A basic xylanase mutant with improved specific activity and thermostability and a preparation method thereof

Abstract

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

Description

TECHNICAL FIELD

[0001] The present application relates to a basic xylanase mutant with improved specific activity and thermal stability and a preparation method thereof, and belongs to the field of genetic engineering and enzyme engineering. BACKGROUND

[0002] Xylan is the second most abundant polysaccharide in nature, next to cellulose. Xylans from different sources differ greatly in composition and structure. The main chain of natural xylan is composed of β-1, 4-pyranoid D-xylose, which can also be substituted by arabinose, coumaric acid and other sugars and organic acids. The chemical composition of the branched substituent group of xylan is usually related to its source, such as O-acetyl, α-L-arabinofuranosyl, α-D-glucuronopyranosyl, etc. The branched substituent group can be further connected with ferulic acid, lignin, etc., to form a more complex structure. Therefore, a series of hydrolytic enzymes with different specificity are often needed for the degradation of xylan, such as xylanase, β-xylosidase, α-l-arabinosidase, etc.

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

[0004] In many industrial fields such as papermaking and feed industry, textile process, basic heat-resistant xylanase has important application. The catalytic activity and heat resistance of xylanase have high requirements under the conditions of alkali treatment and high temperature in the environment. Patent CN201010256129.0 provides a high-temperature-resistant and alkali-resistant xylanase, which can be applied in pulp bleaching and other fields, and higher bleaching temperature is beneficial to accelerate the reaction rate and improve the bleaching efficiency. Generally speaking, the bleaching speed is accelerated by 1 time when the temperature is increased by 7℃. Therefore, it is of great significance to provide a basic xylanase with high specific activity and stability at extreme temperature for the promotion of its industrial production and application. SUMMARY

[0005] In order to overcome the defects and deficiencies of the prior art, the purpose of the present application is to provide a basic xylanase mutant with improved specific 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 application is achieved by the following technical scheme:

[0007] The application improves the mutant of heat-resistant and alkali-resistant xylanase (amino acid sequence SEQ ID NO: 1, nucleotide sequence SEQ ID NO: 2) derived from Bacillus halodurans by semi-rational design method, and the specific enzyme activity and stability of the mutant are improved.

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

[0009] The second object of the application is to provide a gene encoding the alkali xylanase mutant.

[0010] In an embodiment of the application, the nucleotide sequence of the gene encoding the alkali xylanase mutant is shown as SEQ ID NO: 4.

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

[0012] In an embodiment of the application, the starting vector of the recombinant vector includes but is not limited to pET series of vectors or pPICZ alpha vectors; preferably pET-28a(+) vector or pPICZ alpha A vector.

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

[0014] In an embodiment of the application, the host bacterium of the recombinant engineering bacterium is bacteria, fungi, etc.; the bacteria include Escherichia and other bacteria; the fungi include Pichia and other yeasts.

[0015] Preferably, in an embodiment of the application, the recombinant engineering bacterium takes E. coli BL21 (DE3) as the host.

[0016] Preferably, in an embodiment of the application, the host cell is Pichia pastoris.

[0017] The fifth object of the present application is to provide a method for enhancing the specific activity and thermal stability of alkaline xylanase, which is to mutate the 96th histidine (H) of 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 application is to provide a method for obtaining the mutant alkaline xylanase.

[0019] In one embodiment of the present application, the gene encoding alkaline xylanase with the amino acid sequence shown in SEQ ID NO: 1 is subjected to site-directed mutagenesis by designing primers containing mutation sites, and then expressed to obtain the mutant alkaline xylanase with enhanced specific activity and thermal stability.

[0020] Further, the gene encoding alkaline xylanase with the amino acid sequence shown in SEQ ID NO: 1 is introduced with mutations by designing primers containing mutation sites, and then expressed in E. coli BL21 (DE3) after sequencing to obtain the mutant alkaline xylanase with enhanced specific activity and thermal stability.

[0021] Still further, the gene engineering bacteria with correct mutations verified by colony PCR are inoculated into LB medium with a final concentration of 50 μg / mL kanamycin, cultured at 37°C on a shaker at 200 rpm for 12-16 h to obtain seed liquid. The seed liquid is inoculated into LB medium containing 50 μg / mL kanamycin at an initial OD 600 = 0.1, and cultured at 37°C on a shaker at 200 rpm until the OD 600 = 0.6-0.8, and then induced to express by adding IPTG with a final concentration of 0.4 mmol / L and culturing at 16°C on a shaker at 200 rpm for 16 h.

[0022] The seventh object of the present application is to provide the use of the mutant alkaline xylanase, the encoding gene, the expression cassette, the recombinant vector or the recombinant engineering bacteria in the preparation of the mutant alkaline xylanase with enhanced specific activity and thermal stability.

[0023] The eighth object of the present application is to provide the use of the mutant alkaline xylanase, the encoding gene, the expression cassette, the recombinant vector or the recombinant engineering bacteria in the field of biomass resource utilization, food or pharmaceuticals.

[0024] The present application has the following advantages and effects compared with the prior art:

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

[0026] Figure 1 is the relative residual activity of the basic xylanase combined mutant M22 and the wild type after treatment at 75 DEG C, 80 DEG C, 85 DEG C and 90 DEG C for 30 min. DETAILED DESCRIPTION

[0027] The application will be further described in detail below in combination with examples and drawings, but the embodiments of the application are not limited thereto.

[0028] The application discloses a basic xylanase mutant with high specific enzyme activity and thermal stability, a preparation method and application of the basic xylanase mutant, a DNA molecule coding the enzyme mutant, a corresponding vector and a host cell. The technology can provide a reference for professionals in the field. Specific examples and drawings will further describe the application in detail, but are not limited to the description. Any operation step or condition not described in detail in the examples meets the conventional technical standard in the field, and any other examples obtained by any ordinary skilled person in the field without creative labor are within the protection scope of the application.

[0029] Unless otherwise specified, the reagents, methods and equipment used in the application 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 culture 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): 2.38 g of isopropyl thioglycolate-β-D-galactoside (IPTG) was dissolved in 8 mL of distilled water, and distilled water was added to 10 mL, 0.22 μm filter membrane was sterilized, and 1 mL was divided into small parts and stored at -20℃.

[0034] Gly-NaOH buffer: 7.47 g of glycine was dissolved in 900 mL of distilled water, and the pH was adjusted to 9.0 with NaOH, and water was added to 1 L, and it was used at room temperature.

[0035] Example 1: Construction of a recombinant plasmid of xylanase

[0036] The recombinant plasmid pET-28a(+)-Xyn was constructed by homologous recombination using a primer designed with a homologous arm on the pET-28a(+) vector and the nucleotide sequence (SEQ ID NO: 2) of the gene encoding xylanase Xyn (SEQ ID NO: 1).

[0037] The primer on the pET-28a(+) vector is:

[0038] The upstream primer is 5'-TGAGATCCGGCTGCTAACAA-3';

[0039] The downstream primer is 5'-GTGATGATGATGATGATGGCT-3';

[0040] The primer on the nucleotide sequence of the gene of xylanase is:

[0041] The upstream primer is 5'-AGCCATCATCATCATCATCACATGGCTCAAGGTGGTCCACCAAAGT-3';

[0042] The downstream primer is 5'-GCTTTGTTAGCAGCCGGATCTCAATCAATAATTCTCCAGTAAGCT-3'.

[0043] Example 2: Construction of a mutant recombinant plasmid

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

[0045] Table 1 PCR reaction system

[0046]

[0047] Table 2 Mutant primer design (lower case is the replaced codon)

[0048]

[0049] Product recovery: 50 μL sterile water was added to the PCR product, then 200 μL Buffer GDP was added, mixed, centrifuged at 12000 rpm for 30 s; the filtrate was discarded, washed twice with 700 μL Buffer PW2 (diluted with absolute ethanol); finally eluted with 30 μL water and measured the concentration.

[0050] Template digestion: Dpn I was added to the recovered product to remove the template plasmid. The digestion reaction was 600-1200 ng of PCR recovery product, 1 μL of 10x buffer, 0.5 μL of Dpn I digestion enzyme, and ddH2O was added to a final system of 10 μL. The reaction condition was 37°C for 30 min.

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

[0052] Verification of transformed clones: 1-2 single colonies were randomly picked and sequenced for verification.

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

[0054] 5 μL of the plasmid successfully sequenced was transformed into 100 μL of E. coli BL21 (DE3) competent cells, and plated on LB solid plates containing a final concentration of 50 μg / mL kanamycin, and incubated in a 37°C incubator for 16 h.

[0055] The colonies were picked and verified by colony PCR, then inoculated in LB medium containing a final concentration of 50 μg / mL kanamycin, with a liquid volume of 10 mL / 50 mL, and cultured at 37°C on a shaker at 200 r / min for 12-16 h to obtain seed liquid.

[0056] The seed liquid was transferred to LB medium containing a final concentration of 50 μg / mL kanamycin with an initial OD 600 = 0.1 inoculation amount, with a liquid volume of 100 mL / 250 mL, and cultured at 37°C on a shaker at 200 r / min until the OD 600= 0.6-0.8, IPTG was added to a final concentration of 0.4 mmol / L, and the culture was induced for 16 h at 16°C and 200 r / min.

[0057] The same optical density of fermentation broth was placed in a 4°C condition, centrifuged at 7000 rpm per minute for 5 minutes. The centrifuged bacteria were washed once with 15 mL of pH 9.0 Gly-NaOH buffer, and then the bacteria were resuspended with 10 mL of pH 9.0 buffer. The cells were broken in an ice water bath using an ultrasonic cell disrupter for a total of 10 minutes (3 seconds of work and 3 seconds of interval). Then, centrifugation was performed at 10000 rpm per minute for 30 minutes at 4°C, and filtration was performed using a filter membrane with a pore size of 0.22 μm to obtain the fermentation supernatant of the mutant. The mutant M22 (H96N / E145I / V168A) was activated, transferred, and induced in combination with the wild type, and the bacteria breaking conditions were consistent.

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

[0059] Example 4: Determination method of specific activity and relative residual activity of alkaline xylanase

[0060] The enzyme activity determination method was as follows: the alkaline xylanase was determined by a 540 nm light absorption method using beechwood xylan as a substrate. One enzyme activity unit refers to the reaction efficiency of producing 1 μmol of reducing sugar per minute under the conditions of pH 9.0 and 70°C. The reaction system was prepared by adding 200 μL of a substrate solution (1% beechwood xylan solution) to a 1.5 mL EP tube, preheating in a constant temperature mixer at 70°C for 5 min, adding 20 μL of appropriately diluted enzyme solution, reacting for 10 min, immediately adding 300 μL of DNS solution, boiling for 5 min to terminate the reaction, and measuring the absorbance value at 540 nm and calculating the enzyme activity. The protein concentration was determined by the Coomassie brilliant blue method, and the specific activity of the purified combination mutant M22 and the wild type was obtained by dividing the enzyme activity by the protein concentration.

[0061] In an embodiment of the present application, the specific activity of the combination mutant M22 was 211 U / mg, which was increased by 181.33% compared to the wild type (75 U / mg).

[0062] The fermentation supernatant of the wild type and the mutant M22 is treated at 75℃, 80℃, 85℃ and 90℃ for 30 minutes respectively, and the residual activity after different incubation time is measured respectively; the enzyme activity of the wild type treated at 75℃ for 30 minutes is recorded as 100%, and the result is shown in Table 1. Figure 1 As shown in the figure, the relative residual activity of the combined mutant M22 is 169.30% relative to the wild type treated at 75℃ for 30 minutes, and the relative residual activity of M22 after treatment at 80℃, 85℃ and 90℃ is 153.94%, 127.84% and 115.02% respectively. It can be seen that the combined mutant M22 of the present application has good thermal stability.

[0063] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement methods, and all shall be included in the protection scope of the present application.

Claims

1. An alkaline xylanase mutant with improved specific enzymatic activity and thermostability, 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 enzymatic activity and thermostability according to 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 vector described in (2) or (3).

5. The biomaterial according to claim 4, characterized in that: The starting vector of the recombinant vectors described in (2) and (3) is the pET-28a(+) vector; The host bacteria of the recombinant engineered bacteria described in (4), (5) and (6) is Escherichia coli.

6. Use of the gene according to any one of claims 2 to 3 or the biomaterial according to any one of claims 4 to 5 in preparing an alkaline xylanase mutant with improved specific enzymatic activity and thermal stability.

7. Use of the alkaline xylanase mutant with improved specific enzyme activity and thermal stability according to claim 1 in degrading beech xylan.

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 modified by mutating the histidine at position 96 to asparagine, mutating the glutamic acid at position 145 to isoleucine, and mutating the valine at position 168 to alanine.

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