Difunctional xylanase fusion protein, preparation method thereof and application of difunctional xylanase fusion protein in preparation of feruloyl oligosaccharide

Abstract

The invention discloses a bifunctional xylanase fusion protein, a preparation method thereof and application of the bifunctional xylanase fusion protein in preparation of feruloyl oligosaccharide, and belongs to the technical field of genetic engineering and enzyme engineering. The fusion protein is composed of GH10 xylanase derived from bacillus altitudinis, a connecting peptide and GH11 xylanase derived from bacillus amyloliquefaciens. According to the invention, through an elaborately designed linker, the GH10 family xylanase and the GH11 family xylanase are fused through a specific connecting peptide, so that mutual interference between structural domains can be effectively overcome, the synergistic catalysis of GH10 and GH11 is fully played, and the novel bifunctional xylanase with synergistic interaction, strong specificity, high catalytic efficiency and high thermal stability is obtained. Compared with a single enzyme or a physical compound enzyme, the compound enzyme has better hydrolysis efficiency and application performance, and can be widely applied to the fields of food, textile, paper pulp bleaching, feed industry and the like.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and enzyme engineering, specifically to a bifunctional xylanase fusion protein, its preparation method, and its application in the preparation of ferulic acid oligosaccharides. Background Technology

[0002] Feruloyl oligosaccharides (FOs) are natural active substances formed by the ester bond between xylooligosaccharides arabino-oligosaccharides and ferulic acid. They not only possess the prebiotic function of xylooligosaccharides, promoting the proliferation of beneficial bacteria such as Bifidobacterium in the gut (Feruloylated and nonferuloylated arabino-oligosaccharides from sugar beet pectin selectively stimulate the growth of Bifidobacterium spp. in human fecal in vitro fermentations. J Agric FoodChem, 2011, 59 (12), 6511-6519.), but also exhibit strong antioxidant, anti-inflammatory, antitumor, antithrombotic, and hepatoprotective activities due to their ferulic acid content (Feruloylated arabinoxylan andoligosaccharides: chemistry, nutritional functions, and options for enzymatic modification. Annu Rev Food Sci Technol, 2021, 12, 331-354.). Due to their excellent functional properties, FOs have extremely broad application prospects in functional foods, health products, pharmaceuticals and cosmetics, and are regarded as a high-value-added bioactive ingredient.

[0003] Currently, FOs are mainly prepared by hydrolyzing natural plant materials (such as corn bran, wheat bran, and buckwheat straw) using physical, chemical, or enzymatic methods. Among biological methods, enzymatic hydrolysis has become the mainstream due to its mild conditions, high specificity, and pure products. The key to this process is the use of endoxylanases that can specifically cleave the xylan backbone. Commercially available xylanases fall into two categories: one is a mixed enzyme preparation containing several xylanases, which is inexpensive but yields more complex products; the other is an enzyme preparation containing only one xylanase, which is more expensive. Furthermore, due to the complexity and heterogeneity of xylan structures, a single xylanase often fails to achieve efficient and complete degradation. Combining xylanases from different families is a common solution, but this involves complex formulations, unstable enzyme ratios, and high costs. Furthermore, while GH11 xylanase, obtained through genetic engineering, has a cost advantage and is a single xylanase, it suffers from the common problem of poor thermal stability. Therefore, even a single GH11 xylanase is difficult to hydrolyze xylan to obtain feruloyl oligosaccharides.

[0004] According to the Carbohydrate Active Enzyme Database (CAZy), most xylanases are distributed in the glycoside hydrolase (GH) families 10 and 11. In addition, a few xylanases are distributed in GH families 5, 7, 8, 30, 43, 51 and 141. GH10 and GH11 family xylanases are key enzymes for hydrolyzing xylan (Feruloylatedarabinoxylan and oligosaccharides: chemistry, nutritional functions, and options for enzymatic modification. Annu Rev Food Sci Technol, 2021, 12, 331-354.). GH10 and GH11 xylanases differ significantly in structure and function: GH10 xylanase has a larger molecular weight and a (β / α)8 barrel-shaped structure, with more open catalytic active sites. It can hydrolyze not only long-chain xylans but also has high hydrolytic activity for xylooligosaccharides and better tolerance to substituted xylans (such as arabinoyl xylan) (Insights into the catalytic mechanism of a novel XynA and structure-based engineering for improving bifunctional activities. Biochemistry-Us 2021, 60 (26), 2071-2083.). GH11 xylanase has a smaller molecular weight and a β-sheet "right-hand" structure. Its catalytic active site is deeper and narrower, mainly acting on long-chain xylans to generate more xylobiose and xylo-oligosaccharides. Its specific activity is usually higher than that of the GH10 family (Improving the thermal stability of GH11 Xylanase XynASP through cord region engineering. J Agr Food Chem, 2025, 73 (2), 1516-1528.). If an enzyme could combine GH10 and GH11 xylanases to make up for the shortcomings of single enzymes, it would certainly provide a richer theoretical basis for the application of xylanases.

[0005] Constructing a single multifunctional enzyme from GH10 and GH11 xylanases using protein fusion technology theoretically allows for "one enzyme, multiple uses," synergistic effects, and improved thermostability. However, simple head-to-tail fusion often leads to steric hindrance between the two domains, affecting their folding and catalytic efficiency, and even causing loss of enzyme activity. Therefore, designing the linker that connects the two enzyme domains is crucial to the success of this technique. The length, rigidity, flexibility, and sequence of the linker directly affect the conformational freedom, stability, and catalytic performance of the fusion protein.

[0006] Therefore, how to construct highly active and highly synergistic fusion enzymes through rational molecular design, especially by designing a linker that can effectively separate two domains and endow them with sufficient conformational freedom, is the core key and a technical challenge that urgently needs to be solved in this field. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention aims to provide a bifunctional xylanase fusion protein, its preparation method, and its application in the preparation of feruloyl oligosaccharides. This fusion enzyme, through a carefully designed flexible linker, fuses GH10 family xylanases and GH11 family xylanases via a specific linker. This effectively overcomes interdomain interference and fully leverages the synergistic catalytic effect of GH10 and GH11, resulting in a novel bifunctional xylanase with synergistic effects, high specificity, high catalytic efficiency, and high thermal stability. It exhibits superior hydrolysis efficiency and application performance compared to single enzymes or physically combined enzymes. This invention aims to overcome existing technological bottlenecks and meet the urgent market demand for efficient, low-cost production of high-purity FOs.

[0008] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0009] A bifunctional xylanase fusion protein, wherein the fusion protein is linked to GH10 xylanase and GH11 xylanase via a linker peptide. The amino acid composition of the fusion protein consists of GH10 and amino acids with a similarity greater than 70%, a linker, and GH11 and amino acids with a similarity greater than 70%. The linker includes a flexible linker peptide or a rigid linker peptide, and the amino acid sequence of the flexible linker peptide is (GGGGS). n The amino acid sequence of the rigid linker peptide is (EAAAK). n , where n is 1 or 2.

[0010] The rigid linker peptide has the base sequence GAAGCCGCCGCCAAG, while the flexible linker peptide has the base sequence GGTGGTGGCGGTAGC.

[0011] Preferably, GH10 xylanase is derived from Bacillus hygroscopicus, and GH11 xylanase is derived from Bacillus hygroscopicus (GH11-1) or Bacillus amyloliquefaciens (GH11-2).

[0012] Preferably, the amino acid sequence of GH10 is as shown in SEQ ID NO.1, comprising 408 amino acids and a theoretical molecular weight of 47.7 kDa; the amino acid sequence of GH11 is GH11-1, as shown in SEQ ID NO.2, comprising 291 amino acids and a theoretical molecular weight of 31.9 kDa; the amino acid sequence of GH11 is GH11-2, as shown in SEQ ID NO.3, comprising 213 amino acids and a theoretical molecular weight of 23.4 kDa.

[0013] SEQ ID NO.1 MVNKKEFMNHSSNGGSQEQENQSWRKEANNRILQHRQRELVINVINKEQKPVAGIEVEIKQIRHEFAFGSAMNDQVLFNQTYADFFVQHFNWAVFENEAKW YANEPERGKITYEKADAMLNFANRHQIPVRGHALFWEVEDANPNWLKSLPNHEVYEAMKWRLEHAGNHFKGKFRWDVNNEMMHGSFFKDRFGKQIWKWMYEE TKKIDPQALLFVNDYNVISYGEHHAYKAHINELRQLGAPVEAIGVQGHFEDRVDPVVVKQRLDVLAELGLPIWVTEYDSVHPDANRRADNLEALYRVAFSHP AVKGVLMWGFWAGAHWRGEHAAIVNHDWSLNEAGRRYEKLLQEWTTQRVEKTDANGQVTCPAFHGTYEVRIGEVSKMLQQQTIELDSTKQTPLQLDIIVPVE

[0014] SEQ ID NO.2

[0015] MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEAARTITNNEMGNHSGYDYELWKDYGNTSMTLNNNGGAFSAGWNNIGNALFRKGKKFDS TRTHHQLGNISINYNASFNPGGNSYLCVYGWTQSPLAEYYIVDSWGTYRPTGAYKGSFYADGGTYDIYETTRVNQPSIIGIATFKQYWSVRQTKRTSGTVSVSAHFRKWESLGMPMGKMYETAFTVEGYQSSGSANVMTNQLFIGN

[0016] SEQ ID NO.3

[0017] MFKFKKNFLVGLSAALMSISLFSATASVASTDYWQNWTDGGGIVNAVNGSGGNYSVNWSNTGNFVVGKGWTTGSPFRTINYNAGVWAPNGNGYLTLYGWTRSPLIE YYVVDSWGTYRPTGTYKGTVKSDGGTYDIYTTTRYNAPSIDGDRTTFTQYWSVRQTKRPTGSNATITFSNHVNAWKSHGMNLGSNWAYQVMATEGYQSSGSSNVTVW

[0018] Specifically, the nucleotide sequence encoding GH10 xylanase is SEQ ID NO. 4, 1227 bases. The nucleotide sequence encoding GH11-1 xylanase is SEQ ID NO. 5, 600 bases. The nucleotide sequence encoding GH11-2 xylanase is SEQ ID NO. 6, 555 bases.

[0019] SEQ ID NO.4

[0020]

[0021] SEQ ID NO.5

[0022] ATTACGAATAATGAAATGGGCAACCACAGCGGGTACGATTATGAACTATGGAAGGACTACGGAAACACTTCGATGACACTCAATAACGGCGGGGCATTTAGTGCAGGCTGGAACAATATCGGAAATGCTTTATTTAGAAAAGGGAAAAAGTTTGATTCTACTAGAACTCACCATCAGCTTGGCAACATCTCCATCAATTACAACGCAAGTTTTAACCCAGGCGGGAATTCCTATCTATGTGTCTATGGCTGGACACAATCTCCATTAGCAGAATACTACATTGTTGATTCATGGGGCACGTATCGTCCAACAGGAGCGTATAAAGGATCATTTTATGCTGATGGAGGCACATATGACATTTATGAAACAACCCGTGTCAATCAGCCTTCCATTATCGGGATCGCAACCTTCAAGCAATATTGGAGTGTACGTCAAACGAAACGTACAAGCGGAACGGTCTCCGTCAGTGCGCATTTTAGAAAATGGGAAAGCTTAGGGATGCCAATGGGGAAAATGTATGAAACGGCATTTACTGTAGAAGGCTACCAAAGCAGCGGAAGTGCAAATGTGATGACCAATCAGCTGTTTATTGGCAACTAA

[0023] SEQ ID NO.6

[0024] GGCACAGATTACTGGCAAAATTGGACTGATGGGGGCGGAACAGTCAACGCTGTCAATGGATCTGGCGGGAATTACAGTGTTAATTGGTCTAATACCGGAAATTTCGTTGTTGGTAAAGGCTGGACTACAGGCTCGCCA TTTAGAACAATAAACTATAATGCCGGAGTCTGGGCGCCGAATGGCAATGGATATTTGACTTTATATGGCTGGACGAGAGCACCTCTCATCGAATATTATGTAGTGGATTCATGGGGTACTTACAGACCTACCGGAACGT ATAAAGGTACTGTAAACAGTGATGGAGGTACATATGACATATATACAACGACACGTTATAACGCACCTTCCATTGATGGCGATAACACTACTTTTACGCAGTACTGGAGTGTTCGCCAGTCGAAGAGACCGACCGGAAG CAACGCTGCAATCACTTTCAGTAATCATGTTAACGCATGGAAGAGCCATGGAATGAATCTGGGCAGTAATTGGGCTTATCAAGTCTTAGCGACAGAAGGATATAAAAGCAGCGGAAGTTCTAATGTAACAGTGTGGTAA

[0025] Preferably, the amino acid sequence of the fusion protein consists of GH10, linker, and GH11, as shown in SEQ ID NO.1+linker+SEQ ID NO.2 and SEQ ID NO.1+linker+SEQ ID NO.3.

[0026] A DNA gene sequence encoding a nucleotide of the bifunctional xylanase fusion protein according to any one of the preceding claims.

[0027] A recombinant expression vector containing the above-mentioned genes, wherein the vector is a prokaryotic expression vector pET-28a.

[0028] A host cell comprising the above-mentioned recombinant expression vector, wherein the host cell is Escherichiacoli BL21(DE3).

[0029] A method for expressing the above-mentioned bifunctional xylanase fusion protein includes the following steps: cloning the gene encoding the fusion protein according to any one of claims 1 to 4 into an expression vector to construct a recombinant expression vector; transforming the recombinant expression vector into a host cell to obtain a recombinant strain; culturing the recombinant strain to induce expression of the fusion protein; isolating the recombinant bacterial cells from the culture and obtaining the cell-free bifunctional xylanase fusion protein by lysis.

[0030] Preferably, the bifunctional xylanase fusion protein obtained by the method has a molecular weight of 68.4 kDa.

[0031] Preferably, the method yields fusion proteins B, N, P, K, and L, and the thermal stability of B, N, P, K, or L is increased several times compared to GH11 xylanase.

[0032] Preferably, the fusion protein B (T 1 / 2 =360 min) thermal stability is better than GH11-2 (T =360 min) 1 / 2 =50 min) increased by 7.2 times, the optimal reaction temperature of the fusion protein was 50℃, and the optimal reaction pH was 6.0.

[0033] The application of the bifunctional xylanase fusion protein prepared by any of the above methods in the preparation of feruloyl oligosaccharides, wherein the feruloyl oligosaccharides have a small molecular weight and a narrow degree of polymerization distribution, with a degree of polymerization of 3 being the main component.

[0034] Beneficial effects:

[0035] Compared with existing technologies, the present invention provides a bifunctional xylanase fusion protein, its preparation method, and its application in the preparation of ferulic acid oligosaccharides, which have the following advantages:

[0036] Synergistic effect: This invention, for the first time, rationally designs and fuses GH10 and GH11 xylanases with different substrate specificities through a linker. This fusion enzyme can simultaneously exert the hydrolytic ability of GH10 on substituted xylans and the highly efficient endonuclease activity of GH11 on long-chain xylans. The total hydrolysis efficiency of natural xylan substrates is significantly higher than that of either parent enzyme and also higher than that of a simple physical combination of the two.

[0037] Superior performance: Due to the synergistic effect of the two domains, this fusion enzyme exhibits higher hydrolytic activity and higher thermal stability (optimal temperature 50℃, with a prolonged half-life at 50℃), making it more suitable for industrial applications.

[0038] One enzyme, multiple uses: As a single protein, it solves the problems of complex formulations, high costs, and poor stability of physical compound formulations, and simplifies downstream application processes. Attached Figure Description

[0039] Figure 1 This is a schematic diagram of the structure of the bifunctional xylanase fusion protein of the present invention.

[0040] Figure 2 SDS-PAGE analysis of bifunctional xylanase fusion protein.

[0041] Figure 3 This is a comparison of the relative enzyme activities of the fusion enzyme of the present invention with those of the parental xylanases GH10 and GH11, wherein G1 is GH11-1; G2 is GH11-2; G3 is GH10+GH11-2; G4 is GH10+GH11-1; G5 is GH10+GH11-1+GH11-2, and the fusion proteins are A~P.

[0042] Figure 4 The enzymatic properties of GH10 and GH11 xylanases are shown in Figure 1. A represents the optimal reaction temperature, B represents the optimal reaction pH, C represents the thermal stability analysis of the enzymes, and D represents the pH stability analysis of the enzymes.

[0043] Figure 5 The enzymatic properties of fusion proteins B, N, P, K, and L are shown in Figure 1. A represents the optimal reaction temperature, B represents the optimal reaction pH, C represents the thermal stability analysis of the enzyme, and D represents the pH stability analysis of the enzyme.

[0044] Figure 6 ESI-MS analysis of insoluble dietary fiber hydrolyzed by GH10 and GH11 complex enzymes: A represents insoluble dietary fiber FOs from wheat bran, B represents insoluble dietary fiber FOs from corn cob, and C represents insoluble dietary fiber FOs from buckwheat straw.

[0045] Figure 7 ESI-MS analysis of hydrolyzed insoluble dietary fiber of fusion protein B: A represents insoluble dietary fiber FOs from wheat bran, B represents insoluble dietary fiber FOs from corn cob, and C represents insoluble dietary fiber FOs from buckwheat straw.

[0046] Figure 8 The predicted structure for fusion xylanase B. Detailed Implementation

[0047] This invention provides a fusion protein comprising GH10 xylanase and GH11 xylanase linked by a linker peptide.

[0048] The GH10 xylanase described in this invention was cloned using primers designed based on the gene sequence (SEQ ID NO.4) and the Bacillus hygroscopicus genome as a DNA template. The cloned DNA fragment was ligated into the vector pET-28a with EcoR I and Hind III restriction sites. The recombinant expression vector pET-28a-GH10 was then transformed into the host E. coli BL21(DE3).

[0049] The GH11-1 xylanase described in this invention was cloned using primers designed based on the gene sequence (SEQ ID NO.5) and the Bacillus amyloliquefaciens genome as a DNA template. The cloned DNA fragment was ligated into the vector pET-28a with EcoR I and Hind III restriction sites. The recombinant expression vector pET-28a-GH11-1 was then transformed into the host E. coli BL21(DE3).

[0050] The GH11-2 xylanase described in this invention was cloned using primers designed based on the gene sequence (SEQ ID NO.6) and the Bacillus hygroscopicus genome as a DNA template. The cloned DNA fragment was ligated into the vector pET-28a with EcoR I and Hind III restriction sites. The recombinant expression vector pET-28a-GH11-2 was then transformed into the host E. coli BL21(DE3).

[0051] This invention utilizes flexible or rigid linker peptides to link GH10 xylanase and GH11 xylanase. During linking, GH10 xylanase can be fused to the C-terminus or N-terminus of GH11 xylanase. GH11 xylanase is either GH11-1 or GH11-2 xylanase. The linker peptide can be flexible or rigid, wherein the flexible linker peptide includes S1 (GGGGS)1 or S2 (GGGGS)2, and the rigid linker peptide includes R1 (EAAAK)1 or R2 (EAAAK)2. Based on different combinations, 16 fusion proteins were constructed and named fusion proteins A…P (…). Figure 1 ).

[0052] Table 1 Primers for fusing xylanase and xylanase

[0053]

[0054]

[0055]

[0056] In one embodiment of the present invention, using pET28a-GH10, pET28a-GH11-1, and pET28a-GH11-2 plasmids as templates, DNA fragments containing linker peptides GH10, GH11-1, and GH11-2 were cloned using the primers in Table 1. The GH10 and GH11 fragments were fused using homologous recombinase and then transformed into the host E. coli BL21(DE3) to obtain recombinant strains RB-A to RB-P as shown in Table 2.

[0057] Table 2 Design features of fused xylanase

[0058]

[0059]

[0060]

[0061] In one embodiment of the present invention, the amino acid sequence of the obtained fusion protein A…P includes SEQ ID NO.1+linker+ SEQ ID NO.2, SEQ ID NO: 1+linker+ SEQ ID NO.3, SEQ ID NO.2+linker+ SEQ ID NO.1, SEQ ID NO.3+linker+ SEQ ID NO.1, the amino acid sequence of the rigid linker is (EAAAK)n (n=1-2), and the amino acid sequence of the flexible linker is (GGGGS)n (n=1-2), for a total of 16 amino acid sequences.

[0062] In one embodiment of the present invention, the gene sequence of the obtained fusion protein A…P includes SEQ ID NO. 4 + linker base + SEQ ID NO. 5, SEQ ID NO. 4 + linker base + SEQ ID NO. 6, SEQ ID NO. 5 + linker base + SEQ ID NO. 4, SEQ ID NO. 6 + linker base + SEQ ID NO. 4, and the rigid linker base sequence is (GAAGCCGCCGCCAAG). n (n=1-2), the base sequence of the flexible linker is (GGTGGTGGCGGTAGC). n (n=1-2), a total of 16 DNA gene sequences.

[0063] The expression of the fusion protein RB-A ~ RB-P in recombinant bacteria was induced using IPTG. The specific method was as follows: Recombinant bacteria were inoculated into LB medium at a 1‰ inoculum rate and cultured at 37℃ and 200 rpm for 12 h; then, 2% inoculum was added to LB medium and cultured at 37℃ and 200 rpm until the expression of the fusion protein was induced. 600 When the concentration reached approximately 0.8, 0.4 mM IPTG was added, and the mixture was incubated at 20°C and 200 rpm for 24 h. The bacterial cells were collected by centrifugation, and the supernatant was collected after lysis. The supernatant contained the target fusion protein, and its expression was analyzed by SDS-PAGE. The results are as follows: Figure 2 As shown, fusion proteins A through P were successfully expressed.

[0064] Using beech xylan as a substrate, with an addition amount of 0.5% (w / v), 1.5 mL of substrate and 0.5 mL of enzyme solution were reacted at pH 6.0 and 50℃ for 10 min. The amount of reducing sugar produced was determined by the DNS method. The amount of enzyme required to produce 1 μmol xylose per minute is defined as one enzyme activity unit (U).

[0065] The relative enzyme activity of the above fusion proteins was measured. The results showed that, compared with the enzyme activities of GH10 xylanase, GH11 xylanase, and the combination of GH10 and GH11, the relative enzyme activity of fusion protein B was increased by 1.22 times. Figure 3 Furthermore, the optimal temperature for this fused xylanase is 50°C, the optimal pH is 6.0, and it can maintain more than 50% of its relative enzyme activity within a pH range of 4.0-9.0. Figure 5 ).

[0066] This invention determined the thermostability of several fusion proteins B, N, P, K, and L. The thermostability of the fusion xylanases was evaluated by measuring the remaining enzyme activity after incubation at 50°C for different times (0-360 min). Compared to GH11 xylanase ( Figure 4 ), fusion protein B (T 1 / 2 =360 min) thermal stability is better than GH11-2 (T =360 min) 1 / 2 =50 min) increased by 7.2 times, and the thermal stability of other fusion proteins also showed a significant improvement ( Figure 5 ).

[0067] This invention uses insoluble dietary fiber from wheat bran, corn cob, and buckwheat straw as substrates to determine the hydrolysis efficiency of fused xylanase B (224 U, calculated based on beech xylan as substrate), GH10 enzyme, and GH11 enzyme (added at 102 U and 122 U respectively, calculated based on beech xylan as substrate). The insoluble dietary fiber addition ratio is 1:20 (w:v). Hydrolysis is carried out at 50℃ and pH 6 for 6 h. After the reaction is completed, the yield of FOs is measured.

[0068] The results showed that the FOs yields from the hydrolysis of insoluble dietary fiber from wheat bran, corn cob, and buckwheat straw by the fusion xylanase B were 1.38 mM, 1.43 mM, and 0.76 mM, respectively; while the FOs yields from the hydrolysis of the same insoluble dietary fiber by the compound enzyme were 1.12 mM, 1.16 mM, and 0.59 mM, respectively. This demonstrates a significant synergistic effect between the two domains within the fusion xylanase B. The FOs produced by the fusion xylanase B had degrees of polymerization of 3-7, 3-7, and 3-4, respectively. The FOs produced by the compound enzyme had degrees of polymerization of 3-9, 3-10, and 3-5, respectively. The FOs obtained from the hydrolysis of the fusion protein B had lower molecular weights and narrower degrees of polymerization, with a predominance of degree of polymerization of 3.

[0069] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A bifunctional xylanase fusion protein, characterized in that, The fusion protein is linked to GH10 xylanase and GH11 xylanase via a linker peptide. The amino acid composition of the fusion protein consists of GH10 and amino acids with a similarity greater than 70%, a linker, and GH11 and amino acids with a similarity greater than 70%. The linker includes a flexible linker peptide or a rigid linker peptide. The amino acid sequence of the flexible linker peptide is (GGGGS). n The amino acid sequence of the rigid linker peptide is (EAAAK). n , where n is 1 or 2.

2. The bifunctional xylanase fusion protein according to claim 1, characterized in that, GH10 xylanase is derived from Bacillus hygroscopicus, while GH11 xylanase is derived from either Bacillus hygroscopicus or Bacillus amyloliquefaciens.

3. The bifunctional xylanase fusion protein according to claim 2, characterized in that, The amino acid sequence of GH10 xylanase is shown in SEQ ID NO.1, and the amino acid sequence of GH11 xylanase is GH11-1 or GH11-2, as shown in SEQ ID NO.2 or SEQ ID NO.

3.

4. The bifunctional xylanase fusion protein according to any one of claims 3, characterized in that, The amino acid sequence of the fusion protein consists of the amino acid sequence of GH10 xylanase, the linker, and the amino acid sequence of GH11 xylanase, as shown in SEQ ID NO.1+linker+SEQ ID NO.2 and SEQ ID NO.1+linker+SEQ ID NO.

3.

5. A DNA gene sequence, characterized in that, Nucleotides encoding the bifunctional xylanase fusion protein according to any one of claims 1 to 4.

6. A method for expressing the bifunctional xylanase fusion protein according to any one of claims 1 to 4, characterized in that, The method includes the following steps: cloning the gene encoding the fusion protein according to any one of claims 1 to 4 into an expression vector to construct a recombinant expression vector; transforming the recombinant expression vector into a host cell to obtain a recombinant strain; culturing the recombinant strain to induce expression of the fusion protein; isolating the recombinant bacterial cells from the culture and obtaining a cell-free bifunctional xylanase fusion protein by lysis.

7. The method according to claim 6, characterized in that, The bifunctional xylanase fusion protein obtained by the method has a molecular weight of 68.4 kDa.

8. The method according to claim 7, characterized in that, The method yields fusion proteins of fusion protein B, fusion protein N, fusion protein P, fusion protein K, or fusion protein L, and the thermal stability of fusion proteins B, N, P, K, and L is increased several times compared to GH11 xylanase.

9. The method according to claim 8, characterized in that, Fusion protein B (T) 1 / 2 =360 min) thermal stability is better than GH11-2 (T =360 min) 1 / 2 =50 min) increased by 7.2 times, the optimal reaction temperature of the fusion protein was 50℃, and the optimal reaction pH was 6.

0.

10. The use of the bifunctional xylanase fusion protein prepared by any one of claims 6-9 in the preparation of ferulic acid oligosaccharides, characterized in that, The feruloyl oligosaccharide has a relatively small molecular weight and a narrow degree of polymerization distribution, with a degree of polymerization of 3 being the most common.

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