Alkaline xylanase mutant with high enzyme activity and preparation method thereof

Abstract

The invention discloses a high-enzyme-activity alkaline xylanase mutant and a preparation method thereof, and belongs to the field of gene engineering and enzyme engineering. According to the invention, heat-resistant and alkali-resistant xylanase derived from Bacillus halodurans is modified by a semi-rational design method, so that a series of combined mutants M1-M3 with high enzyme activity are obtained. The enzyme activity of the combined mutants M1-M3 is more than 3.5 times that of a wild type, and the enzyme activity of the optimal mutant M1 is 432.6% that of the wild type under the conditions of 70 DEG C and pH 9.0. The combined mutant provided by the invention can promote application in the fields of biomass resource utilization, food or papermaking industry, effectively solves the core problem of low enzyme activity in current xylanase application, and is more beneficial to application in industrial scenes.

Description

A high-activity alkaline xylanase mutant and its preparation method Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme engineering, and specifically relates to a high-activity alkaline xylanase mutant and its preparation method. Background Technology

[0002] Xylan is a major component of hemicellulose in plant cell walls, accounting for 15%-35% of the dry weight of lignocellulose biomass. It is the second most abundant renewable polysaccharide in nature, composed of xylose units linked by β-1,4-glycosidic bonds. The functional products obtained from its efficient degradation, such as xylooligosaccharides, are of significant value in the food, feed, bioenergy, and paper industries, and are crucial for the sustainable utilization of resources. Xylanases, due to their high catalytic efficiency and strong substrate specificity, have been widely used in pulp bleaching, feed additives, and biorefining; however, the poor enzyme activity of natural enzymes makes them difficult to function under industrial conditions, becoming a bottleneck for industrialization. Therefore, screening for xylanase mutants with significantly enhanced activity is crucial for improving their industrial efficiency and promoting the development of related industries.

[0003] To overcome the aforementioned shortcomings, protein engineering strategies such as rational design and directed evolution have been widely applied to the improvement of xylanases. While existing technologies have reported improving specific enzyme properties through mutations, these improvements often have limitations, and there is still a lack of combinatorial mutation schemes that can significantly break through the limits of their natural catalytic efficiency. Therefore, there is an urgent need in this field to develop novel xylanase combinatorial mutants that can not only withstand high-temperature and alkaline environments but also fundamentally and significantly enhance enzyme activity, thereby meeting the comprehensive requirements of industrial applications for efficient, stable, and economical enzyme preparations. Summary of the Invention

[0004] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of this invention is to provide a high-enzyme-activity alkaline xylanase mutant and its preparation method.

[0005] The objective of this invention is achieved through the following technical solution: This invention modifies the thermostable and alkali-resistant xylanase (amino acid sequence SEQ ID NO.1, nucleotide sequence SEQ ID NO.2) derived from Bacillus halodurans using a semi-rational design method to obtain a mutant with high enzyme activity.

[0006] The first objective of this invention is to provide a series of high-activity alkaline xylanase mutants, wherein the amino acid sequence of the mutant is obtained by any one of the following mutations: H96N, R123W, A135W, E145I, V168A, E177P, V191K, V215N, Q246S, and W249Y.

[0007] Preferably, the mutant is obtained by any combination of SEQ ID NO.1 as follows: 1) R123W / A135W / E145I / V168A / V191K / V215N / W249Y (M1), 2) R123W / V168A / V191K / Q246S (M2), 3) H96N / A135W / E145I / V168A / E177P / V191K / V215N (M3), wherein the most preferred mutant M1 has a relative enzyme activity of 432.6% of the wild type at pH 9.0 and 70℃.

[0008] The amino acid sequence of mutant M1 is shown in SEQ ID NO.3, the amino acid sequence of mutant M2 is shown in SEQ ID NO.5, and the amino acid sequence of mutant M3 is shown in SEQ ID NO.7.

[0009] A second objective of this invention is to provide a gene encoding the aforementioned basic xylanase mutant.

[0010] Preferably, the nucleotide sequence of the gene encoding mutant M1 is shown in SEQ ID NO.4, the nucleotide sequence of the gene encoding mutant M2 is shown in SEQ ID NO.6, and the nucleotide sequence of the gene encoding mutant M3 is shown in SEQ ID NO.8.

[0011] A third objective of this invention is to provide expression cassettes and recombinant expression plasmids for the aforementioned genes.

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

[0013] A fourth objective of this invention is to provide a genetically engineered bacterium containing the above-described recombinant expression plasmid.

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

[0016] Preferably, in one embodiment of the present invention, the recombinant engineered bacteria uses Pichia pastoris as the host.

[0017] The fifth object of the present invention is to provide the use of the above-mentioned alkaline xylanase mutant, gene, expression cassette, recombinant expression plasmid or genetically engineered bacteria in any of the following: (1) in the preparation of alkaline xylanase mutant with high enzyme activity; (2) in the degradation of xylan; (3) in the application of biomass resource utilization, food or paper industry.

[0018] The sixth objective of this invention is to provide a method for obtaining the above-mentioned high-activity alkaline xylanase mutant, comprising the following steps: by designing primers containing mutation sites to perform site-directed mutagenesis on the gene encoding alkaline xylanase with an amino acid sequence as shown in SEQ ID NO.1, and then expressing the mutant to obtain a high-activity alkaline xylanase mutant.

[0019] Furthermore, primers containing mutation sites were designed to introduce mutations into the gene encoding alkaline xylanase, as shown in SEQ ID NO.1. After correct sequencing, the mutant was transformed into Escherichia coli BL21(DE3) for expression, resulting in a high-activity alkaline xylanase mutant.

[0020] The present invention has the following advantages and effects compared with the prior art: Compared with existing mutants, the combined mutants obtained by the present invention through semi-rational design achieve a leap in enzyme activity compared with wild type. The enzyme activities of the three preferred combined mutants are all more than 3.5 times that of wild type, among which combined mutant M1 has the highest relative enzyme activity, which is 432.6% of wild type. The combined mutants of the present invention can promote the hydrolysis of xylan in lignocellulose, effectively improve the utilization of renewable polysaccharides, effectively solve the core problem of low enzyme activity in current xylanase applications, and are more conducive to its application in industrial scenarios. Attached Figure Description

[0021] Figure 1 shows the relative enzyme activities of a series of alkaline xylanase mutants after reacting at 70°C for 10 minutes. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0023] This invention discloses a series of high-activity basic xylanase mutants, their preparation methods, applications, the DNA molecules encoding these mutants, corresponding vectors, and host cells. This technology can provide a reference for those skilled in the art. Specific embodiments and accompanying drawings will further describe the invention in detail, but are not limited thereto. Any operational steps or conditions not described in detail in the embodiments conform to conventional technical standards in the art, and any other embodiments obtained by those skilled in the art without inventive effort are within the protection scope of this invention.

[0024] Unless otherwise specified, the reagents, methods and equipment used in this invention are conventional reagents, methods and equipment in this technical field, and the reagents and materials used are all commercially available or can be prepared by known methods.

[0025] The culture media and required solutions involved in the following examples are as follows: LB liquid culture medium: yeast extract 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L.

[0026] LB solid medium: yeast extract 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L, agar powder 2% (m / v).

[0027] IPTG (1 mol / L): Dissolve 2.38 g of isopropyl thio-β-D-galactoside (IPTG) in 8 mL of distilled water, bring the volume to 10 mL with distilled water, filter through a 0.22 μm filter membrane for sterilization, aliquot into 1 mL portions and store at -20 °C.

[0028] Gly-NaOH buffer: Dissolve 7.47g glycine in 900mL distilled water, adjust the pH to 9.0 with NaOH, add water to a final volume of 1L, and keep warm at room temperature for later use.

[0029] The plasmid pET-28a(+)-Xyn used in the examples is disclosed in the literature “CN119842670A, A basic xylanase mutant with improved enzyme activity and thermostability and its preparation method”.

[0030] Example 1: Construction of recombinant plasmid and mutant recombinant plasmid of xylanase. The recombinant plasmid pET-28a(+)-Xyn was 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), and then constructing it by homologous recombination.

[0031] Using the wild-type strain's recombinant plasmid pET-28a(+)-Xyn nucleotide sequence as a template, whole-plasmid PCR polymerase chain reaction was performed using primers containing the mutation site. PCR reaction conditions: 94℃ denaturation for 5 min, 98℃ denaturation for 10 s, 55℃ annealing for 30 s, 68℃ extension for 195 s, 30 cycles, 68℃ extension for 10 min, and storage at 4℃. The PCR reaction system is shown in Table 1, and the mutation site and codons before and after mutation are shown in Table 2.

[0032] Table 1 PCR reaction system

[0033] Table 2. Mutation sites and codons before and after the mutation.

[0034] Note: The characters in parentheses are codons.

[0035] Product recovery: Add 50 μL of sterile water to the PCR product, then add 200 μL of Buffer GDP, mix well, and centrifuge at 12000 rpm for 30 s; discard the filtrate, wash twice with 700 μL of Buffer PW2 (diluted with anhydrous ethanol); finally, elute with 30 μL of water and measure the concentration.

[0036] Template digestion: Add restriction endonuclease DpnI to the recovered product to remove the template plasmid. The digestion reaction is as follows: 600–1200 ng of PCR recovered product, 1 μL of 10× buffer, 0.5 μL of DpnI digestion enzyme, and ddH2O added to a final volume of 10 μL. The reaction conditions are 37℃ for 30 min.

[0037] Recombinant plasmid transformation: 10 μL of the digested product was transformed into 100 μL of Escherichia coli Top10 competent cells, plated on LB agar plates containing a final concentration of 50 μg / mL kanamycin, and incubated overnight at 37°C for 16 h.

[0038] Transformed clone validation: Randomly select 1-2 single-clone colonies for sequencing validation.

[0039] Example 2: Xylanase fermentation and induced expression. 5 μL of plasmid that was successfully sequenced was transformed into 100 μL of E. coli BL21(DE3) competent cells, plated on LB agar plates containing a final concentration of 50 μg / mL kanamycin, and incubated overnight at 37°C for 16 h.

[0040] After selecting colonies and verifying their correctness by colony PCR, inoculate them into LB medium containing a final concentration of 50 μg / mL kanamycin at a volume of 10 mL / 50 mL. Incubate at 37°C on a shaker at 200 r / min for 12–16 h to obtain the seed culture.

[0041] With initial OD 600 The seed culture was transferred to LB medium containing a final concentration of 50 μg / mL kanamycin at an inoculum size of 0.1, with a volume of 100 mL / 250 mL. The medium was then incubated at 37°C with a shaker at 200 rpm until OD reached its limit. 600 =0.6~0.8, add IPTG to a final concentration of 0.4 mmol / L, and incubate at 16℃ and 200 r / min for 16 h to induce expression.

[0042] Fermentation broths with the same optical density were centrifuged at 7000 rpm for 5 minutes at 4°C. The centrifuged cells were washed once with 15 mL of pH 9.0 Gly-NaOH buffer, and then resuspended in 10 mL of the same pH 9.0 buffer. Cells were then disrupted using an ultrasonic cell disruptor in an ice-water bath for a total of 10 minutes (3 seconds per cycle, 3-second intervals). Next, the cells were centrifuged at 10000 rpm for 30 minutes at 4°C and filtered through a 0.22 μm pore size membrane to obtain the fermentation supernatant of the mutant. The activation, transfer, induction, and harvesting / disruption conditions for the mutant were identical to those for the wild type.

[0043] Example 3: Method for Determining the Relative Enzyme Activity of Alkaline Xylanase The relative enzyme activity of alkaline xylanase was determined using the 540 nm light absorption method with beech xylan as the substrate. One unit of enzyme activity refers to the reaction efficiency of producing 1 μmol of reducing sugar within 1 min under conditions of pH 9.0 and 70℃. The reaction system consisted of 200 μL of substrate solution (1% beech xylan solution) added to a 1.5 mL EP tube. The tube was preheated at 70℃ for 5 min in a constant temperature mixer. 20 μL of appropriately diluted enzyme solution was added, and the reaction was allowed to proceed for 10 min. Immediately afterwards, 300 μL of DNS solution was added, and the reaction was terminated by boiling for 5 min. 200 μL of the reaction solution was taken, and its absorbance was measured at 540 nm to calculate the enzyme activity. The relative activity of the hybrid mutant was calculated by dividing the enzyme activity by the wild-type enzyme activity. The results are shown in Figure 1 and Table 3. The mutant R123W / A135W / E145I / V168A / V191K / V215N / W249Y had the best relative enzyme activity, reaching 432.6%. The mutants R123W / V168A / V191K / Q246S and H96N / A135W / E145I / V168A / E177P / V191K / V215N had relative enzyme activities of 373.8% and 351.4%, respectively.

[0044] Table 3. Relative activity of combined mutants relative to wild type

[0045] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A high-activity alkaline xylanase mutant, characterized in that, The amino acid sequence of the mutant is obtained by any one of the following mutations: H96N, R123W, A135W, E145I, V168A, E177P, V191K, V215N, Q246S, and W249Y.

2. The high-activity alkaline xylanase mutant according to claim 1, characterized in that: The amino acid sequence of the mutant is obtained by any one of the following mutations of SEQ ID NO.1: 1) R123W / A135W / E145I / V168A / V191K / V215N / W249Y, 2) R123W / V168A / V191K / Q246S, 3) H96N / A135W / E145I / V168A / E177P / V191K / V215N, wherein the amino acid sequence of the mutant R123W / A135W / E145I / V168A / V191K / V215N / W249Y is shown in SEQ ID NO.

3.

3. The gene encoding the high-activity basic xylanase mutant according to any one of claims 1 to 2.

4. The gene according to claim 3, characterized in that, The nucleotide sequence of the gene encoding the mutant R123W / A135W / E145I / V168A / V191K / V215N / W249Y is shown in SEQ ID NO.

4.

5. The biomaterial related to the high-enzyme-activity alkaline xylanase mutant according to any one of claims 1 to 2, characterized in that: The biological material is any one or more combinations of the following: (a) an expression cassette containing the gene of claim 3 or 4; (b) a recombinant expression plasmid containing the gene of claim 3 or 4; (c) a recombinant expression plasmid containing the expression cassette of (a); (d) a recombinant engineered bacterium containing the gene of claim 3 or 4; (e) a recombinant engineered bacterium containing the expression cassette of (a); (f) a recombinant engineered bacterium containing the recombinant expression vector of (b) or (c).

6. The biomaterial according to claim 5, characterized in that: The starting plasmids of the recombinant expression plasmids described in (b) and (c) are pET series plasmids or pPICZα plasmids; the host bacteria of the recombinant engineered bacteria described in (d), (e) and (f) are bacteria or fungi.

7. The use of the gene according to any one of claims 3 to 4 or the biological material according to any one of claims 5 to 6 in the preparation of a highly active alkaline xylanase mutant.

8. The use of the high-enzyme-activity alkaline xylanase mutant according to any one of claims 1 to 2, the gene according to any one of claims 3 to 4, or the biomaterial according to any one of claims 5 to 6 in the degradation of xylan.

9. The application of the high-enzyme-activity alkaline xylanase mutant according to any one of claims 1 to 2, the gene according to any one of claims 3 to 4, or the biomaterial according to any one of claims 5 to 6 in the fields of biomass resource utilization, food, or papermaking industry.

10. A method for obtaining the high-enzyme-activity alkaline xylanase mutant according to any one of claims 1 to 2, characterized in that: Includes the following steps: By designing primers containing mutation sites to perform site-directed mutagenesis on the gene encoding alkaline xylanase with the amino acid sequence shown in SEQ ID NO.1, the alkaline xylanase mutant with high enzyme activity as described in any one of claims 1 to 2 is obtained.

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