) show that the linear plasmids that have undergone successful enzyme digestion migrate significantly slower during gel electrophoresis than those that have not. In gel electrophoresis, the migration speed of molecules is closely related to their molecular conformation and size. Circular, undigested plasmids migrate relatively quickly in an electric field due to their compact structure; however, after plasmids are digested into linear structures, their molecular conformation changes, resulting in greater resistance to migration in the gel medium and thus a slower migration speed. Therefore, based on this gel electrophoresis pattern, it can be inferred that the plasmid has been successfully linearized.

[0074] 3.4 p ET32a - BsxynA Transformed into E. coli clonal competent cells Eight single colonies were selected from the plate, and colony PCR was performed using cross-primers for verification. The PCR amplification products were then subjected to 0.8% agarose gel electrophoresis. The target gene size after transformation into competent cloned cells was expected to be 1054 bp. Results are as follows... Figure 7 As shown, analysis of the gel electrophoresis bands revealed that bands of colonies 2, 3, 4, 7, 8, and 9 were of the expected size, proving that these single colonies were successfully transformed.

[0075] 3.5 p ET32a - BsxynA Transformed into E. coli expression competent cells Plasmids extracted from successfully sequenced single colonies were transformed into competent *E. coli* expression cells. Three single colonies were selected, and colony PCR was performed on these three single colonies using cross-primers for verification. After the PCR reaction, the obtained PCR products were subjected to 0.8% agarose gel electrophoresis, and the verification results are as follows: Figure 8 As shown. By Figure 8 It can be seen that all three single colonies were successfully transformed.

[0076] 3.6 SDS-polyacrylamide gel electrophoresis After binding with nickel ion packing material, you elute the nickel ion packing material with imidazole solutions of different concentrations to determine the concentration of imidazole solution in which the target protein is predominantly present. Figure 9The target protein was found to be mainly present in imidazole solutions at concentrations of 80 mmol / L, 100 mmol / L, 150 mmol / L, 200 mmol / L, and 250 mmol / L. The actual protein band was between 50 kDa and 70 kDa, consistent with the predicted protein size of 63.58 kDa. This demonstrates successful expression of the pET32a-BsXynA recombinant protein, and subsequent enzymatic studies will be referred to as BsXynA.

[0077] 3.7 Optimal reaction pH and stability of BsXynA Optimal pH analysis of the BsXynA enzyme showed that, at 50°C, the enzyme exhibited the highest xylanase activity at pH 6.0. Furthermore, the enzyme maintained over 60% of its maximum activity within the pH range of 5.0 to 8.0. However, at pH 9.0, the enzyme activity dropped sharply to below 50%, but still retained 28.72% of its activity. In contrast, xylanase activity was almost undetectable within the pH range of 2.0 to 4.0, with activity values ​​below 10%. Figure 10 a). These results indicate that BsXynA exhibits optimal catalytic performance under weakly acidic to weakly basic conditions and has a relatively wide pH adaptability.

[0078] Subsequent pH stability assessment ( Figure 10 (b) and (c) show that after incubation at the optimal reaction temperature of 60°C for 60 minutes, BsXynA retains over 60% of its relative activity within the pH range of 5.0–8.0; and within the pH range of 6.0–9.0, BsXynA retains over 80% of its relative activity after 14 days. Even at pH 5.0, it retains over 60% of its activity. These results collectively demonstrate that BsXynA exhibits strong colloidal stability under weakly acidic to weakly alkaline conditions, making it highly suitable for biotechnological and industrial applications requiring robust enzymatic performance in these environments.

[0079] 3.8 Optimal reaction temperature and stability of BsXynA Optimal temperature analysis of the BsXynA enzyme showed that, under pH 6.0 conditions, the enzyme exhibited the highest xylanase activity at 60°C. Furthermore, the enzyme maintained over 70% of its maximum activity within a temperature range of 50°C to 70°C. Notably, even at 40°C, the enzyme retained 50.80% of its maximum activity. Moreover, at lower (30°C) and higher (80°C) temperatures, the enzyme still exhibited 30.36% and 41.01% of its maximum activity, respectively, with the relative activity at 80°C being higher than that at 30°C. Figure 11a). These results indicate that the optimal catalytic temperature range of BsXynA falls within the moderately thermophilic range, demonstrating its robustness and application potential in moderately high temperature environments.

[0080] Thermal stability assessments showed that BsXynA exhibited significant stability at moderate temperatures. Specifically, the enzyme retained over 80% of its residual activity after 5 days of incubation at 50°C, and maintained over 55% of its initial activity even after 16 days of incubation at the same temperature. However, rapid inactivation was observed at higher temperatures. The enzyme's half-lives at 60°C and 70°C were approximately 2 hours, and it completely lost its activity within 8 hours and 24 hours, respectively (11b). These thermostability characteristics indicate that BsXynA possesses operational stability under moderate temperature conditions (50°C), suggesting its potential biotechnological application value in industrial processes requiring sustained activity at this temperature.

[0081] 3.9 Effect of Chemical Reagents on Xylanase Activity The catalytic activity of BsXynA is affected by a variety of chemical reagents (including metal ions and organic solvents). For example... Figure 12 As shown in Figure a, Mn²⁺, Zn²⁺, Cu²⁺, Co²⁺, and Fe²⁺ exhibited concentration-dependent inhibitory effects. Except for Mn²⁺ and Fe²⁺, which showed no significant difference in enzyme activity between 2 mmol / L and the absence of metal ions, high concentrations of metal ions (5 mmol / L) showed a stronger inhibitory effect on the catalytic activity of BsXynA than low concentrations (2 mmol / L). Figure 12 a). Conversely, K 2+ Na 2+ Ca 2+ Ba 2+ Mg 2+ Both concentrations can promote the catalytic activity of BsXynA. p <0.05), but the increase in enzyme activity is not higher than 25%.

[0082] Notably, organic solvents exhibited varying effects: 2 mmol / L acetic acid had almost no effect, while a concentration of 5 mmol / L resulted in significant inhibition of enzyme activity. The effect of detergents was particularly noteworthy: SDS completely inhibited BsXynA activity at both tested concentrations (2 mmol / L and 5 mmol / L). In contrast, EDTA only showed significant inhibitory effects at higher concentrations. p <0.05; Figure 12(b) Furthermore, low concentrations of acetonitrile (2 mmol / L) had a slight promoting effect on the activity of BsXynA. These findings highlight the need to monitor the ionic environment and solvent composition in industrial applications to maximize the operating efficiency of BsXynA, and in particular, the use of Zn²⁺, Cu²⁺, and SDS should be avoided in the application of BsXynA.

[0083] 3.10 Substrate Specificity Substrate-specific analysis of BsXynA revealed varying hydrolytic abilities towards different polysaccharides. Among the tested substrates, BsXynA exhibited high specific activity towards beech xylan (BX), bagasse xylan (SBX), and corn cob xylan (CX). In contrast, its catalytic activity against cellulose substrates (including filter paper (FP), Avicel, and carboxymethyl cellulose (CMC)) and non-xylan polysaccharides (such as sucrose and maltose) was extremely low (<1.0 U / mg). Figure 13 This strict substrate preference confirms that BsXynA has high specificity for xylan polymers.

[0084] 3.11 Dynamic Parameters Depend on Figure 14 It can be seen that the kinetic parameters of BsXynA conform to the nonlinear Michaelis-Menten model, and from this, the enzymatic kinetic constants of BsXynA on beech xylan can be derived. K m It was 6.61 mg / mL. V max It is 24.24 μmol / (min·mg). k cat and k cat / K m The concentrations were 15.71 s⁻¹ and 2.38 mL·s⁻¹·mg⁻¹, respectively. (BsXynA) K m The value is significantly lower than previously reported. Cellulomonas sp. B6 rCsXyn10A、 Microbacterium imperiale YD-01Z Xyn1923 and Trichodermaasperellum ND-1 XynTR10 indicates that it has a higher affinity for the substrate.

[0085] 3.12 Analysis of hydrolysis products of BsXynA hydrolyzed beech wood xylan like Figure 15Thin-layer chromatography (TLC) analysis showed that in the initial stage (0–1.5 h), xylotriose (X3) and xylotetraose (X4) were the main degradation products of BsXynA, along with a small amount of xylobiose (X2). After a longer incubation period of 1.5–6 h, X2, X3, and X4 coexisted. After further hydrolysis for 10–24 h, X2 and X4 became the main products in the hydrolysate mixture. Interestingly, the content of X3 decreased as the xylan hydrolysis time was extended to 6 h, possibly due to further hydrolysis forming xylobiose and xylose (bands of these products are not shown in the figure). Notably, the content of X4 remained almost constant throughout the 0–24 h period, indicating that BsXynA has a relatively weak ability to hydrolyze X4. In contrast, T. asperellum XynTR10 of ND-1 rapidly degrades X4 to X2 in the early stages (1 hour). Current results indicate that the xylanase hydrolyzes the substrate primarily to release xylo-oligosaccharides, but cannot act on the resulting oligosaccharides to form xylose, suggesting that it is an endoxylanase suitable for XOS production.

[0086] 3.13 Research on the Application of BsXynA in Feed Considering the differences in feed digestion processes between chickens and pigs (approximately 2-4 hours for chickens and a relatively longer time for pigs), the enzymatic hydrolysis time for chicken feed was set at 4 hours, and for pig feed at 6 hours. The addition of xylanase altered the xylose content in the feed; the role of xylanase in the feed could be analyzed by measuring the xylose content. The research results are as follows... Figure 16 As shown, the xylose content in pig feed increased by 10%; the xylose content in chicken feed increased by 6%.

[0087] 4. Discussion and Conclusion 4.1 Discussion Although the specific activity of BsXynA involved in this study was only 17.34 U / mg, which is relatively low, its high substrate specificity and high affinity for xylan ( K m The value is lower than that reported in the literature. Cellulomonas sp. B6 rCsXyn10A (7.87±1.03 mg / mL), Microbacterium imperiale YD-01Z's Xyn1923 Zobelliagalactanivorans gXyn10A and Cellulophagaalgicola CaXyn10B, Malbrancheacinnamomea F408's McXyn0243 (GH11) and TrichodermaasperellumThe presence of XynTR10 in ND-1 gives it potential for application in the preparation of xylooligosaccharides from xylan hydrolysis. Furthermore, BsXynA, involved in this study, exhibited over 60% relative activity after 1 hour of incubation at 60°C over a wide pH range (5.0-8.0). After 14 days at 4°C within a pH range of 6.0-9.0, it still retained over 80% relative activity. Its high thermal and acid / alkali stability makes it suitable for industrial applications and long-term storage.

[0088] The fact that BsXynA retains 89% of its specific enzyme activity after 14 days at 50°C indicates that this xylanase possesses excellent thermostability. Compared to many common xylanases, this stability allows it to function effectively for extended periods during industrial processing at relatively high temperatures, reducing the frequency of enzyme replenishment. At the molecular level, its amino acid sequence and spatial structure may contain unique stabilizing factors, such as numerous hydrogen bonds and disulfide bonds, which help maintain the enzyme's three-dimensional structure, making it less prone to denaturation and inactivation under heating conditions. This provides an excellent sample for further research into the structure-function relationship of enzymes. The thermostability of this enzyme may also suggest good stability during storage. Under suitable storage conditions, its shelf life may be longer than that of common xylanases, reducing waste caused by enzyme inactivation and potentially lowering inventory management costs for companies engaged in large-scale production and long-term use.

[0089] The inhibitory effects of Mn²⁺, Zn²⁺, Cu²⁺, Co²⁺, and Fe²⁺ may be attributed to their potential interactions with residues at the active site of BsXynA, thereby disrupting the enzyme's catalytic efficiency. Conversely, the enhancing effects of K⁺, Na⁺, Ca²⁺, Ba²⁺, and Mg²⁺ are likely due to their roles in stabilizing the enzyme's structure or promoting substrate binding. To elucidate the detailed molecular mechanisms underlying the differential effects of these metal ions on BsXynA enzyme activity, further investigation of the binding sites of these metal ions on BsXynA and their mechanisms of action is necessary.

[0090] 4.2 Conclusion This study investigated a strain of *Bacillus sabolicii* that, under induction with xylanase activity of 83.913 U / mL in an enzyme-producing liquid culture medium, was previously screened. Bacillus safensis L7, via " Bacillus safensis / pumilus A search of the NCBI database using the keywords "xylanase" revealed two β-1,4-endoxylanase genes that met the screening criteria. Degenerate primers were designed based on their sequences, and xylanase gene amplification was performed using *Bacillus salsafras* L7 genomic DNA as a template. Sequencing confirmed the xylanase coding sequence of *Bacillus salsafras* L7, which was then named the β-1,4-endoxylanase gene. BsxynA And also the β-1,4-endoxylanase gene BsxynA Add the His tag, BsxynA With p ET32a(+) After plasmid ligation, the xylanase was cloned into competent E. coli cells, induced to express using isopropyl-β-D-thiogalactoside, and purified using an affinity chromatography column containing nickel ions to finally obtain purified xylanase.

[0091] Enzymatic studies revealed that the optimal reaction pH for BsXynA enzyme is 6.0. After incubation at 60°C for 60 minutes, it retains over 60% relative activity within a pH range of 5.0-8.0. At 4°C, within a pH range of 6.0-9.0, it maintains over 80% relative activity after 14 days, demonstrating good pH stability. The optimal reaction temperature is 60°C. After treatment at 50°C for 16 days, its activity remains above 55%, indicating it is a mesophilically stable enzyme. This enzyme is sensitive to metal ions and surfactants; 5 mmol / L of Mn²⁺, Zn²⁺, Cu²⁺, Co²⁺, and Fe²⁺ strongly inhibit its activity, while 2 mmol / L of SDS completely inhibits it. Kinetic parameter analysis showed that when beech wood polysaccharide is used as a substrate, BsXynA… K m It was 6.61 mg / mL. V max It is 24.24 μmol / (min·mg). k cat and k cat / K m The values ​​were 15.71 s⁻¹ and 2.38 mL·s⁻¹·mg⁻¹, respectively. These parameters reflect the high efficiency and affinity of the enzyme in catalyzing the hydrolysis of xylan, making it suitable for application in the production of xylo-oligosaccharides and animal feed industries. It can improve hydrolysis yield and feed utilization, and reduce production costs.

[0092] Finally, it should be noted that the above embodiments are merely representative examples of this application. Obviously, the technical solutions of this application are not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the content disclosed in this application should be considered within the scope of protection of this application.

Images