Beta-1,4-endoxylanase mutants and use thereof

Abstract

The application provides a beta-1,4-endoxylanase mutant and application thereof. The amino acid sequence of the beta-1,4-endoxylanase mutant is shown as SEQ ID NO: 3. The application firstly discloses that the C terminal of a xylanase of family 11 has important significance for the thermal stability of the xylanase, by amino acid sequence alignment and scientific design, the C terminal of two thermophilic bacteria (Thermopolyspora flexuosa and Nesterenkonia xinjiangensis) derived xylanase is replaced with the C terminal of xynA, so that the thermal stability of the xylanase is significantly improved. The result shows that not only the N terminal has important influence on the thermal stability of the xylanase, but also the modification of the C terminal has important reference significance. The application also lays a theoretical foundation for the elucidation of the thermal stability mechanism of the xylanase of family 11 and the protein engineering modification of the enzyme.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a beta-1, 4-endoxylanase mutant and application thereof. BACKGROUND

[0002] Beta-1, 4-D-xylanase (EC 3.2.1.8) is the most important component in xylanase enzyme system, which breaks the beta-1, 4-D-glycosidic bond of xylan in an endo way to form oligomeric xylan, and is widely used in food, medicine, feed and new energy. However, due to its poor thermal stability, it cannot maintain high enzyme activity under high temperature conditions, thereby limiting its use. The most representative 11 family xylanase shows wide pH tolerance and substrate specificity, but poor thermal stability is a problem to be solved.

[0003] The modification of xylanase is mainly in the N terminal. According to sequence alignment, the N terminal is replaced by the N terminal of thermophilic xylanase, so as to improve its thermal stability. The introduction of aromatic amino acids in the N terminal by Bai Wenqin et al. makes the optimum reaction temperature increase by 5℃, and the half-life at 65℃ increases from 22min to 106min. The replacement of xylanase AoXyn11A from Aspergillus oryzae by He Yao et al. with the N terminal of Thermobifida fusca shows that the optimum temperature increases by 15℃, and the half-life increases by 41.5 times. Although the N terminal is the key region affecting the thermal stability of 11 family xylanase, the modification of N terminal may reduce the expression level and catalytic activity of the enzyme, and is not suitable for all xylanases. SUMMARY

[0004] The purpose of the present application is to provide a novel beta-1, 4-endoxylanase mutant and application thereof.

[0005] The present application conceives as follows: since the C terminal of xylanase is close to the N terminal in structure, there are more interaction forces. The modification of C terminal not only affects the N terminal from the structure, so as to improve the thermal stability, but also has less influence on the expression level and catalytic activity of the enzyme. Through C terminal replacement, the thermal stability of 11 family Aspergillus xylanase is improved.

[0006] In order to achieve the purpose of the present application, in the first aspect, the present application provides a beta-1, 4-endoxylanase mutant, the amino acid sequence of the mutant is shown as SEQ ID NO: 3.

[0007] In the second aspect, the present application provides a nucleic acid molecule encoding the beta-1, 4-endoxylanase mutant.

[0008] In a third aspect, the present application provides a biological material containing the nucleic acid molecule, which includes but is not limited to recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, engineered bacteria or transgenic cell line.

[0009] In a fourth aspect, the present application provides an application of the beta-1, 4-endoxylanase mutant in xylan degradation.

[0010] In a fifth aspect, the present application provides an application of the beta-1, 4-endoxylanase mutant in preparing a xylan degradation agent.

[0011] In a sixth aspect, the present application provides a xylan degradation agent, the effective component of which is the beta-1, 4-endoxylanase mutant.

[0012] In a seventh aspect, the present application provides a method for high-temperature degradation of xylan, which adds the beta-1, 4-endoxylanase mutant to xylan as a substrate and reacts at 40-60℃.

[0013] Preferably, the reaction temperature is 50-58℃, more preferably 55-58℃.

[0014] In the present application, the molecular weight of the xylan is 20-21kDa.

[0015] In an eighth aspect, the present application provides a method for improving the thermal stability of xylanase by replacing C-terminal amino acids, which comprises: replacing the C-terminal of xynA (SEQ ID NO: 2) with the C-terminal of xylanase from thermophilic bacteria to improve the thermal stability of xylanase.

[0016] Preferably, the thermophilic bacteria is Nesterenkonia xinjiangensis.

[0017] The present application first discloses that the C-terminal has important significance for the thermal stability of xylanase of family 11, and by amino acid sequence alignment and scientific design, the C-terminal of xylanase from two thermophilic bacteria (Thermopolyspora flexuosa and Nesterenkonia xinjiangensis) is replaced with the C-terminal of xynA, which significantly improves the thermal stability of xylanase. The results show that not only the N-terminal has important influence on the thermal stability of xylanase, but also the modification of the C-terminal has important reference significance. The present application also lays a theoretical foundation for the elucidation of the thermal stability mechanism of xylanase of family 11 and the protein engineering modification of enzymes. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The figure is a structural schematic diagram of the recombinant plasmid pMD19-xynA / N.xinjiangensis-C in the preferred embodiment of the present application.

[0019] Figure 2 Figure 1 is a graph of the thermal stability of xynA (WT) and mutants (N. xinjiangensis-C) in preferred embodiments of the present application.

[0020] Figure 3 Figure 2 is a result of alignment of xynA with the amino acid sequences of seven xylanases with higher homology in preferred embodiments of the present application. DETAILED DESCRIPTION

[0021] The present application provides a β-1, 4-endoxylanase mutant derived from Aspergillus niger AG11, the amino acid sequence of which is shown in SEQ ID NO: 3.

[0022] The mutant is constructed by gene engineering means of alignment with the C-terminal sequence of thermophilic xylanase and by large fragment primer PCR, and the enzymatic properties of the mutant are determined. Through the enzymatic property determination, the optimal reaction temperature of the xynA / N. xinjiangensis-C mutant is increased from the initial 50°C to 58°C. The half-life t 1 / 2 50℃ The t of xynA / N. xinjiangensis-C is increased from 18 min to 290 min, and the t of xynA is increased from 18 min to 290 min. 1 / 2 55℃ is 15 min, while the enzyme activity of xynA is only 10% after incubation at 55°C for 5 min. The above results show that not only does the N-terminal have an important influence on the thermal stability of xylanase, but the modification of the C-terminal is also of great significance.

[0023] The present application adopts the following technical solutions:

[0024] 1. Alignment of C-terminal sequence: xynA (SEQ ID NO: 2) is aligned with the amino acid sequences of seven xylanases with higher homology (SEQ ID NO: 1-7), and it is found that the C-terminal of five xylanases is the same, while the other two has great difference. They are Thermopolyspora flexuosa and Nesterenkonia xinjiangensis. Figure 3

[0025] ​2. Construction of the vector: The xynA expression frame was amplified by PCR using the genome of A. niger AG11 as template. Meanwhile, the vector pMD19 was amplified and the product was recovered and ligated with xynA by one-step cloning to obtain the vector pMD19-xynA. After sequencing, the pMD19-xynA and hygromycin (hyg) expression frame were amplified and ligated to obtain the vector pMD19-xynA / hyg, and finally the insert was obtained by PCR amplification. The mutant xynA / T.flexuosa-C and xynA / N.xinjiangensis-C were constructed by designing mutant primers, and the insert of each mutant was obtained by amplification with the same primers. The primer information is shown in Table 1.

[0026] 3. The strain A. niger / pMD19-xynA / hyg was used to express xynA as a control, and each mutant protein was expressed. After protein purification and enzymatic property determination, the thermal stability of the mutant xynA / T.flexuosa-C was unchanged, and the optimum reaction temperature of the mutant xynA / N.xinjiangensis-C was increased from the original 50°C to 58°C. The half-life t 1 / 2 50℃ The half-life t 1 / 2 55℃ was increased from the original 18 min to 290 min, and the half-life t

[0027] , wherein t 1 / 2 50℃ is the half-life of xynA at 50°C, t 1 / 2 55℃ is the half-life of the mutant xynA / N.xinjiangensis-C at 55°C.

[0028] The following examples are used to illustrate the present application, but are not used to limit the scope of the present application. If not specifically indicated, the examples are carried out according to the conventional experimental conditions, such as Sambrook et al. Molecular Cloning: a Laboratory Manual (2001), or according to the conditions suggested by the manufacturer's instructions.

[0029] Example 1 Preparation of β-1,4-endoxylanase mutants

[0030] The present embodiment performs sequence alignment on heat-resistant xylanases with homology greater than 50% derived from the xylanase xynA of Aspergillus niger 11 family, and finds that the C-terminal of xynA has low homology. The C-terminal of xynA is replaced by long fragment primer PCR amplification. The thermal stability is obviously improved, and the specific enzyme activity is unchanged. The nucleotide sequence of the xynA gene is shown in SEQ ID NO: 1.

[0031] The amino acid sequence of xynA is shown in SEQ ID NO: 2, and the amino acid sequence of the C-terminal replaced xynA is shown in SEQ ID NO: 3.

[0032] (1) Xylanase enzyme activity determination

[0033] The xylanase enzyme activity is determined by the 3,5-dinitrosalicylic acid method. 500 μL of enzyme solution diluted to an appropriate concentration is mixed with 500 μL of birch xylan substrate (pH 4.0, 10 mg / mL, substrate molecular weight is 150.13 Da), and the reaction is terminated with 3,5-dinitrosalicylic acid reagent after 10 min of reaction at 50°C. The sample is immediately cooled with water after color development in boiling water for 5 min, and the absorbance is determined at 545 nm. The enzyme activity unit (U / mL) is defined as the amount of enzyme required to hydrolyze 1 μmol of reducing sugar per minute.

[0034] (2) Determination of optimal reaction temperature

[0035] The optimal reaction temperature is the enzyme solution after purification diluted to an appropriate concentration with NaH2PO4-Na2HPO4 buffer (50 mM, pH 7.5), and the enzyme activity is determined at 40-60°C. The relative enzyme activity at each temperature is calculated based on the highest enzyme activity as 100%.

[0036] (3) Determination of temperature stability

[0037] The temperature stability is the enzyme activity under each condition after the above diluted enzyme solution is incubated at 40-60°C for 0-90 min and stored on ice. The relative enzyme activity at each incubation time is calculated based on the enzyme activity at 0 min incubation as 100%. The regression equation at different temperatures is fitted based on the above temperature stability determination, and the half-life of xylanase at each temperature is calculated.

[0038] The mutation, expression and enzyme property determination of xylanase gene are as follows:

[0039] 1. Alignment of C-terminal sequences: The xynA was aligned with seven xylanase amino acid sequences (Table 1) with higher homology, and it was found that the C-terminal of xynA was identical to five xylanases, while the other two had large differences. They were Thermopolyspora flexuosa and Nesterenkonia xinjiangensis.

[0040] 2. Construction of vector and mutants: The xynA expression frame was amplified by PCR using the genomic DNA of A. niger AG11 as template and primers F1 and R1 (Table 1). Meanwhile, the vector pMD19 was amplified by primers F2 and R2, and the product was recovered and ligated with xynA to obtain the vector pMD19-xynA. The PCR amplification program was as follows: 94°C pre-denaturation for 3 min; 94°C denaturation for 10 s, 55°C annealing for 15 s, 72°C extension for 50 s, 34 cycles; and finally 72°C extension for 5 min. After sequencing, the pMD19-xynA and hygromycin (hyg) expression frames were amplified by primers F3 and R3, and F4 and R4, respectively, and then ligated to obtain the vector pMD19-xynA / hyg. Finally, the insert fragment was amplified by primers F1 and R4. The mutant primers (Table 1) were designed to construct the mutants xynA / T.flexuosa-C and xynA / N.xinjiangensis-C, and the insert fragments of the mutants were amplified by the same primers. The structure of the recombinant plasmid pMD19-xynA / N.xinjiangensis-C is shown in Figure 1 . The amino acid sequence of the mutant xynA / T.flexuosa-C is shown in SEQ ID NO: 4.

[0041] 3. Expression, purification and determination of enzymatic properties of xynA and mutants: The strain A. niger / pMD19-xynA / hyg was used to express xynA as a control, and the mutant proteins were also expressed. After fermentation for a certain time, the bacterial cells were collected by centrifugation, filtered through a filter membrane, purified by HisTrap TM FF, desalted by a desalting column Sephadex G25, and the optimal reaction temperature and temperature stability were determined. The optimal reaction temperature of the mutant xynA / N.xinjiangensis-C was increased from the original 50°C to 58°C. 1 / 2 50℃ The half-life t 1 / 2 55℃ was increased from the original 18 min to 290 min, and the t Figure 2 of the mutant xynA / N.xinjiangensis-C was 15 min, while the enzyme activity of xynA was only 10% after incubation at 55°C for 5 min.

[0042] Table 1 Primer table for constructing each plasmid

[0043]

[0044]

[0045] Although the present application has been described in detail with general description and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present application. Therefore, these modifications or improvements made without departing from the spirit of the present application are within the scope of the present application. SEQUENCE LISTING <110> China Petroleum & Chemical Corporation China Petroleum & Chemical Corporation Dalian Research Institute of Petroleum & Chemical Industry Jiangnan University <120> Beta-1, 4-endoxylanase mutants and uses thereof <130> KHP211119260.3 <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 564 <212> DNA <213> Aspergillus niger <400> 1 tcgaccccga gctcgaccgg cgagaacaac ggcttctact actccttctg gaccgacggc 60 ggtggcgacg tgacctacac caacggagat gctggtgcct acactgttga gtggtccaac 120 gtgggcaact ttgtcggtgg aaagggctgg aaccccggaa gtgcgcagga catcacctac 180 agcggcacct tcacccctag cggcaacggc tatctctccg tctatggctg gaccactgac 240 cccctgatcg agtactacat cgtcgagtcc tacggcgact acaaccccgg cagtggaggc 300 acatacaagg gcaccgtcac ctcggacgga tccgtttacg atatctacac ggctacccgt 360 accaatgctg cttccattca gggaaccgct accttcactc agtactggtc cgtccgccag 420 aacaagagag ttggcggaac tgttaccacc tccaaccact tcaatgcttg ggctaagctg 480 ggaatgaacc tgggtactca caactaccag atcgtggcta ccgagggtta ccagagcagt 540 ggatcttcgt ccatcactgt tcag 564 <210> 2 <211> 188 <212> PRT <213> Aspergillus niger <400> 2 Ser Thr Pro Ser Ser Thr Gly Glu Asn Asn Gly Phe Tyr Tyr Ser Phe 1 5 10 15 Trp Thr Asp Gly Gly Gly Asp Val Thr Tyr Thr Asn Gly Asp Ala Gly 20 25 30 Ala Tyr Thr Val Glu Trp Ser Asn Val Gly Asn Phe Val Gly Gly Lys 35 40 45 Gly Trp Asn Pro Gly Ser Ala Gln Asp Ile Thr Tyr Ser Gly Thr Phe 50 55 60 Thr Pro Ser Gly Asn Gly Tyr Leu Ser Val Tyr Gly Trp Thr Thr Asp 65 70 75 80 Pro Leu Ile Glu Tyr Tyr Ile Val Glu Ser Tyr Gly Asp Tyr Asn Pro 85 90 95 Gly Ser Gly Gly Thr Tyr Lys Gly Thr Val Thr Ser Asp Gly Ser Val 100 105 110 Tyr Asp Ile Tyr Thr Ala Thr Arg Thr Asn Ala Ala Ser Ile Gln Gly 115 120 125 Thr Ala Thr Phe Thr Gln Tyr Trp Ser Val Arg Gln Asn Lys Arg Val 130 135 140 Gly Gly Thr Val Thr Thr Ser Asn His Phe Asn Ala Trp Ala Lys Leu 145 150 155 160 Gly Met Asn Leu Gly Thr His Asn Tyr Gln Ile Val Ala Thr Glu Gly 165 170 175 Tyr Gln Ser Ser Gly Ser Ser Ser Ile Thr Val Gln 180 185 <210> 3 <211> 191 <212> PRT <213> Artificial Sequence <400> 3 Ser Thr Pro Ser Ser Thr Gly Glu Asn Asn Gly Phe Tyr Tyr Ser Phe 1 5 10 15 Trp Thr Asp Gly Gly Gly Asp Val Thr Tyr Thr Asn Gly Asp Ala Gly 20 25 30 Ala Tyr Thr Val Glu Trp Ser Asn Val Gly Asn Phe Val Gly Gly Lys 35 40 45 Gly Trp Asn Pro Gly Ser Ala Gln Asp Ile Thr Tyr Ser Gly Thr Phe 50 55 60 Thr Pro Ser Gly Asn Gly Tyr Leu Ser Val Tyr Gly Trp Thr Thr Asp 65 70 75 80 Pro Leu Ile Glu Tyr Tyr Ile Val Glu Ser Tyr Gly Asp Tyr Asn Pro 85 90 95 Gly Ser Gly Gly Thr Tyr Lys Gly Thr Val Thr Ser Asp Gly Ser Val 100 105 110 Tyr Asp Ile Tyr Thr Ala Thr Arg Thr Asn Ala Ala Ser Ile Gln Gly 115 120 125 Thr Ala Thr Phe Thr Gln Tyr Trp Ser Val Arg Gln Asn Lys Arg Val 130 135 140 Gly Gly Thr Val Thr Thr Ser Asn His Phe Asn Ala Trp Ala Lys Leu 145 150 155 160 Gly Met Asn Leu Gly Thr His Asn Tyr Gln Ile Val Ala Thr Glu Gly 165 170 175 Tyr Gln Ser Ser Gly Ser Ser Ser Ile Thr Val His Thr Ala Pro 180 185 190 <210> 4 <211> 178 <212> PRT <213> Artificial Sequence <400> 4 Ser Thr Pro Ser Ser Thr Gly Glu Asn Asn Gly Phe Tyr Tyr Ser Phe 1 5 10 15 Trp Thr Asp Gly Gly Gly Asp Val Thr Tyr Thr Asn Gly Asp Ala Gly 20 25 30 Ala Tyr Thr Val Glu Trp Ser Asn Val Gly Asn Phe Val Gly Gly Lys 35 40 45 Gly Trp Asn Pro Gly Ser Ala Gln Asp Ile Thr Tyr Ser Gly Thr Phe 50 55 60 Thr Pro Ser Gly Asn Gly Tyr Leu Ser Val Tyr Gly Trp Thr Thr Asp 65 70 75 80 Pro Leu Ile Glu Tyr Tyr Ile Val Glu Ser Tyr Gly Asp Tyr Asn Pro 85 90 95 Gly Ser Gly Gly Thr Tyr Lys Gly Thr Val Thr Ser Asp Gly Ser Val 100 105 110 Tyr Asp Ile Tyr Thr Ala Thr Arg Thr Asn Ala Ala Ser Ile Gln Gly 115 120 125 Thr Ala Thr Phe Thr Gln Tyr Trp Ser Val Arg Gln Asn Lys Arg Val 130 135 140 Gly Gly Thr Val Thr Thr Ser Asn His Phe Asn Ala Trp Ala Lys Leu 145 150 155 160 Gly Met Asn Leu Gly Thr His Asn Tyr Gln Ile Val Ala Thr Glu Gly 165 170 175 Tyr Gln

Claims

1. A β-1,4-endoxylanase mutant, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID NO:

3.

2. A nucleic acid molecule encoding the mutant of claim 1.

3. A biomaterial containing the nucleic acid molecule of claim 2, characterized in that, The biological materials are recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, or transgenic cell lines.

4. The application of the mutant of claim 1 in xylan degradation.

5. The use of the mutant of claim 1 in the preparation of xylan degrading agents.

6. A xylan degrading agent, characterized in that, The active ingredient is the mutant described in claim 1.

7. A method for high-temperature degradation of xylan, characterized in that, Using xylan as a substrate, the mutant described in claim 1 was added, and the reaction was carried out at 40-60°C.

8. The method according to claim 7, characterized in that, The reaction temperature is 50-58℃.

9. The method according to claim 8, characterized in that, The reaction temperature is 55-58℃.

10. The method according to any one of claims 7-9, characterized in that, The xylan has a molecular weight of 20-21 kDa.

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