Xylanase mutant with remarkably improved acid stability as well as preparation method and application of xylanase mutant

Abstract

The invention relates to a xylanase mutant with remarkably improved acid stability and a preparation method and application thereof, and belongs to the technical field of xylanase, the site of N75F of xylanase XynASP is mutated, and the amino acid sequence of the obtained mutant is shown as SEQ ID NO.1 or comprises an amino acid sequence which has at least 90% sequence homology with SEQ ID NO.1 and contains N75F mutation. The detection result shows that the optimal pH value of the mutant XynN75F is 3.0 and is reduced to a certain extent compared with that of a wild type, and the mutant XynN75F is closer to an acid environment. Besides, the acid tolerance of the XynN75F is excellent, the residual enzyme activity of the XynN75F under each pH gradient is superior to that of a wild type after heat preservation is carried out for 1 hour under the condition that the pH is 2.0-8.0 and the temperature is 50 DEG C, particularly, the acid tolerance of the XynN75F is more prominent in an acid interval, and it is further proved that the acid resistance of the mutant is remarkably improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of xylanase, and particularly relates to a xylanase mutant with significantly improved acid stability and a preparation method and application thereof. BACKGROUND

[0002] As an important member of glycoside hydrolase family, endo-beta-1,4-xylanase (EC 3.2.1.8) can specifically recognize and cut the beta-1,4-glycosidic bond in xylan molecules to produce bioactive xylo-oligosaccharides. According to the classification standard of the international carbohydrate active enzyme database, this kind of enzyme is mainly distributed in GH5, GH7, GH10 and other families. It is worth noting that the two subfamilies of GH10 and GH11 have important application value in the field of industrial biological catalysis due to their unique structural characteristics and high catalytic capacity. In particular, the xylanase of GH11 subfamily is often regarded as an ideal model for studying the mechanism of xylan degradation due to its highly specific substrate recognition ability, and the acid-resistant GH11 xylanase has particularly outstanding application potential in acidic environments.

[0003] Although the xylanase of GH11 family exhibits excellent catalytic performance, it still faces severe challenges in actual industrial applications, especially in the field of feed additives. The optimal action pH of this kind of enzyme is usually in the neutral to weakly acidic range, while the feed will undergo a strong acidic environment in the animal digestive tract (such as the pH in the pig stomach is as low as 2.0-3.5, and the pH in the chicken crop is 3.0-4.5), and the xylanase as a feed additive will easily be irreversibly inactivated under acidic conditions after entering the digestive tract. In addition, the pelleting process in the feed processing process (often accompanied by a short-term acidic environment) and the slight acidification during feed storage will also destroy the active center conformation of the enzyme through protonation, significantly reducing its catalytic efficiency, resulting in the inability to effectively degrade non-starch polysaccharides (such as xylan in corn and wheat feed) in feed, and affecting the absorption and utilization of nutrients by animals. Current research shows that although enhancing the structural stability of proteins in acidic conditions through molecular modification can improve acid resistance, it often comes at the cost of catalytic activity. This trade-off between acid resistance and activity directly restricts the application effect of xylanase in the field of feed additives, and has become a key scientific problem that needs to be solved in the current cross-field of enzyme engineering and feed science.

[0004] In the three-dimensional structure of GH11 xylanase, the key acidic stable region can maintain the conformational stability of the enzyme in low pH environment through charge distribution and hydrogen bond network, and is involved in substrate recognition and catalytic process under acidic conditions. Recent studies have shown that rational design of this region can significantly improve the acid resistance of the enzyme. When researchers modified xylanase from fungi, they found that a single point mutation (E129K) in the acidic stable region could increase the residual activity of the enzyme by 50% at pH 3.0. This breakthrough discovery provides important clues for understanding the functional mechanism of the acidic stable domain. However, there is still a lack of systematic molecular mechanism explanation on how the mutation of this region affects the acid resistance and catalytic activity of the enzyme, which will be an important direction for future research. SUMMARY

[0005] Technical problem solved: Based on the above problems, the present application provides a xylanase mutant with significantly improved acid stability, as well as a preparation method and application thereof. Experimental data show that the mutant exhibits obvious advantages in acid resistance and thermal stability. In terms of acid environment adaptability, XynN75F shows significant advantages. When the pH value decreases to 3.0, the relative enzyme activity of the mutant is maintained at more than 70%, which is increased by 30% compared with the wild type. Temperature adaptability test shows that the optimal catalytic temperature of XynN75F is increased by 5℃ compared with the wild type, reaching 50℃. After 1 hour of continuous treatment at 45℃, the mutant can still retain more than 70% of the initial activity, while the activity retention rate of the wild type enzyme under the same conditions is less than 30%. This result confirms the enhancement of the mutation on the thermal stability of the enzyme molecule, showing good industrial application potential.

[0006] Technical solution: A xylanase mutant, which is a site mutation of N75F of xylanase XynASP, the amino acid sequence of the obtained mutant is shown in SEQ ID NO. 1, or an amino acid sequence having at least 90% sequence homology with SEQ ID NO. 1 and containing N75F mutation.

[0007] Preferably, the above sequence homology is at least 95%.

[0008] A nucleic acid molecule encoding the xylanase mutant, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO. 2; or a nucleic acid sequence having at least 90% sequence homology with SEQ ID NO. 2 and encoding an amino acid sequence containing N75F mutation.

[0009] An expression vector comprising the nucleic acid molecule, the expression vector is plasmid pET-28a(+).

[0010] A host cell comprising the expression vector, wherein the host cell is Escherichia coli or Pichia pastoris.

[0011] A method for producing the xylanase mutant, comprising the following steps: (a) culturing the host cell; (b) inducing the host cell to express the xylanase mutant; (c) collecting the xylanase mutant from the culture.

[0012] An enzyme preparation comprising the xylanase mutant or a carrier thereof.

[0013] Use of the xylanase mutant in preparing an animal feed additive.

[0014] Beneficial effects: The detection results show that the optimal pH of the mutant XynN75F obtained by the application is 3.0, which is reduced to a certain extent compared with the wild type (optimal pH 6.0), and is more inclined to acidic environment. In addition, the acid tolerance of XynN75F is excellent, after incubation at 50℃ for 1 hour in the pH range of 2.0-8.0, the residual enzyme activity of XynN75F at each pH gradient is better than that of the wild type, especially in the acidic range, which further confirms the significant improvement of the mutant in acid resistance. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is a schematic diagram of the three-dimensional model of the xylanase XynN75F.

[0016] Figure 2 is the optimal temperature and the optimal pH of the xylanase XynN75F, wherein A is the optimal temperature, and B is the optimal pH.

[0017] Figure 3 is the thermal stability of the xylanase XynN75F.

[0018] Figure 4 is the pH stability of the xylanase XynN75F.

[0019] Figure 5 is the metal ion and organic reagent tolerance of the xylanase XynN75F. DETAILED DESCRIPTION

[0020] The application belongs to the technical field of genetic engineering and enzyme engineering, and specifically provides a modified xylanase mutant, a coding gene thereof, an expression vector, a host cell, and a preparation method and application of the mutant. The application optimizes natural xylanase by means of molecular modification technology, and successfully obtains a mutant with improved performance. The mutant can be efficiently expressed in host cells such as Escherichia coli and yeast, and is suitable for industrial production scenarios. In addition, the application also provides a preparation process of the enzyme and application modes in different fields.

[0021] The technical scheme of the application has been verified by preferred embodiments, and the related experimental data show that the mutant has significant advantages in enzyme activity, thermal stability and pH adaptability. A person skilled in the art can adjust the culture conditions, expression system and purification process according to actual needs to obtain the best application effect.

[0022] Example 1

[0023] Materials required for the experiment:

[0024] In the present study, the xylanase XynASP gene was derived from Aspergillus saccharolyticus JOP 1030-1. Escherichia coli DH5α, Escherichia coli BL21(DE3), and vector pET-28a(+) were purchased from Novagen company; the recombinant plasmid pET-28a-xynASP was synthesized by GenScript Biotech (Shanghai) Co., Ltd. High-fidelity DNA polymerase, deoxyribonucleotide triphosphates (dNTPs), and restriction endonuclease DpnI were purchased from New England Biolabs company; isopropyl-β-D-thiogalactopyranoside (IPTG) and kanamycin (Kan) were purchased from Takara company. The used beechwood xylan was purchased from Megazyme company, and SYPRO dye was purchased from Sigma-Aldrich company. The rest of the reagents were all of domestic analytical purity.

[0025] The specific preparation methods of the main reagents are as follows:

[0026] LB medium: 0.5 g of yeast powder, 1 g of peptone and 1 g of NaCl were weighed, dissolved in 70 mL of deionized water, and then diluted to 100 mL. After sterilization at 121℃ for 20 minutes, the solution was obtained.

[0027] Solid LB medium: 2% agar powder was added to the liquid LB medium.

[0028] TB medium: weigh 6 g of tryptone, 12 g of yeast extract and 2 mL of glycerol, dissolve in distilled water and make up to 450 mL, divide into 500 mL flasks, 90 mL per flask, a total of 5 flasks.

[0029] 3,5-dinitrosalicylic acid (DNS reagent): first dissolve 91 g of potassium sodium tartrate in 300 mL of hot water, then add 10.48 g of NaOH, 3.15 g of 3,5-dinitrosalicylic acid, 2.5 g of phenol and 2.5 g of anhydrous sodium sulfite in sequence, heat with constant temperature water bath and stir constantly throughout the process to ensure complete dissolution of all ingredients. After cooling, transfer to a volumetric flask, make up to 500 mL with distilled water, store in the dark at room temperature, and use after one week.

[0030] Disodium hydrogen phosphate-citric acid buffer (pH 5.0): weigh 33.5 g of disodium hydrogen phosphate and 11.2 g of citric acid, dissolve in 800 mL of double distilled water (ddH2O), make up to 1000 mL, and adjust the pH to 5.0.

[0031] Mass fraction 0.5% beechwood xylan solution: take 100 mL of disodium hydrogen phosphate-citric acid buffer (pH 4.6), weigh 0.5 g of beechwood xylan into a flask, heat to boiling and maintain for 5 minutes, cool, and make up to 100 mL with distilled water, and store at 4°C.

[0032] Construction of mutant models and determination of mutation sites

[0033] The three-dimensional structure of recombinant xylanase XynASP was analyzed by homology modeling using the Alphafold2 protein structure prediction platform Alphafold2 (https: / / alphafold2.biodesign.ac.cn / ). The specific implementation process includes: first, submit the target amino acid sequence to the prediction system to obtain 5 candidate models; then, evaluate the quality of each model by predicting local distance difference test (PLDDT) and draw a model quality distribution map, and select the most reasonable structure model according to the evaluation results. The above structure analysis process is performed according to the default parameter settings of the software.

[0034] Based on the analysis of the structure of the homologous enzyme protein, it is found that the amino acid substitution of the Loop segment (flexible domain) adjacent to the active center can significantly affect the functional characteristics of the enzyme, including substrate binding ability, thermal stability and catalytic activity. Therefore, the Loop region of the target enzyme is selected as the key modification site. Through preliminary screening experiments, it is observed that the mutant of asparagine (Asn75) in the Loop region shows the trend of enhanced enzyme activity. Combined with the analysis of the physicochemical properties of amino acids, specific amino acids are selected for site-directed mutagenesis to systematically study the regulation of enzyme characteristics by modifying the domain. The wild-type plasmid pET-28a-xynASP is used as a template, and single-point mutation primers are used to carry out PCR amplification. The recombinant mutant plasmid pET-28a-xynN75F is constructed. After PCR amplification, DpnI is used to remove the template. Then, the recombinant plasmid after removing the template is introduced into E. coli DH5α competent cells by chemical transformation. The transformed cells are selected on the medium containing Kan resistance, and positive monoclonal colonies are picked and sent to Hefei Yikang Company for gene sequencing.

[0035] Enzyme activity determination method

[0036] The DNS method is used to determine the enzyme activity: 2.4 mL of 0.5% beech xylan solution is taken and mixed with 0.1 mL of appropriately diluted enzyme solution. After reacting at a suitable temperature for 15 min, 2.5 mL of DNS reagent is added to terminate the reaction, and it is placed in a water bath for boiling for 7 min. After cooling, 5 mL of double distilled water (ddH2O) is added, mixed well, and the absorbance value is measured at a wavelength of 540 nm.

[0037] The activity unit (U) of xylanase is defined as: the amount of enzyme required to catalyze the hydrolysis of xylan to generate 1 μmol of xylose per minute under the above activity determination conditions.

[0038] Site-directed mutagenesis method

[0039] Using pET-28a-xynASP as a template, N75F-F as an upstream primer (Table 1), and T7-R as a downstream primer, full-plasmid PCR is used to perform site-directed mutagenesis on the Asn75 codon. The PCR product is digested with DpnI, and E. coli BL21 (DE3) is transformed to construct the mutant site. Positive transformants are selected from LB solid medium and inoculated in 1 mL of LB medium containing Kan (final concentration 50 μg / mL) at 37°C, 220 r / min for 12 h. The culture is transferred to 1 mL of the same medium at a 2% inoculum volume fraction, and cultured until the OD 600When the ratio is about 0.6-0.8, 0.5 mmol / L IPTG is added, expression is induced at 28°C for 5 h, the bacterial cells are collected by centrifugation at 4°C and 8000 r / min for 5 min, 200 μL of 1 mg / mL lysozyme solution is added, and the bacterial cells are incubated at 37°C for 2 h to lyse the cells, and then the supernatant is collected by centrifugation to obtain the crude enzyme solution. 5 μL of the enzyme solution is diluted and added to 95 μL of 0.5 g / mL beechwood xylan solution (pH 5.5, 50 mmol / L citric acid-sodium dihydrogen phosphate buffer), and catalysis and color development are performed by means of a PCR instrument (75°C for 10 min, addition of 50 μL of DNS reagent, 95°C for 5 min, cooling to 10°C), and then the mixture is added to a 96-well plate, and OD 540 The mutant xylanase with an enzyme activity greater than 30% of the original xylanase is defined as a positive mutant, and DNA sequencing is performed on the mutant. The sites with a relatively obvious increase in enzyme activity are subjected to combined mutation, and the optimal mutant is screened by means of the DNS method.

[0040] Table 1 design of mutation primers

[0041] Primer Primer sequence (5'-3') N75F-F GGGGGCAAAGGCTGG TTT CCGGGCAGTGCCAGA]]>

[0042] Expression and purification of the mutant

[0043] The constructed recombinant expression plasmid is introduced into competent cells of Escherichia coli BL21 (DE3) by means of chemical transformation. The transformed product is spread on an LB solid culture medium containing 50 μg / mL kanamycin, and is cultured at 37°C for 12-16 hours. A single colony is selected and inoculated into 2 mL of LB liquid culture medium containing an equal concentration of antibiotic, and is cultured at 37°C and 180 rpm for 12 hours to the logarithmic growth phase.

[0044] 1 mL of the overnight culture is transferred into 100 mL of TB culture medium containing 50 μg / mL kanamycin, and is cultured at 37°C and 180 rpm for 3 hours. After the addition of IPTG, the temperature is lowered to 30°C, and the induction culture is continued for 2 hours. After the induction, the bacterial cells are collected by centrifugation at 4°C and 6000 rpm for 10 minutes.

[0045] The bacterial cell pellet is resuspended with pre-cooled citric acid buffer, and is subjected to crushing treatment by means of a pressure disrupter. The cell crushing solution is centrifuged at 4°C and 12000 rpm for 20 minutes, and the collected supernatant is the crude enzyme solution of the recombinant xylanase.

[0046] The crude enzyme solution is purified by means of nickel affinity chromatography, and the purified enzyme sample is subjected to SDS-PAGE electrophoresis analysis. The concentration of the separation gel used for electrophoresis is 12%, the sample loading amount is 10 μg, the electrophoresis conditions are 80 V for the concentrated gel and 120 V for the separation gel, and the protein bands are analyzed after staining with Coomassie brilliant blue R-250.

[0047] Characterization of mutant enzyme properties

[0048] Determination of optimum pH: The optimum pH of the xylanase was determined by measuring the enzyme activity at a range of pH 2.0-9.0 at 50 °C. Determination of pH stability: The residual enzyme activity was measured after incubation of the enzyme in buffers at pH 2.0-9.0 at 50 °C for 15 min. Further evaluation of the pH stability was performed by incubation at pH 2.0-9.0 at 50 °C for 60 min.

[0049] Determination of optimum temperature: The optimum temperature of the enzyme was determined by measuring the enzyme activity at a range of 35-60 °C at 5 °C intervals in 50 mM citrate buffer for 15 min. Evaluation of thermal stability: Incubation experiments at different time and temperature were performed. The purified enzyme protein was first diluted to an appropriate level with citrate buffer at pH 5.0, and then the enzyme was incubated at 35-60 °C for 60 min, followed by immediate cooling in an ice-water bath. The residual enzyme activity of mutant XynN75F was calculated with the untreated enzyme activity as 100%.

[0050] Calculation of half-life: According to the formula y = Ae -kt (where A is the initial enzyme activity, t is the time, and k is the decay constant), the half-life of mutant XynN75F at 50 °C was calculated according to the half-life t1 / 2 = ln2 / k.

[0051] Determination of metal ion and chemical reagent tolerance: The residual enzyme activity was measured after incubation of the enzyme with different substances at 50 °C for 1 h, and the tolerance was calculated with the untreated initial enzyme activity as 100%. Specifically, 5 mM metal ions (K + , Ca 2+ , Na + , Mg 2+ , Al 3+ , Mn 2+ , Fe 2+ , Fe 3+ , Ni 2+ , Cu 2+ , Zn 2+ , Ba 2+ ), 5 mM chemical reagents (sodium dodecyl sulfate SDS, ethylenediaminetetraacetic acid EDTA), and 5% (v / v) final concentration of methanol, ethanol, and isopropanol.

[0052] All experiments were repeated 3 times.

[0053] Method for applying animal feed additive

[0054] Acid-resistant xylanase as a feed additive, by maintaining stability and catalytic activity in acidic environment, degrades feed xylan to eliminate the anti-nutritional effect, and improves the nutritional value of feed. Fermentation by engineering bacteria, concentrated into liquid or solid preparation, adapt to processing environment. In the design of feed formula, for corn, wheat and other xylan content of 5%-10% raw materials, add 200-500U per kilogram of feed. Solid enzyme is directly mixed (stirring for 10-15 minutes, coefficient of variation ≤7%), liquid enzyme is added by spraying, and the effect can be improved by 20%-30% by compounding with phytase. When processing, control the granulation temperature to be 75-85℃, the conditioning pH to be 5.5-6.5, shorten the high-temperature acid residence time to be ≤30 seconds, and more than 70% of the activity can be retained by microencapsulating the enzyme. The enzyme-containing feed needs to be stored in an environment with a temperature of ≤25℃ and a humidity of ≤60%, and the shelf life is extended to 3-6 months. In the animal digestive tract, the enzyme degrades xylan into xylooligosaccharides in the acidic environment of the stomach or crop, reduces the chyme viscosity by 40%-60%, and promotes nutrient absorption. The application effect is remarkable, the xylan residue in mouse feces is reduced by more than 50%, and the intestinal volatile fatty acid is increased by 20%-30%. At the same time, the residual activity and safety are detected to ensure that the standards are met. The enzyme effectively overcomes the limitation of the acidic environment, improves the feed utilization rate, reduces the breeding cost, and helps efficient and healthy breeding.

[0055] The results show

[0056] Some enzyme property data are shown in Table 2. The optimum pH of mutant XynN75F is 3.0, which is lower than that of the wild type (optimum pH 6.0). Under the condition of pH 2.0-8.0 and 50℃, incubate for 1 hour, the activity of wild type and mutant enzyme remains relatively stable with pH fluctuation, showing a wide pH adaptation range; especially at pH 3.0, the residual enzyme activity of XynN75F is 76.13%, which is much higher than that of the wild type (45.86%), indicating that the activity of the mutant is better in this pH range.

[0057] The optimum temperature of XynN75F is 50℃, which is 5℃ higher than that of the wild type (45℃). Under the condition of 50℃ and pH 5.0, the activity of the wild type decreases rapidly to only 30.3% of the original activity, while the mutant XynN75F still maintains a high activity of 73.92%.

[0058] Table 2 Enzyme property parameters

[0059] XynASP XynASP-N75F Specific activity (U / mg) 51.51±2.39 70.86±3.10 t 1 / 2 45 °C (min) 22.34 25.65 t 1 / 2 50 °C (min) 5.31 10.07 Optimum pH 6 3 Optimum temperature (°C) 45 50

[0060] In summary, the present application successfully constructs a mutant XynN75F with significantly improved enzyme properties in the XynASP loop structure of xylanase. The mutant has significantly improved acid resistance, and the thermal stability is also improved compared with the wild type enzyme.

Claims

1. A xylanase mutant, characterized in that, A mutation at the N75F site of xylanase XynASP, the amino acid sequence of the resulting mutant is shown in SEQ ID NO.1, or contains an amino acid sequence that has at least 90% sequence homology with SEQ ID NO.1 and contains the N75F mutation.

2. The xylanase mutant according to claim 1, characterized in that, The sequence homology is at least 95%.

3. A nucleic acid molecule encoding a xylanase mutant according to claim 1 or 2, characterized in that, The nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.2; or a nucleic acid sequence containing at least 90% sequence homology with SEQ ID NO.2 and encoding an amino acid sequence containing the N75F mutation.

4. An expression carrier, characterized in that, It includes the nucleic acid molecule as described in claim 3.

5. The expression vector according to claim 4, characterized in that, The expression vector is plasmid pET-28a(+).

6. A host cell, characterized in that, It includes the expression vector as described in claim 4 or 5.

7. The host cell according to claim 6, characterized in that, The host cell is Escherichia coli (E. coli) Escherichia coli ) or Pichia pastoris ( Pichia pastoris ).

8. A method for producing the xylanase mutant of claim 1, characterized in that, Includes the following steps: (a) Culturing the host cells as described in claim 6 or 7; (b) Inducing the host cells to express the xylanase mutant; (c) Collecting the xylanase mutant from the culture.

9. An enzyme preparation, characterized in that, It includes the xylanase mutant or its vector as described in claim 1 or 2.

10. The use of the xylanase mutant of claim 1 or 2 in the preparation of animal feed additives.

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