High-activity nattokinase mutant and application thereof

Abstract

The invention discloses N365S mutant nattokinase with high activity and application of the N365S mutant nattokinase, aiming at wild type nattokinase, N-S mutation is introduced at the 365 amino acid position of the wild type nattokinase, the enzyme activity of the obtained nattokinase mutant is improved by 110.60%, and compared with the wild type nattokinase, the mutant shows higher fibrinolytic activity. The invention provides a theoretical basis for large-scale production of nattokinase.

Description

Technical Field

[0001] The invention relates to the field of enzyme genetic engineering, in particular to a high-activity nattokinase mutant and application thereof. Background Art

[0002] Nattokinase (NK) is a fibrinolytic enzyme that has the function of dissolving blood clots and preventing thrombosis. Urokinase, tissue plasminogen activator and streptokinase are traditional thrombolytic drugs. Although they have certain efficacy, they are usually accompanied by high costs, lack of specificity and the risk of serious bleeding complications. As a natural protein bioactive substance that can be taken orally and absorbed by the human body, nattokinase has low cost, low toxicity, long-lasting efficacy and no obvious side effects. It shows potential as a treatment and prevention method and is gradually used in thrombosis prevention and functional foods. With the widespread application of nattokinase, higher requirements are placed on its efficient production. The production of nattokinase by microorganisms can achieve higher yields in a shorter time and effectively reduce production costs. Summary of the invention

[0003] The object of the present invention is to provide a highly active nattokinase mutant to solve the problems raised in the above-mentioned background technology, which has the advantage of showing a significant improvement in enzyme activity.

[0004] To achieve the above object, the present invention provides the following technical solution: a highly active nattokinase mutant, wherein the mutant is a nattokinase mutant having an amino acid sequence of SEQ ID NO: 2, wherein the nattokinase mutant is mutated at the 365th position from the N-terminus, and the mutation is a mutation from amino acid N to S.

[0005] As a further embodiment of the present invention: a gene encoding the nattokinase mutant according to claim 1.

[0006] As a further embodiment of the present invention: an expression vector comprising the gene according to claim 2.

[0007] As a further solution of the present invention: a recombinant microorganism is used to express the protein shown in SEQ ID NO. 2.

[0008] Another object of the present invention is to provide a method for preparing a nattokinase mutant, comprising inserting a gene encoding a protein shown in SEQ ID NO. 2 into an expression vector and transforming the vector into Bacillus subtilis 168 for fermentation expression.

[0009] Another object of the present invention is to provide the application of nattokinase single point mutant.

[0010] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention proposes a nattokinase mutant, which introduces the N365S mutation into the Bacillus subtilis WB800N expression system to obtain a nattokinase variant with significantly improved enzyme activity. It has been determined that the fibrinolytic activity of the mutant is 110.60% higher than that of the wild type; 2. The enzyme activity of the N365S mutant was comprehensively evaluated by measuring the diameter of the fibrinolytic zone and detecting the amount of pNA released. The more pNA released, the higher the enzyme's efficiency in hydrolyzing the substrate, and therefore the stronger the activity. The results showed that the enzyme activity of the N365S mutant was 1.1 times higher than that of the wild type, showing significant potential in thrombolytic applications; 3. The fibrinolytic activity data of each mutant were statistically analyzed by GraphPad Prism software, including the diameter of the fibrinolytic zone, and the amount of pNA generated was measured by the change in absorbance at 405 nm. The amount of pNA released directly reflects the strength of enzyme activity, so as to verify the significant effect of the N365S mutant in improving enzyme activity. The results showed that the N365S mutant increased its fibrinolytic activity by 1.1 times, laying a foundation for the further application of plasmin. 4. Significantly improved enzyme activity: The N365S mutation increased the enzyme activity by 1.1 times, effectively enhancing the fibrinolytic activity of nattokinase; 5. Broad application potential: Since the mutant has advantages in fibrinolytic effect, it is suitable for thrombolysis and helps expand the application of nattokinase in the field of thrombolytic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The figure shows the enzyme activity performance of the N365S mutant and the wild type. DETAILED DESCRIPTION

[0012] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0013] The present invention utilizes conventional techniques and methods used in the field of genetic engineering and molecular biology. Those skilled in the art may, based on the technical solutions described in the present invention, adopt other conventional methods, experimental schemes and reagents in the art without being limited to the specific embodiments of the present invention.

[0014] In the embodiment of the present invention, A highly active nattokinase mutant, wherein the mutant is a nattokinase mutant having an amino acid sequence of SEQ ID NO: 2, wherein the nattokinase mutant is mutated at the 365th position from the N-terminus, and the mutation is a mutation of amino acid N to S, wherein N is asparagine instead of S and serine.

[0015] Gene encoding a mutant of nattokinase.

[0016] An expression vector comprising a gene encoding a nattokinase mutant.

[0017] Used to express a recombinant microorganism containing the protein shown in SEQ ID NO. 2.

[0018] A method for preparing a nattokinase mutant comprises inserting a gene encoding a protein shown in SEQ ID NO. 1 into an expression vector and transforming the gene into Bacillus subtilis 168 for fermentation expression.

[0019] Application of single-point mutants of nattokinase.

[0020] Example 1: Preparation of N365S mutant Using the Bacillus subtilis nattokinase aprN gene (Gene ID: 14100838) as a template, the 365th amino acid sequence was replaced from asparagine (N) to serine (S) using site-directed mutagenesis.

[0021] Step 1: Site-directed mutagenesis and PCR amplification: To introduce the N365S mutation, design directional primers Forward: CAACATATCTTGGAAgCTCTTTCTACTATGG, Reverse: CCATAGTAGAAAGAGcTTCCAAGATATGTTGC.

[0022] Plasmid DNA containing the nattokinase aprN gene was used as a template for site-directed mutagenesis PCR amplification. The PCR reaction system included 20-50 ng template DNA, 0.5 μM directional primers, 200 μM dNTPs, 2.5 U DNA polymerase and 1X PCR buffer, and the total reaction volume was 50 μL. The PCR reaction conditions were: initial denaturation at 94°C for 2 minutes, followed by 30 cycles, each cycle including 94°C for 30 seconds, 55°C annealing for 30 seconds, 72°C extension for 1 minute, and finally 72°C extension for 5 minutes to complete the amplification.

[0023] Step 2: Construction of mutant gene vector: The amplified mutant fragment was purified by a DNA purification kit, and the mutant gene fragment was cloned into the pHT01 expression vector using a recombinase to construct the pHT01-N365S expression plasmid. The ligation reaction was performed according to the instructions of the recombinase kit to ensure that the vector and the target fragment were correctly connected. The cloned product was electrotransformed into Bacillus subtilis 168, and the correct construction was subsequently verified by PCR and sequencing.

[0024] Step 3: Transformation and screening: The constructed pHT01-N365S plasmid was transformed into the Bacillus subtilis WB800N strain. The specific electroporation steps are as follows: First, prepare competent cells, dilute the culture solution and culture it to OD600 of 0.2, add 1% DL-threonine, 2% glycine, 0.1% tryptophan and 0.03% Tween 80, and incubate for 1 hour to induce competence. The cells were placed on ice for 20 minutes, centrifuged and washed, and then suspended in electroporation buffer and concentrated to 1 / 100 of the original volume. The pHT01-N365S plasmid was electroporated into competent cells using a 2 mm electroporation cup with parameters set to 2,500V voltage, 25 μF capacitance and 200 Ω resistance. After electroporation, immediately add 1 mL of room temperature 2x YT medium (16 g / L peptone, 10 g / L yeast extract, 5 g / L sodium chloride) + 0.5 M sorbitol and 0.38 M mannitol, and shake for 3 hours before plating.

[0025] Step 4: Screening positive clones: Inoculate the transformed bacterial solution on a YT plate containing 0.5xK and 0.5xChlo and culture at 37°C. Screen positive clones containing the N365S mutation by resistance.

[0026] Example 2: Fermentation and expression of N365S mutant Specifically, the mutant was cloned into the pHT01 expression vector and transformed into Bacillus subtilis WB800N for fermentation expression.

[0027] After verification, the WB800N transformants were cultured overnight in LB medium, transferred to 2x YT medium, and induced with 1mM β-D-1-thiogalactopyranoside (IPTG) for 24 hours. Since NK protein is a secreted protease, the bacterial culture supernatant was collected by centrifugation and protein was extracted by standard ammonium sulfate precipitation. The resulting precipitate was dialyzed in TBS buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.6) for 16 hours to remove ammonium sulfate and other residues, using a 3.5 kDa dialysis bag (by Beyotime Biotech). Subsequently, the samples were centrifuged and filtered through a 0.22 μm filter membrane, and the concentration of all protein samples was determined using a BCA kit to ensure accurate quantification in subsequent analysis and applications.

[0028] Example 3: Determination of mutant enzyme activity The plasmin activity of the N365S mutant was determined by the fibrin plate method and the tetrapeptide substrate method: Preparation of fibrin plates: Mix 10 mL of 1.5 mg / mL bovine fibrinogen solution, 50 μL of 20 U / mL thrombin, and 1% (w / v) agar. Heat the mixture until it melts and prepare the fibrin plates under sterile conditions. Cool it to room temperature to solidify.

[0029] Sample loading and incubation: A well (6 mm in diameter) was punched on the solidified fibrin plate, 20 μL of the N365S mutant fermentation supernatant was added to each well, and the plates were incubated at 37°C for 12 h. The diameter of the fibrinolytic zone was measured to evaluate the enzyme activity.

[0030] Tetrapeptide substrate method: 50 μg of protein sample was adjusted to a total volume of 100 μL, mixed with 50 μL of 0.5 mmol / L N-succinyl-Ala-Ala-Pro-Phe-pNA substrate, and incubated at 37°C for 6 hours. The OD value of the reaction solution was measured at 405 nm every 5 minutes using a Thermo Fisher Varioskan™ LUX microplate reader.

[0031] Measurement and recording: The enzyme activity of the N365S mutant was comprehensively evaluated by measuring the diameter of the fibrinolytic zone and detecting the amount of pNA released. The more pNA released, the more efficient the enzyme is in hydrolyzing the substrate, and therefore the stronger the activity. The results showed that the enzyme activity of the N365S mutant was 1.1 times higher than that of the wild type, showing significant potential in clinical thrombolytic applications.

[0032] Example 4: Screening and verification of N365S mutant Screening and exclusion: 1) Establishing standards for screening mutants 2) Preparation of mutants 3) Inducing expression of mutants 4) Enzyme activity testing and data analysis 10 μL of overnight culture of Bacillus was taken from the experimental bacterial library and added to 10% (w / v) soybean liquid for three days of fermentation. Then, all fermentation broths were spotted on fibrin plates and the diameter of the fibrinolytic zone was measured. We defined strains with a diameter ≥ 15.0 mm as strains with high thrombolytic activity, cloned their nk genes into the pHT01 expression vector, and calculated the DEUT-ΔΔG values ​​of all mutation sites. Subsequently, all candidates with DUET-ΔΔG higher than -0.5 kcal / mol were tested for enzyme activity using the tetrapeptide substrate method, and finally mutants with significantly improved enzyme activity (such as N365S) were screened.

[0033] Data analysis and verification: GraphPad Prism software was used to perform statistical analysis on the fibrinolytic activity assay data of each mutant, including the diameter of the measured fibrinolytic zone, and the amount of pNA generated by measuring the absorbance change at 405 nm. The amount of pNA released directly reflects the strength of the enzyme activity, in order to verify the significant effect of the N365S mutant in improving enzyme activity. The results showed that the N365S mutant increased its fibrinolytic activity by 1.1 times, laying the foundation for the further application of plasmin.

[0034] Table 1 shows the enzyme activity performance of the N365S mutant and the wild type

[0035] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A highly active nattokinase mutant, characterized in that: The mutant is a nattokinase mutant having an amino acid sequence of SEQ ID NO: 2, and the nattokinase mutant is a mutation from N to S at position 365 from the N-terminus.

2. A gene encoding the nattokinase mutant according to claim 1.

3. An expression vector comprising the gene according to claim 2.

4. A recombinant microorganism for expressing a protein represented by SEQ ID NO.

2.

5. A method for preparing the nattokinase mutant according to claim 1, comprising inserting the gene encoding the protein shown in SEQ ID NO. 1 into an expression vector and transforming it into Bacillus subtilis 168 for fermentation expression.

6. The use of the nattokinase single point mutant according to claim 1.

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