A xylanase XynZT-2 mutant and its application

Abstract

The present invention belongs to the technical field of molecular biology, and specifically relates to a xylanase XynZT-2 mutant and its application. The xylanase XynZT-2 mutant is obtained by site-directed mutagenesis of the xylanase XynZT-2 with the amino acid sequence shown in SEQ ID No. 2. The specific mutation sites are as follows: T28I, A33V, M97L, A102C, S148T, A238G, S239P, M241L, T287S or I291V. The optimal temperature and optimal pH of 10 xylanase XynZT-2 mutants have changed, and the obtained xylanase XynZT-2 mutants show application value in the fields of pulp brewing, bioenergy, food manufacturing, etc.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and particularly relates to a xylanase XynZT-2 mutant and its application. Background Art

[0002] Xylanase (endo-1,4-β-d-xylan xylanohydrolase EC3.2.1.8) is a group of complex enzyme systems that degrade hemicellulose xylan into oligosaccharides and xylose. Xylanase mainly includes endo-β-1,4-xylanase and β-xylanase, which can degrade high-molecular-weight water-insoluble xylan into water-soluble xylooligosaccharides and xylose. Xylanase is derived from families such as GH10, GH11, and GH43. Currently, extensive research has been conducted on the GH10 and GH11 families, while less research has been done on the GH43 family. β-Xylosidase is a type of xylanase. According to the amino acid sequence similarity, the glycoside hydrolase families containing this enzyme activity include GH5, GH43, etc. Population genetics analysis of the GH43 family (Glycoside Hydrolase Family 43) genes from sources such as bacteria, fungi, animals, and plants was carried out in terms of nucleotide polymorphism, selection pressure, conserved sequences, etc. to determine its evolutionary pattern. It was found that the GH43 gene originated before the differentiation of bacteria, archaea, fungi, and plants and does not exist in animals. Xylanase has important application values in food industries such as delaying the aging of bread and steamed buns, juice clarification, and producing functional xylooligosaccharides.

[0003] Regarding the mutation of xylanase to obtain xylanase mutants with altered optimal temperature and optimal pH, many studies have been done by those skilled in the art. For example, the invention patent CN104911163B discloses a thermostable xylanase mutant and its application, and the thermostable ability of the mutated xylanase mutant is significantly improved; the invention patent CN103343113B discloses a xylanase XynAS9-m mutant V81P / G82E with improved thermal stability and its gene and application, and the thermal stability of the mutated xylanase is significantly improved; the invention patent CN110607291A discloses a heat-resistant xylanase mutant, and the heat-resistant ability of the mutated xylanase is significantly improved. However, there is currently no research on the mutation of xylanase XynZT-2.

[0004] In the previous research of this laboratory, a strain of bacteria was isolated from the coastal waters of Yantai, Shandong. After identification, it was identified as Alteromonas macleodii. This strain has no toxic side effects, no pollution to the environment, and has a good nitrogen removal effect. It can be used as an ideal strain for removing inorganic nitrogen in aquaculture. The inventors also isolated xylanase XynZT-2 from Alteromonas macleodii. It was found that xylanase XynZT-2 is a member of the GH43 family. During the research process, the inventors unexpectedly discovered the active center sites and conserved sites of xylanase XynZT-2 from Alteromonas macleodii through sequence analysis and structural modeling; selected amino acid sites closely related to the enzyme catalytic function for homologous site mutation to obtain the mutated gene sequence. The recombinant gene was expressed in Escherichia coli, and the enzymatic properties of the recombinant enzyme and the original enzyme were studied to further explore the evolutionary relationship between the gene sequence of the GH43 family and its function. The experimental results showed that after amino acid homology mutation in related species, the optimal temperature and pH of the mutant enzyme changed compared with the original enzyme, expanding its application range. Summary of the Invention

[0005] The primary object of the present invention is to provide a mutant of xylanase XynZT-2, and the amino acid sequence of the xylanase XynZT-2 mutant is as follows:

[0006] (1) Mutate the 28th threonine of xylanase XynZT-2 with the amino acid sequence shown in SEQ ID No.2 to isoleucine;

[0007] (2) Mutate the 33rd alanine of xylanase XynZT-2 with the amino acid sequence shown in SEQ ID No.2 to valine;

[0008] (3) Mutate the 97th methionine of xylanase XynZT-2 with the amino acid sequence shown in SEQ ID No.2 to leucine;

[0009] (4) Mutate the 102nd alanine of xylanase XynZT-2 with the amino acid sequence shown in SEQ ID No.2 to cysteine;

[0010] (5) Mutate the 148th serine of xylanase XynZT-2 with the amino acid sequence shown in SEQ ID No.2 to threonine;

[0011] (6) Mutate the 238th alanine of xylanase XynZT-2 with the amino acid sequence shown in SEQ ID No.2 to glycine;

[0012] (7) Mutate the 239th serine of xylanase XynZT-2 with the amino acid sequence shown in SEQ ID No.2 to proline;

[0013] (8) Mutate the methionine at position 241 of xylanase XynZT-2 with the amino acid sequence as shown in SEQ ID No. 2 to leucine;

[0014] (9) Mutate the threonine at position 287 of xylanase XynZT-2 with the amino acid sequence as shown in SEQ ID No. 2 to serine;

[0015] (10) Mutate the isoleucine at position 291 of xylanase XynZT-2 with the amino acid sequence as shown in SEQ ID No. 2 to valine.

[0016] The second object of the present invention is to provide a xylanase XynZT-2 mutant gene, which encodes the xylanase XynZT-2 mutant described above.

[0017] The third object of the present invention is to provide a recombinant expression vector containing the xylanase XynZT-2 mutant gene.

[0018] The fourth object of the present invention is to provide a recombinant strain containing the xylanase XynZT-2 mutant gene.

[0019] Preferably, the recombinant strain is Escherichia coli BL21.

[0020] The fifth object of the present invention is to provide the application of the xylanase XynZT-2 mutant in hydrolyzing xylan.

[0021] The sixth object of the present invention is to provide the application of the xylanase XynZT-2 mutant in the preparation of feed / feed additives.

[0022] The seventh object of the present invention is to provide the application of the xylanase XynZT-2 mutant in food, brewing and bioenergy.

[0023] The beneficial effects of the present invention are as follows: The present invention provides a xylanase XynZT-2 mutant, which is obtained by site-directed mutagenesis of xylanase XynZT-2. The site-directed mutation sites are any one of T28I, A33V, M97L, A102C, S148T, A238G, S239P, M241L, T287S or I291V. The optimal temperature of the obtained mutant enzyme has changed compared with that of the original enzyme. Among them, the optimal temperatures of mutant enzymes M97L, A102C, A152G, M241L and I291V have increased, rising by about 15 - 35 °C, with mutant enzyme M97L showing the largest increase, by 35 °C. Among them, the optimal temperatures of mutant enzymes A33V, A238G and T287S have decreased, by 5 °C, and the optimal temperatures of mutant enzymes T28I, S148T and S239P remain unchanged compared with the original enzyme. The optimal pH of the mutant enzyme has changed compared with that of the original enzyme. Among them, the optimal pH values of mutant enzymes T28I, A33V, M97L, A102C, S148T, M241L and T287S have increased; the optimal pH values of mutant enzymes A152G, A238G and S239P have decreased; the optimal pH of mutant enzyme I291V remains unchanged. In summary, among the 10 xylanase XynZT-2 mutants obtained by mutation in the present invention, the optimal temperatures and optimal pH values of 9 xylanase XynZT-2 mutants have changed, and the obtained xylanase XynZT-2 mutants show application values in the fields of pulp brewing, bioenergy, food manufacturing, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Optimal Temperatures and Optimal pH Values of Xylanase XynZT-2 and Its 10 Mutant Enzymes DETAILED DESCRIPTION OF THE EMBODIMENTS

[0025] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure content of the present invention more thorough and comprehensive.

[0026] For each reaction or detection condition described in the content of the present invention, it can be combined or changed according to common knowledge in the art and can be verified through experiments.

[0027] The DNS method mentioned in the following examples is the 3,5-dinitrosalicylic acid colorimetric method. The principle is that 3,5-dinitrosalicylic acid is reduced to a brownish-red substance after being heated together with the reducing sugar obtained from the hydrolysis of polysaccharides under neutral or slightly alkaline conditions.

[0028] Escherichia coli BL21 described in the following examples is from our laboratory.

[0029] Plasmid pET28a described in the following examples is stored in our laboratory, and restriction endonucleases EcoRⅠ and HindⅢ are purchased from TaKaRa Company.

[0030] Example 1. Mutation of xylanase XynZT-2 from Alteromonas macleodii

[0031] 1. Bioinformatics analysis

[0032] Search for species information of the GH43 family in the CAZy database and download and save it. Predict and screen GH43 family genes from sources such as bacteria, fungi, animals, and plants. Conduct in-depth analysis from aspects such as conserved sequences to determine its phylogenetic law. Download the GH43 family genes and corresponding amino acid reference sequences from NCBI and find the model organisms. Perform homologous sequence alignment of the GH43 family domain by BLASTP, construct the LG maximum likelihood phylogenetic tree using multiple alignments, and visualize it in ITOL. Combining the above phylogenetic analysis results of the GH43 family, find the conserved region of xylanase XynZT-2 from Alteromonas macleodii through alignment of related species and bioinformatics analysis. The nucleotide sequence of the xylanase XynZT-2 is shown in SEQ ID No.1, and the amino acid sequence is shown in SEQ ID No.2.

[0033] Perform homologous site mutations on non-strictly conserved sites near the conserved region respectively, design primers, and obtain the corresponding mutants by overlap extension PCR. Specifically as follows:

[0034] 2. Primer design

[0035] Select 10 amino acid sites closely related to the enzyme catalytic function for homologous site replacement to obtain the replaced gene sequence; use the upstream and downstream primer design rules to design the upstream and downstream primers for 10 mutation sites. Send them to Genewiz for synthesis.

[0036] Table 1 Primer information

[0037]

[0038] 3. Obtaining of mutant genes

[0039] Adopt the overlap extension PCR method, use xynZT-2 as the template, and other conditions are shown in Table B to obtain the upper and lower segments of the mutant gene. Then use the upper and lower segments containing the mutation points as templates and ZT-2-F and ZT-2-R as primers (Table C) to obtain 11 mutant genes xynZT-2T28I / A33V / M97L / A102C / S148T / A152G respectively.

[0040] / A238G / S239P / M241 / T287S / I291V was used to construct pET-28a-xynZT-2T28I / A33V / M97L / A102C / S148T / A152G / A238G / S239P / M241L / T287S / I291V. After agarose gel electrophoresis of the constructed product, the gel was cut and recovered.

[0041] Table 2 PCR reaction system for the upper and lower segments of 10 mutant genes

[0042]

[0043]

[0044] Table 3 PCR reaction system for 10 mutant genes

[0045]

[0046] 4. Construction of recombinant engineering bacteria

[0047] Ten mutant genes and the empty plasmid pET-28a were double-digested with restriction endonucleases EcoRⅠ and HindⅢ under the reaction conditions of 37 °C for 2.5 h. The double-digested products were recovered by cutting the gel. The double-digested products were ligated with T4 DNA ligase, transformed into Escherichia coli BL21(DE3), and spread on LB solid medium containing Kan resistance. The positive colonies verified by sequencing were successfully constructed into 10 recombinant Escherichia coli strains containing mutant genes.

[0048] The amino acid sequence of the xylanase XynZT-2 mutant is as follows:

[0049] (1) The 28th threonine of xylanase XynZT-2 with the amino acid sequence shown in SEQ ID No. 2 was mutated to isoleucine;

[0050] (2) The 33rd alanine of xylanase XynZT-2 with the amino acid sequence shown in SEQ ID No. 2 was mutated to valine;

[0051] (3) The 97th methionine of xylanase XynZT-2 with the amino acid sequence shown in SEQ ID No. 2 was mutated to leucine;

[0052] (4) The 102nd alanine of xylanase XynZT-2 with the amino acid sequence shown in SEQ ID No. 2 was mutated to cysteine;

[0053] (5) The 148th serine of xylanase XynZT-2 with the amino acid sequence shown in SEQ ID No. 2 was mutated to threonine;

[0054] (6) Mutate alanine at position 238 of xylanase XynZT-2 with the amino acid sequence as shown in SEQ ID No.2 into glycine;

[0055] (7) Mutate serine at position 239 of xylanase XynZT-2 with the amino acid sequence as shown in SEQ ID No.2 into proline;

[0056] (8) Mutate methionine at position 241 of xylanase XynZT-2 with the amino acid sequence as shown in SEQ ID No.2 into leucine;

[0057] (9) Mutate threonine at position 287 of xylanase XynZT-2 with the amino acid sequence as shown in SEQ ID No.2 into serine;

[0058] (10) Mutate isoleucine at position I291V of xylanase XynZT-2 with the amino acid sequence as shown in SEQ ID No.2 into valine.

[0059] By overlap extension PCR method, first obtain the xylanase XynZT-2 gene from the plasmid. Design 10 primers each containing a mutation site, use the xylanase XynZT-2 gene as a template to obtain 10 pairs of upper and lower fragments containing mutation points respectively. Then use the 10 pairs of upper and lower fragments as templates respectively to obtain the corresponding 10 mutant genes. Double digest the 10 mutant genes and plasmid pET28a with restriction enzymes EcoRⅠ and HindⅢ respectively, the reaction conditions are 37°C for 2.5 h, perform agarose gel electrophoresis on the double-digested products, and cut and recover the gel. Connect the double-digested products with T4 DNA ligase overnight at 16°C, transform Escherichia coli BL21(DE3), coat it on the LB solid medium containing Kan resistance, screen to obtain positive colonies, and verify by sequencing, which are the constructed mutant strains.

[0060] Example 2: Determination of the optimal temperature of xylanase XynZT-2 mutant enzyme

[0061] Method: Under the same pH condition, the mutant enzyme and the substrate react at different temperatures (35°C to 65°C, at intervals of 5°C) for 15 min, and use the DNS method to measure the enzyme activities of the original enzyme and the mutant enzyme respectively. Among them, for mutant M97L, select points near the optimal temperature due to its higher optimal temperature.

[0062] The experimental results are shown in Table 1. The optimal temperature of the mutant enzymes changed compared with that of the original enzyme. Among them, the optimal temperatures of mutant enzymes M97L, A102C, A152G, M241L, and I291V increased by about 15 - 35 °C, with mutant enzyme M97L showing the largest increase of 35 °C. Among them, the optimal temperatures of mutant enzymes A33V, A238G, and T287S decreased by 5 °C, and the optimal temperatures of mutant enzymes T28I, S148T, and S239P remained unchanged compared with the original enzyme.

[0063] Table 1 Comparison of the Optimal Temperatures of the Original Enzyme and Mutant Enzymes

[0064]

[0065] Example 3. Determination of the Optimal pH of the Xylanase XynZT-2 Mutant Enzyme

[0066] Method: At the optimal temperature, measure the enzyme activities of the original enzyme and mutant enzymes at different pH values to obtain the optimal pH. Buffer solutions: Disodium hydrogen phosphate - citric acid buffer (pH 3.0 - 5.5); Disodium hydrogen phosphate - sodium dihydrogen phosphate buffer (pH 6.0 - 7.5); Tris - hydrochloric acid buffer (pH 8.0, pH 8.5); Glycine - sodium hydroxide buffer (pH 9.0). Use the DNS method to determine the optimal pH of the mutant enzyme and the original xylanase XynZT-2.

[0067] The results are shown in Table 2. The optimal pH of the mutant enzymes changed compared with that of the original enzyme. Among them, the optimal pH values of mutant enzymes T28I, A33V, M97L, A102C, S148T, M241L, and T287S increased; the optimal pH values of mutant enzymes A152G, A238G, and S239P decreased; the optimal pH of mutant enzyme I291V remained unchanged.

[0068] The optimal temperatures and optimal pH values of xylanase XynZT-2 and its 11 mutant enzymes are as Figure 1 shown.

[0069] Table 2 Comparison of the Optimal pH of the Original Enzyme and Mutant Enzymes

[0070]

[0071] In summary, among the 10 xylanase XynZT-2 mutants obtained by mutation in the present invention, the optimal temperatures and optimal pH values of 9 xylanase XynZT-2 mutants changed, and the obtained xylanase XynZT-2 mutants showed application values in the fields of pulp brewing, bioenergy, food manufacturing, etc. Sequence Listing <110> Xinxiang Medical University <120> A Xylanase XynZT-2 Mutant and Its Application <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1029 <212> DNA <213> Alteromonas macleodii <400> 1 atgccggaga aacagtacga gaacgagatc gaaacgtaca aggagagcgc tatcagcaag 60 ccgctggtcg agcatatcta tacggccgat ccgagcgccc atgtcttcga gggcaagatc 120 tacatctacc cgagccacga tatcgaagcc ggaatcccgt tcaacgataa cggcgaccac 180 ttcgccatgt gcgactacca cgttctgagc atggattctc ttacgtcccc ggttcaagat 240 catggcgtcg ctcttcacgt tgataacgtc aagtgggctg aacgccaaat gtgggccccg 300 gacgctgcca caaagaacgg caagtacttt ctttacttcc cggccaagaa gagcgatggc 360 atcttccaaa tcggcgtcgc cgttagcaat acaccgcatg gcccgtttat ggcccagcca 420 gaaccgatcg gaggctccta cagcatcgat ccggctgttt tcgaagacga tgacggcacg 480 cactacatgt acttcggcgg aatttggggc ggccagctgc agaactaccg cgagaacaaa 540 tacaacgcca gctttgccga accgcaagat ggagaaccag ctctgggccc gatcgtcgcc 600 tacaacgcca gctttgccga accgcaagat ggagaaccag ctctgggccc gatcgtcgcc 600 aaaatgtccg gcgacatgct ggagtttgat catacgccgc gcgaaattct tattgtcgat 660 aaaatgtccg gcgacatgct ggagtttgat catacgccgc gcgaaattct tattgtcgat 660 gagggcggca aaccgctgct tgctggcgac catgaacgcc gcttcttcga agccagctgg 720 gagggcggca aaccgctgct tgctggcgac catgaacgcc gcttcttcga agccagctgg 720 atgcacaagc acaagggaaa gtactatttc agctactcca cgggcaacac gcactacctt 780 atgcacaagc acaagggaaa gtactatttc agctactcca cgggcaacac gcactacctt 780 tgttatgcca tcggcgactc cccgtatggc ccattcgttt acgccggccg cattcttgaa 840 tgttatgcca tcggcgactc cccgtatggc ccattcgttt acgccggccg cattcttgaa 840 ccggttgttg gatggacgac gcatcatagc atctgcgagt ttgagggcaa gcactatctt 900 ccggttgttg gatggacgac gcatcatagc atctgcgagt ttgagggcaa gcactatctt 900 ttctaccacg acagcacgct gtccggaggc gtcacacacc ttcgctccgt caaggtcgcc 960 ttctaccacg acagcacgct gtccggaggc gtcacacacc ttcgctccgt caaggtcgcc 960 ccgattgagt acgatgacga tggcaagatc atcacgctgt ccccgtacgg cgatgtccgc 1020 ccgattgagt acgatgacga tggcaagatc atcacgctgt ccccgtacgg cgatgtccgc 1020 gttgaataa 1029 gttgaataa 1029 <210> 2<210> 2 <211> 342<211> 342 <212> PRT<212> PRT <213> Alteromonas macleodii<213> Alteromonas macleodii <400> 2<400> 2 Met Pro Glu Lys Gln Tyr Glu Asn Glu Ile Glu Thr Tyr Lys Glu Ser Met Pro Glu Lys Gln Tyr Glu Asn Glu Ile Glu Thr Tyr Lys Glu Ser 1 5 10 15 1 5 10 15 Ala Ile Ser Lys Pro Leu Val Glu His Ile Tyr Thr Ala Asp Pro Ser Ala Ile Ser Lys Pro Leu Val Glu His Ile Tyr Thr Ala Asp Pro Ser 20 25 30 Ala His Val Phe Glu Gly Lys Ile Tyr Ile Tyr Pro Ser His Asp Ile 35 40 45 Glu Ala Gly Ile Pro Phe Asn Asp Asn Gly Asp His Phe Ala Met Cys 50 55 60 Asp Tyr His Val Leu Ser Met Asp Ser Leu Thr Ser Pro Val Gln Asp 65 70 75 80 His Gly Val Ala Leu His Val Asp Asn Val Lys Trp Ala Glu Arg Gln 85 90 95 Met Trp Ala Pro Asp Ala Ala Thr Lys Asn Gly Lys Tyr Phe Leu Tyr 100 105 110 Phe Pro Ala Lys Lys Ser Asp Gly Ile Phe Gln Ile Gly Val Ala Val 115 120 125 Ser Asn Thr Pro His Gly Pro Phe Met Ala Gln Pro Glu Pro Ile Gly 130 135 140 Gly Ser Tyr Ser Ile Asp Pro Ala Val Phe Glu Asp Asp Asp Gly Thr 145 150 155 160 His Tyr Met Tyr Phe Gly Gly Ile Trp Gly Gly Gln Leu Gln Asn Tyr 165 170 175 Arg Glu Asn Lys Tyr Asn Ala Ser Phe Ala Glu Pro Gln Asp Gly Glu 180 185 190 Pro Ala Leu Gly Pro Ile Val Ala Lys Met Ser Gly Asp Met Leu Glu 195 200 205 Phe Asp His Thr Pro Arg Glu Ile Leu Ile Val Asp Glu Gly Gly Lys 210 215 220 Pro Leu Leu Ala Gly Asp His Glu Arg Arg Phe Phe Glu Ala Ser Trp 225 230 235 240 Met His Lys His Lys Gly Lys Tyr Tyr Phe Ser Tyr Ser Thr Gly Asn 245 250 255 Thr His Tyr Leu Cys Tyr Ala Ile Gly Asp Ser Pro Tyr Gly Pro Phe 260 265 270 Val Tyr Ala Gly Arg Ile Leu Glu Pro Val Val Gly Trp Thr Thr His 275 280 285 His Ser Ile Cys Glu Phe Glu Gly Lys His Tyr Leu Phe Tyr His Asp 290 295 300 Ser Thr Leu Ser Gly Gly Val Thr His Leu Arg Ser Val Lys Val Ala 305 310 315 320 Pro Ile Glu Tyr Asp Asp Asp Gly Lys Ile Ile Thr Leu Ser Pro Tyr 325 330 335 Gly Asp Val Arg Val Glu 340

Claims

1. A mutant of xylanase XynZT-2, characterized in that, The amino acid sequence of the xylanase XynZT-2 mutant is as follows: (1) Mutate methionine at position 97 of xylanase XynZT-2 with the amino acid sequence as shown in SEQ ID No.2 to leucine; (2) Mutate alanine at position 102 of xylanase XynZT-2 with the amino acid sequence as shown in SEQ ID No.2 to cysteine; (3) Mutate methionine at position 241 of xylanase XynZT-2 with the amino acid sequence as shown in SEQ ID No.2 to leucine; (4) Mutate threonine at position 287 of xylanase XynZT-2 with the amino acid sequence as shown in SEQ ID No.2 to serine; (5) Mutate isoleucine at position 291 of xylanase XynZT-2 with the amino acid sequence as shown in SEQ ID No.2 to valine.

2. A xylanase XynZT-2 mutant gene, characterized in that, It encodes the xylanase XynZT-2 mutant as described in claim 1.

3. A recombinant expression vector containing the gene of the xylanase XynZT-2 mutant as described in claim 2.

4. A recombinant strain containing the gene of the xylanase XynZT-2 mutant as described in claim 2.

5. The recombinant strain according to claim 4, wherein The recombinant strain is Escherichia coli BL21.

6. The application of the xylanase XynZT-2 mutant as described in claim 1 in hydrolyzing xylan.

7. The application of the xylanase XynZT-2 mutant as described in claim 1 in the preparation of feed / feed additives.

8. The application of the xylanase XynZT-2 mutant as described in claim 1 in food, brewing and bioenergy.

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