Alkaline xylanase mutant with improved specific enzyme activity and thermal stability and preparation method thereof

Abstract

The invention discloses an alkaline xylanase mutant with improved specific enzyme activity and thermal stability and a preparation method thereof, and belongs to the field of gene engineering and enzyme engineering. The alkaline xylanase mutant M22 is provided through a semi-rational design method, and the specific enzyme activity of the alkaline xylanase mutant M22 is 211 U / mg and is improved by 181.33% compared with that of a wild type. After the mutant M22 is subjected to heat treatment at 75 DEG C for 30 minutes, the residual activity of the mutant M22 relative to a wild type is 169.30%, and relative residual activities of the mutant M22 relative to the wild type which is subjected to heat treatment at 75 DEG C for 30 minutes are 153.94%, 127.84% and 115.02% respectively after the mutant M22 is subjected to heat treatment at 80 DEG C, 85 DEG C and 90 DEG C. Compared with a wild type, the alkaline xylanase mutant M22 obtained by the invention has the advantages that the specific enzyme activity and stability are obviously improved, and the alkaline xylanase mutant M22 has higher application value and potential in industrial production.

Description

Technical Field

[0001] The invention relates to an alkaline xylanase mutant with improved specific enzyme activity and thermal stability and a preparation method thereof, and belongs to the field of gene engineering and enzyme engineering. Background Art

[0002] Xylan is the second most abundant polysaccharide in nature, second only to cellulose. Xylans from different sources vary greatly in composition and structure. The main chain of natural xylan is composed of β-1,4-pyranose D-xylose, and can also be replaced by sugars and organic acids such as arabinose and coumaric acid. The chemical composition of the branched substituents of xylan is usually related to its source, such as O-acetyl, α-L-furanosyl, α-D-pyranoglucuronic acid, etc. Its branched substituents can be further connected with ferulic acid, lignin, etc. to form a more complex structure. Therefore, for the degradation of xylan, a series of hydrolases with different specificities are often required, such as xylanase, β-xylosidase, α-l-arabinosidase, etc.

[0003] Xylanase endo-1,4-β-xylanase (EC 3.2.1.8) belongs to the glycoside hydrolase (GH) family, catalyzes β-1-4-glycosidic bonds, realizes the degradation of the main chain skeleton to form oligosaccharides, and plays a major role in the degradation of xylan. At present, most of the research on xylanases focuses on GH10 and GH11 family xylanases. GH10 xylanases have (α / β)8 barrel-shaped results and can effectively degrade pretreated biomass, which has great potential in the fields of industry and biotechnology.

[0004] In many industrial fields such as papermaking and feed industry, and textile process, alkaline thermostable xylanase has important applications. Alkaline treatment and high temperature conditions in the environment have high requirements for the catalytic activity and heat resistance of xylanase. Patent CN201010256129.0 provides a high temperature and alkali resistant xylanase, which can be used in pulp bleaching and other fields. Higher bleaching temperature is conducive to accelerating the reaction rate and improving the bleaching efficiency. Generally speaking, the bleaching speed is doubled for every 7°C increase in temperature. Therefore, it is of great significance to provide an alkaline xylanase with high specific enzyme activity and stability under extreme temperatures to promote its industrial production application. Summary of the invention

[0005] In order to overcome the shortcomings and deficiencies of the prior art, the object of the present invention is to provide an alkaline xylanase mutant with improved enzyme activity and thermal stability and a preparation method thereof, which is beneficial to its many applications in the industrial field.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] The present invention transforms the heat-resistant and alkali-resistant xylanase (amino acid sequence SEQ ID NO: 1, nucleotide sequence SEQ ID NO: 2) from Bacillus halodurans through a semi-rational design method to obtain a mutant with improved enzyme activity and stability. The present invention also provides a recombinant vector and a recombinant engineering bacterium containing the mutant gene.

[0008] Therefore, the first object of the present invention is to provide an alkaline xylanase mutant with improved specific enzymatic activity and thermal stability, the amino acid sequence of which is shown in SEQ ID NO: 3. The mutant is obtained by mutating the 96th histidine (H) of the alkaline xylanase shown in SEQ ID NO: 1 to asparagine (N), the 145th glutamic acid (E) to isoleucine (I), and the 168th valine (V) to alanine (A). The mutant is named M22.

[0009] The second object of the present invention is to provide a gene encoding the alkaline xylanase mutant.

[0010] In one embodiment of the present invention, the nucleotide sequence of the gene encoding the alkaline xylanase mutant is shown in SEQ ID NO:4.

[0011] The third object of the present invention is to provide an expression cassette and a recombinant vector containing the above gene.

[0012] In one embodiment of the present invention, the starting vector of the recombinant vector includes but is not limited to pET series vectors or pPICZα vectors; preferably pET-28a(+) vectors or pPICZαA vectors.

[0013] The fourth object of the present invention is to provide a recombinant engineering bacterium expressing the xylanase mutant.

[0014] In one embodiment of the present invention, the host bacteria of the recombinant engineered bacteria are bacteria, fungi, etc.; the bacteria include bacteria of the genus Escherichia, etc.; the fungi include yeasts such as Pichia pastoris.

[0015] Preferably, in one embodiment of the present invention, the recombinant engineered bacteria uses E. coli BL21 (DE3) as a host.

[0016] Preferably, in one embodiment of the present invention, the host cell is Pichia pastoris.

[0017] The fifth object of the present invention is to provide a method for enhancing the specific enzymatic activity and thermal stability of alkaline xylanase, which is to mutate the 96th histidine (H) of the xylanase to asparagine (N), the 145th glutamic acid (E) to isoleucine (I), and the 168th valine (V) to alanine (A).

[0018] The sixth object of the present invention is to provide a method for obtaining the alkaline xylanase mutant.

[0019] In one embodiment of the present invention, primers containing mutation sites are designed to perform site-directed mutagenesis on the gene encoding the alkaline xylanase with an amino acid sequence as shown in SEQ ID NO: 1 and then expressed to obtain an alkaline xylanase mutant with improved enzyme activity and thermal stability.

[0020] Furthermore, primers containing mutation sites were designed to introduce mutations into the gene encoding the alkaline xylanase having an amino acid sequence as shown in SEQ ID NO: 1. After correct sequencing, the gene was transformed into Escherichia coli BL21 (DE3) for expression to obtain an alkaline xylanase mutant with improved enzyme activity and thermal stability.

[0021] Furthermore, the single clone of the genetically engineered bacteria verified by colony PCR was inoculated into LB medium with a final concentration of 50 μg / mL kanamycin, and cultured at 37°C, 200 rpm for 12-16 h to obtain seed solution. 600 =0.1, transfer the seed solution to LB medium containing kanamycin at a final concentration of 50 μg / mL, and culture at 37°C with a shaker at 200 rpm until OD 600 =0.6-0.8, add IPTG with a final concentration of 0.4 mmol / L, and culture in a shaking incubator at 16°C and 200 rpm for 16 h to induce expression.

[0022] The seventh object of the present invention is to provide the use of the alkaline xylanase mutant, encoding gene, expression cassette, recombinant vector or recombinant engineered bacteria in preparing an alkaline xylanase mutant with improved specific enzyme activity and thermal stability.

[0023] The eighth objective of the present invention is to provide the application of the alkaline xylanase mutant, encoding gene, expression cassette, recombinant vector or recombinant engineered bacteria in the fields of biomass resource utilization, food or pharmaceuticals.

[0024] Compared with the prior art, the present invention has the following advantages and effects:

[0025] The present invention provides an alkaline xylanase mutant M22 by a semi-rational design method, and its specific enzyme activity is 211U / mg, which is 181.33% higher than that of the wild type. After heat treatment at 75°C for 30 minutes, the residual activity of the mutant M22 relative to the wild type is 169.30%; relative to the wild type treated at 75°C for 30 minutes, the relative residual activities of M22 after treatment at 80°C, 85°C, and 90°C are 153.94%, 127.84%, and 115.02%, respectively. Compared with the wild type, the alkaline xylanase mutant M22 obtained by the present invention has significantly improved specific enzyme activity and stability, and has higher application value and potential in industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the relative residual activity of the alkaline xylanase combination mutant M22 and the wild type after treatment at 75℃, 80℃, 85℃, and 90℃ for 30 minutes. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0028] The present invention discloses an alkaline xylanase mutant with high specific enzyme activity and thermal stability, as well as a preparation method and application thereof, a DNA molecule encoding the enzyme mutant, a corresponding vector and a host cell. The technology can provide a reference for professionals in the field. Specific embodiments and drawings will further describe the present invention in detail, but are not limited to this description. Any operating steps or conditions not described in detail in the embodiments are in accordance with the conventional technical standards in the field, and any other embodiments obtained by any ordinary technician in the field without creative work are within the scope of protection of the present invention.

[0029] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field, and the reagents and materials used are commercially available or can be prepared by known methods.

[0030] The culture medium and required solutions involved in the following examples are as follows:

[0031] LB liquid culture medium: yeast powder 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L.

[0032] LB solid medium: yeast powder 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L, agar powder 2% (m / v).

[0033] IPTG (1 mol / L): Dissolve 2.38 g of isopropylthio-β-D-galactoside (IPTG) in 8 mL of distilled water, make up to 10 mL with distilled water, filter and sterilize with a 0.22 μm filter membrane, and divide into 1 mL portions and store at -20°C.

[0034] Gly-NaOH buffer: dissolve 7.47 g of glycine in 900 mL of distilled water, adjust the pH to 9.0 with NaOH, add water to make up to 1 L, and keep warm at room temperature for later use.

[0035] Example 1: Construction of xylanase recombinant plasmid

[0036] The recombinant plasmid pET-28a(+)-Xyn is constructed by designing homologous arm recombination primers on the pET-28a(+) vector and the nucleotide sequence (SEQ ID NO: 2) of the gene encoding xylanase Xyn (SEQ ID NO: 1) through a homologous recombination method.

[0037] The primers on the pET-28a(+) vector are:

[0038] The upstream primer was: 5′-TGAGATCCGGCTGCTAACAA-3′;

[0039] The downstream primer was: 5′-GTGATGATGATGATGATGGCT-3′;

[0040] The primers on the nucleotide sequence of the xylanase gene are:

[0041] Upstream primer: 5′-AGCCATCATCATCATCATCACATGGCTCAAGGTGGTCCACCAAAGT-3′;

[0042] Downstream primer: 5′-GCTTTGTTAGCAGCCGGATCTCAATCAATAATTCTCCAGTAAGCT-3′.

[0043] Example 2: Construction of mutant recombinant plasmid

[0044] The wild-type strain recombinant plasmid pET-28a(+)-Xyn nucleotide sequence was used as a template, and the whole plasmid PCR polymerase chain reaction was performed using primers containing mutation sites. PCR reaction conditions: 94°C denaturation for 5 min, 98°C denaturation for 10 s, 55°C annealing for 30 s, 68°C extension for 195 s, 30 cycles, 68°C extension for 10 min, and storage at 4°C. The PCR reaction system is shown in Table 1, and the mutant primers are shown in Table 2.

[0045] Table 1 PCR reaction system

[0046]

[0047] Table 2 Mutation primer design (lowercase indicates replaced codons)

[0048]

[0049] Product recovery: add 50 μL sterile water to the above PCR product, then add 200 μL Buffer GDP, mix well, and centrifuge at 12000 rpm for 30 seconds; discard the filtrate, wash twice with 700 μL Buffer PW2 (diluted with anhydrous ethanol); finally elute with 30 μL water and measure the concentration.

[0050] Template digestion: Add restriction endonuclease Dpn I to the above-recovered product to remove the template plasmid. The digestion reaction is 600-1200ng of PCR recovered product, 1μL of 10× buffer, 0.5μL of Dpn I digestion enzyme, and ddH2O is added to the final system of 10μL. The reaction conditions are 37℃, 30min.

[0051] Recombinant plasmid transformation: 10 μL of the above digested product was transformed into 100 μL of E. coli Top10 competent cells, spread on LB solid plates containing kanamycin at a final concentration of 50 μg / mL, and cultured overnight in a 37°C constant temperature incubator for 16 h.

[0052] Verification of transformed clones: Randomly pick 1 to 2 monoclonal colonies for sequencing verification.

[0053] Example 3: Fermentation, induced expression and purification of combined mutant M22

[0054] Take 5 μL of the plasmid after successful sequencing verification and transform it into 100 μL of Escherichia coli BL21 (DE3) competent cells, spread it on an LB solid plate containing a final concentration of 50 μg / mL kanamycin, and culture it overnight in a 37°C constant temperature incubator for 16 hours.

[0055] Pick the colonies, and after verification by colony PCR, inoculate them into LB medium containing kanamycin at a final concentration of 50 μg / mL, with a liquid volume of 10 mL / 50 mL, and culture them at 37°C, 200 r / min for 12 to 16 h to obtain seed solution.

[0056] Initial OD 600 =0.1, transfer the seed solution to LB medium containing a final concentration of 50 μg / mL kanamycin, fill the solution in 100mL / 250mL, and culture at 37℃ shaker, 200r / min until OD 600=0.6-0.8, add IPTG with a final concentration of 0.4 mmol / L, and culture in a shaking incubator at 16°C and 200 r / min for 16 h to induce expression.

[0057] The fermentation broth of the same optical density was placed at 4°C and centrifuged at 7000 rpm for 5 minutes. The centrifuged bacteria were washed once with 15mL of Gly-NaOH buffer at pH 9.0, and then resuspended with 10mL of the same buffer at pH 9.0. In an ice water bath, the cells were broken using an ultrasonic cell crusher for a total of 10 minutes (3 seconds each time, 3 seconds interval). Then, at 4°C, centrifuged at 10,000 rpm for 30 minutes, and filtered using a filter membrane with a pore size of 0.22 μm to obtain the fermentation supernatant of the mutant. The combined mutant M22 (H96N / E145I / V168A) was activated, transferred, induced, and harvested under the same conditions as the wild type.

[0058] Use Ni + The protein in the supernatant was purified by affinity chromatography column, and gradient elution was performed using imidazole. The purified eluate was collected, replaced with Gly-NaOH buffer using a 10 kDa ultrafiltration tube, and concentrated.

[0059] Example 4: Method for determining specific enzymatic activity and relative residual activity of alkaline xylanase

[0060] Alkaline xylanase activity determination method: The enzyme activity was determined by 540nm light absorption method, and alkaline xylanase was determined with beech xylan as substrate. One enzyme activity unit refers to the reaction efficiency of producing 1μmoL reducing sugar within 1min under the conditions of pH 9.0 and 70℃. The reaction system added 200μL of substrate solution into a 1.5mL EP tube, and the substrate solution was 1% beech xylan solution. It was preheated at 70℃ for 5min in a constant temperature mixer, and 20μL of appropriately diluted enzyme solution was added. The reaction was allowed to react for 10min, and 300μL of DNS solution was immediately added. The reaction was terminated by boiling for 5min. 200μL of the solution after the reaction was taken and its absorbance value was measured at 540nm to calculate the enzyme activity. The protein concentration was determined by the Coomassie brilliant blue method, and the specific enzyme activity of the purified combined mutant M22 and the wild type was obtained by dividing the enzyme activity by the protein concentration.

[0061] In one embodiment of the present invention, the specific enzyme activity of the combined mutant M22 is 211 U / mg, which is 181.33% higher than that of the wild type (75 U / mg).

[0062] The fermentation supernatants of the wild type and mutant M22 were treated at 75°C, 80°C, 85°C, and 90°C for 30 min, and their residual activities after different incubation time periods were measured. The enzyme activity of the wild type treated at 75°C for 30 min was recorded as 100%. The results are shown in Figure 1 As shown in the figure, compared with the wild type treated at 75°C for 30 min, the relative residual activity of the combined mutant M22 is 169.30%, and after treatment at 80°C, 85°C, and 90°C, the relative residual activities of M22 are 153.94%, 127.84%, and 115.02%, respectively. It can be seen that the combined mutant M22 of the present invention has good thermal stability.

[0063] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. An alkaline xylanase mutant with improved specific enzymatic activity and thermal stability, characterized in that: The amino acid sequence of the mutant is shown in SEQ ID NO:

3.

2. A gene encoding the alkaline xylanase mutant with improved specific enzyme activity and thermal stability as claimed in claim 1.

3. The gene according to claim 2, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO:

4.

4. The biomaterial related to the alkaline xylanase mutant with improved specific enzyme activity and thermal stability according to claim 1, characterized in that: Any one or more combinations of the following biological materials: (1) An expression cassette containing the gene according to claim 2 or 3; (2) A recombinant vector containing the gene according to claim 2 or 3; (3) a recombinant vector containing the expression cassette described in (1); (4) A recombinant engineered bacterium containing the gene of claim 2 or 3; (5) A recombinant engineered bacterium containing the expression cassette described in (1); (6) A recombinant engineered bacterium containing the recombinant expression vector described in (2) or (3).

5. The biomaterial according to claim 4, characterized in that: The starting vector of the recombinant vector in (2) and (3) is a pET series vector or a pPICZα vector; The host bacteria of the recombinant engineered bacteria described in (4), (5) and (6) are bacteria or fungi.

6. Use of the alkaline xylanase mutant with improved specific enzyme activity and thermal stability according to claim 1, the gene according to any one of claims 2 to 3, or the biomaterial according to any one of claims 4 to 5 in the preparation of the alkaline xylanase mutant with improved specific enzyme activity and thermal stability.

7. Use of the alkaline xylanase mutant with improved specific enzyme activity and thermal stability as claimed in claim 1, the gene as claimed in any one of claims 2 to 3, or the biomaterial as claimed in any one of claims 4 to 5 in the fields of biomass resource utilization, food or pharmaceuticals.

8. A method for enhancing the specific enzymatic activity and thermal stability of alkaline xylanase, characterized in that: The alkaline xylanase shown in SEQ ID NO: 1 is mutated by changing the histidine at position 96 to asparagine, the glutamic acid at position 145 to isoleucine, and the valine at position 168 to alanine.

9. A method for obtaining the alkaline xylanase mutant with improved specific enzyme activity and thermal stability as claimed in claim 1, characterized in that: The method comprises the following steps: performing site-directed mutation on a gene encoding an alkaline xylanase having an amino acid sequence as shown in SEQ ID NO: 1 by designing primers containing mutation sites, and then expressing the gene to obtain the alkaline xylanase mutant with improved specific enzyme activity and thermal stability as claimed in claim 1.

10. The method according to claim 9, characterized in that: Primers containing mutation sites are designed to introduce mutations into the gene encoding the alkaline xylanase whose amino acid sequence is shown in SEQ ID NO: 1, and after correct sequencing, the gene is transformed into Escherichia coli BL21 (DE3) for expression to obtain the alkaline xylanase mutant with improved specific enzyme activity and thermal stability as described in claim 1.

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