Chimeric xylanase XynZT-1-Z6 as well as construction method and application thereof

Abstract

The invention provides a chimeric xylanase XynZT-1-Z6 as well as a construction method and application of the chimeric xylanase XynZT-1-Z6. A strain Alteromonas macleodii HY35 capable of degrading xylan is separated from a marine environment, new xylanase XynZT-1 is excavated by comprehensively analyzing genomics and transcriptomics data, the specific sequence structure of the xylanase XynZT-1 is analyzed, recombinant expression is performed on the xylanase XynZT-1, and it is proved that the xylanase XynZT-1 can hydrolyze xylanase. In addition, the xylanase is subjected to mutation evolution through a method based on rational design, and the xylanase mutant XynZT-1-Z6 of which the enzyme catalytic activity and the thermal stability are greatly improved is obtained. In conclusion, the brand-new chimeric xylanase is obtained and has remarkable novelty, innovativeness and practicability, and the application potential of the enzyme is greatly improved due to the fact that the mutant with high thermal stability is obtained.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial enzymes, and in particular relates to a chimeric xylanase XynZT-1-Z6, a construction method and application thereof. Background Art

[0002] Xylan is a heterogeneous polysaccharide present in plant cell walls, accounting for about 15% to 35% of the dry weight of plant cells, and is the main component of plant hemicellulose. Most xylans are complex, highly branched heterogeneous polysaccharides containing many different substituents. The biodegradation of xylan therefore requires a complex enzyme system to degrade xylan through the synergistic effects of various components.

[0003] Xylanases are widely distributed in nature and can be obtained from animals, plants and microorganisms. For example, xylanases are found in marine and terrestrial bacteria, marine algae, fungi, yeasts, rumen and ruminant bacteria, snails, crustaceans, terrestrial plant tissues and various invertebrates. Xylanases from microbial sources are ubiquitous in nature, with a wide variety of species and a wide range of applications. Therefore, there are many reports on the research of microbial xylanases. Xylanases are mostly single domains in structure, with a right-handed half-grip shape, a relative molecular mass of less than 30 kDa, and an optimal pH value of generally acidic to neutral range, but they are specific to xylan substrates from different sources, and the hydrolysis products are mainly oligoxylose, with less monosaccharide content. Among them, endo-1,4-β-xylanases belonging to the GH11 family (Glycoside Hydrolase Family 11) can hydrolyze the β-1,4-glycosidic bonds in the xylan backbone and convert polymeric xylanases into oligoxylose. GH11 xylanases play an important role in sugar metabolism and are one of the most widely used enzymes in various industries such as pulp and paper, food and feed, biorefining, textiles and pharmaceuticals.

[0004] Since many industrial production processes need to be carried out under high temperature conditions, poor thermal stability has become a bottleneck for most natural xylanases in industrial applications. Compared with GH10 family xylanases, GH11 family xylanases have the advantages of high catalytic efficiency and good substrate specificity. In order to make the application of GH11 family xylanases with excellent enzymatic properties unrestricted under high temperature conditions, the improvement of thermal stability of GH11 family xylanases has become one of the important issues to be solved in this field. Summary of the invention

[0005] In view of the deficiencies in the prior art, the object of the present invention is to provide a chimeric xylanase XynZT-1-Z6 with good thermal stability, a construction method and application thereof.

[0006] In a first aspect of the present invention, a chimeric xylanase XynZT-1-Z6 is provided.

[0007] In another preferred embodiment, the amino acid sequence of the chimeric xylanase has more than 95% homology with the amino acid sequence shown in SEQ ID NO.3, preferably 96% homology, more preferably 97%, 98%, 99% or 100% homology, and has the ability to hydrolyze xylan.

[0008] In another preferred example, the amino acid sequence of the chimeric xylanase is shown in SEQ ID NO.3.

[0009] In a second aspect of the present invention, a biomaterial related to the chimeric xylanase of the first aspect of the present invention is provided, wherein the biomaterial comprises at least one of the following 1)-5):

[0010] 1) A gene encoding the chimeric xylanase according to the first aspect of the present invention;

[0011] 2) an expression cassette containing the coding gene described in 1);

[0012] 3) a recombinant vector containing the coding gene described in 1);

[0013] 4) A recombinant microorganism or recombinant cell containing the coding gene described in 1);

[0014] 5) A recombinant microorganism or recombinant cell containing the recombinant vector described in 3).

[0015] Further preferably, in 1), the nucleotide sequence of the encoding gene is a nucleotide sequence having more than 95% homology with the nucleotide sequence shown in SEQ ID NO.4, preferably 96% homology, more preferably 97%, 98%, 99% or 100% homology.

[0016] Further preferably, in 1), the nucleotide sequence of the encoding gene is as shown in SEQ ID NO.4.

[0017] More preferably, in 3), the recombinant vector uses pET-28a(+) vector as an expression vector.

[0018] More preferably, in 4) or 5), the recombinant microorganism is recombinant Escherichia coli BL21 (DE3).

[0019] More preferably, in 4) or 5), the recombinant cell is a recombinant animal cell or a recombinant human cell.

[0020] In the third aspect of the present invention, a method for constructing the chimeric xylanase described in the first aspect of the present invention is provided, characterized in that, based on the xylanase XynZT-1 derived from Alteromonas macleodii, the structural sequence of XynZT-1 is compared with the heat-resistant enzyme derived from thermophilic bacteria; and according to the conservation of amino acids, the original amino groups of β10-β11 are replaced with the corresponding amino acids that are relatively conserved in the heat-resistant enzyme to construct the chimeric xylanase XynZT-1-Z6, and the amino acid sequence of the xylanase XynZT-1 is shown in SEQ ID NO.1.

[0021] In a fourth aspect of the present invention, provided is the use of the chimeric xylanase according to the first aspect of the present invention or the biomaterial according to the second aspect of the present invention in hydrolyzing xylan.

[0022] In a fifth aspect of the present invention, provided is the use of the chimeric xylanase according to the first aspect of the present invention or the biomaterial according to the second aspect of the present invention in xylose production.

[0023] In the sixth aspect of the present invention, there is provided the use of the chimeric xylanase of the first aspect of the present invention or the biomaterial of the second aspect of the present invention in the fields of pulp and paper, food and feed, biorefining, textile and pharmaceutical.

[0024] In a seventh aspect of the present invention, a method for hydrolyzing xylan is provided, characterized in that the method comprises the step of contacting or mixing xylan with the chimeric xylanase according to the first aspect of the present invention.

[0025] Further preferably, the contacting and mixing are carried out under temperature and pH conditions suitable for the chimeric xylanase of the first aspect of the invention.

[0026] Further preferably, the contacting and mixing are performed at the optimal temperature and optimal pH of the chimeric xylanase according to the first aspect of the present invention.

[0027] More preferably, the optimum temperature is 75°C.

[0028] More preferably, the optimum pH is 6.

[0029] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

[0030] Compared with the prior art, the present invention has the following advantages and progress:

[0031] The present invention isolates a strain of xylan-degrading strain Alteromonas macleodiiHY35 from the marine environment, and through comprehensive analysis of genomic and transcriptomic data, discovers a new xylanase XynZT-1, analyzes its specific sequence structure, and recombinantly expresses it, proving that it can hydrolyze xylan. In addition, the ambient temperature of xylanase is generally high, and there is a high demand for the heat resistance of the enzyme. Therefore, the present invention performs mutation evolution on xylanase based on a rational design method, and obtains a xylanase mutant XynZT-1-Z6 with greatly improved enzyme catalytic activity and thermal stability. In summary, the present invention obtains a brand-new chimeric xylanase with significant novelty, innovation and practicality. The acquisition of this high thermal stability mutant greatly enhances the application potential of the enzyme. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0033] Figure 1 is a flow chart of the method for constructing the chimeric xylanase XynZT-1-Z6 in an embodiment of the present invention;

[0034] Figure 2 is a sequence comparison diagram of XynZT-1 and a thermostable enzyme in an embodiment of the present invention;

[0035] Figure 3 This is the double restriction enzyme digestion map of pET28a-xynZT-1 in the embodiment of the present invention;

[0036] Figure 4 This is the double restriction enzyme digestion map of pET28a-xynZT-1-Z6 in the embodiment of the present invention;

[0037] Figure 5 It is a protein purification diagram of XynZT-1-Z6 and XynZT-1 in the embodiment of the present invention;

[0038] Figure 6 is a comparison chart of the enzyme activities of XynZT-1-Z6 and XynZT-1 in the embodiments of the present invention;

[0039] Figure 7 is a comparison of the optimum temperatures for the action of XynZT-1-Z6 and XynZT-1 in the embodiments of the present invention;

[0040] Figure 8 It is a comparison of the thermal stability of XynZT-1-Z6 and XynZT-1 at 50°C in the examples of the present invention;

[0041] Fig. 9is the thermal stability of XynZT-1-Z6 at 85°C in the embodiment of the present invention;

[0042] Fig.10 The optimal pH comparison between XynZT-1-Z6 and XynZT-1 in the embodiments of the present invention;

[0043] Fig.11 It is a comparison of the pH stability of XynZT-1-Z6 and XynZT-1 in the examples of the present invention. DETAILED DESCRIPTION

[0044] The present invention provides a chimeric xylanase XynZT-1-Z6, a construction method and application thereof. Specifically, the present invention screens Alteromonas macleodii from the ocean, and performs genome sequencing on Alteromonas macleodii, finding that it contains a xylanase gene XynZT-1, and analyzing the sequence finds that it belongs to the GH11 family.

[0045] The structural sequence of XynZT-1 was compared with that of 8 thermostable enzymes from thermophilic bacteria ( Figure 2 The PDB accession numbers of thermostable enzymes are given in the , and the thermophilic bacteria and related literature of the corresponding enzyme sources can be found in the PDB database through the accession numbers). According to the conservation of amino acids, the original amino groups of β10-β11 were replaced with the corresponding amino acids that are relatively conservative for thermostable enzymes ( Figure 2 and Table 1), to construct the chimeric xylanase XynZT-1-Z6.

[0046] Table 1 XynZT-1-Z6 replacement sequence

[0047]

[0048] The present invention is specifically described below in conjunction with the embodiments to facilitate further understanding of the present invention by those skilled in the art. However, the embodiments described below are only a part of the embodiments of the present invention and should not be regarded as any form of limitation of the present invention. It should be pointed out that the adjustments and improvements made by those of ordinary skill in the art based on the concept of the present invention should be regarded as the protection scope of the present invention. The specific technical operation steps and operators are not indicated in the embodiments, and are carried out according to the general technical conditions described in the literature of the art or the relevant product specifications.

[0049] Example 1 Construction of recombinant expression plasmid of xylanase XynZT-1

[0050] From the genome information of Alteromonas macleodii HY35, a GH11 family xylanase was mined through bioinformatics technology and named XynZT-1. According to the gene sequence of xylanase xynZT-1, EcoRI and HindШ restriction sites were added at both ends, and then synthesized by a gene company according to the method of chemical total synthesis. Then, it was connected with the pET-28a(+) expression vector linearized by double restriction enzymes to construct the recombinant expression plasmid pET-28a(+)-xynZT-1 of xylanase XynZT-1, which was transferred into Escherichia coli Top10 for amplification. After the plasmid was extracted, it was verified to be completely correct by sequencing.

[0051] Xylanase XynZT-1 amino acid sequence:

[0052] ISLDNDSEAFKPQGITKFKAYTKQFDDDIHMSKNHATGHVGDLFFTHWKDGGEASLK

[0053] LSQSGEFEVSWEGGGYNYVGGPGWHYGDVSREIGYRFEQESGASYIALYGWGYDSN

[0054] MSKDNPAHLVEYYVLQRWTYDPSQNGIFGKTFVSNGVEYSTYRTIRERKPSINNTATF

[0055] YQYWSKPPSKPMPLGEDHKIIFADHVAAWADTGWILPDMNNFDASDDPTYQVMAVEVFNPAKDGKATGKVWDASAQF (SEQ ID NO. 1).

[0056] Xylanase XynZT-1 gene DNA sequence:

[0057] ATAAGTCTTGATAATGACTCTGAAGCTTTCAAGCCGCAAGGTATTACTAAATTTAA

[0058] AGCCTATACTAAGCAATTCGACGATGACATACACATGTCTAAAAACCACGCAACA

[0059] GGACATGTTGGGGATCTCTTTTTTACCCACTGGAAAGACGGTGGCGAAGCCTCTCT

[0060] CAAACTAAGTCAAAGCGGAGAATTTGAAGTGAGTTGGGAGGGCGGCGGATATAAT

[0061] TATGTTGGGGGCCCCGGGTGGCATTACGGAGATGTGAGCCGCGAAATTGGCTATC

[0062] GCTTTGAACAGGAGAGTGGCGCTAGTTATATTGCACTCTATGGATGGGGATACGA

[0063] CAGCAACATGTCTAAAGACAATCCTGCTCACTTAGTTGAATATTATGTTCTTCAGC

[0064] GCTGGACCTATGATCCTAGCCAAAACGGCATTTTCGGTAAGACCTTTGTGAGTAAC

[0065] GGCGTTGAATACTCAACTTATCGAACTATACGAGAGCGAAAGCCCTCTATAAATA

[0066] ATACGGCCACGTTTTATCAGTATTGGAGTAAACCGTCTAAACCCATGCCTCTAGGC

[0067] GAAGATCATAAAATTATCTTTGCCGATCACGTGGCAGCTTGGGCTGACACAGGCTG

[0068] GATTCTTCCTGATATGAATAATTTTGACGCAAGCGACGACCCTACATATCAAGTTA

[0069] TGGCAGTAGAAGTATTTAATCCTGCAAAGGACGGTAAGGCGACTGGTAAAGTGTGGGATGCTAGTGCTCAATTTTAA(SEQ ID NO.2).

[0070] Primer sequences for amplifying the xynZT-1 gene:

[0071] ZT-1-F: CCGGAATTCATCTCTCTTGATAACG

[0072] ZT-1-R: CCCAAGCTTTTAGAACTGGGCGGAGGCAT

[0073] Table 1 PCR reaction process

[0074]

[0075] Table 2 PCR reaction system

[0076]

[0077] The genomic DNA of Alteromonas macleodii HY35 was extracted as a template, and PCR amplification was performed using primers ZT-1-F and ZT-1-R. The amplified product was detected by 1% agarose gel electrophoresis. The results are shown in Figure 3 As shown in lane 1, the detection band is about 744bp, which is consistent with the theoretical size. The clone constructed by the company was double-digested and the results are as follows Figure 3 As shown in lane 2, they are consistent with the prediction, indicating that the construction is successful.

[0078] Example 2 Construction of the recombinant expression plasmid of the xylanase chimera XynZT-1-Z6

[0079] According to the gene sequence of xylanase xynZT-1-Z6, EcoRI and HindШ restriction sites were added to both ends of the gene, and then it was synthesized by a gene company according to the method of chemical total synthesis. Then, it was connected with the pET-28a(+) expression vector linearized by double restriction enzyme digestion to construct the recombinant expression plasmid pET-28a(+)-xynZT-1-Z6 of chimeric xylanase XynZT-1-Z6, and transferred into Escherichia coli Top10 for amplification. After the plasmid was extracted, double restriction enzyme digestion was performed. The results of PCR amplification and double restriction enzyme digestion are shown in Figure 4 , which was verified by sequencing to be completely correct.

[0080] Chimeric xylanase XynZT-1-Z6 amino acid sequence:

[0081] ISLDNDSEAFKPQGITKFKAYTKQFDDDIHMSKNHATGHVGDLFFTHWKDGGEASLK

[0082] LSQSGEFEVSWEGGGYNYVGGPGWHYGDVSREIGYRFEQESGASYIALYGWGYDSN

[0083] MSKDNPAHLVEYYVLQRWTYDPSQNGTYMGTVTTDGGTYDIYRTTRYNAPSIEGTAT

[0084] FYQYWSKPSKPMPLGEDHKIIFADHVAAWADTGWILPDMNNFDASDDPTYQVMAVEVFNPAKDGKATGKVWDASAQF(SEQ ID NO.3).

[0085] DNA sequence of the chimeric xylanase gene xynZT-1-Z6:

[0086] ATAAGTCTTGATAATGACTCTGAAGCTTTCAAGCCGCAAGGTATTACTAAATTTAA

[0087] AGCCTATACTAAGCAATTCGACGATGACATACACATGTCTAAAAACCACGCAACA

[0088] GGACATGTTGGGGATCTCTTTTTTACCCACTGGAAAGACGGTGGCGAAGCCTCTCT

[0089] CAAACTAAGTCAAAGCGGAGAATTTGAAGTGAGTTGGGAGGGCGGCGGATATAAT

[0090] TATGTTGGGGGCCCCGGGTGGCATTACGGAGATGTGAGCCGCGAAATTGGCTATC

[0091] GCTTTGAACAGGAGAGTGGCGCTAGTTATATTGCACTCTATGGATGGGGATACGA

[0092] CAGCAACATGTCTAAAGACAATCCTGCTCACTTAGTTGAATATTATGTTCTTCAGC

[0093] GCTGGACCTATGATCCTAGCCAAAACGGCACGTACATGGGCACGGTCACGACGGA

[0094] CGGCGGCACGTACGACATCTACCGCACGACGCGCTACAACGCTCCGTCCATCGAG

[0095] GGCACGGCCACGTTTTATCAGTATTGGAGTAAACCGTCTAAACCCATGCCTCTAGG

[0096] CGAAGATCATAAAATTATCTTTGCCGATCACGTGGCAGCTTGGGCTGACACAGGCT

[0097] GGATTCTTCCTGATATGAATAATTTTGACGCAAGCGACGACCTACATATCAAGTT

[0098] ATGGCAGTAGAAGTATTTAATCCTGCAAAGGACGGTAAGGCGACTGGTAAAGTGTGGGATGCTAGTGCTCAATTTTAA (SEQ ID NO. 4).

[0099] Example 3 Inducible expression of XynZT-1 and XynZT-1-Z6 in Escherichia coli

[0100] The plasmids pET-28a(+)-xynZT-1 and pET-28a(+)-xynZT-1-Z6 were transformed into competent cells of E. coli BL21(DE3) according to the following method to obtain XynZT-1 and XynZT-1-Z6 mutant engineering strains E. coli BL21(DE3) / pET-28a(+)-xynZT-1 and E. coli BL21(DE3) / pET-28a(+)-xynZT-1-Z6. The specific steps are:

[0101] (1) Take 5 μL of recombinant plasmid sample and add it to 50-100 μL of competent cells, mix slowly and place on ice for 30 min.

[0102] (2) Heat shock for 90 seconds (42°C water bath), then quickly return to the ice bath and wait for 3 to 5 minutes.

[0103] (3) Add 500 μL LB liquid culture medium (without antibiotics), mix gently, and culture with shaking for 1 h (37°C).

[0104] (4) Centrifuge the bacterial solution (5000 rpm, 1 min) to precipitate the bacteria and aspirate most of the supernatant (about 50-100 μL of the remaining resuspended bacteria).

[0105] (5) Spread the bacterial solution evenly on LB plates (containing Kana antibiotics) and culture in a 37°C incubator overnight.

[0106] (6) Pick a single colony on the LB plate and culture it. Take 50 μL of the bacterial solution and send it for testing to determine whether the recombinant strain is correct.

[0107] The successfully constructed genetically engineered strains E. coli BL21 (DE3) / pET28a-xynZT-1 and E. coli BL21 (DE3) / pET28a-xynZT-1-Z6 were inoculated into 5 mL of LB medium containing Kana and cultured overnight at 37 ° C, 220 rpm. As seed liquid, they were inoculated into 200 mL of LB medium containing Kana and cultured at 37 ° C, 220 rpm until OD 600 When the p-value reaches about 0.6, add 200 μL IPTG (0.5 M) and induce expression at 12°C for 20-24 h.

[0108] Take the induced culture and centrifuge it at 4500rpm for 20min, and discard the supernatant. Add 15mL of lysis buffer (pH7.0) to the precipitate and oscillate it to fully suspend it. Place it on ice for ultrasonic disruption (power 260W, total disruption time 25min, working time 2s, interval time 4s). After ultrasonic disruption, centrifuge at 4℃ for 40min (8000rpm), and the resulting supernatant is the crude enzyme solution. Use nickel column HisTrap TM HP-5mL to purify the recombinant enzyme.

[0109] The enzyme solution was added with 5×SDS loading buffer, mixed, boiled for 10 min, and then centrifuged at 12000 rpm for 10 min. The supernatant was subjected to SDS-PAGE protein electrophoresis with 5% concentrated gel and 12% separation gel, stained with Coomassie Brilliant Blue R-250 for 2 h, and destained with decolorant for 3 h, changing the decolorant once in the middle, and observing the results. Figure 5 As shown in the figure, the size of the overexpressed xylanase XynZT-1 and XynZT-1-Z6 protein is about 26kD, which is consistent with the expected size, indicating that the overexpression of xylanase enzyme genes xynZT-1 and xynZT-1-Z6 in E. coli was successfully achieved, and the expression was soluble. The results of SDS-PAGE showed that the purified recombinant proteins XynZT-1 and XynZT-1-Z6 reached electrophoretic purity ( Figure 5 , lane 1: XynZT-1, lane 2: XynZT-1-Z6).

[0110] Example 4 Enzyme activity determination and xylanase degradation activity evaluation

[0111] The two enzymes XynZT-1 and XynZT-1-Z6 were diluted to 0.0005 mg·mL-1, and 1% birch xylan was used as substrate for the reaction. The OD value at 540 nm was measured by the DNS method, and the amount of xylose was converted. We define the amount of enzyme required to hydrolyze xylan to produce 1 μmol of xylose per unit time (per minute) as an enzyme activity unit U. By calculation, it can be found that the enzyme activities of XynZT-1 and XynZT-1-Z6 are 0.046U and 1.12U, respectively. Using the origin software to draw a graph, the relative enzyme activity relationship between the two can be obtained as follows Figure 6 It can be clearly seen that the specific enzyme activity of XynZT-1-Z6 is higher, 24.35 times that of XynZT-1, which significantly improves the ability of xylanase to catalyze the decomposition of xylan.

[0112] Example 5 Evaluation of thermal and pH stability of XynZT-1 and XynZT-1-Z6

[0113] The optimum temperature and temperature stability of the recombinant enzyme were determined: in an environment of pH = 4.6, the enzyme solution and the substrate were reacted at different temperatures (35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C) for 15 minutes, and the enzyme activity of the recombinant enzyme was determined respectively, and its relative enzyme activity was calculated to determine the optimum reaction temperature of the xylanase. The results are shown in Figure 7 The recombinant enzyme solution was incubated at different temperatures (45°C, 75°C) for 1 hour (pH = 4.6). Samples were taken every 5 minutes, and then the residual enzyme activity was measured. The activity of the unincubated enzyme sample was taken as 100% to determine the stability of the recombinant enzyme solution. The results are shown in Figure 8 and Fig. 9 .

[0114] Depend on Figure 7 It can be seen that the optimum temperature of XynZT-1 is 45°C, and the optimum temperature of XynZT-1-Z6 is 75°C. Figure 8 The results showed that the relative enzyme activity of XynZT-1 was basically lost after being kept at 45℃ for 10 minutes, but the enzyme activity of XynZT-1-Z6 was still maintained above 90% after being kept at 45℃ for 25 minutes. In addition, the enzyme activity of XynZT-1-Z6 was still maintained above 75% after being kept at 75℃ for 60 minutes, showing high thermal stability ( Fig. 9 ).

[0115] The optimal pH and pH stability of the recombinant enzyme were determined: the relative enzyme activity was determined at different pH environments (pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0) and at the optimal reaction temperature (XynZT-1 45°C, XynZT-1-Z6 75°C), and the optimal pH of the recombinant enzyme was obtained. The results are shown in Fig.10 The recombinant enzyme was incubated at the optimal temperature for 1 hour under different pH conditions, and its residual enzyme activity was measured and compared with the enzyme activity without incubation to determine the pH stability of the recombinant enzyme. The results are shown in Fig.11 .

[0116] Depend on Fig.10 It can be seen that the optimal pH of XynZT-1 is 5, and the optimal pH of XynZT-1-Z6 is 6. Fig.11 The results showed that after the two enzymes were kept in different pH buffers for 60 minutes, the two enzymes exhibited different relative enzyme activities. XynZT-1 showed the best stability at pH 6, while XynZT-1-Z6 had better stability at pH 7.

[0117] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. For those skilled in the art, any modification and change made to the above embodiment according to the technical essence of the present invention shall fall within the protection scope of the technical solution of the present invention.

Claims

1. A chimeric xylanase XynZT-1-Z6, characterized in that The amino acid sequence of the chimeric xylanase has more than 95% homology with the amino acid sequence shown in SEQ ID NO.3, preferably 96% homology, more preferably 97%, 98%, 99% or 100% homology, and has the ability to hydrolyze xylan.

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

3.

3. A biological material related to the chimeric xylanase according to claim 1 or 2, wherein the biological material comprises at least one of the following 1)-5): 1) A gene encoding the chimeric xylanase according to claim 1 or 2; 2) an expression cassette containing the coding gene described in 1); 3) a recombinant vector containing the coding gene described in 1); 4) A recombinant microorganism or recombinant cell containing the coding gene described in 1); 5) A recombinant microorganism or recombinant cell containing the recombinant vector described in 3).

4. The biomaterial according to claim 3, characterized in that The nucleotide sequence encoding the gene in 1) is a nucleotide sequence having more than 95% homology with the nucleotide sequence shown in SEQ ID NO.4, preferably 96% homology, more preferably 97%, 98%, 99% or 100% homology.

5. The method for constructing a chimeric xylanase according to claim 1 or 2, characterized in that: Based on the xylanase XynZT-1 from Alteromonas macleodii, the structural sequence of XynZT-1 was compared with the heat-resistant enzyme from thermophilic bacteria; and according to the conservation of amino acids, the original amino groups of β10-β11 were replaced with the corresponding amino acids that are relatively conservative for the heat-resistant enzyme to construct a chimeric xylanase XynZT-1-Z6. The amino acid sequence of the xylanase XynZT-1 is shown in SEQ ID NO.

1.

6. Use of the chimeric xylanase according to claim 1 or 2, or the biomaterial according to claim 3 or 4 in hydrolyzing xylan.

7. Use of the chimeric xylanase according to claim 1 or 2 and the biomaterial according to claim 3 or 4 in xylose production.

8. Use of the chimeric xylanase according to claim 1 or 2, and the biomaterial according to claim 3 or 4 in the fields of pulp and paper, food and feed, biorefining, textile and pharmaceutical.

9. A method for hydrolyzing xylan, characterized in that: The method comprises the step of contacting or mixing xylan with the chimeric xylanase of claim 1 or 2.

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