Alkaline xylanase and its multi-site mutants and applications

Abstract

The present invention discloses an alkaline xylanase and its multi-site mutants and applications. The present invention provides an alkaline xylanase that can exhibit good stability under alkaline conditions, the amino acid sequence of which is shown in SEQ ID No. 1; in order to further improve the stability and catalytic efficiency of the alkaline xylanase under alkaline conditions, the present invention performs multi-site mutations on the alkaline xylanase to obtain multiple multi-site mutants, each of which can maintain a high enzyme activity within the range of 50-55°C, exhibit better stability and catalytic efficiency under alkaline conditions, and can more efficiently convert the substrate xylan into the corresponding product while improving the substrate binding capacity. The present invention has application prospects in the large-scale production of xylan decomposition.

Description

Technical Field

[0001] The invention relates to enzymes and mutants thereof, in particular to alkaline xylanase and multi-site mutants thereof and applications in decomposing xylan, belonging to the field of xylanase and mutants thereof and applications. Background Art

[0002] Xylanase (EC3.2.1.8), as an enzyme that can decompose xylan, has shown important application value in many fields such as biomass conversion, food industry processing, feed production and pulp bleaching. Its sources are quite wide, covering microorganisms, plants and animals.

[0003] Traditional methods for discovering and modifying high-temperature alkaline xylanases have primarily relied on directed evolution and rational design. While these methods have achieved some success, the process is not only time-consuming and labor-intensive, but also requires strong technical expertise. The rapid development of artificial intelligence (AI) technology has brought new opportunities and challenges to the field of protein engineering.

[0004] Existing xylanases have poor stability and catalytic efficiency under alkaline conditions, resulting in low efficiency in decomposing xylan under alkaline conditions, which urgently needs to be improved. Summary of the Invention

[0005] One of the objects of the present invention is to provide an alkaline xylanase that exhibits good stability under alkaline conditions;

[0006] The second object of the present invention is to provide a codon-optimized coding gene for alkaline xylanase;

[0007] The third object of the present invention is to provide a multi-site mutant of the alkaline xylanase and a gene encoding the same;

[0008] The fourth object of the present invention is to apply the alkaline xylanase and its multi-site mutants to the enzymatic decomposition of xylan.

[0009] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0010] One aspect of the present invention is to provide an alkaline xylanase that exhibits good stability under alkaline conditions, the amino acid sequence of which is shown in SEQ ID No. 1.

[0011] Another aspect of the present invention is to codon-optimize the coding gene of the alkaline xylanase to obtain the coding gene shown in SEQ ID No. 2; accordingly, the present invention provides a recombinant expression vector containing the coding gene and a recombinant host cell containing the recombinant expression vector.

[0012] Another aspect of the present invention is to provide a multi-site mutant of alkaline xylanase, wherein the multi-site mutant is selected from any one of the multi-site mutants described in (1) to (3):

[0013] (1) Multi-site mutant E20-21: A multi-site mutant obtained by simultaneously subjecting the amino acid sequence shown in SEQ ID No. 1 to multi-site mutations of S11A, R58E, K181L, S197E, V228I, G380E, T393E, and G398L;

[0014] (2) Multi-site mutant E20-158: A multi-site mutant obtained by simultaneously subjecting the amino acid sequence shown in SEQ ID No. 1 to multi-site mutations of S3Q, T33V, D139E, R185G, L218I, L273I, T393E, and G398L;

[0015] (3) Multi-site mutant E20-178: A multi-site mutant obtained by simultaneously subjecting the amino acid sequence shown in SEQ ID No. 1 to multi-site mutations of R25N, T33V, D189E, V228I, L273I, V319I, C328A, and G398L.

[0016] The specific meaning of "simultaneously performing multiple mutations of S11A, R58E, K181L, S197E, V228I, G380E, T393E and G398L on the amino acid sequence shown in SEQ ID NO.1" in the present invention is to simultaneously mutate the 11th amino acid of the amino acid sequence shown in SEQ ID NO.1 from serine (Ser, S) to alanine (Ala, A), mutate the 58th arginine (Arg, R) to glutamic acid (Glu, E), and mutate the 181st lysine (Lys, K ) is mutated to leucine (Leu, L), the serine (Ser, S) at position 197 is mutated to glutamic acid (Glu, E), the valine (Val, V) at position 228 is mutated to isoleucine (Ile, I), the glycine (Gly, G) at position 380 is mutated to glutamic acid (Glu, E), the threonine (Thr, T) at position 393 is mutated to glutamic acid (Glu, E), and the glycine (Gly, G) at position 398 is mutated to leucine (Leu, L); the description of the remaining multi-site mutants of the present invention is similar.

[0017] Another aspect of the present invention is to apply the alkaline xylanase and its multi-site mutants to the enzymatic decomposition of xylan, comprising: using xylan as a substrate and the alkaline xylanase or its multi-site mutants as a catalytic enzyme to carry out an enzymatic catalytic reaction to decompose xylan.

[0018] The present invention measured the optimal temperature of the wild type and the multi-site mutants at their respective optimal pH values. The results showed that the three multi-site mutants could maintain relatively high activity within the range of 50-55°C.

[0019] To evaluate the stability of multi-site mutants under alkaline conditions, the multi-site mutants were subjected to long-term incubation experiments under different pH conditions. The results showed that the enzyme activity of wild-type xylanase E20 rapidly decreased to 57% of the original activity after 2 hours of incubation, and further decreased to 46% after 8 hours. In contrast, the multi-site mutant E20-21 had a residual activity of 87% after 2 hours and still maintained 75% after 8 hours; the multi-site mutant E20-158 had a residual activity of 84% after 2 hours and 74% after 8 hours; the multi-site mutant E20-178 had a residual activity of 87% after 2 hours and still maintained above 85% after 8 hours.

[0020] At pH 9.0, the activity of wild-type xylanase E20 dropped to 63% after 2 hours and to 55% after 8 hours. Multi-site mutant E20-21 maintained 79% activity after 2 hours, but this activity dropped to 59% after 8 hours. Mutant E20-158 retained 62% activity after 2 hours, but only 48% after 8 hours. Multi-site mutant E20-178 maintained 88% activity within 2-4 hours and remained at 77% after 8 hours, demonstrating superior stability. This result further confirmed that among the three multi-site mutants, mutant E20-178 exhibited the most significant improvement in alkaline stability.

[0021] Under incubation conditions of pH 10.0, the results showed that the activity of wild-type xylanase E20 was only 46% after 8 hours; in comparison, among the three multi-site mutants, the multi-site mutant E20-21 had a remaining activity of 73% after 2 hours and 54% after 8 hours; the multi-site mutant E20-158 had a remaining activity of 62% after 2 hours and 38% after 8 hours; the multi-site mutant E20-178 maintained its activity above 60% after 2-4 hours and 50% after 8 hours.

[0022] Under incubation conditions of pH 11.0, the results showed that the activity of wild-type xylanase E20 dropped to 48% after 8 h; in comparison, among the three multi-site mutants, the multi-site mutant E20-158 had a residual activity of 40% after 8 h; the multi-site mutant E20-21 had a residual activity of 74% after 2 h, which gradually decreased after 4 h; the multi-site mutant E20-178 had a residual activity of 74% after 2 h, and remained above 70% after 4 h; in summary, the multi-site mutant E20-178 exhibited the most significant stability and catalytic efficiency under alkaline conditions.

[0023] Under the optimal reaction conditions, the present invention further used beech xylan as a substrate to measure the kinetic parameters of xylanase E20 and its multi-site mutants. The results showed that the multi-site mutants E20-21 and E20-158 had enhanced affinity for the substrate, while the multi-site mutant E20-178 had K m The value is close to that of wild-type xylanase E20, indicating that its affinity for the substrate is similar to that of wild-type xylanase E20; in addition, the k cat / K m The value was 10.12±0.37 mL / mg -1 min -1 , while the multi-site mutant E20-21 k cat / K m The value was 31.92±1.23 mL / mg -1 min -1 , which is about 3 times that of wild-type xylanase E20. k cat / K m The value was 19.44±0.20 mL / mg -1 min -1 , which is about 2 times that of wild-type xylanase E20. The test results show that the three multi-site mutants provided by the present invention can not only improve the substrate binding ability but also more efficiently convert the substrate into product.

[0024] Definitions of terms used in this invention

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described.

[0026] The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in single or double stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, which include PNA (peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioates, phosphamidates, etc.). Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including but not limited to degenerate codon substitutions) and complementary sequences as well as explicitly specified sequences. In particular, degenerate codon substitutions ( Mol Cell. Probes 8:91-98 (1994)).

[0027] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. That is, a description directed to a polypeptide equally applies to describing a peptide and describing a protein, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturally encoded amino acid. As used herein, the terms encompass amino acid chains of any length, including full-length proteins (i.e., antigens), in which the amino acid residues are linked via covalent peptide bonds.

[0028] The terms "mutation" and "mutant" have their ordinary meanings herein and refer to genetic, naturally occurring or introduced changes in nucleic acid or polypeptide sequences, and their meanings are the same as those generally understood by those skilled in the art.

[0029] The term "recombinant host cell strain" or "host cell" refers to a cell comprising a polynucleotide of the present invention, regardless of the method used for insertion to produce the recombinant host cell, such as direct uptake, transduction, f-mating, or other methods known in the art. The exogenous polynucleotide may be maintained as a non-integrating vector, such as a plasmid, or may be integrated into the host genome. The host cell may be a prokaryotic cell or a eukaryotic cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Results of enzyme property analysis of xylanase E20; (A): Optimal reaction temperature of xylanase E20; (B): Stability of xylanase E20 at different temperatures; (C): Optimal reaction pH of xylanase E20; (D): Stability of xylanase E20 at different pH levels.

[0031] Figure 2Results of the optimal conditions determination for xylanase E20 multi-site mutants; (A) Optimal pH for xylanase E20 and its mutants; (B) Optimal temperature for xylanase E20 and its mutants.

[0032] Figure 3 The stability of xylanase E20 and its mutants at different pH conditions; (A) Stability of xylanase E20 and its multi-site mutants at pH 8.0; (B) Stability of xylanase E20 and its multi-site mutants at pH 9.0; (C) Stability of xylanase E20 and its multi-site mutants at pH 10.0; (D) Stability of xylanase E20 and its multi-site mutants at pH 11.0. DETAILED DESCRIPTION

[0033] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are merely exemplary and do not limit the scope of the present invention in any way. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but such modifications or replacements fall within the scope of protection of the present invention.

[0034] Experimental Example 1 Screening of alkaline xylanase and its multi-site mutants and determination of their enzymatic properties, alkaline resistance, and kinetic parameters

[0035] 1 Test method

[0036] 1.1 Construction of xylanase gene

[0037] The xylanase was obtained by screening the Uniparc database, and the selected xylanase gene was optimized according to the Escherichia coli codon, synthesized by General Biotechnology Co., Ltd. and constructed into the plasmid pET-28a(+) vector, with two ends added. Eco RI and Xho I restriction endonuclease cutting site.

[0038] The amino acid sequence of the screened xylanase is shown in SEQ ID No. 1:

[0039] MDSLAHRRANSRLQVLNPDGTPASRRPVLIDQTSHSFLFGCGAFDTVPLLSLRDWGRDFLRRRMEKWLALFNYGTLPFYWGRYEPEEGHTMQEETLAAARWLGERGVKVKGHPLCWHTACAPWLMQYSNEEILRRQLDRIRREVTAFRGVIDMWDVINEVVIMPVFDKYDNAVTRICREKGRIRLVKDVFAAARESNPGATLL INDFNTSVSYEILLEGLLEAGVPVSAIGIQSHQHQGYWGLEKLNDVLERFSRFGLPIHFTENTLISGELMPAHIVDLNDWQVPSWPTTPEGEERQAREISEMYTTLFSHPLVEAVTTWDFNDGCWLGAPSGLVRADNSEKPAYEALMGLIHGAWETHERLVTDEEGFVSFTGFKGGYALTAENRRTAFTLKGDGTQTLTL (SEQ ID No.1).

[0040] The nucleotide sequence of the xylanase gene after optimization according to E. coli codons is shown in SEQ ID No. 2:

[0041]

[0042] 1.2 Heat shock transformation and induced expression in Escherichia coli BL21 (DE3)

[0043] Will E. coli After thawing on ice, BL21 (DE3) competent cells were gently mixed with purified plasmid DNA, adsorbed on ice for 30 min, heat-shocked at 42°C for 90 s and chilled on ice, and then revived and cultured in LB-free medium for 45 min. Single clones were plated on LB plates containing kanamycin (50 μg / mL) to obtain single clones. Single clones were picked and cultured in LB liquid medium containing kanamycin until the OD 600 ≈ 1.5, transfer to fresh culture medium at 1% inoculum volume, and wait until OD 600 When the protein expression reaches 0.6-0.8, IPTG is added to a final concentration of 0.3 mM and expression is induced at 16°C and 180 rpm for 18-20 hours. After induction, the cells are harvested by centrifugation at 6000 rpm, resuspended in ice-cold 20 mM Tris-HCl buffer, and sonicated in an ice bath (40% power, 4s on / 3s off, 30 cycles). The supernatant is then centrifuged at 8000 rpm and the supernatant is collected. The supernatant is loaded onto a pre-equilibrated Ni-NTA column and eluted with NTA-0, NTA-20, and NTA-40 buffers, followed by NTA-200, to remove contaminants and the target protein. The eluate is dialyzed (4°C, 16 hours, with buffer exchanged regularly) to remove salt and imidazole, concentrated with PEG 8000, and aliquoted by centrifugation for storage at -80°C (containing 5% glycerol). The purified protein was detected by SDS-PAGE (12% separating gel, 5% stacking gel, electrophoresis at 80 V for 3 h) and quantified by the BCA assay (incubation at 37°C for 30 min, detection at 562 nm).

[0044] 1.3 Determination of xylanase activity properties

[0045] The DNS method was used to determine the activity of xylanase. First, 100 μL of xylanase solution (reaction group) or buffer (blank group) was mixed with 900 μL of 1% beechwood xylan solution for 10 min at 37°C. 1.5 mL of DNS reagent was added to terminate the reaction and the mixture was heated in a boiling water bath for 5 min. After cooling, the absorbance at 540 nm (OD) was measured. 540 The enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the production of 1 μmol of reducing sugar per minute under optimal conditions.

[0046] The enzyme activity calculation formula is: A=[(A1-A0)×K+C0]×V1×n / (V2×t), where A is the enzyme activity (U / mL), A1 and A0 are the absorbance of the sample and blank respectively, K and C0 are the parameters of the p-nitrophenol standard curve, n is the dilution factor, V1 is the reaction volume (mL), V2 is the volume of the enzyme solution (mL), and t is the reaction time (min). Each group has 3 parallels. At the same time, draw a xylose standard curve: prepare a xylose solution with a concentration gradient of 0-7 mg / mL, take 1 mL of each solution and add 1.5 mL of DNS reagent in a boiling water bath for 5 minutes, and measure the OD after cooling. 540 The values ​​(three replicates per group) were plotted with xylose concentration (mg / mL) as the horizontal axis and OD 540 Draw a standard curve for the ordinate.

[0047] 1.4 Optimal reaction conditions and stability determination of recombinant enzymes

[0048] Enzyme activity was determined using the DNS reducing sugar product method. The enzyme solution to be tested was diluted appropriately to ensure that the absorbance reading fell between 0.5 and 2.0.

[0049] Optimum temperature: The enzyme activity is measured at different temperatures. The enzyme activity at the optimum temperature is set as 100% and the relative enzyme activity at other temperature conditions is calculated based on this.

[0050] Optimum pH: 10.0 mg·mL prepared in BR buffer (pH 5.0-12.0) -1 The enzyme activity was measured after the reaction at the optimum temperature using beech wood xylan substrate. The enzyme activity at the optimum pH was set as 100%, and the relative enzyme activity at other pH values ​​was calculated.

[0051] Thermal stability: After diluting the enzyme solution, heat it in a water bath at the set test temperature. The enzyme activity is measured at regular intervals. The initial enzyme activity is set as 100% of the baseline value, and the residual enzyme activity is then calculated at different heat treatment times.

[0052] pH stability: The enzyme was incubated at the optimal temperature for 1 h in different pH environments. The enzyme activity was then measured and the highest measured enzyme activity was set as 100%. The residual enzyme activity at each pH value was calculated based on this.

[0053] 1.5 Kinetic parameter detection

[0054] The enzyme activity was measured at the optimal reaction temperature using xylan solutions (pH 8.0-10.0) of different mass concentrations (1.0-50.0 mg / mL) as substrates. Graphpad prism software was used to plot the activity of the recombinant enzyme. K m andV max .

[0055] 2 Test results

[0056] 2.1 Enzymatic properties of xylanase E20

[0057] By measuring the enzyme activity of xylanase E20 (hereinafter referred to as "E20") at different temperatures, it was found that E20 had the highest activity at 55℃ ( Figure 1 -A). Within the range of 45°C to 55°C, E20 can still maintain over 60% of its activity, and even at 60°C, it can still maintain 30% of its activity. Further research on the temperature stability of E20 found that E20 can still retain 80% of its residual activity after being treated at 50°C for 1 hour, and still maintain a residual activity of 65% after being treated at 55°C for 20 minutes. Further increasing the temperature, when the temperature exceeds 60°C for 30 minutes, the activity of E20 is almost completely lost ( Figure 1 -B).

[0058] The results of enzyme activity assays of E20 in different pH buffers showed that E20 had the highest activity at pH 8.0 in BR buffer and could maintain more than 65% of its activity within the pH range of 6.0-8.5 ( Figure 1 -C). To further evaluate the pH stability of E20, it was mixed with BR buffer at pH levels ranging from 5.0 to 12.0, incubated on ice for 8 hours, and then its residual enzyme activity was measured. The results showed that when the pH was ≤ 5.0 or ≥ 12.0, E20 activity was almost completely lost; however, in the pH range of 6.0-11.0, E20 still retained more than 30% of its residual activity after 8 hours of treatment ( Figure 1 -D). It is worth noting that E20 exhibits better stability under alkaline conditions.

[0059] 2.2 Screening of xylanase E20 and its multi-site mutants and determination of alkaline resistance

[0060] To further improve the alkali and heat resistance of E20, an ODM generation model was constructed based on the homologous sequence of the target protein. A random mask re-prediction method was used to generate 100,000 mutant sequences. 3,000 candidate mutant sequences were screened using the minimum mask principle. The candidate mutant sequences were predicted using the expression model (MPBEXP-model), the heat resistance model (MPBTm-model), and the alkali resistance model (AA-model), respectively, and each mutant was evaluated. Using weighted random selection (weight ratio 1:3:6), the mutant sequences were sorted from highest to lowest. After screening and experimental verification, 200 mutant sequences were identified, resulting in the multi-site mutants E20-21, E20-158, and E20-178.

[0061] Table 1 Display of mutation sites of wild-type and multi-site mutant proteins

[0062]

[0063] Pure enzyme solutions of wild-type (i.e., xylanase E20) and multi-site mutants were prepared by protein purification using nickel affinity chromatography. Enzyme activity was measured at 55°C within the pH range of 8.0-12.0. The results showed that the optimal pH for multi-site mutants E20-21 and E20-158 was 9.0, while that for multi-site mutant E20-178 was 10.0. Compared to wild-type xylanase E20, the peak activity point was shifted to the right ( Figure 2 -A). Subsequently, the optimal temperature of the multi-site mutants was determined at their respective optimal pH values, and the results showed that ( Figure 2 -B): The optimum temperature of the multi-site mutants E20-21 and E20-158 is 50°C, and that of xylanase E20 and the multi-site mutant E20-178 is 55°C. Although the mutants are more sensitive to heat than the wild type, the three multi-site mutants (E20-21, E20-178 and E20-158) can maintain high activity within the range of 50-55°C.

[0064] In order to evaluate the stability of the multi-site mutants under alkali-resistant conditions, the multi-site mutants were subjected to long-term incubation experiments (0 h, 2 h, 4 h, and 8 h) under different pH conditions. Figure 3 -A. Wild-type xylanase E20 and multi-site mutants were incubated at pH 8.0 for the same time and then measured for residual enzyme activity. The results showed that the activity of wild-type xylanase E20 rapidly decreased to 57% of its original activity after 2 hours of incubation, and further decreased to 46% after 8 hours. In contrast, the multi-site mutant E20-21 had a residual activity of 87% after 2 hours and maintained 75% after 8 hours. The multi-site mutant E20-158 had a residual activity of 84% after 2 hours and 74% after 8 hours. The multi-site mutant E20-178 had a residual activity of 87% after 2 hours and maintained over 85% after 8 hours.

[0065] At pH 9.0, the activity of wild-type xylanase E20 dropped to 63% after 2 hours and to 55% after 8 hours. The performance of various multi-site mutants varied: multi-site mutant E20-21 had 79% remaining activity after 2 hours, but this dropped to 59% after 8 hours; multi-site mutant E20-158 had 62% remaining activity after 2 hours, but only 48% after 8 hours; and multi-site mutant E20-178 maintained 88% activity within 2-4 hours and remained at 77% after 8 hours, demonstrating superior stability. Figure 3-B). This result further confirmed that among the three multi-site mutants, the multi-site mutant E20-178 had the most significant improvement in stability in alkaline environment.

[0066] Incubate at pH 10.0. Figure 3 As shown in Figure 3-C, wild-type xylanase E20 retained only 46% of its activity after 8 hours. Among the three multi-site mutants, E20-21 retained 73% of its activity after 2 hours and 54% after 8 hours; E20-158 retained 62% of its activity after 2 hours and 38% after 8 hours; and E20-178 maintained over 60% of its activity after 2-4 hours and 50% after 8 hours.

[0067] At pH 11.0, Figure 3 As shown in Figure 3-D, the activity of wild-type xylanase E20 dropped to 48% after 8 hours. Among the three multi-site mutants, multi-site mutant E20-158 had a residual activity of 40% after 8 hours; multi-site mutant E20-21 had a residual activity of 74% after 2 hours, which gradually decreased after 4 hours; and multi-site mutant E20-178 had a residual activity of 74% after 2 hours and remained above 70% after 4 hours.

[0068] In summary, the multi-site mutant E20-178 showed the most significant stability and catalytic efficiency under alkaline conditions.

[0069] 2.3 Kinetic parameters of xylanase E20 and its multi-site mutants

[0070] Under the optimal reaction conditions, beech xylan was used as a substrate to determine the kinetic parameters of xylanase E20 and its multi-site mutants. The results are shown in Table 2.

[0071] Table 2 Kinetic parameter determination of xylanase E20 and its multi-site mutants

[0072]

[0073] According to the test results in Table 2, the Michaelis constant of wild-type xylan E20 is K m was 8.33 ± 0.25 mg / mL, while the multi-site mutants E20-21, E20-158 and E20-178 K mThe values ​​were 6.78 ± 0.31 mg / mL, 6.56 ± 0.56 mg / mL and 8.13 ± 0.13 mg / mL respectively. This shows that the affinity of the two multi-site mutants E20-21 and E20-158 to the substrate is enhanced, while the affinity of the multi-site mutant E20-178 is K m The value is close to that of wild-type xylan E20, indicating that its affinity for the substrate is similar to that of wild-type xylan E20.

[0074] In addition, the wild-type xylan E20 k cat / K m The value was 10.12±0.37 mL / mg -1 min -1 , while the multi-site mutant E20-21 k cat / K m The value was 31.92±1.23 mL / mg -1 min -1 , which is about 3 times that of wild-type xylan E20.

[0075] At the same time, the multi-site mutant E20-178 k cat / K m The value was 19.44±0.20 mL / mg -1 min -1 , which is about twice that of wild-type xylan E20. This shows that these three multi-site mutants can not only improve substrate binding ability, but also more efficiently convert xylan substrate into corresponding products.

Claims

1. A multi-site mutant of alkaline xylanase, characterized in that The multi-site mutant is selected from any one of the following multi-site mutants (1) to (3): (1) A multi-site mutant obtained by simultaneously performing multi-site mutations of S11A, R58E, K181L, S197E, V228I, G380E, T393E and G398L on the amino acid sequence shown in SEQ ID No. 1; (2) A multi-site mutant obtained by simultaneously performing multi-site mutations of S3Q, T33V, D139E, R185G, L218I, L273I, T393E, and G398L on the amino acid sequence shown in SEQ ID No. 1; (3) A multi-site mutant obtained by simultaneously performing multi-site mutations of R25N, T33V, D189E, V228I, L273I, V319I, C328A and G398L on the amino acid sequence shown in SEQ ID No.

1.

2. The gene encoding the multi-site mutant according to claim 1.

3. A recombinant expression vector containing the coding gene according to claim 2.

4. A recombinant host cell containing the recombinant expression vector according to claim 3.

5. Use of the multi-site mutant according to claim 1 in the enzymatic decomposition of xylan.

6. The use according to claim 5, characterized in that include: The method uses xylan as a substrate and the multi-site mutant as a catalytic enzyme to carry out an enzymatic catalytic reaction and decompose the substrate xylan.

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