Xylanase mutants with thermostability

Abstract

The present application relates to a kind of xylanase Xyn10 mutant and its application.The modified xylanase mutant Xyn10 has better thermal stability, and exhibits excellent enzymatic properties, and can be applied in the process of hydrolyzing xylan to release xylo-oligosaccharide and xylose.The obtained xylanase mutant Xyn10 gene can be cloned into suitable host to realize heterologous expression, realize industrial production of xylanase mutant Xyn10, and provide low-cost xylanase starting material for subsequent industrial application.Xylanase mutant Xyn10 can show important economic and social value in xylo-oligosaccharide production.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a thermostable xylanase mutant and its applications. Background Technology

[0002] Hemicellulose is a highly abundant biomass found in plants, with a wide range of sources. It is a mixture of polysaccharides composed of xylan, xyloglucan, and galactoglucomannan. Xylan is the main component of hemicellulose and is a complex polypentose sugar. The xylan content varies among different plants and even within the same plant at different growth stages. Xylooligosaccharides are the most important products of xylan degradation, possessing various beneficial biological effects and serving as important functional substances. Xylooligosaccharides can inhibit inflammation and regulate the gut microbiota, have antioxidant properties, enhance the expression of immune-related genes in the gut of fish, promote the proliferation of beneficial bacteria (Bifidobacteria) in the human gut to aid digestion and absorption, regulate cellular immunity, and have anti-tumor effects. How to efficiently obtain xylooligosaccharides has become a hot research topic.

[0003] Traditional methods for preparing xylooligosaccharides include hot water extraction, acid hydrolysis, and alkaline hydrolysis. These methods often suffer from high energy consumption, significant environmental impact, complex processes, and demanding equipment requirements. To overcome these shortcomings, enzymatic hydrolysis has emerged. Under suitable conditions, biological enzymes hydrolyze xylan into xylooligosaccharides and other sugars. Current major enzymatic hydrolysis technologies include complex enzymatic hydrolysis and immobilized enzyme hydrolysis. Complex enzymatic hydrolysis, being a green and environmentally friendly process, is currently the mainstream method and a hot research topic. Xylanase is the most important functional enzyme for hydrolyzing xylan.

[0004] Xylanases are a class of enzymes that hydrolyze xylan to release xylooligosaccharides and xylose. They include endo-β-1,4-D-xylanases, exo-β-1,4-D-xylanases, and β-xylosidases. Based on the similarity of the amino acid sequences of their catalytic domains, most xylanases belong to the GH10 and GH11 families of glycoside hydrolases. In recent years, some xylanases belonging to other families, such as GH5, GH30, and GH43, have also been discovered. Current research on xylanases mainly focuses on the GH10 and GH11 families. The GH11 family has a simpler structure, lower molecular weight (usually not exceeding 20 kDa), and its molecular structure consists only of β-sheets, containing a single catalytic domain and possessing only xylanase activity. Xylanases of the GH10 family generally have larger molecular weights than those of the GH11 family, and their molecular structures are more complex, consisting of both α-helices and β-sheets. The overall structure is a single-layered bowl-shaped structure, with the inner part being a bowl formed by β-sheets and the outer part by α-helices. Compared to GH11 family xylanases, the GH10 family has more than one catalytic domain, potentially possessing other catalytic activities besides xylanase activity. Furthermore, due to its more complex structure, GH10 family xylanases are more structurally stable and exhibit better thermal stability. Current research focuses on identifying xylanases with high activity, modifying enzymes to improve activity or stability, enzyme immobilization, and the efficient enzymatic hydrolysis of substrates by complex enzymes. In the modification of xylanases, Harit Boonyaputthikul increased the activity of the GH11 family xylanase Xyn11A by adding disulfide bonds through mutation; Li, Yangyang modified the flexible region of the GH11 family xylanase XynA through molecular dynamics simulation by mutating some amino acids, significantly enhancing the enzyme's thermostability; Li Zhihong modified the GH11 family xylanases using both semi-rational gene design and computer-aided techniques, improving their thermostability. Among the currently discovered xylanases, the GH11 family xylanases have higher enzyme activities than the GH10 family xylanases, but some GH10 xylanases with high activity have also been discovered. For example, one GH10 family xylanase was discovered in Usami trichomoniasis, with an exogenous expression activity of 6267 U / mg in Pichia pastoris, which increased to 8870 U / mg after modification. However, the modified xylanase has a heat inactivation half-life of only 72 minutes, which is insufficient to meet the requirements of industrial production.

[0005] Therefore, it is necessary to search for enzyme mutants with higher thermal stability based on existing technologies. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a xylanase mutant with higher thermal stability, which can be used to hydrolyze xylan to release xylooligosaccharides and xylose.

[0007] The first aspect of this invention is to provide a xylanase mutant Xyn10 with higher thermal stability, the sequence of which is shown in SEQ ID NO: 1:

[0008] QASVSIDTKFKAHGKKYLGNIGDQYRLTTGKNAAIIKADFGALTPENSMKWDATEPSRGQFSFSGSDYLVNFAQSNNKLIRGHTLVWHSQLPSWVQSITDKNTLIEVMKNHITTVMQHYKGKIYAWDVVNEIFNEDGSLRDSVFYKVIGEDYVRIAFETARAA DPNAKLYINDYNLDSASYPKLTGMVSHVKKWIAAGIPIDGIGSQTHLSAGGGAGISGALNALAGAGTKEIAVTELDIAGASSTDYVEVVEACLNQPKCIGITVWGVADPDSWRSSSTPLLFDGNYNPKPAYTAIANALSGSGTTTTTTTSTTTGGTDPT(SEQ ID NO:1)

[0009] The xylanase mutant Xyn10 provided by this invention is obtained by adding 22 amino acids to the C-terminus of wild-type xylanase. Studies have shown that the modified xylanase exhibits improved tolerance to temperature and pH, particularly in terms of thermal stability.

[0010] Amino acid sequence analysis revealed that the catalytic center of the xylanase mutant Xyn10 is a barrel-shaped structure formed by β-sheets in a three-dimensional structure, with glutamate (E) at positions 131 and 237 serving as catalytic sites. Without affecting the activity of the xylanase mutant Xyn10, various substitutions, additions, and / or deletions of one or more amino acids at positions away from the catalytic center (preferably away from glutamate (E) at positions 131 and 237) as shown in SEQ ID NO:1 can be made to obtain derived proteins containing the xylanase mutant Xyn10. According to common knowledge in the art, the biological activity of a protein is closely related to its functional domains. Generally, only site mutations occurring in functional domains can affect the two-dimensional and three-dimensional structure of a protein, thereby affecting its biological activity. For amino acid sites located far from the functional domain (preferably at glutamate positions 131 and 237), since this region does not participate in protein functional conformation, individual point mutations in amino acids will not have a substantial impact on the protein's biological activity, thus essentially preserving the original protein's biological function. The preferred xylanase mutant Xyn10 has at least 90% homology to the amino acid sequence shown in SEQ ID NO:1, more preferably at least 95% homology, and most preferably at least 99% homology.

[0011] Similarly, the present invention also provides a gene sequence encoding the amino acid sequence shown in SEQ ID NO: 1, which is consistent with the nucleotide sequence shown in SEQ ID NO: 2.

[0012] caggcttcagtgagtattgataccaaattcaaggctcacgggaagaaatatcttggaaacattggtgatcagtaccggctgacgaccggcaaga

[0013] atgcggccattatcaaggccgattttggcgcgttgactccagagaacagcatgaagtggggatgctactgaacccagccgtggacagttctctttct

[0014] caggatcggactacctggtcaactttgcccagtctaacaacaagctgatccgcggacatactctcgtgtggcactcgcagctcccctcctgggtc

[0015] caatccatcacggacaagaatacactgatcgaagtcatgaagaatcacatcaccacagtgatgcaacactataagggcaagatttatgcctggg

[0016] atgttgtcaatgaaatcttcaacgaagacggctccctacgcgacagcgtcttttacaaggtcatcggcgaggactacgtgcggatcgccttcgag

[0017] actgctcgggctgcagatcccaatgcaaagctctacatcaatgattacaacctggattccgcctcctaccctaaattgaccggcatggttagccat

[0018] gtcaagaagtggatcgcagctggcatccctatcgatggaatcggttcccaaacccacttgagcgctggtggaggtgctggaatttctggagctct

[0019] caatgctctcgcaggtgccggcaccaaggagattgctgtcaccgagcttgacatcgctggcgccagctcgaccgactacgtggaggtcgtcga

[0020] agcctgcctgaaccagcccaagtgtatcggtatcaccgtttggggagttgctgacccggattcctggcgctccagctccactcctctgctgttcga

[0021] cggcaactacaacccgaagcctgcatacactgctatcgcaaatgctctcagcggctccggcaccacaacgaccactactactacttctactacgacaggaggtacggaccctact(SEQ ID NO:2)

[0022] This invention also provides a mutant gene encoding a protein that substantially retains the biological activity of the xylanase mutant Xyn10 by replacing, adding, and / or deleting one or more nucleotides in the nucleotide sequence shown in SEQ ID NO: 2, except for nucleotides 391-393 and 709-711. Preferably, the xylanase mutant Xyn10 gene has at least 90% homology to the nucleotide sequence shown in SEQ ID NO: 2, more preferably at least 95% homology, and most preferably at least 99% homology.

[0023] Using gene cloning technology, the cloned xylanase mutant Xyn10 gene sequence (SEQ ID NO:2) can be ligated into a suitable vector and transformed or transfected into a prokaryotic or eukaryotic host for expression to prepare recombinant xylanase mutant Xyn10 (SEQ ID NO:1). Suitable prokaryotic hosts include various bacteria such as E. coli, and suitable eukaryotic hosts include yeast and mammalian cells (such as Chinese hamster ovary cells), with yeast expression systems being preferred, and Pichia pastoris, Saccharomyces cerevisiae, and Saccharomyces hansenii being more preferred. The target protein can be present in the host cell or secreted from the host, preferably secreted from the host. The signal peptide used for secretion is preferably the yeast MFα signal peptide. The nucleic acid encoding the target protein can be inserted into the host chromosome or exist in the form of a free plasmid.

[0024] Suitable vectors are various commercially available prokaryotic or eukaryotic expression vectors well known to those skilled in the art, such as the pET series vectors and pQE series vectors; and eukaryotic expression vectors such as the pPIC series vectors and pGAPZ series vectors. A preferred example is the ligation of the xylanase mutant Xyn10 gene sequence (SEQ ID NO: 2) screened in this invention into the yeast expression vector pPIC9H, followed by transformation into yeast strain GS115, which induces the expression of the highly active xylanase mutant Xyn10. Transformation of the vector into host cells can be performed using conventional methods, such as electroporation and preparation of competent protoplasts. Successfully transformed cells, i.e., cells containing the DNA construct of this invention, can be identified using well-known techniques, such as cell collection and lysis, DNA extraction, and subsequent PCR identification.

[0025] A second aspect of this invention provides a method for preparing a xylanase mutant Xyn10 with higher thermal stability, comprising the following steps:

[0026] (a) A recombinant expression vector carrying the gene encoding the xylanase mutant Xyn10 was introduced into yeast host cells, and the recombinant host cells were cultured under conditions conducive to the production of the target protein; and

[0027] (b) The xylanase mutant Xyn10 was recovered.

[0028] In the preparation method of this invention, a host containing the DNA construct of this invention is cultured using methods known in the art, such as recombinant yeast to produce the xylanase mutant Xyn10 of this invention. Specific culture methods can include shake flasks or bioreactors, with bioreactors being preferred for production. The culture medium should provide the substances required for bacterial (or cell) growth and product expression, and should contain nitrogen sources, carbon sources, pH buffers, etc. The culture medium formulation should generally be obtained experimentally based on different culture subjects. The culture can be divided into two stages: the first stage is mainly used for bacterial (or cell) growth, and the second stage is mainly used for inducing the expression of the target product. The constructed genetically engineered bacteria are subjected to liquid culture and fermentation, induced by methanol. The methanol-induced culture medium is centrifuged, and the supernatant is collected.

[0029] The xylanase mutant Xyn10 can be isolated and purified using various protein separation methods, such as salting out, precipitation, ultrafiltration, liquid chromatography, and combinations thereof. Liquid chromatography can utilize techniques such as gel size exclusion, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, and reversed-phase chromatography.

[0030] In another embodiment, a second aspect of the present invention provides a method for preparing a xylanase mutant Xyn10 with higher thermal stability, comprising the following steps:

[0031] (a) A recombinant expression vector carrying the gene encoding the xylanase mutant Xyn10 was introduced into yeast host cells, and the recombinant yeast cells were cultured under conditions that would help produce the target protein.

[0032] (b) Collect the supernatant after centrifuging the yeast culture;

[0033] (c) The supernatant was purified by His affinity chromatography to prepare the xylanase mutant Xyn10.

[0034] In another embodiment, in step (c) of the method, the His affinity chromatography column purification process is as follows: first, the chromatography column is washed with a washbuffer containing 10-40 mmol / L imidazole, 100-400 mmol / L NaCl, 10-40 mmol / L Tris, and pH 7.3-9.0 until the chromatography resin is free of culture medium color; finally, elution is performed with a buffer containing 250-450 mmol / L imidazole, 100-400 mmol / L NaCl, 10-40 mmol / L Tris, and pH 7.3-9.0 to obtain the target protein xylanase mutant Xyn10. The purity of the protein is determined to be greater than 90%, more preferably greater than 95%.

[0035] In another embodiment, in step (c) of the method, the His affinity chromatography column purification process is as follows: first, the chromatography column is washed with a washbuffer containing 25-35 mmol / L imidazole, 150-350 mmol / L NaCl, 25-35 mmol / L Tris, and pH 8.0-9.0 until the chromatography resin is free of culture medium color; finally, elution is performed with a buffer containing 250-350 mmol / L imidazole, 150-350 mmol / L NaCl, 25-35 mmol / L Tris, and pH 8.0-9.0 to obtain the target protein xylanase mutant Xyn10. The purity of the protein is determined to be greater than 90%, more preferably greater than 95%.

[0036] In another embodiment, in step (c) of the method, the His affinity chromatography column purification process is as follows: first, the chromatography column is washed with a wash buffer containing 30 mmol / L imidazole, 300 mmol / L NaCl, 30 mmol / L Tris, and pH 8.5 until the chromatography resin is free of culture medium color; finally, elution is performed with a buffer containing 300 mmol / L imidazole, 300 mmol / L NaCl, 30 mmol / L Tris, and pH 8.5 to obtain the target protein xylanase mutant Xyn10. The purity of the protein is determined to be greater than 90%, more preferably greater than 95%.

[0037] A third aspect of the present invention is to provide the industrial application of the xylanase mutant Xyn10, which has higher thermal stability.

[0038] This invention also provides the industrial application of the xylanase mutant Xyn10, for example, it can be used to hydrolyze xylan to release xylooligosaccharides and xylose. Enzyme activity assays showed that comparing the enzymatic data of wild-type and mutant Xyn10 xylanase in the same graph, the mutant, with 22 amino acids added at the C-terminus, had the same optimal temperature and pH as the wild-type. At other temperatures and pH values, the relative enzyme activity increased to varying degrees. At pH 5.0 and 50°C, the relative enzyme activity of the mutant was higher than that of the wild-type xylanase at different incubation times. At the optimal temperature and pH, after a reaction time of 20 minutes, the mutant xylanase had an enzyme activity of 132 U / mg, slightly higher than the wild-type xylanase activity of 128 U / mg.

[0039] The xylanase mutant Xyn10 obtained in this invention exhibits better thermal stability and superior enzymatic properties, making it applicable to the hydrolysis of xylan to release xylooligosaccharides and the production of xylose. The obtained xylanase mutant Xyn10 gene can be cloned into a suitable host for heterologous expression, enabling the industrial production of xylanase mutant Xyn10 and providing a low-cost starting material for subsequent industrial applications. Xylanase mutant Xyn10 demonstrates significant economic and social value in xylooligosaccharide production. Attached Figure Description

[0040] Figure 1 Construction and sequencing comparison of wild-type and mutant xylanase gene vectors.

[0041] Figure 2 Expression of xylanase protein in wild type and mutant.

[0042] Figure 3 Wild-type and mutant xylanase protein affinity chromatography purification.

[0043] Figure 4 The relationship between wild-type xylanase activity and temperature.

[0044] Figure 5 The relationship between the activity of mutant xylanase and temperature.

[0045] Figure 6 The relationship between wild-type xylanase activity and pH.

[0046] Figure 7 The relationship between mutant xylanase activity and pH.

[0047] Figure 8 Thermostability of wild-type xylanase.

[0048] Figure 9 Thermostability of mutant xylanase.

[0049] Figure 10 : Fitting curve of Michaelis-Menten equation for xylanase.

[0050] Figure 11 Comparison of relative enzyme activities between wild type and mutant at different temperatures.

[0051] Figure 12 Comparison of relative enzyme activity between wild-type and mutant at different pH levels.

[0052] Figure 13 Comparison of relative enzyme activity after the same heat treatment. Detailed Implementation

[0053] Example 1: Construction and site-directed mutagenesis of recombinant Xyn10 vector

[0054] The xylanase gene Xyn10, derived from Aspergillus Usami, was synthesized by Beijing Qingke Biotechnology Co., Ltd. and constructed in the pET-28a vector. The gene was amplified using upstream primer CATGCCATGGATCAGGCTTCAGTGAGTATTGATACC (SEQ ID NO: 3) and downstream primer AAGGAAAAAAGCGGCCGCGAGAGCATTTGCGATAGCAGT (SEQ ID NO: 4). The vector and target fragment were then digested with NcoI and Not I restriction enzymes, and ligated into the pPIC9H vector plasmid. This plasmid was then transformed into DH5α competent cells. After sequencing verification, the correct recombinant plasmid was extracted and stored at -20℃ for further research.

[0055] The mutant sequence design is shown in SEQ ID NO: 1. It was constructed into the pET-28a vector through gene synthesis (SEQ ID NO: 2). An upstream primer (SEQ ID NO: 3) and a downstream primer AAGGAAAAAAGCGGCCGCAGTAGGGTCCGTACCTCCTGT (SEQ ID NO: 7) were designed, and the mutant was constructed into pPIC9H according to the wild-type gene vector construction method. The plasmid was extracted and stored at -20℃ for subsequent research.

[0056] The recombinant vector of the Xyn10 wild-type gene (SEQ ID NO: 5) and the mutant gene was successfully constructed on the Pichia pastoris expression vector pPIC9H. Sequencing results are as follows: Figure 1 As shown.

[0057] The wild-type and mutant genes were analyzed using software and websites such as DNAMAN 6.0 and Expasy (https: / / web.expasy.org / protparam / ), including physicochemical properties such as the number of encoded amino acids, molecular weight, isoelectric point, hydrophilicity, and hydrophobicity. The wild-type Xyn10, after removing the signal peptide, encodes 301 amino acids (SEQ ID NO: 6), with a molecular weight of 32.6 kDa and an isoelectric point of 6.63; its instability index is 26.86, far below 40, indicating a stable protein, and its hydrophilicity index is -0.203. The mutant encodes 323 amino acids (SEQ ID NO: 1), with a molecular weight of 34.6 kDa and an isoelectric point of 6.40; its instability index is 25.31, also indicating a stable protein, and its hydrophilicity index is -0.246, showing higher hydrophilicity than the wild-type. Based on the isoelectric point analysis, the protein can be purified using a buffer with a pH of 8.0-8.5.

[0058] The wild-type Xyn10 gene and amino acid sequence are shown below:

[0059] caggcttcagtgagtattgataccaaattcaaggctcacgggaagaaatatcttggaaacattggtgatcagtaccggctgacgaccggcaaga

[0060] atgcggccattatcaaggccgattttggcgcgttgactccagagaacagcatgaagtgggatgctactgaacccagccgtggacagttctctttct

[0061] caggatcggactacctggtcaactttgcccagtctaacaacaagctgatccgcggacatactctcgtgtggcactcgcagctcccctcctgggtc

[0062] caatccatcacggacaagaatacactgatcgaagtcatgaagaatcacatcaccacagtgatgcaacactataagggcaagatttatgcctggg

[0063] atgttgtcaatgaaatcttcaacgaagacggctccctacgcgacagcgtcttttacaaggtcatcggcgaggactacgtgcggatcgccttcgag

[0064] actgctcgggctgcagatcccaatgcaaagctctacatcaatgattacaacctggattccgcctcctaccctaaattgaccggcatggttagccat

[0065] gtcaagaagtggatcgcagctggcatccctatcgatggaatcggttcccaaacccacttgagcgctggtggaggtgctggaatttctggagctct

[0066] caatgctctcgcaggtgccggcaccaaggagattgctgtcaccgagcttgacatcgctggcgccagctcgaccgactacgtggaggtcgtcga

[0067] agcctgcctgaaccagcccaagtgtatcggtatcaccgtttggggagttgctgacccggattcctggcgctccagctccactcctctgctgttcgacggcaactacaacccgaagcctgcatacactgctatcgcaaatgctctc (SEQ ID NO: 5)

[0068] QASVSIDTKFKAHGKKYLGNIGDQYRLTTGKNAAIIKADFGALTPENSMKWDATEPSRGQF

[0069] SFSGSDYLVNFAQSNNKLIRGHTLVWHSQLPSWVQSITDKNTLIEVMKNHITTVMQHYKGK

[0070] IYAWDVVNEIFNEDGSLRDSVFYKVIGEDYVRIAFETARAADPNAKLYINDYNLDSASYPKL

[0071] TGMVSHVKKWIAAGIPIDGIGSQTHLSAGGGAGISGALNALAGAGTKEIAVTELDIAGASSTDYVEVVEACLNQPKCIGITVWGVADPDSWRSSSTPLLFDGNYNPKPAYTAIANAL (SEQ ID NO: 6)

[0072] Example 2 Wild-type and mutant protein expression

[0073] Wild-type and mutant plasmids were linearized using the restriction endonuclease salⅠ. Complete linearization was verified by electrophoresis, and the plasmids were then recovered using a kit. The recovered linearized plasmids were electroporated into GS115 competent cells. The electroporation parameters were set as follows: 1.5 kV, 6 ms. After electroporation, the plasmids were plated on MD medium. Single colonies were picked onto YPD plates containing 0.6 mg / mL G418 and cultured for 3-4 days. Larger colonies were then picked onto YPD plates containing 1.0 mg / mL G418 and cultured for 3-4 days. Larger plaques were then transferred to BMGY liquid medium and cultured at 30°C and 250 rpm until OD500. 600 Approximately 1.0-1.5, let stand for 3 hours, slowly pour out the supernatant, resuspend the precipitate in BMMY medium, and then... 600Adjust the concentration to around 0.5, maintain a temperature of 30℃ and a rotation speed of 250 rpm, and replenish methanol every 24 hours until the final methanol concentration in the culture medium reaches 1%. Take samples and analyze the changes in protein production using SDS-PAGE.

[0074] After linearization, the recombinant plasmid was electroporated into GS115 competent cells. High-cloning strains were then selected using G418 screening. Finally, SDS-PAGE confirmed that the induced protein expression time was 84-96 hours. The wild-type Xyn10 encodes a protein with a molecular weight of approximately 33 kDa, while the mutant reXyn10 encodes a protein with a molecular weight slightly greater than 43 kDa. Figure 2 The mutant protein was about 8 kDa larger than the predicted 34.6 kDa, possibly due to additional modifications caused by the 22 added amino acids in the yeast expression system.

[0075] Example 3: Purification of Wild-Type and Mutant Proteins

[0076] The pPIC9H vector is a secretory expression vector with a His tag. The protein expressed by strain GS115 is secreted in the culture medium and can be purified by His affinity chromatography (electrophoresis image shown). Figure 3 (As shown). After BMMY culture, the supernatant was collected at 4℃, 11000 rpm, and 5 min. The supernatant was poured into a His affinity chromatography column and refluxed 3-4 times. The column was washed with a wash buffer containing 30 mmol / L imidazole, 300 mmol / L NaCl, 30 mmol / L Tris, and pH 8.5 until the resin was free of culture medium color. Finally, the column was eluted with a buffer containing 300 mmol / L imidazole, 300 mmol / L NaCl, 30 mmol / L Tris, and pH 8.5. After elution, the protein purity was determined to be above 90% by NanoDrop. Finally, the column was flash-frozen in liquid nitrogen and stored at -80℃ for subsequent studies.

[0077] Example 4 Determination of Enzymatic Properties

[0078] 4.1 Preparation of the xylose standard curve

[0079] Take six clean test tubes and label them 0, 1, 2, 3, 4, and 5 respectively. Add 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of 1% xylose solution to each tube respectively. Then add pure water to bring the volume to 1.0 mL. Next, accurately add 2.0 mL of DNS reagent to each test tube, place them in boiling water for 10 minutes, cool to room temperature, and then dilute with 10 mL of pure water and shake well. Zero the tube with 0 and measure the absorbance at 540 nm. Plot the absorbance value on the ordinate and the xylose concentration (mg / mL) of each standard on the abscissa to obtain a standard curve.

[0080] 4.2 Determination of the optimal reaction temperature of xylanase

[0081] Prepare a 1% xylan solution with pH 5.5 as the substrate for the enzymatic hydrolysis reaction; dilute xylanase with a certain factor using disodium hydrogen phosphate-citric acid buffer (pH 5.5) to obtain the enzyme solution for the reaction; assay procedure: equilibrate the xylan solution at different temperatures (35℃-70℃) for 20 min, and equilibrate the diluted enzyme solution at different temperatures (35℃-70℃) for 10 min. Pipette 1 mL of the enzyme solution into a glass test tube, add 4 mL of DNS reagent, shake well, then add 1 mL of xylan solution, incubate at different temperatures for 20 min, then immediately boil in a water bath for 10 min, cool to room temperature with tap water, dilute with 20 mL of pure water, and shake well. Use this tube as a blank control; zero the test when measuring the Abs value. Take 1 mL of enzyme solution and add it to a test tube, then add 1 mL of xylan solution. Incubate at different temperatures for 20 min, then remove and add 4 mL of DNS reagent. Immediately boil in a water bath for 10 min, cool to room temperature, dilute with 20 mL of pure water and shake well. Measure the Abs value at 540 nm. Perform three replicates for each temperature sample and take the average value. Calculate the enzyme activity according to national standards, defining the highest enzyme activity as 100%. The ratio of enzyme activity at other temperatures to this enzyme activity is the relative enzyme activity, and plot a temperature-relative enzyme activity curve. 4.3 Determination of the optimal pH of xylanase

[0082] Prepare 1% xylan solutions with different pH values ​​(3.0-8.0) using disodium hydrogen phosphate-citrate buffer. Dilute the xylanase with these different pH (3.0-8.0) disodium hydrogen phosphate-citrate buffer solutions. Equilibrate the xylan solutions at the optimum temperature for 20 min, and then equilibrate the diluted enzyme solution at the same temperature for 10 min. Pipette 1 mL of the equilibrated enzyme solution into a glass test tube, add 4 mL of DNS reagent, shake well, then add 1 mL of the equilibrated xylan solution. Incubate at the optimum temperature for 20 min, then immediately remove and boil in a water bath for 10 min. Cool to room temperature with tap water, dilute with 20 mL of pure water, and shake well. Use this tube as a blank control; zero the test tube when measuring the Abs value. Pipette 1 mL of equilibrated enzyme solution into a glass test tube, then add 1 mL of equilibrated xylan solution. Incubate at the optimal temperature for 20 min, then remove and add 4 mL of DNS reagent. Immediately boil in a water bath for 10 min, cool to room temperature with tap water, dilute with 20 mL of pure water, and shake well. Measure the Abs value at 540 nm. Perform three replicates for each pH sample and take the average value. Calculate the enzyme activity, defining the highest enzyme activity as 100%. The ratio of enzyme activity at other pH values ​​to this highest enzyme activity is the relative enzyme activity, and plot a pH-relative enzyme activity curve.

[0083] 4.4 Determination of the thermal stability of xylanase

[0084] Based on the determination of the optimum temperature, the optimum temperature was selected for the determination of thermal stability. The determination procedure was as follows: The diluted enzyme solution was incubated at the optimum temperature for different times (0 min, 10 min, 20 min, 0.5 h, 1 h, 2 h, 4 h, 8 h), and then cooled in ice. The xylan solution was equilibrated at the optimum temperature for 20 min, and the diluted enzyme solution was equilibrated at the optimum temperature for 10 min. 1 mL of the equilibrated enzyme solution was added to a glass test tube, followed by 4 mL of DNS reagent, and the mixture was shaken well. Then, 1 mL of the equilibrated xylan solution was added, and the mixture was incubated at the optimum temperature for 20 min. Immediately after incubation, the mixture was placed in a boiling water bath for 10 min, cooled to room temperature with tap water, diluted with 20 mL of pure water, and shaken well. This tube served as a blank control, and the Abs value was zeroed using this tube. Pipette 1 mL of equilibrated enzyme solution into a glass test tube, then add 1 mL of equilibrated xylan solution. Incubate at the optimal temperature for 20 min, then remove and add 4 mL of DNS reagent. Immediately boil in a water bath for 10 min, cool to room temperature with tap water, dilute with 20 mL of pure water, and shake well. Measure the Abs value at 540 nm and calculate the enzyme activity. Perform three replicates for each experimental group and take the average value. The enzyme activity at 0 min of incubation is taken as 100%. The ratio of enzyme activity at other incubation times to enzyme activity at 0 min is the relative enzyme activity, and plot the incubation time-relative enzyme activity curve.

[0085] 4.5 Determination of xylanase activity

[0086] Based on the results of the optimal temperature and pH measurements, the enzyme activity and maximum reaction rate of xylanase, and other enzymatic reaction constants, were determined under the optimal temperature and pH conditions. Enzyme activity assay: The xylan solution was equilibrated at the optimal temperature for 20 min, and the diluted enzyme solution was equilibrated at the same temperature for 10 min. 1 mL of the equilibrated enzyme solution was added to a glass test tube, followed by 4 mL of DNS reagent. The mixture was shaken well, and then 1 mL of the equilibrated xylan solution was added. The tube was incubated at the optimal temperature for 20 min, then immediately placed in a boiling water bath for 10 min. It was then cooled to room temperature with tap water, diluted with 20 mL of pure water, and shaken well. This tube served as a blank control, and the Abs value was zeroed using this tube. Pipette 1 mL of equilibrated enzyme solution into a glass test tube, then add 1 mL of equilibrated xylan solution. Incubate at the optimal temperature for 20 min, then remove and add 4 mL of DNS reagent. Immediately boil in a water bath for 10 min, cool to room temperature with tap water, dilute with 20 mL of pure water, and shake well. Measure the Abs value at 540 nm. Perform three replicates for each pH sample and take the average value to calculate enzyme activity. Determination of enzyme-catalyzed reaction kinetic parameters: Prepare a series of xylan solutions of different concentrations as reaction substrates. Add a certain concentration of xylanase solution and measure the product production rate per unit time. Then input the measured rate and the corresponding substrate concentration into GraphPad Prism 8.5 software, select the Michaelis equation to fit the curve, and directly calculate the enzyme-catalyzed reaction kinetic parameters of the corresponding xylanase.

[0087] 4.6 Results

[0088] 4.6.1 Optimal reaction temperature of xylanase

[0089] Wild-type Xyn10 xylanase was tested, and the relative enzyme activity increased with increasing temperature within the range of 35℃-50℃, with 50℃ being the optimal temperature. Above 50℃, the relative enzyme activity decreased with increasing temperature, and the relative enzyme activity was very low at 70℃. The results are as follows. Figure 4 As shown, at pH below 5.0, the relative enzyme activity of wild-type Xyn10 xylanase increases with increasing pH; at pH above 5.0, the relative enzyme activity decreases with increasing pH; and at pH 7.5, enzyme activity is almost completely lost. The mutant Xyn10 xylanase, after testing, showed that the relative enzyme activity increased with increasing temperature between 35℃ and 50℃, with 50℃ being the optimal temperature. Above 55℃, the relative enzyme activity decreased rapidly, and at 70℃, enzyme activity was almost completely lost. The results are as follows. Figure 5 As shown;

[0090] 4.6.2 Optimal pH for xylanase reaction

[0091] At pH below 5.0, the relative activity of wild-type Xyn10 xylanase increases with increasing pH; above pH above 5.0, the relative activity decreases with increasing pH, and at pH 7.5, the enzyme activity is essentially lost. The optimal pH for wild-type xylanase is 5.0. Figure 6 As shown. At pH below 5.0, the relative enzyme activity of the mutant Xyn10 xylanase increases with increasing pH; at pH above 5.0, the relative enzyme activity decreases with increasing pH; and at pH 8.0, the enzyme activity is essentially lost. Figure 7 As shown.

[0092] 4.6.3 Thermostability of Xylanase

[0093] Under optimal conditions of 50℃ and 5.0, and after incubation for different durations, the thermostability of wild-type Xyn10 xylanase is as follows: Figure 8 As shown, the relative enzyme activity gradually decreased with increasing incubation time, reaching only 45.47% after 8 hours of incubation. The thermostability of the mutant Xyn10 xylanase at its optimal temperature of 50℃ and optimal pH of 5.0 for different incubation times is as follows: Figure 9 As shown, the relative enzyme activity gradually and slowly decreased with the increase of incubation time. After 8 hours of incubation, the relative enzyme activity was only 69.72%.

[0094] 4.6.4 Determination of xylanase activity and enzyme-catalyzed reaction constant

[0095] Wild-type Xyn10 xylanase, reacting for 20 min under optimal conditions, showed an enzyme activity of 128 U / mg. The mutant xylanase, reacting for 20 min at optimal temperature and pH, showed an enzyme activity of 132 U / mg, slightly higher than the wild-type xylanase activity of 128 U / mg. The kinetic parameters of the wild-type xylanase enzymatic reaction are as follows: Figure 10 As shown, Vmax is 0.2973 and Km is 52.94. The kinetic parameters of the mutant xylanase enzymatic reaction are as follows: Figure 10 As shown, Vmax is 0.4390 and Km is 45.59.

[0096] 4.7 Comparison of enzymatic properties between mutant and wild type

[0097] The enzymatic data of wild-type and mutant Xyn10 xylanase were compared in the same figure. From the following... Figure 11-13As can be seen, the mutant with 22 added amino acids at the C-terminus has the same optimal temperature and pH as the wild type, while the relative enzyme activity increases to varying degrees at other temperatures and pH values. At pH 5.0 and a temperature of 50℃, the relative enzyme activity of the mutant is higher than that of the wild-type xylanase at different incubation times. At the optimal temperature and pH, after a reaction time of 20 minutes, the mutant xylanase has an enzyme activity of 132 U / mg, slightly higher than the wild-type xylanase activity of 128 U / mg.

[0098] The experimental results above show that adding 22 amino acids to the C-terminus of the mutant xylanase had little effect on enzyme activity, only slightly increasing it. However, the modified xylanase showed some improvement in its tolerance to temperature and pH, especially in thermal stability. The modified Xyn10 had a molecular weight increase of approximately 10 kDa compared to the original, far exceeding the expected 2 kDa, possibly indicating additional modification during the expression and translation process in Pichia pastoris.

Claims

1. A xylanase mutant, Xyn10, with the amino acid sequence shown in Seq ID NO:

1.

2. The gene encoding the xylanase mutant Xyn10 of claim 1, the nucleotide sequence of which is shown in SEQ ID NO:

2.

3. A vector carrying the gene of claim 2.

4. The carrier according to claim 3, characterized in that: The vector is selected from pET series vectors, pQE series vectors, pPIC series vectors or pGAPZ series vectors.

5. A host cell obtained by transforming or transfecting a prokaryotic or eukaryotic host with the vector described in any one of claims 3-4.

6. The host cell according to claim 5, wherein it is a bacterial, yeast, or mammalian cell.

7. The host cell according to claim 6 is yeast, selected from Pichia pastoris, Saccharomyces cerevisiae or Hansenula polymorpha.

8. The application of the xylanase mutant Xyn10 according to claim 1 in the catalytic hydrolysis of xylan.

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