A gene encoding xylanase and a vector and application thereof

Abstract

The application provides a xylanase coding gene, a carrier and application thereof, and belongs to the technical field of genetic engineering and enzyme engineering. The nucleotide sequence of the xylanase coding gene is shown as SEQ ID NO: 3, and the application solves the problems of low enzyme catalytic activity of the existing xylanase and difficulty in giving consideration to high enzyme activity and good environmental tolerance.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme engineering technology, specifically relating to a gene encoding xylanase, its vector, and its applications. Background Technology

[0002] Xylan is a major component of plant hemicellulose, widely found in agricultural and forestry raw materials such as grain straw and bran, and is a naturally abundant polysaccharide resource. As a non-starch polysaccharide, the presence of large amounts of xylan in the feed, food, and paper industries can have several adverse effects: In feed, xylan increases digesta viscosity, producing significant anti-nutritional effects, hindering the digestion and absorption of nutrients in the animal intestines, and restricting livestock growth performance; in food processing, excessively high xylan content in dough deteriorates dough processing characteristics, negatively impacting the quality of baked goods; in the paper industry, xylan in raw materials increases pulp viscosity, increases the consumption of chemical bleaching agents, and exacerbates the pollution load in production. Xylanase can specifically hydrolyze xylan, destroying its polysaccharide structure, and is a core industrial enzyme preparation for solving the above problems.

[0003] Xylanase is widely used in various fields such as feed, food, and pulp bleaching, with different applications placing varying demands on its performance. In the feed industry, enzyme preparations need to withstand the acidic environment of the animal's gastrointestinal tract and high-temperature granulation processes; in food baking, enzymes need to withstand temperature fluctuations; and pulp bleaching also requires enzymes with good temperature and pH tolerance. Wild-derived xylanases often suffer from insufficient heat resistance and poor acid stability, resulting in significant enzyme activity loss and decreased catalytic efficiency under actual working conditions, thus limiting their industrial application.

[0004] Relevant patent documents retrieved: The publication, published in China (CN122081282A) on May 26, 2026, discloses five mutant xylanases obtained by targeted amino acid site mutations in wild-type xylanase Xyn-WT. Compared with wild-type xylanase, the enzyme activities of all five mutants were significantly improved. Among them, the Xyn-4 mutant had the best enzyme activity, reaching 245.17 U / mL, which is 90.60% higher than that of wild-type. The expression level was increased by 20.74% after codon optimization, exhibiting both high expression level and excellent catalytic activity.

[0005] This document, published in China (CN120098970A) on May 29, 2026, discloses a xylanase mutant. The mutant is a single-point mutant based on the wild-type xylanase Xyn, exhibiting a generally increased specific activity of 12.4%-63.4%. Among them, the xylanase mutant containing the I128E single-point mutation has the highest specific activity, reaching 1273.9 U / mg, which is beneficial for reducing the production cost of this enzyme and promoting its widespread application in the feed industry.

[0006] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: In existing technologies, xylanase mutants are often obtained through protein molecule modification to improve enzyme properties. However, many of the resulting mutant enzymes have low catalytic activity, making it difficult to balance high enzyme activity with good environmental tolerance, and thus unable to adapt to the actual production conditions of multiple industries such as feed, food, and papermaking. Summary of the Invention

[0007] The purpose of this invention is to provide: A gene encoding a xylanase, its vector and application, and related technologies, to solve existing technical problems such as low enzyme catalytic activity and difficulty in balancing high enzyme activity with good environmental tolerance, or a combination thereof.

[0008] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0009] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0010] The definitions of standard chemical terms can be found in the references "Introduction to Enzymology and Enzyme Engineering" (Tsinghua University Press, 2021-08-01) and "Enzyme Engineering" (Science Press, 2024-01-01, Edition 5).

[0011] Unless otherwise specified, conventional methods within the scope of the art, such as enzyme digestion and electroporation, shall be used.

[0012] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0013] The term "codon bias" used in this article refers to the process of performing synonymous substitutions on the original gene sequence based on the distribution of frequently used synonymous codons in the Aspergillus niger genome to improve translation efficiency.

[0014] The term "codon optimization" used in this article refers to adjusting the gene sequence to conform to the codon preferences of the host cell, without changing the coding amino acid sequence.

[0015] The term "whole-genome synthesis" as used in this article refers to the de novo construction of a target gene through chemical synthesis methods.

[0016] As used herein, the term "vector" refers to a vector that can autonomously replicate within a host cell, preferably a multi-copy vector. Furthermore, vectors typically possess markers such as antibiotic resistance genes for selecting transformants. Additionally, vectors may have promoters and / or terminators for expressing the introduced gene. Vectors can be, for example, viral vectors, vectors derived from bacterial plasmids, vectors derived from yeast plasmids, vectors derived from bacteriophages, granules, phage particles, etc. Genetically engineered vectors are vectors that enable the expression of a target gene in cells, and are typically linear or circular DNA molecules comprising polynucleotides encoding proteins or polypeptides and operatively linked to an expression control sequence.

[0017] The term "enzyme" as used in this article refers to a class of biological macromolecules in living organisms that possess catalytic activity and a specific spatial conformation. Enzymes are generally proteins or ribonucleic acids produced by living cells that have high specificity and high catalytic efficiency towards their substrates. Their core function is to lower the activation energy of chemical reactions, accelerate metabolic reactions in living organisms, and are not consumed before or after the reaction. Their catalytic activity depends on their specific spatial structure and can function under mild conditions.

[0018] In a first aspect, the present invention provides a gene encoding xylanase, the nucleotide sequence of which is shown in SEQ ID NO:3.

[0019] SEQ ID NO:3: .

[0020] According to some embodiments of the present invention, the gene encoding the xylanase may have at least 90% sequence identity with the sequence shown in SEQ ID NO:3.

[0021] Specifically, the sequence of the gene encoding xylanase has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, and 99.9% sequence identity with the sequence shown in SEQ ID NO:3.

[0022] In a second aspect, the present invention provides a xylanase encoded by the aforementioned coding gene, wherein the amino acid sequence of the xylanase is shown in SEQ ID NO:4.

[0023] SEQ ID NO:4: SFRSSASHSGQSVKVTGNKVGTIGGWGYELWADSGNNSATFYSDGSSFSCTFQNAGDYLCRSGLSFDSTKTPSQIGRMKADFKLVKQNSSNVGYSYVGVYGWTRSPLVEYYIV DNWLSPFPGDWVGNKKFGSFTIDGAQYTVYENTRTGPSIDGDTTFNQYFSIRQQARDCGTIDISAHFDQWEKLGMTMGKLHEAKVLGEAGNNVNGGASGTADFPYAKVYIG.

[0024] Thirdly, the present invention provides a recombinant vector comprising the encoding gene of the aforementioned xylanase.

[0025] Specifically, the carrier is selected from any one or more of pAN7-1 and pPHT.

[0026] Preferably, the carrier is pAN7-1.

[0027] According to some embodiments of the present invention, the present invention provides a method for preparing a recombinant vector, comprising: digesting the encoding gene with an enzyme, ligating it with the enzyme-digested expression vector, and obtaining a recombinant plasmid containing a gene encoding xylanase.

[0028] According to some embodiments of the present invention, the enzymatic digestion is performed using... Hind III and Nar I. Double enzyme digestion.

[0029] Fourthly, the present invention provides a host cell comprising the above-described recombinant vector.

[0030] Specifically, the host cell is a fungal host cell.

[0031] Furthermore, the fungal host cell is selected from any one of Aspergillus, Pichia pastoris, or Saccharomyces cerevisiae.

[0032] Furthermore, the Aspergillus host is Aspergillus niger.

[0033] Fifthly, the present invention provides a recombinant engineered bacterium, which transforms the aforementioned recombinant vector.

[0034] According to some embodiments of the present invention, the recombinant engineered bacteria is a recombinant Aspergillus niger engineered bacteria.

[0035] According to some embodiments of the present invention, the method for constructing the recombinant Aspergillus niger engineered strain includes transferring the recombinant plasmid into Aspergillus niger, thereby obtaining the strain.

[0036] In a sixth aspect, the present invention provides a method for the fermentation production of xylanase, wherein the above-mentioned recombinant engineered bacteria are inoculated, and after seed culture, fermentation culture is carried out in a fermenter, and the fermentation broth is collected.

[0037] According to some embodiments of the present invention, the culture medium for seed culture is 6% sucrose, 3.5% soybean meal, 2.5% corn steep liquor, and 0.12% magnesium sulfate.

[0038] According to some embodiments of the present invention, the seed culture conditions are as follows: the inoculum is inoculated into a culture medium and cultured at 32°C and 250-280 rpm for 72 hours, and then transferred to a secondary seed bottle and cultured at 32°C and 250-280 rpm for 48 hours.

[0039] According to some embodiments of the present invention, the culture medium for fermentation is: glucose (digested) 2.5%; corn steep liquor 4.0%; soybean meal 2.8%; ammonium sulfate 1.2%; disodium hydrogen phosphate 0.15%; potassium dihydrogen phosphate 0.38%; magnesium sulfate 0.36%; potassium sulfate 0.39%; citric acid 0.13%; calcium chloride 0.03%; zinc sulfate 0.006%; ferrous sulfate 0.004%.

[0040] According to some embodiments of the present invention, the culture medium in the feed tank for fermentation culture is 63% glucose (distilled); 1.2% ammonium sulfate; 4.3% corn steep liquor; 0.63% disodium hydrogen phosphate; 0.42% potassium dihydrogen phosphate; and the pH is adjusted to 4.0 with phosphoric acid.

[0041] In a seventh aspect, the present invention provides the application of the above-mentioned coding gene, the above-mentioned xylanase, the above-mentioned recombinant vector, and the above-mentioned recombinant Aspergillus niger engineered strain in the preparation of feed, food processing, and papermaking.

[0042] Specifically, when applied in the feed industry, it can catalyze the degradation reaction of xylan, effectively reduce the viscosity of the feed system, alleviate the anti-nutritional effects caused by high viscosity, improve the digestion and absorption efficiency of nutrients by animals, and thus promote animal growth.

[0043] Specifically, when applied to food processing, it can improve the stability and extensibility of dough systems; when used to process bread and other baked goods, the product volume increases, the taste is improved, and the shelf life of baked goods can be extended.

[0044] Specifically, when applied to the paper industry, it can decompose the xylan components in papermaking raw materials, reduce pulp viscosity, and improve paper strength and surface smoothness.

[0045] The present invention has at least the following beneficial effects: 1. Compared with the prior art, the present invention has better technical effects in terms of heat resistance and acid resistance.

[0046] According to experimental tests, the present invention increases enzyme activity from 57% in the prior art to over 88% after treatment with pH 3.0 for 30 minutes.

[0047] According to experimental tests, the present invention increases enzyme activity from 66% in the prior art to over 95% after 80℃ water bath treatment for 90 seconds.

[0048] (1) In this invention, the xylanase gene is optimized according to the codon preference of Aspergillus niger, and a single-point mutation of C→R at position 3 is introduced. The modified gene is obtained in one go through whole-gene synthesis, which significantly improves the expression level. The fermentation activity of xylanase is increased from 93476.67 U / mL of the original strain to 143856.33 U / mL, and the fermentation efficiency is increased by 53.9%, which is higher than the existing fermentation level. (2) The C→R mutation at position 3 significantly improves the thermal stability of xylanase. After treatment in an 80℃ water bath for 90s, the enzyme activity retention rate is as high as 95%, which can meet the requirements of high-temperature pelleting of feed and has good temperature resistance. (3) The C→R mutation at position 3 significantly improved the acid resistance of xylanase. After treatment at pH 3.0 for 30 min, the enzyme activity retention rate was as high as 87%, which showed strong resistance to gastric acid. (4) Using Aspergillus niger as the expression host, the engineered bacteria are genetically stable and the fermentation process is mature, which is conducive to industrial-scale production. Attached Figure Description

[0049] Figure 1 The comparison results are for SEQ ID NO.1 and SEQ ID NO.3.

[0050] Figure 2 The comparison results are for SEQ ID NO.2 and SEQ ID NO.4.

[0051] Figure 3 This is a PCR electrophoresis image of the xylanase gene from Aspergillus niger; where M is the DL2000 DNA Marker, and numbers 1 and 2 are xylanase gene amplification bands using XylT-up / XylT-dn as primers.

[0052] Figure 4 This is the pH curve for xylanase.

[0053] Figure 5 This is the temperature curve of xylanase.

[0054] Figure 6 The results are for evaluating xylanase in a water bath at 80℃ for 90 seconds. Detailed Implementation

[0055] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0056] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0057] Example 1: Optimization of xylanase gene and construction of recombinant expression vector 1. Optimization and synthesis of xylanase gene The original xylanase gene was derived from a Pichia pastoris strain that already expressed xylanase in the laboratory. Its nucleotide sequence is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2. Under the premise of fine-tuning the encoded amino acid sequence (changing only one amino acid), and following the codon preference of *Aspergillus niger*, the xylanase gene was codon optimized, and the complete xylanase gene sequence XylT was synthesized using a whole-gene synthesis method. The nucleotide sequence of the artificially synthesized xylanase gene XylT is shown in SEQ ID NO.3, and the encoded amino acid sequence is shown in SEQ ID NO.4. The synthesized XylT gene also has nucleotides at both ends. Hind III and Nar I restriction enzyme sites were provided to facilitate ligation with the expression vector. The xylanase XylT gene was amplified using the XylT gene sequence as a template. The primers used were: XylT-up: 5'-CCC AAGCTT AGCTTTCGCAGCTCCGCCAGCCACTCTGGC-3' (SEQ ID NO.5); XylT-dn: 5'-CCC GGCGCC CCCGATGTAGACCTTAGCGTAAGGAAAGTC-3' (SEQ ID NO. 6); The underlined parts are respectively Hind III and Nar I restriction site.

[0058] The amino acid sequence of xylanase from Pichia pastoris differs from that of Aspergillus niger from only one of the 223 amino acids in their amino acid sequences. Specifically, the third amino acid is different: C-cysteine ​​in Pichia pastoris and R-arginine in Aspergillus niger.

[0059] Introducing arginine at specific sites on the protein surface can enhance intramolecular hydrogen bonds or ionic interactions and improve thermal stability; if the mutated cysteine ​​is close to the active site, replacing it with a positively charged arginine may directly affect the binding of the enzyme to the substrate and improve enzyme activity.

[0060] 1.1 The sequencing results of the yeast xylanase gene are as follows (669bp). SEQ ID NO.1: .

[0061] 1.2 Yeast amino acid sequence (5'-3' sequence) SEQ ID NO.2: SFCSSASHSGQSVKVTGNKVGTIGGWGYELWADSGNNSATFYSDGSSFSCTFQNAGDYLCRSGLSFDSTKTPSQIGRMKADFKLVKQNSSNVGYSYVGVYGWTRSPLVEYYIV DNWLSPFPGDWVGNKKFGSFTIDGAQYTVYENTRTGPSIDGDTTFNQYFSIRQQARDCGTIDISAHFDQWEKLGMTMGKLHEAKVLGEAGNNVNGGASGTADFPYAKVYIG.

[0062] 1.3 The sequencing results of the xylanase gene of Aspergillus niger are as follows (669bp): SEQ ID NO.3: AGCTTTCGCAGCTCCGCCAGCCACTCTGGCCAGAGCGTTAAGGTTACCGGCAACAAGGTCGGCACCATCGGCGGCTGGGGTTACGAGCTGTGGGCCGACAGCGGCAACAACTCCGCGACCTTCTATTCTGACGGCTCCTTCTCCTGTACTTTCCAGAACGCCGGCGACTACCTCTGCCGCTCCGGCCTCTCCTTCGATTCCACAAAGACCCCCTCCCAGATCGGCCGCATGAAGGCCGATTTCAAGCTGGTCAAGCAAAACAGCTCGAACGTGGGTTACTCCTACGTCGGTGTCTATGGCTGGACGAGGTCCCCTTTGGTCGAGTATTACATCGTCGATAACTGGTTGTCCCCCTTCCCCCCTGGTGACTGGGTCGGTAACAAGAAGTTCGGCTCCTTTACCATCGACGGTGCCCAATATACCGTTTACGAGAACACTCGTACGGGTCCAAGCATTGATGGTGACACCACCTTCAACCAGTACTTCTCGATCCGCCAGCAGGCCAGGGATTGTGGCACCATCGATATCTCCGCCCACTTCGACCAGTGGGAGAAGCTCGGAATGACCATGGGCAAGCTCCACGAGGCCAAGGTGCTCGGCGAAGCCGGCAACGTCAACGGCGGCGCCTCGGGCACCGCCGACTTTCCTTACGCTAAGGTCTACATCGGG。

[0063] 1.4 Amino acid sequence of Aspergillus niger SEQ ID NO.4: SFRSSASHSGQSVKVTGNKVGTIGGWGYELWADSGNNSATFYSDGSFSCTFQNAGDYLCRSGLSFDSTKTPSQIGRMKADFKLVKQNSSNVGYSYVGVYGWTRSPLVEYYIVDNWLSPFPPGDWVGNKKFGSFTIDGAQYTVYENTRTGPSIDGDTTFNQYFSIRQQARDCGTIDISAHFDQWEKLGMTMGKLHEAKVLGEAGNVNGGASGTADFPYAKVYIG。

[0064] 1.5 Sequence Comparison Results Alignment results of SEQ ID NO.1 and SEQ ID NO.3 (see) Figure 1 ), SEQ ID NO.1 is Figure 1 In the query, SEQ ID NO.3 is Figure 1 Sbjct in.

[0065] The alignment results of SEQ ID NO.2 and SEQ ID NO.4 (see) Figure 2 ), SEQ ID NO.2 is Figure 2 In the query, SEQ ID NO.4 is Figure 2 Sbjct in.

[0066] 2. Construction of recombinant expression vectors The XylT gene was used Hind III and Nar After double digestion with enzyme I, and Hind III and Nar The expression vector pAN7-1, digested with enzyme I, was ligated to obtain the recombinant plasmid pAN7-1-XylT containing the xylanase gene. The recombinant plasmid pAN7-1-XylT was transformed into *E. coli* competent cells JM109, and positive clones were screened by LB agar culture. Plasmids from positive clones were extracted and sent to BGI Genomics for sequencing. Sequencing results showed that the xylanase gene XylT is 669 bp in length and encodes 223 amino acids.

[0067] Example 2: Obtaining and Identifying Xylanase-Enhanced Bacteria The recombinant plasmid pAN7-1-XylT was transformed into Aspergillus niger using conventional electroporation. Aspergillus niger The transformed individuals were screened, and their genomes were extracted using a fungal genomic DNA extraction kit. PCR amplification was performed using XylT-up / XylT-dn primers. After the reaction, the PCR amplification products were identified by 1% agarose gel electrophoresis. Figure 3 The PCR product was extracted and purified by gel extraction, and sent to BGI for sequencing to confirm the sequence was correct and free of mutations. The recombinant strain was stored at -70℃ for later use.

[0068] Example 3: High-efficiency expression of xylanase recombinant strains 1. Seed liquid preparation Culture medium: 6% sucrose, 3.5% soybean meal, 2.5% corn steep liquor, 0.12% magnesium sulfate, adjust pH to around 6.0, autoclave at 121℃ for 30 min.

[0069] Culture conditions: Inoculate the inoculum into 50mL primary seed culture flasks and culture at 32℃ and 250-280rpm for 72 hours, then transfer to 200mL secondary seed bottles (4-5 bottles) and culture at 32℃ and 250-280rpm for 48 hours.

[0070] 2. 15L fermenter cultivation (feed volume 9L) Fermentation tank culture medium: glucose (digested) 2.5%; corn steep liquor 4.0%; soybean meal 2.8%; ammonium sulfate 1.2%; disodium hydrogen phosphate 0.15%; potassium dihydrogen phosphate 0.38%; magnesium sulfate 0.36%; potassium sulfate 0.39%; citric acid 0.13%; calcium chloride 0.03%; zinc sulfate 0.006%; ferrous sulfate 0.004%.

[0071] Culture medium for feeder: 63% glucose (digested); 1.2% ammonium sulfate; 4.3% corn steep liquor; 0.63% disodium hydrogen phosphate; 0.42% potassium dihydrogen phosphate; pH adjusted to 4.0 with phosphoric acid.

[0072] Sterilization conditions: 121℃, 30 min, adjust pH to 4.5 before transfer, and cool to 34℃.

[0073] Culture conditions: Inoculum size 10%, culture temperature 32℃, aeration ratio maintained at 1:1.5 throughout fermentation, and stirring speed 140 rpm. Feeding was initiated when the reducing sugar content in the fermentation broth fell below 0.1% (w / w). During fermentation, online monitoring of temperature, pH, dissolved oxygen, tank pressure, aeration rate, and stirring speed was required to ensure environmental stability. Samples were taken every 12 hours during fermentation to detect xylanase activity and Aspergillus niger concentration (OD600).

[0074] Under the same fermentation conditions, after 120 hours of cultivation, the xylanase activity of the *Pichia pastoris* strain containing the original xylanase gene was 93476.67 U / mL, while that of the *Aspergillus niger* genetically engineered strain was 143856.33 U / mL. This indicates that the xylanase activity of the *Aspergillus niger* genetically engineered strain (strain 2) is 1.54 times that of the *Pichia pastoris* strain (strain 1) compared to the *Pichia pastoris* strain containing the original xylanase gene.

[0075] The construction method of strain 2 is described in Example 2; the construction method of strain 1 is as follows: strain 1 is based on a laboratory-preserved strain, specifically a Pichia pastoris genetically engineered strain, which uses the pPICZα A plasmid. A recombinant plasmid was constructed by ligating the xylanase gene (SEQ ID NO.1) to the pPICZα A plasmid, and then transformed into Pichia pastoris GS115 by electroporation. The resulting transformants were then screened to obtain strain 1.

[0076] Table 1

[0077] Example 4: Determination of xylanase activity and evaluation of enzymatic properties of recombinant strains (1) Method for detecting xylanase activity Xylanase activity was determined according to GB / T 23874-2009 "Determination of Xylanase Activity in Feed Additives - Spectrophotometric Method".

[0078] (2) Xylanase pH activity curve 0.1M citric acid solution and 0.2M disodium hydrogen phosphate solution were mixed at a certain volume ratio to achieve pH values ​​of 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, and 7. Xylanase activity was measured under different pH conditions. The results showed that ( Figure 4 (See Table 2) The optimal pH for the recombinant strain xylanase is 5.0–6.0. ​​Within the pH range of 4.0–7.5, the xylanase activity retention rate of the recombinant strain is above 60%, indicating that the xylanase of the recombinant strain has a broad pH range of action.

[0079] Table 2. Xylanase activity values ​​detected at different pH values

[0080] (3) Xylanase temperature activity curve The reaction temperatures in the enzyme activity assay were set to 30℃, 37℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, and 80℃, respectively. Xylanase activity was measured under different reaction temperature conditions. The results showed that ( Figure 5 (As shown in Table 3) The optimal temperature for the recombinant strain xylanase is 65℃.

[0081] Table 3. Xylanase activity values ​​detected at different temperatures

[0082] (4) Evaluation of the heat resistance of xylanase The enzyme solution to be tested was diluted to approximately 20 U / mL with acetate-sodium acetate buffer at pH 5.5. After treatment at 80℃ for 90 s, the residual enzyme activity was measured. The enzyme activity of the untreated sample was taken as 100%, and the relative enzyme activity was calculated. The results showed that the enzyme activity retention rate of xylanase after treatment at 80℃ for 90 s was 75%. Figure 6 (See Table 4). After treatment in an 80℃ water bath for 90 seconds, the recombinant strain of xylanase retained 95% of its enzyme activity (e.g., ...). Figure 6(See Table 5). This indicates that the thermostability of the xylanase expressed by the recombinant strain is significantly improved.

[0083] Table 4. Evaluation of the heat resistance of xylanase in the original strain

[0084] Table 5. Evaluation of the heat resistance of xylanase in recombinant strains

[0085] (5) Evaluation of xylanase acid resistance The enzyme solutions to be tested were diluted 20-fold with citrate-disodium hydrogen phosphate solution (pH 3.0) and placed in a 40℃ thermostatic water bath for 30 min. After being removed from the water bath, the enzyme solutions were immediately diluted appropriately with acetate-sodium acetate buffer (pH 5.5). The enzyme activity after acid-resistant treatment was measured, with the enzyme activity of the untreated sample taken as 100%, and the relative enzyme activity was calculated. The results showed that the xylanase of the recombinant strain retained 87% of its activity after 30 min treatment at pH 3.0, indicating that the xylanase of the recombinant strain has strong acid resistance.

[0086] Table 6. Evaluation of Xylanase Acid Resistance

[0087] Comparative analysis of the enzymatic properties of xylanases obtained using other mutant forms in Comparative Example 1 and the xylanase of this invention. This comparative study used similar heat-resistant xylanase products on the market (products from Tianjin Bofeide Technology Co., Ltd.) and the xylanase sample described in this invention (same as the test sample in Example 4) as the research objects. Their heat resistance and acid resistance were examined. The specific test methods and parameter settings for heat resistance were the same as in Example 4 (4) xylanase heat resistance assessment, examining the enzyme activity retention rate after "80℃ water bath treatment for 90s"; the specific test methods and parameter settings for acid resistance were the same as in Example 4 (5) xylanase acid resistance assessment, examining the enzyme activity retention rate after "pH 3.0 treatment for 30min". The specific results are as follows: Table 7 Comparative analysis of the enzymatic properties of existing xylanases and the xylanase of this invention

[0088] The results above show that the xylanase of the present invention has better temperature resistance and acid resistance than existing xylanase samples on the market.

[0089] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A gene encoding a xylanase, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO:

3.

2. The xylanase encoded by the gene according to claim 1, characterized in that, The amino acid sequence of the xylanase is shown in SEQ ID NO:

4.

3. A recombinant vector comprising the gene encoding the xylanase as described in claim 1.

4. The recombinant vector according to claim 3, characterized in that, The vector is selected from any one or more of pAN7-1 and pPHT.

5. The method for preparing the recombinant vector according to any one of claims 3-4, characterized in that, include: The encoding gene is digested with enzymes and ligated with the digested expression vector to obtain a recombinant plasmid containing the gene encoding xylanase.

6. A host cell, characterized in that, It includes the recombinant vector according to any one of claims 3-4.

7. A recombinant engineered bacterium, characterized in that, The recombinant engineered bacteria are used to transform the recombinant vector according to any one of claims 3-4.

8. The method for constructing recombinant engineered bacteria according to claim 7, characterized in that, This involves transferring the recombinant vector into Aspergillus niger, which yields the product.

9. A fermentation method for producing xylanase, characterized in that, The recombinant engineered bacteria described in claim 7 are inoculated, and after seed culture, they are fermented in a fermenter, and the fermentation broth is collected.

10. The application of the encoding gene of claim 1, the xylanase of claim 2, the recombinant vector of any one of claims 3-4, the host cell of claim 6, and the recombinant engineered bacteria of claim 7 in the preparation of feed, food processing, and papermaking.

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