Xylanase gene BaXynA, xylanase BaXynA and application of xylanase BaXynA

Abstract

The invention relates to a xylanase gene BaXynA, xylanase BaXynA and application of the xylanase BaXynA, and belongs to the technical field of gene engineering. The invention provides a xylanase gene BaXynA, and the nucleotide sequence of the xylanase gene BaXynA is as shown in SEQ ID NO: 1. The xylanase BaXynA coded according to the xylanase gene BaXynA has the effect of hydrolyzing wheat arabinoxylan and beech xylan, can keep good enzyme activity at 10-70 DEG C, is not influenced by chemical reagents and part of metal ions, has a promoting effect on the xylanase BaXynA at 10-30 DEG C, still has stable enzyme activity, and can be used for preparing the xylanase. The method can be used for decomposing xylan in industrial production of food, feed, papermaking, detergent, medicine and the like and aquaculture.

Description

Technical Field

[0001] The present invention relates to the technical field of gene engineering, and in particular to a xylanase gene BaXynA, a xylanase BaXynA and applications thereof. Background Art

[0002] Xylanases can degrade xylan into oligoxylose and xylose, mainly acting on enzymes at the terminal or internal glycosidic bonds, such as β-1,4-endo-xylanase, β-xylosidase, α-L-arabinofuranosidase, acetyl-xylan esterase and α-D-glucuronidase, etc. The xylanase system that has been discovered is commonly found in fungi, bacteria, and actinomycetes.

[0003] Industry has special working environment requirements for xylanases, such as high temperature, low temperature, acidity, alkalinity, high pressure, etc. Exploring xylanases in special environments has become a major research hotspot, and they are usually screened from alkaline environments, industrial wastewater and other environments. Alkaline microorganisms usually live in environments with a pH greater than 9, but the optimum temperature for the xylanases they produce is often neutral, and they can still maintain high activity in alkaline environments. However, these xylanases are difficult to have stable enzyme activity in low-temperature scenarios of 15°C to 30°C, such as low-temperature dough fermentation and aquatic feed, and may even lose their activity, making it difficult to meet production needs. Therefore, there is an urgent need to discover more xylanases with stable enzyme activity in low-temperature environments.

[0004] Based on this, the present invention is proposed. Summary of the invention

[0005] The object of the present invention is to provide a xylanase gene BaXynA, a xylanase BaXynA and an application thereof, so as to solve the problem in the prior art that the xylanase has low enzyme activity and unstable enzyme activity at medium and low temperatures.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a xylanase gene BaXynA, and the nucleotide sequence of the xylanase gene BaXynA is shown in SEQ ID NO:1.

[0008] The present invention provides a primer pair for amplifying the xylanase gene BaXynA. The nucleotide sequence of the primer pair is shown in SEQ ID NOs: 2-3.

[0009] The present invention provides a xylanase BaXynA encoded by the xylanase gene BaXynA. The amino acid sequence of the xylanase BaXynA is shown in SEQ ID NO:4.

[0010] The invention provides a recombinant vector for expressing xylanase BaXynA. The recombinant vector comprises the xylanase gene BaXynA and an empty vector; the empty vector is pET-28a.

[0011] The invention provides a recombinant bacterium expressing xylanase BaXynA. The recombinant bacterium comprises the xylanase gene BaXynA or the recombinant vector and a host bacterium; the host bacterium is Escherichia coli BL21 (DE3).

[0012] The invention provides application of the xylanase BaXynA in hydrolyzing xylan.

[0013] The present invention provides the use of the recombinant vector or the recombinant bacteria in preparing products for degrading xylan.

[0014] The present invention provides a method for degrading xylan, comprising the following steps:

[0015] (1) mixing a xylanase BaXynA solution with a substrate to obtain a mixed solution;

[0016] (2) hydrolyzing the mixed solution obtained in step (1) at a pH value of 5 to 8 and a temperature of 10 to 70° C.;

[0017] The xylanase BaXynA in step (1) is the xylanase BaXynA or the xylanase BaXynA expressed by the recombinant vector or the xylanase BaXynA expressed by the recombinant bacteria.

[0018] Preferably, the volume ratio of the xylanase BaXynA solution to the substrate in step (1) is 1:8-10;

[0019] The concentration of the xylanase BaXynA solution is 50-200 mg / L; the concentration of the substrate is 5-20 mg / mL.

[0020] Preferably, the substrate in step (1) is a substance containing xylan;

[0021] The xylan-containing material includes wheat arabinoxylan and beech xylan.

[0022] The present invention has the following technical effects and advantages:

[0023] The present invention provides a xylanase gene BaXynA, the nucleotide sequence of the xylanase gene BaXynA is shown in SEQ ID NO: 1. The xylanase BaXynA encoded by the xylanase gene BaXynA of the present invention has the function of hydrolyzing wheat arabinoxylan and beech xylan, can maintain good enzyme activity at 10-70°C, is not affected by chemical reagents and some metal ions, and Mg 2+ and Mn 2+ Ions have a promoting effect on it, and it still has stable enzyme activity at 10-30°C. It can be used to decompose xylan in industrial production of food, feed, papermaking, detergents, medicines, and aquaculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The gel electrophoresis diagram of the purified protein solution eluted with NTA buffer of different concentrations;

[0025] Figure 2 is the chromatogram of the product of xylan hydrolysis by xylanase BaXynA;

[0026] Figure 3 It is an enzymatic analysis diagram of xylanase BaXynA, Figure A shows the relative enzyme activity of xylanase BaXynA at different temperatures; Figure B shows the stability of xylanase BaXynA at different temperatures; Figure C shows the relative enzyme activity of xylanase BaXynA under different pH conditions of substrate; Figure D shows the stability of xylanase BaXynA treated with different pH;

[0027] Figure 4 Relative enzyme activity of xylanase BaXynA under different metal ion and chemical reagent conditions. DETAILED DESCRIPTION

[0028] The present invention provides a xylanase gene BaXynA, the nucleotide sequence of the xylanase gene BaXynA is shown in SEQ ID NO: 1;

[0029] The nucleotide sequence of SEQ ID NO:1 is as follows: ATGGTTCAGAAAGACGGGGCGTTTTATACCGGACAATATAGGAACTTATTTAAAGAGTATGGTTACAGCGATGCAGAAATTGCCGCGAAAGTCAAAAATACATGGGAAGAGTTATTTATTCTTGAAGGTCCGAACCGAATCTATTTCGAAGTGGGCGATGACCTTGGATACATGCTCGATACAGGTAACCATGATGTACGTACTGAAGGGATGAGTTACGGAATGATGATGGCGGTACAGATGGACGATAAAGACATCTTTGATCGCCTCTGGCGCTGGACCATGAAATATATGTATATGACAGAGGGAAAGCACGCTGGATATTTCGCCTGGTCATGTAGCCCCGAAGGGAAGAAGAATGCTTACGGTCCCGCTCCAGACGGCGAAGAGTACTTTGCCCTGGCCCTGTTCTTTGCAGCGCATAGGTGGGGCGATGGAGAGGACGAGGTGTTCAATTACAGCAAGCACGCAAAGGACCTTCTGCATACCTGTATTCATAATGGTGAAGATGGGGAAGGGTACCCAATGTGGAACCCTGATAACAAGTTAATCAAGTTTGTCCCTGAGTTCGAGTATTCAGATCCTTCTTATCATCTGCCTCACTTTTATGAACTCTTTGCCCTGTGGGCAAACGAGGAAGACCGCCCTTTCTGGAAGGAGGCCGCTGCCGCATCAAGAGAATACTTAAAGATCGCCTGTCACCCCGAGACGGGGCTTGCACCGGAATACGCCTTCTACGACGGGACACCGAATGACGAGCGGGGCTTTGGTCACTTTTTCAGTGACTCTTATCGGGTAGCGGCTAATATCGGACTTGATTATGAATGGTTTCGGGGATGAGTGGGAAACGGAGCTAGCTAATAAAATTCAAGGTTTCTTTGCTGATAAGGAACCGAGTGATTACCGGCGTTACAAGATTGACGGTACACCTTTTGAAGAAAAATCCCTTCACCCAGTAGGGCTTGTTGCCACCAATGCTA TGGCATCCTTGGCCACAGTTGACGGGCCTTATGCCCGACAGAACGTTGAACTTTTCTGGAACACGCCTGTGCGTTCCGGTGATAGACGCTATTATGATAATTGTTTATATCTCTTTGCCGTCCTCGCGCTGTCCGGGCAATTTTAAAATATGGTAA.

[0030] The present invention provides a primer pair for amplifying the xylanase gene BaXynA, the nucleotide sequence of the primer pair is shown in SEQ ID NO: 2-3;

[0031] Upstream primer (BX-F): 5′-ATATACCATGGGCATGGTTCAGAAAGACG-3′ (SEQ ID NO: 2);

[0032] Downstream primer (BX-R): 5'-GTGGTGCTCGAGCCATATTTTAAAATTGCC-3' (SEQ ID NO: 3).

[0033] The present invention provides a xylanase BaXynA encoded by the xylanase gene BaXynA, and the amino acid sequence of the xylanase BaXynA is shown in SEQ ID NO: 4;

[0034] The SEQ ID The amino acid sequence of NO:4 is: MVQKDGAFYTGQYRNLFKEYGYSDAEIAAKVKNTWEELFILEGPNRIYFEVGDDLGYMLDTGNHDVRTEGMSYGMMMAVQMDDKD IFDRLWRWTMKYMYMTEGKHAGYFAWSCSPEGKKNAYGPAPDGEEYFALALLFFAAHRWGDGEDEVFNYSKHAKDLLHTCIHNGEDGEGYPMWNPDNKL IKFVPEFEYSDPSYHLPHFYELFALWANEEDRPFWKEAAAASREYLKIACHPETGLAPEYAFYDGTPNDERGFGHFFSDSYRVAANIGLDYEWFRGDEWETELANKIQGFFADKEPSDYRRYKIDGTPFEEKSLHPVGLVATNAMASLATVDGPYARQNVELFWNTPVRSGDRRYYDNCLYLFAVLALSGNFKIW.

[0035] The invention provides a recombinant vector for expressing xylanase BaXynA. The recombinant vector comprises the xylanase gene BaXynA and an empty vector; the empty vector is pET-28a.

[0036] The invention provides a recombinant bacterium expressing xylanase BaXynA. The recombinant bacterium comprises the xylanase gene BaXynA or the recombinant vector and a host bacterium; the host bacterium is Escherichia coli BL21 (DE3).

[0037] The invention provides application of the xylanase BaXynA in hydrolyzing xylan.

[0038] The present invention provides the use of the recombinant vector or the recombinant bacteria in preparing products for degrading xylan.

[0039] The present invention provides a method for degrading xylan, comprising the following steps:

[0040] (1) mixing a xylanase BaXynA solution with a substrate to obtain a mixed solution;

[0041] (2) The mixed solution obtained in step (1) is hydrolyzed at a pH value of 5 to 8 and a temperature of 10 to 70° C.; the pH value is preferably 6 and the temperature is preferably 70° C.

[0042] The xylanase BaXynA in step (1) is the xylanase BaXynA or the xylanase BaXynA expressed by the recombinant vector or the xylanase BaXynA expressed by the recombinant bacteria.

[0043] In the present invention, the volume ratio of the xylanase BaXynA solution to the substrate in step (1) is 1:8-10, preferably 1:9;

[0044] The concentration of the xylanase BaXynA solution is 50-200 mg / L, preferably 80-120 mg / L, and more preferably 100 mg / L; the concentration of the substrate is 8-12 mg / mL, preferably 8-12 mg / mL, and more preferably 10 mg / mL.

[0045] In the present invention, the substrate in step (1) is a substance containing xylan;

[0046] The xylan-containing material includes wheat arabinoxylan and beech xylan.

[0047] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0048] The plasmid described in the example is pET28a, which was purchased from Merck Life Science Division, Germany; Escherichia coli BL21 (DE3) and 2×Phanta Max MasterMix reagent were purchased from Beijing Novozyme Biotechnology Co., Ltd., and NTA buffer was purchased from Sangon Biotech (Shanghai) Co., Ltd., with the serial number C600304-0500.

[0049] Example 1: Construction of a recombinant bacterium expressing the xylanase BaXynA gene

[0050] 1. The xylanase gene of the present invention was found in the Xinjiang salt lake silt metagenome, and its gene size is 1137bp. Sequencing revealed that its nucleotide sequence is as shown in SEQ ID NO: 1, and it is named as the xylanase gene BaXynA. The gene encodes xylanase BaXynA, which contains 378 amino acids, and the amino acid sequence is as shown in SEQ ID NO: 4.

[0051] A pair of specific primers BX-F and BX-F were designed based on the nucleotide sequence of the xylanase gene BaXynA. The nucleotide sequences of the specific primers BX-F and BX-F are as follows:

[0052] Upstream primer (BX-F): 5′-ATATACCATGGGCATGGTTCAGAAAGACG-3′ (SEQ ID NO: 2);

[0053] Downstream primer (BX-R): 5'-GTGGTGCTCGAGCCATATTTTAAAATTGCC-3' (SEQ ID NO: 3).

[0054] 2. The xylanase gene BaXynA was amplified by PCR. The PCR reaction system was 20 μL, specifically 10 μL of 2×Phanta Max MasterMix, 1 μL of upstream primer BX-F with an initial concentration of 10 μM, 1 μL of downstream primer BX-R with an initial concentration of 10 μM, 1 μL of DNA template and 7 μL of ddH2O. The PCR reaction program is shown in Table 1.

[0055] Table 1. PCR reaction program

[0056]

[0057] 3. The PCR amplification product was purified and recovered by silica gel column, and then the PCR amplified xylanase gene BaXynA was connected to the pET-28a vector containing sticky ends obtained by double digestion with Nco I / Xho I by recombinase. The His-Tag coding sequence exists downstream of the XhoI site on the vector pET28a. When the gene BaxynA is connected to the vector, the gene integrates the His-Tag coding sequence on the vector with the gene. The C-terminus of the xylanase BaXynA after induced expression carries a fused His-Tag. Thus, the recombinant vector pET28a-BaXynA expressing the xylanase BaXynA with a His-Tag tag is constructed.

[0058] 4. The recombinant vector pET28a-BaXynA was transformed into Escherichia coli BL21 (DE3) to obtain a recombinant bacterium expressing the xylanase BaXynA. The recombinant bacterium was tested by PCR using primers BX-F and BX-R to detect whether the strain was correct, and the plasmid was extracted from the correct strain, and double enzyme digestion was performed using Nco I / Xho I to verify that the inserted sequence was correct. The recombinant bacterium was named BL21-BaXynA.

[0059] Example 2: In vitro expression and purification of recombinant bacterial xylanase BaXynA

[0060] 1. Experimental Materials

[0061] The recombinant bacteria BL21-BaXynA expressing the xylanase BaXynA obtained in Example 1.

[0062] 2. Experimental Methods

[0063] 1. Inducible expression of xylanase BaXynA

[0064] (1) The recombinant bacteria BL21-BaXynA was inoculated into 20 mL of LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C in a shaking incubator;

[0065] (2) OD the next day 600 The bacterial solution was inoculated into 500 mL of LB liquid medium containing 50 μg / mL kanamycin, with an inoculum volume of 1 mL. The medium was cultured at 37°C until the bacterial solution OD 600 The value is 0.7;

[0066] (3) To OD 600 1 mol / L IPTG solution was added to the bacterial solution with a pH value of 0.7 to make the final concentration of IPTG 0.5 mmol / L. The recombinant bacteria BL21-BaXynA was induced to express xylanase BaXynA at a temperature of 16°C for 20 hours to obtain a bacterial solution containing xylanase BaXynA.

[0067] 2. Purification of xylanase BaXynA by affinity chromatography

[0068] (1) centrifuging the obtained bacterial solution containing xylanase BaXynA at 5000 rpm for 10 min, removing the liquid to collect the bacterial cells, adding NTA buffer without imidazole to the centrifuge tube to resuspend the bacterial cells to obtain a bacterial suspension;

[0069] (2) Ultrasonic disruption of bacterial suspension: Place the centrifuge tube containing the bacterial suspension in a beaker of ice-water mixture, and place the beaker in an ultrasonic instrument for 8 min to obtain ultrasonically disrupted bacterial suspension. The setting program of the ultrasonic instrument is: ultrasonic for 3 s, stop for 5 s, and the power is 400 W.

[0070] (3) centrifuging the ultrasonically disrupted bacterial solution at 12000 g for 30 min, and collecting the supernatant to obtain a crude xylanase BaXynA solution;

[0071] (4) Purifying the crude xylanase BaXynA solution by chromatography, taking out the nickel column stored in 20% ethanol, washing the nickel column twice with deionized water after the ethanol flows out, and then balancing the nickel column with NTA buffer without imidazole, and maintaining the flow rate at 1 mL / min; then eluting the crude xylanase BaXynA solution twice at a flow rate of 1 mL / min, so that the crude xylanase BaXynA solution components are adsorbed on the nickel column;

[0072] (5) performing gradient elution using NTA buffer without imidazole and NTA buffer with imidazole concentrations of 5 mM, 10 mM, 20 mM, 30 mM, 50 mM, 100 mM, 150 mM, and 200 mM, collecting the eluate, detecting the protein in the eluate of different concentrations using a protein detection solution, and collecting the protein solution; replacing the buffer of the protein solution using ultrafiltration centrifugation to remove the imidazole in the protein solution, and obtaining a purified protein solution obtained by elution with different buffer concentrations.

[0073] 3. Experimental Results

[0074] The purified protein solution eluted with different NTA buffer concentrations was subjected to gel electrophoresis using the SDS-PAGE method. The gel electrophoresis diagram is shown in Figure 1 As shown in the figure, M represents the crude xylanase BaXynA solution; 1 represents an eluent without imidazole, 2 represents an eluent with a concentration of 5 mM imidazole, 3 represents an eluent with a concentration of 10 mM imidazole, 4 represents an eluent with a concentration of 20 mM imidazole, 5 represents an eluent with a concentration of 30 mM imidazole, 6 represents an eluent with a concentration of 50 mM imidazole, 7 represents an eluent with a concentration of 100 mM imidazole, 8 represents an eluent with a concentration of 150 mM imidazole, and 9 represents an eluent with a concentration of 200 mM imidazole. Figure 1 It can be seen that the gel electrophoresis of the purified protein solution has a clear background, high protein purity, and a large expression level. When eluted with 50mM, 100mM and 150mM imidazole eluents, a single protein band appears at 45kDa, which is close to the size of the predicted xylanase BaXynA fused His-Tag, indicating that the xylanase BaXynA is eluted in a buffer containing 50-150mM imidazole. The concentration of the purified xylanase BaXynA solution eluted with 100mM imidazole buffer was determined to be 5mg / mL.

[0075] Example 3: Analysis of enzymatic properties of xylanase BaXynA

[0076] 1. Experimental Materials

[0077] The purified xylanase BaXynA solution obtained in Example 2 has a concentration of 5 mg / mL.

[0078] 2. Test methods

[0079] 1. Enzyme Activity Assay

[0080] (1) The hydrolase activity was determined by the dinitrosalicylic acid (DNS) method. The purified xylanase BaXynA solution was diluted at a ratio of 1:50 to obtain a xylanase BaXynA solution with a concentration of 100 mg / L, and 40 μL of the xylanase BaXynA solution with a concentration of 100 mg / L was mixed with 360 μL of a beech xylan substrate solution with a concentration of 10 mg / mL and a pH of 6 dissolved in 0.1 M Tris-HCl buffer to obtain a mixed solution.

[0081] (2) The mixed solution was incubated at 70°C for 10 min, and 600 μL of DNS was added to terminate the hydrolysis to obtain a hydrolyzate. The hydrolyzate was then placed in a metal bath at 100°C for 10 min for color development. The solution was cooled to room temperature and the reading was recorded at 540 nm to calculate the enzyme activity. The enzyme activity unit (U / mg) was defined as the amount of xylanase required to decompose xylan to produce 1 μmol of xylose per minute under certain conditions.

[0082] The calculation formula of enzyme activity is as follows:

[0083] Enzyme activity (U / mg) = (y+0.0568) / 0.6085 / t / V / N;

[0084] In the formula, y represents OD 540 The reading value of

[0085] t represents the hydrolysis reaction time (min);

[0086] V represents the volume of xylanase BaXynA solution added to the reaction system (mL);

[0087] N represents the final concentration of the xylanase BaXynA solution in the reaction system (mg / mL).

[0088] The enzyme activity of xylanase BaXynA in hydrolyzing beech xylan was determined to be 1715U / mg.

[0089] 2. Substrate Specificity Determination

[0090] (1) 10 mg / L beech xylan, wheat arabinoxylan, pullulan, amylopectin, amylose, arabinogalactose, D-cellobiose, microcrystalline cellulose, and sodium carboxymethyl cellulose were used as substrates to determine the enzyme activity of xylanase BaXynA. The enzyme activity was determined in the same manner as in “1. Enzyme activity determination”. The enzyme activity of xylanase BaXynA hydrolyzing beech xylan obtained in “1. Enzyme activity determination” was used as the standard enzyme activity to calculate the relative enzyme activity of different substrates.

[0091] The relative enzyme activity calculation formula is:

[0092] Relative enzyme activity (%) = substrate enzyme activity / standard enzyme activity × 100%.

[0093] The relative enzyme activity results of different substrates are shown in Table 2. According to Table 2, xylanase BaXynA has strict substrate specificity and has enzyme activity only on two types of xylans, wheat arabinoxylan and beech xylan, and has no activity on pullulan, amylose, amylopectin, arabinogalactose, D-cellobiose, microcrystalline cellulose, and sodium carboxymethyl cellulose.

[0094] Table 2 Relative enzyme activities of xylanase BaXynA on different substrates

[0095] Substrate Relative enzyme activity (%) Beech Xylan 100±2.7 Wheat arabinoxylan 60±2.0 Microcrystalline Cellulose 3.1±1.7 Pullulan 3.2±0.7 Amylose 3.0±4.5 Sodium Carboxymethyl Cellulose 3.1±0.5 Amylopectin 3.0±0.4 D-Cellobiose 3.2±0.4

[0096] (2) Using equal volumes of 10 mg / mL beech xylan and wheat arabinoxylan solutions as substrates, 40 μL of 100 mg / L xylanase BaXynA solution was mixed with 360 μL of pH 6 substrate solution to obtain a mixed solution, which was then hydrolyzed at 70°C for 10 min to obtain a substrate hydrolyzate. The substrate hydrolyzate was analyzed by thin layer chromatography (TLC). The results are as follows: Figure 2 As shown, in the figure, X1 represents xylose, X2 represents xylose, X3 represents xylose, X4 represents xylotetraose, X5 represents xylopentaose, M represents a mixed solution of X1 to X5 standards, and B represents a hydrolyzate.

[0097] according to Figure 2 It can be seen that the hydrolyzate contains xylose, xylobiose, xylotetrose and xylopentose, indicating that the products of the hydrolysis of beech xylan and wheat arabinoxylan by xylanase BaXynA are xylose, xylobiose, xylotetrose and xylopentose.

[0098] 3. Enzyme Characterization Analysis

[0099] (1) 40 μL of a 100 mg / L xylanase BaXynA solution was mixed with 360 μL of a 10 mg / mL, pH 6, beech xylan substrate solution dissolved in 0.1 M Tris-HCl buffer to obtain a mixed solution, which was then hydrolyzed at 10°C, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C and 90°C for 10 min, respectively. The enzyme activity of the xylanase BaXynA hydrolyzing beech xylan obtained in "1. Enzyme activity determination" was used as the standard enzyme activity, and the relative enzyme activity of the xylanase BaXynA at different temperatures was determined. The results are as follows: Figure 3 As shown in A, the horizontal axis represents temperature (°C) and the vertical axis represents relative enzyme activity (%).

[0100] according to Figure 3A shows that the relative enzyme activity of xylanase BaXynA is highest when the temperature is 70℃.

[0101] (2) 40 μL of 100 mg / L xylanase BaXynA solution was mixed with 360 μL of 10 mg / mL beech xylan substrate solution dissolved in 0.1 M Tris-HCl buffer at a pH of 7 to obtain a mixed solution, which was then hydrolyzed at 10°C, 20°C, 30°C, 50°C and 70°C for 160 min, respectively. The enzyme activity of xylanase BaXynA in hydrolyzing beech xylan obtained in "1. Enzyme activity determination" was used as the standard enzyme activity. The stability of xylanase BaXynA at different temperatures was determined every 20 min. The results are as follows: Figure 3 As shown in B, the horizontal axis represents the hydrolysis time (min), the vertical axis represents the relative enzyme activity (%), and the broken lines of different shapes represent different hydrolysis temperatures.

[0102] according to Figure 3 B shows that the xylanase BaXynA has excellent low-temperature enzyme activity, retaining more than 80% of the enzyme activity after hydrolysis at 30°C for 160 min, and retaining more than 90% of the enzyme activity after hydrolysis at 10°C and 20°C for 160 min.

[0103] (3) 40 μL of 100 mg / L xylanase BaXynA solution was mixed with 360 μL of 10 mg / mL beech xylan solution to obtain a mixed solution (the pH values ​​of the beech xylan substrate were adjusted to 3, 4, 5, 6, 7 and 8 with citric acid-disodium hydrogen phosphate buffer; and the pH values ​​were adjusted to 9, 10 and 11 with glycine-sodium hydroxide buffer), and then hydrolyzed at 70°C for 10 min to obtain hydrolyzates under different pH conditions of the substrate. The enzyme activity of xylanase BaXynA hydrolyzing beech xylan obtained in "1. Enzyme activity determination" was used as the standard enzyme activity, and the relative enzyme activity of xylanase BaXynA under different pH conditions of the substrate was determined. The results are as follows: Figure 3 As shown in C, the horizontal axis represents pH value and the vertical axis represents relative enzyme activity (%).

[0104] according to Figure 3 C shows that the relative enzyme activity of xylanase BaXynA is different under different pH conditions of the substrate. The optimal pH value for xylanase BaXynA to hydrolyze the substrate is 6, and more than 85% of the enzyme activity is retained at pH 5-8.

[0105] (4) The purified xylanase BaXynA solution obtained in Example 2 was diluted with different pH buffers (1:50), and the pH values ​​were adjusted to 3, 4, 5, 6, 7 and 8 respectively with citric acid-disodium hydrogen phosphate buffer; the pH values ​​were adjusted to 9, 10 and 11 respectively with glycine-sodium hydroxide buffer, and the diluted enzyme solution was placed at 30°C for 60 minutes to obtain xylanase BaXynA solutions of different pH with a concentration of 100 mg / L. 40 μL of xylanase BaXynA solutions of different pH values ​​were mixed with 360 μL of beech xylan substrate solution with a concentration of 10 mg / mL and a pH of 6 dissolved in 0.1 M Tris-HCl buffer to obtain a mixed solution, which was then hydrolyzed at 70°C for 10 minutes. The enzyme activity of xylanase BaXynA hydrolyzing beech xylan obtained in "1. Enzyme activity determination" was used as the standard enzyme activity, and the stability of xylanase BaXynA treated with different pH values ​​was determined. The results are as follows Figure 3 As shown in D, the horizontal axis represents pH value and the vertical axis represents relative enzyme activity (%).

[0106] according to Figure 3 D shows that xylanase BaXynA is relatively stable when treated at pH 4-8 for 60 min, with the residual enzyme activity above 70%, and has the highest stability when treated at pH 6 for 60 min.

[0107] (5) Use Mg at a concentration of 10 mM diluted in 0.1 M Tris-HCl pH 6 buffer. 2+ , Cu 2+ , Mn 2+ 、Ni 2+ 、Zn 2+ The five metal ion solutions and 20 μL of 1% SDS, EDTA, and Tween20 solutions diluted with 0.1M Tris-HCl pH 6 buffer were respectively diluted with the purified xylanase BaXynA solution to obtain 100 mg / L xylanase BaXynA diluted solutions diluted with different metal ions and chemical reagents. 40 μL of 100 mg / L xylanase BaXynA diluted solutions were respectively diluted with 360 μL of 10 mg / mL, pH 6 0.1M The beech xylan substrate solution dissolved in Tris-HCl buffer was mixed to obtain a mixed solution, and the mixed solution was hydrolyzed at 70°C for 10 min. The enzyme solution without metal ions and chemical reagents was used as the control (CK), and the enzyme activity of xylanase BaXynA hydrolyzing beech xylan obtained in "1. Enzyme activity determination" was used as the standard enzyme activity. The relative enzyme activity of xylanase BaXynA in the control group and under different metal ion and chemical reagent conditions was determined. The results are shown in Figure 4As shown, the horizontal axis represents metal ions and chemical reagents, and the vertical axis represents relative enzyme activity.

[0108] according to Figure 4 It can be seen that Mn 2+ and Mg 2+ It has a promoting effect on xylanase BaXynA, Cu 2+ 、Ni 2+ 、Zn 2+ Metal ions have an inhibitory effect on the xylanase BaXynA, while SDS, EDTA and Tween20 chemical reagents have no effect on the activity of BaXynA, indicating that BaXynA has great potential for commercial development.

[0109] As can be seen from the above examples, the present invention provides a xylanase gene BaXynA, and the nucleotide sequence of the xylanase gene BaXynA is shown in SEQ ID NO: 1. The xylanase BaXynA encoded by the xylanase gene BaXynA of the present invention has the function of hydrolyzing wheat arabinoxylan and beech xylan, can maintain good enzyme activity at 10-70°C, is not affected by chemical reagents and some metal ions, and Mg 2+ and Mn 2+ Ions have a promoting effect on it, and it still has stable enzyme activity at 10-30°C. It can be used to decompose xylan in industrial production of food, feed, papermaking, detergents, medicines, and aquaculture.

[0110] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

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

1.

2. A primer pair for amplifying the xylanase gene BaXynA according to claim 1, characterized in that: The nucleotide sequences of the primer pairs are shown in SEQ ID NOs: 2-3.

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

4.

4. A recombinant vector expressing xylanase BaXynA, characterized in that: The recombinant vector comprises the xylanase gene BaXynA according to claim 1 and an empty vector; the empty vector is pET-28a.

5. A recombinant bacterium expressing xylanase BaXynA, characterized in that: The recombinant bacteria comprises the xylanase gene BaXynA according to claim 1 or the recombinant vector according to claim 4 and a host bacteria; the host bacteria is Escherichia coli BL21 (DE3).

6. Use of the xylanase BaXynA according to claim 3 in hydrolyzing xylan.

7. Use of the recombinant vector according to claim 4 or the recombinant bacteria according to claim 5 in preparing products for degrading xylan.

8. A method for degrading xylan, characterized in that: The steps include: (1) mixing a xylanase BaXynA solution with a substrate to obtain a mixed solution; (2) hydrolyzing the mixed solution obtained in step (1) at a pH value of 5 to 8 and a temperature of 10 to 70° C.; The xylanase BaXynA in step (1) is the xylanase BaXynA described in claim 3, the xylanase BaXynA expressed by the recombinant vector described in claim 4, or the xylanase BaXynA expressed by the recombinant bacteria described in claim 5.

9. The method according to claim 8, characterized in that The volume ratio of the xylanase BaXynA solution to the substrate in step (1) is 1:8-10; The concentration of the xylanase BaXynA solution is 50-200 mg / L; the concentration of the substrate is 5-20 mg / mL.

10. The method according to claim 8, characterized in that The substrate in step (1) is a substance containing xylan; The xylan-containing material includes wheat arabinoxylan and beech xylan.

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