Xylanase mutant Xyn, coding gene, vector, engineering bacterium, preparation method and application

Abstract

The invention discloses a xylanase mutant Xyn, a coding gene, a carrier, an engineering bacterium as well as a preparation method and application. The xylanase mutant Xyn is obtained by mutating the 56th amino acid, the 152nd amino acid and the 204 amino acid of wild type xylanase Xyn into isoleucine, aspartic acid and glutamic acid from valine, asparagine and lysine respectively; the amino acid sequence and the nucleotide sequence of the gene are SEQ ID NO.1 and SEQ ID NO.2 respectively. Under the conditions that the pH is 10.0 and the temperature is 60 DEG C, the specific enzyme activity of the xylanase mutant Xyn is 9951.5 U / mL and is 2.2 times that of wild enzyme Xyn; after the xylanase mutant Xyn is subjected to heat preservation at 70 DEG C for 40 minutes, the residual enzyme activity of the xylanase mutant Xyn is 8312 U / mL, which is 4.1 times of that of wild enzyme Xyn, so that the xylanase mutant Xyn has important application prospects under alkaline conditions.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, and more specifically, relates to a xylanase mutant △Xyn, its encoding gene, vector, engineered bacteria, preparation method, and application. Background Technology

[0002] Xylanase is a class of hydrolases that catalyze the hydrolysis of 1,4-β-D-xylosidic bonds in xylan. It is widely used in papermaking, food processing, feed, bioenergy, and environmental protection.

[0003] The industrial application of xylanase currently faces multiple challenges: (1) Insufficient thermal stability: Most wild-type xylanases are easily inactivated under high temperature conditions (such as above 60 °C). Some xylanases have an enzyme activity residual rate of less than 50% after treatment at 70 °C for 30 min, which is difficult to meet the requirements of high-temperature industrial processes (such as biobleaching and high-temperature fermentation). (2) Poor alkaline stability: In alkaline environments (pH 9-11), the catalytic efficiency of wild-type xylanases decreases significantly. The specific activity of some xylanases under alkaline conditions is only 30%-50% of that under neutral conditions, which limits their application in high-alkaline scenarios such as biobleaching in the paper industry. (3) Difficulty in synergistic optimization of heat resistance and alkali resistance: In existing studies, the modification of thermal stability or alkali stability is mostly in a single direction, lacking a systematic strategy to improve the performance of both at the same time. Some mutants significantly improve heat resistance through site-directed mutation (such as N165H, H61L), but their performance under alkaline conditions is not clear. Other mutants have improved activity in alkaline environment, but thermal stability has not been optimized at the same time.

[0004] To date, there have been no reports on simultaneously optimizing the thermal and alkaline stability of xylanase. Summary of the Invention

[0005] In view of this, the present invention provides a xylanase mutant △Xyn, its encoding gene, vector, engineered bacteria, preparation method, and application.

[0006] Specifically, this invention designs potential mutant xylanases based on wild-type xylanase sequences in a database using structural biology and energy calculations; and obtains the xylanase mutant gene Δ through site-directed mutagenesis based on the wild-type gene sequence. xyn By employing a eukaryotic expression system for efficient expression, xylanase mutants with improved thermal stability, alkali stability, and specific activity were obtained. This can reduce production costs, expand the scope of application, and enhance its application potential in industrial scenarios such as papermaking, food, and feed.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: This invention provides a xylanase mutant △Xyn, in which the amino acids at positions 56, 152, and 204 of the wild-type xylanase Xyn are mutated from valine, asparagine, and lysine to isoleucine, aspartic acid, and glutamic acid, respectively.

[0008] This invention utilizes combined site-directed mutagenesis technology to molecularly modify wild-type xylanase. Specifically, mutants are designed using molecular dynamics simulations combined with the computational program FoldX, and mutations at sites such as V56I, N152D, and K204E are screened out, which significantly improve the enzyme's enzymatic properties such as thermal stability and alkaline stability.

[0009] In detail, in the above technical solution, valine at position 56 is mutated to isoleucine, making its side chain longer and more hydrophobic. This can enhance the interaction between the β-sheet barrels in the core hydrophobic region of the enzyme molecule, reduce the probability of conformational unfolding at high temperatures, and significantly improve the thermal stability of the mutant xylanase.

[0010] In detail, in the above technical solution, the asparagine at position 152 is mutated to aspartic acid. Introducing a negative charge on the surface can enhance hydrophilicity under alkaline conditions and reduce enzyme protein aggregation. At the same time, the carboxyl group of the Asp side chain can form additional hydrogen bonds with neighboring residues, which can enhance spatial conformational stability and improve the heat resistance of the mutant xylanase.

[0011] In detail, in the above technical solution, the lysine at position 204 is mutated to glutamic acid, which increases the surface negative charge density, enhances the stability of the mutated xylanase in an alkaline environment, and the Glu204 side chain can form a salt bridge with the adjacent Arg180, further stabilizing the conformation at high temperature.

[0012] In the above technical solution, the combined mutation of three key amino acid sites produced a significant synergistic effect, transforming xylanase into a novel industrial enzyme with high enzyme activity under high temperature and alkaline conditions.

[0013] Specifically, the thermophilic bacterium *Thermophyton floccosum* (GenBank database) is used to publish the results of the study. Thermotoga maritima M9) xyn Based on the gene sequence (GenBank accession number: PX724084), the key amino acids encoded by it were replaced.

[0014] Furthermore, in the above technical solution, the amino acid sequence of the xylanase mutant △Xyn is shown in SEQ ID NO.1.

[0015] The present invention also provides the encoding gene △Xyn for the above-mentioned xylanase mutant △Xyn. xyn .

[0016] Furthermore, in the above technical solution, the gene encoding the xylanase mutant △Xyn is △ xyn The nucleotide sequence is shown in SEQ ID NO.2.

[0017] The present invention also provides a gene encoding the xylanase mutant △Xyn. xyn The carrier.

[0018] Furthermore, the present invention also provides a gene encoding the xylanase mutant △Xyn. xyn Or the one containing the coding gene △ xyn The host cell of the vector.

[0019] Furthermore, the present invention also provides a coding gene Δ containing the xylanase mutant described above. xyn Or the one containing the coding gene △ xyn Engineered bacteria on a carrier.

[0020] In another aspect, the present invention provides a method for preparing the xylanase mutant ΔXyn, comprising: The nucleotide sequence shown in SEQ ID NO.2 was expressed using a plasmid capable of expressing the enzyme as an expression vector and a bacterial strain capable of expressing the enzyme as an expression host, thereby achieving efficient expression of the mutant shown in SEQ ID NO.1.

[0021] In detail, in the above technical solution, the nucleotide sequence shown in SEQ ID NO.2 is expressed using a plasmid (preferably pPICZαA) capable of expressing the enzyme, and a strain capable of expressing the enzyme (preferably Pichia pastoris) is used. P. pastoris ) to express the host, achieving the coding gene △ for xylanase mutants xyn V56I / N152D / K204E Highly efficient secretory expression.

[0022] Specifically, in the above technical solution, the wild-type xylanase gene xyn The accession number of the xylanase xyn encoded by the gene is PX724084 in the GenBank database. The pPICZαA expression vector used contains the AOX1 promoter and also inserts elements such as the α-factor signal peptide, the Zeocin resistance gene, and the His4 selector. It can induce the expression of exogenous enzymes with methanol and has controllable expression.

[0023] In another aspect, the present invention provides the encoding gene Δ of the xylanase mutant. xyn The application of the enzymes it encodes in cotton and linen processing, food processing, feed processing, and pulp and paper making.

[0024] Compared with the prior art, the present invention has the following advantages: (1) The xylanase mutant △Xyn provided by the present invention has significantly improved enzyme activity and heat resistance under alkaline conditions, which solves the problems of low catalytic activity and insufficient thermal stability of wild-type xylanase under alkaline conditions, and creates favorable conditions for the application of this enzyme in cotton and linen processing, food processing, feed processing, pulp and paper making and other industries. (2) This invention compares the degradation capacity of wild-type enzyme Xyn and xylanase mutant △Xyn under alkaline conditions. The results show that at pH 10.0 and 60 ℃, the specific enzyme activity of xylanase mutant △Xyn is 9951.5 U / mL, which is 2.2 times that of wild-type enzyme Xyn (4523.4 U / mg). After incubation at 70 ℃ for 40 min, the residual enzyme activity of xylanase mutant △Xyn is 8312 U / mL, which is 4.1 times that of wild-type enzyme Xyn. This indicates that xylanase mutant △Xyn has important application prospects under alkaline conditions. Attached Figure Description

[0025] Figure 1 This is a flowchart illustrating the construction process of the engineered strain of xylanase mutant △Xyn in this embodiment of the invention. Figure 2 This is a construction map of the recombinant plasmid pPICZαA-△xyn for the xylanase mutant △Xyn in this embodiment of the invention; Figure 3 The recombinant plasmid pPICZαA-△ for the xylanase mutant △Xyn in this embodiment of the invention. xyn Linearization detection graph (in the graph: M is DNA Marker; 1 is the recombinant plasmid before linearization; 2 is the recombinant plasmid after linearization). Figure 4 The recombinant plasmid pPICZαA-△ for the xylanase mutant △Xyn in this embodiment of the invention. xyn PCR detection graph (in the graph: M is DL5000 DNA Marker; 1-20 are PCR bands of different clones; - is negative control; + is positive control). Figure 5 This is an SDS-PAGE spectrum of the fermentation supernatant induced by the xylanase mutant △Xyn in this embodiment of the invention (in the figure: M is the protein marker; 1-10 are the genetically engineered strain pPICZαA-△ of the xylanase mutant △Xyn that was positive by PCR detection). xyn / X33; - is the blank plasmid engineered bacteria pPICZαA / X33 without the inserted foreign gene, serving as a negative control); Figure 6This is a Western blot analysis of the fermentation supernatant induced by the xylanase mutant △Xyn in this embodiment of the invention (in the figure: M is the protein marker; 1-10 are the genetically engineered strain pPICZαA-△ of the xylanase mutant △Xyn that tested positive by PCR). xyn / X33; - is the blank plasmid engineered bacteria pPICZαA / X33 without the inserted foreign gene, serving as a negative control). Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments.

[0027] It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0028] In the embodiments, unless otherwise specified, all methods used are conventional methods in the art.

[0029] The terms “comprising,” “including,” or any other variations thereof, as used herein, are intended to cover non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, article, or apparatus.

[0030] Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0031] Materials and reagents: Expression vector pPICZαA, eukaryotic competent cells P. pastoris All X33s were purchased from Novagen. Restriction endonucleases Eco R I and Xho I. Speedy Cut SacI enzyme, Ni-NTA, and goat anti-mouse secondary antibody were purchased from Sangon Biotech. Ultra HiFidelity PCR Kit, Prokaryotic Cloning Competent Cells E. coli TOP10, RNA Simple Total RNA Extraction Kit, Rapid Site-directed Mutagenesis Kit, TIANScript II RT Kit, Agarose Gel DNA Recovery Kit, and Plasmid Rapid Extraction Kit were all purchased from Tiangen Biosciences. Xylan, tryptone, yeast extract and agar powder were purchased from Sigma-Aldrich. LB medium, BMMY medium, and BMGY medium were purchased from Beijing Coolplay Technology Co., Ltd.

[0032] Primer synthesis and nucleic acid sequencing were performed by Sangon Biotech.

[0033] The remaining chemical reagents were all commercially available analytical grade products, purchased from Shanghai Sinopharm Group.

[0034] like Figure 1 The diagram shown is a flowchart illustrating the construction process of the engineered strain of xylanase mutant △Xyn in an embodiment of the present invention; as shown Figure 2 The diagram shows the construction pattern of the recombinant plasmid pPICZαA-△xyn of the xylanase mutant △Xyn in an embodiment of the present invention.

[0035] Example 1: Primitive xylanase gene xyn Construction of recombinant plasmids Will T. maritima M9 (wild-type strain, derived from hot spring water in Yichun, Jiangxi) was cultured to the logarithmic growth phase. 1.5 mL of the bacterial culture was centrifuged at 12000 r / min for 1 min, and the bacterial pellet was collected. Then, genomic DNA was extracted according to the kit instructions to obtain the genomic DNA of the wild-type strain.

[0036] Based on wild-type xylanase gene xyn The sequence (GenBank accession number: PX724084) was used to design the following primers using the bioinformatics software SnapGene: F: 5'-CC GAATTC ATGTCTCAGAATGTATCTCTGAGAGAAC-3' (SEQ ID NO.3, containing Eco R I restriction site) R:5'-CG CTCGAG TTTTCTTTCTTCTATCTTTTTCTCCAGC-3' (SEQ ID NO.4, containing Xho I restriction site).

[0037] by T. maritima Using M9 genomic DNA as a template, PCR amplification was performed.

[0038] The PCR reaction system was as follows: cDNA template, 1 μL (approximately 50 ng); forward primer F (10 µmol / L), 1.0 µL; reverse primer R (10 µmol / L), 1.0 µL; 2×Pfu Master Mix, 25 μL; and sterile ddH2O was added to bring the total volume to 50 μL. After mixing, the mixture was placed on a PCR instrument for PCR reaction.

[0039] The parameters are set as follows: (1) Pre-denaturation at 94 ℃ for 5 min; (2) Denaturation at 94 ℃ for 50 s; (3) Annealing at 58 ℃ for 50 s; (4) Extension at 72 ℃ for 1 min; Repeat steps (2)-(4) for 30 cycles; (5) Extension at 72 ℃ for 10 min.

[0040] The obtained PCR products were detected by 1.0% agarose gel electrophoresis, and the gel was cut. The target fragment was recovered using a DNA gel recovery kit and stored at -20℃ for later use.

[0041] For pPICZαA plasmid and target gene PCR product, use Eco R I and Xho After double digestion with enzyme I, followed by gel purification, the digestion product was ligated with T4 DNA ligase at 16°C overnight to obtain the recombinant plasmid. The plasmid was then transformed into DNA using a heat shock method. E. coli Top 10 were plated on LB medium containing 25 μg / mL Zeocin and cultured overnight; positive transformants were picked, cultured overnight, and plasmids were extracted for PCR identification; the plasmids of positive clones were submitted to Sangon Biotech for sequencing, and the recombinant plasmid with the correct target gene was pPICZαA-xyn.

[0042] Example 2: Site-directed mutagenesis Site-directed mutagenesis principle: The construction of point mutation plasmids uses... Dpn I method.

[0043] The following PCR point mutation primers were designed based on the amino acid sites to be mutated: F V56I :5' GTACATGGAA ATT GCAAGAAGAG3' (SEQ ID NO.5) R V56I :5' CTCTTCTTGC AAT TTCCATGTAC3' (SEQ ID NO.6) F N152D :5' GATGTGGTG GAC GAAGCGGTGA 3' (SEQ ID NO.7) R N152D :5' TCACCGCTTC GTC CACCACATC 3' (SEQ ID NO.8) F K204E :5' ATCAACGCA GAATCGAACTTC 3' (SEQ ID NO.9) R K204E :5' GAAGTTCGA TTC TGCGTTGAT 3' (SEQ ID NO.10) The underlined parts represent the codons corresponding to isoleucine at position 56, aspartic acid at position 152, and glutamic acid at position 204 encoded by the mutant gene.

[0044] Using a rapid site-directed mutagenesis kit with pPICZαA-xyn recombinant plasmid as a template, whole plasmid PCR was performed to introduce mutation sites.

[0045] The PCR reaction system consisted of: 0.5 µL of forward primer (10 µmol / L), 0.5 µL of reverse primer (10 µmol / L), 5 µL of 5×Fast Alteration Buffer, 1 µL of plasmid DNA, 0.5 µL of Fast Alteration DNA Polymerase, and sterile ddH2O to a final volume of 25 µL.

[0046] The parameters are set as follows: (1) Pre-denaturation at 95 °C for 2 min; (2) Denaturation at 94 °C for 20 s; (3) Refolding at 60 °C for 10 s; (4) Extension at 68 °C for 2.5 min; Repeat steps (2)-(4) for 18 cycles; (5) Heat at 68 °C for 5 min, and store the product at 4 °C.

[0047] 0.5 µL of restriction endonuclease Dpn Add I to 25 µL of the mutant PCR product, mix thoroughly, and digest at 37°C for 1 h; take 5 µL Dpn I digestion product transfer E. coli Top 10: Transformed bacterial cultures were evenly spread onto LB selection plates (containing 25 μg / mL Zeocin) and incubated overnight at 37°C to obtain transformants of the relevant mutant enzyme gene; plasmids were extracted and sequenced for verification, yielding the correct recombinant plasmid pPICZαA-△ for the mutant xylanase gene. xyn (like Figure 2 (As shown).

[0048] Example 3 Construction, induction, expression, and electrophoretic analysis of recombinant Pichia pastoris Use the correct mutant plasmid Sac I is linearized (e.g.) Figure 3 As shown), respectively with P. pastorisX33 competent cells were mixed at a volume ratio of 1:8, transferred to a pre-cooled electroporator, incubated on ice for 5 min, and then electroporated for 5 ms; pre-cooled sorbitol was added immediately.

[0049] In addition, the empty vector pPICZαA without the inserted foreign gene was electroporated to P. pastoris X33 was used as a negative control.

[0050] After electroporation, the solution was incubated at 28-30 ℃ for 2 h, then centrifuged. The bacterial cells were plated on YPDS (containing 100 μg / mL Zeocin) plates and incubated upside down at 28-30 ℃. Once single colonies formed, they were detected using PCR. The PCR primers are as follows: Upstream primer 9413-F: 5'-AGAGTACATCGAGAAGGCTTTC-3' (SEQ ID NO.11) Downstream primer 3'AOX: 5'-GCAAATGGCATTCTGACATCC-3' (SEQ ID NO.12) Ten clones were verified as positive by PCR (e.g.) Figure 4 (As shown) Colonies were inoculated into BMGY medium and incubated at 28-30 °C until... OD 600 2-6; replace with BMMY medium for induction (1% methanol), incubate at 28 ℃ for 72 h and then test.

[0051] Centrifuge 100 μL of fermentation broth at 12000 r / min for 5 min, then take 80 μL of supernatant into a 1.5 mL centrifuge tube, add 20 μL of 5× Loading Buffer, and incubate in a boiling water bath for 10 min.

[0052] A negative control was prepared by using the blank plasmid engineered bacteria pPICZαA / X33 without the insertion of foreign genes.

[0053] Using SDS-PAGE (e.g.) Figure 5 (as shown) and Western blot (as shown) Figure 6 (As shown) The protein secretion and expression were detected; the results showed that, compared with the blank control, the supernatant of the mutant xylanase genetically engineered bacteria pPICZαA-△xyn / X33 showed specific target protein bands in all sample wells, indicating that the mutant xylanase was successfully expressed.

[0054] Example 4: Comparison of in vitro catalytic efficiency of wild-type xylanase Xyn and xylanase mutant ΔXyn To compare the biocatalytic activity of wild-type xylanase Xyn and xylanase mutant ΔXyn under alkaline conditions, enzyme activity was measured under the same conditions.

[0055] Enzyme activity assay: Prepare a 10 mg / mL xylan substrate solution using 0.05 mol / L glycine-sodium hydroxide buffer (pH 10.0); preheat 800 μL of the xylan substrate solution to 50°C, add 200 μL of diluted enzyme solution, react accurately at 50°C for 10 min, and immediately add 2 mL of DNS; develop color in a boiling water bath for 5 min, then rapidly cool in an ice water bath; use the same enzyme solution (inactivated by boiling) as a negative control to determine the activity of the sample. OD 540 .

[0056] Xylanase activity is defined as the amount of enzyme required to release the equivalent of 1 μmol of xylose reducing sugar per minute from the substrate, which is denoted as U.

[0057] The results showed that, under conditions of 60℃ and pH 10.0, the specific activity of the mutant xylanase ΔXyn was 9951.5 U / mg, which was 2.2 times that of the wild-type xylanase Xyn (4523.4 U / mg). This indicates that the combined mutation of the three key sites V56I / N152D / K204E enhances the ability of this xylanase to catalyze the degradation of xylan.

[0058] Example 5: Comparison of the heat resistance of wild-type xylanase Xyn and xylanase mutant ΔXyn The crude enzyme solutions of wild-type xylanase Xyn and xylanase mutant △Xyn were incubated at 70 °C for 40 min, and the remaining enzyme activity was measured to characterize the thermostability of the enzymes.

[0059] The results show that: After incubation at 70 °C for 40 min, the residual enzyme activity of the xylanase mutant △Xyn was 8312 U / mL, which is 4.1 times that of the wild-type xylanase Xyn (2027.3 U / mL). This indicates that the combined mutation of the three key sites V56I / N152D / K204E significantly improves the thermostability of this xylanase.

[0060] The above embodiments are merely examples of several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent.

[0061] It should be noted that those skilled in the art can make various modifications and improvements without departing from the inventive concept, and these all fall within the scope of protection of this invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A xylanase mutant ΔXyn, characterized in that, The amino acids at positions 56, 152, and 204 of the wild-type xylanase Xyn were mutated from valine, asparagine, and lysine to isoleucine, aspartic acid, and glutamic acid, respectively.

2. The xylanase mutant ΔXyn according to claim 1, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

1.

3. The gene encoding the xylanase mutant ΔXyn as described in any one of claims 1-2. xyn .

4. The encoding gene △ according to claim 3 xyn Its characteristics are, Its nucleotide sequence is shown in SEQ ID NO.

2.

5. Contains the encoding gene △ as described in any one of claims 3-4 xyn The carrier.

6. Containing the coding gene △ as described in any one of claims 3-4 xyn Or the host cell of the vector as described in claim 5.

7. Contains the coding gene △ as described in any one of claims 3-4 xyn Or the engineered bacteria of the carrier described in claim 5.

8. The method for preparing the xylanase mutant ΔXyn according to any one of claims 1-2, characterized in that, The nucleotide sequence shown in SEQ ID NO.2 was expressed using a plasmid capable of expressing the enzyme as an expression vector and a bacterial strain capable of expressing the enzyme as an expression host, thereby achieving efficient expression of the mutant shown in SEQ ID NO.

1.

9. The method for preparing the xylanase mutant ΔXyn according to claim 8, characterized in that, The expression vector is preferably pPICZαA; And / or, the expression host is preferably Pichia pastoris. P. pastoris .

10. The gene encoding Δ according to any one of claims 3-4 xyn The application of the enzymes it encodes in cotton and linen processing, food processing, feed processing, and pulp and paper making.

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