Deoxyribonuclease mutants and their use in the preparation of pdrn

Abstract

The present application relates to a deoxyribonuclease mutant and its application in preparing PDRN. The amino acid sequence of the deoxyribonuclease mutant is shown as SEQ ID NO. 1, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 2. The present application also provides the application of the deoxyribonuclease mutant and / or the encoding gene of the deoxyribonuclease mutant in preparing PDRN, and the specific application method. The present application is based on the deoxyribonuclease expressed by the DNASE1 gene of bovine pancreas tissue, and the amino acids at positions 69, 132 and 215 are selected for iterative saturation mutation. Compared with the wild-type enzyme, the enzyme activity of the deoxyribonuclease mutant A69H-S132R-Q215C is increased by 2.15 times, reaching 4.1x10 6 U / mL, and the DNA extraction rate of salmon PDRN prepared by using the mutant enzyme prepared by the present application is 15.2%, the DNA content is 99.1%, and the molecular weight is 1.05 million Daltons, which is significantly improved compared with the wild-type enzyme, and the PDRN in this molecular weight range has more significant biological activity.

Description

TECHNICAL FIELD

[0001] The present application relates to a deoxyribonuclease mutant and its application in preparing PDRN, belonging to the field of biotechnology. BACKGROUND

[0002] Polydeoxyribonudeotide (PDRN) is a specific specification salmon fragment extracted from salmon germ cells, generally composed of 50-2000 base pairs, which binds to adenosine A2A receptor, starts multiple signal pathways, increases anti-inflammatory factors, and produces anti-inflammatory effects; it can also provide purines or pyrimidines through the salvage pathway to accelerate DNA synthesis and repair damaged skin cells. Activation of the receptor causes osteoblasts, fibroblasts, and adipose precursor cells to proliferate, activating senescent damaged cells to make them younger. In addition, VEGF, angiogenin, and glutamine transaminase are also released with the activation of the receptor, improving blood circulation and accelerating cell neogenesis. In the field of medical and cosmetic injections, PDRN has a broad application prospect and a huge demand.

[0003] When extracting PDRN from salmon testis tissue, it is necessary to first degrade the high molecular weight genomic DNA to obtain PDRN with appropriate molecular weight distribution. Common DNA degradation methods include acid degradation, alkaline degradation, enzyme degradation, and DNA breaking instrument degradation.

[0004] Chinese patent document CN110747194A discloses a preparation method of PDRN, which requires lysing testis tissue at 90-100℃ and using a DNA breaking instrument to break DNA, which is prone to generate heat and cause Maillard reaction, resulting in yellowing of the product and affecting product quality. Meanwhile, it is not easy to operate in large-scale industrial production.

[0005] Chinese patent document CN112315836A discloses a preparation method of PDRN, which requires pretreatment, cell collection, trypsin digestion, protein removal, alcohol precipitation, and restriction endonuclease Sau3 AI digestion to obtain PDRN product. This method uses mild enzyme digestion conditions, but it uses two-step enzyme digestion with enzyme digestion times of 16-48h and 5-16h, respectively, which is a multi-step and long-cycle process prone to bacterial growth and affecting product quality. Moreover, the cell collection process requires repeated washing with a mixed salt solution, which is a tedious and uneconomical process for industrial production.

[0006] Enzymatic degradation of fish white DNA is a milder method than acid degradation and alkaline degradation, which does not damage the molecular structure of DNA and ensures stable product quality. However, there is currently a lack of enzymes suitable for industrial production, which are efficient, stable, and cost-controllable, to some extent, limiting the development of enzyme preparation of PDRN.

[0007] Therefore, it has important value to study a DNase mutant and improve the enzymatic efficiency of the nuclease on PDRN for preparing the extracted PDRN. SUMMARY

[0008] In view of the deficiencies in the prior art, the present application provides a DNase mutant and its application in the preparation of PDRN.

[0009] The technical scheme of the present application is as follows:

[0010] A DNase mutant, the amino acid sequence of which is shown as SEQ ID NO. 1, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2.

[0011] The DNase mutant provided by the present application is based on the DNase expressed by the DNASE1 gene derived from the pancreas tissue of Bos taurus (NCBI accession number BC142349.1), and the alanine (Ala) at the 69th position is mutated to histidine (His), the serine (Ser) at the 132nd position is mutated to arginine (Arg), and the glutamine (Gln) at the 215th position is mutated to cysteine (Cys).

[0012] A recombinant vector is obtained by inserting the coding gene of the DNase mutant into a plasmid vector.

[0013] According to the present application, the plasmid vector is preferably a pET-28a plasmid.

[0014] A recombinant strain is obtained by transforming the above-mentioned recombinant vector into a host strain.

[0015] According to the present application, the host strain is preferably Escherichia coli.

[0016] The DNase mutant and / or the coding gene of the DNase mutant are used in the preparation of PDRN.

[0017] According to the present application, the specific steps of the application are as follows:

[0018] (1) The salmon fish white is thawed in water bath at 20-30 DEG C, rinsed with purified water, and then crushed by a crusher to obtain a paste-like fish white;

[0019] (2) at 35-60 DEG C, adding purified water to the paste-like fish milt of step (1), stirring uniformly, adjusting pH to 7.0-8.5, then adding DNase mutant, and enzymolysis for 18-90 min; after the enzymolysis, continuously adding urea and sodium sulfate at 35-60 DEG C, stirring and incubating for 0.5-1.5 h to obtain an enzymolysis solution; after alcohol precipitation, washing and drying, PDRN is obtained.

[0020] Further preferably, in step (2), the mass ratio of the paste-like fish milt to purified water is 1:10, g / mL; the mass ratio of the paste-like fish milt to DNase mutant is 1:(0.01-0.08); the mass ratio of the paste-like fish milt to urea is 1:(0.03-0.07); and the mass ratio of the paste-like fish milt to sodium sulfate is 1:(0.2-0.6).

[0021] The present application has the following beneficial effects:

[0022] 1. The DNase mutant A69H-S132R-Q215C provided by the present application is based on the DNase expressed by the DNASE1 gene of Bos taurus pancreas tissue (NCBI accession number BC142349.1), and the amino acids at positions 69, 132 and 215 are subjected to iterative saturation mutation, i.e., the alanine (Ala) at position 69 is mutated to histidine (His), the serine (Ser) at position 132 is mutated to arginine (Arg), and the glutamine (Gln) at position 215 is mutated to cysteine (Cys). Compared with the wild-type DNase (NCBI accession number BC142349.1), the enzyme activity of the DNase mutant A69H-S132R-Q215C provided by the present application is increased by 2.15 times, reaching 4.1 x 10 6 U / mL.

[0023] 2. The DNase mutant A69H-S132R-Q215C provided by the present application can be used for preparing PDRN, and the extraction rate reaches 15.2%, the DNA content is 99.1%, and the molecular weight of PDRN is 1.05 million daltons, which is significantly improved compared with the wild-type enzyme; meanwhile, the Maillard reaction caused by high temperature can be avoided, and the problems of complicated two-step enzymolysis procedure, long production cycle and easy microbial contamination can be overcome. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is a 3D structure model of the DNase mutant A69H-S132R-Q215C.

[0025] Figure 2 is a nucleic acid electropherogram of the DNase mutant A69H-S132R-Q215C and the wild-type enzyme;

[0026] Lane 1 is the deoxyribonuclease mutant A69H-S132R-Q215C, and lane 2 is the wild-type enzyme.

[0027] Figure 3 Electrophoretogram of PDRN extracted from Comparative Example 1 and Example 4;

[0028] Lane 1 is Comparative Example 1, and lane 2 is Example 4. DETAILED DESCRIPTION

[0029] The following examples of the present application only illustrate the specific implementation of the present application, which can not be understood as a limitation of the present application, and any changes made without departing from the principles and essence of the present application, fall within the scope of protection of the present application.

[0030] The experimental techniques and experimental methods used in the present embodiment are all conventional technical methods unless otherwise specified. The materials, reagents, etc. used in the present embodiment can be obtained through the regular commercial channel unless otherwise specified.

[0031] The E. coli used in the present application is a commonly commercially available strain, which can be purchased from the Microbial Preservation Center or a strain sales company.

[0032] Example 1: Selection of Mutation Sites

[0033] According to the homology modeling (100% identity) of the amino acid sequence of the deoxyribonuclease expressed by the DNASE1 gene of the pancreas tissue of Bos taurus (NCBI accession number BC142349.1) on SWISS-MODEL, a PDB file was generated, and the 3D model is shown in Figure 1 According to the conserved regions 66 / 134 / 212 of the deoxyribonuclease expressed by the DNASE1 gene of the pancreas tissue of Bos taurus (NCBI accession number BC142349.1), combined with sequence conservation analysis and 3D model analysis, A69, S132 and Q215 of the deoxyribonuclease DNASE1 were selected as potential mutation sites.

[0034] Example 2: Obtaining of Mutant Genes

[0035] 1. The nucleotide sequence of DNASE1 gene is artificially synthesized by Huada Gene Company according to the information of NCBI accession number BC142349.1, and then the DNASE1 gene is connected to the plasmid vector pET-28a by using restriction enzymes Nhe I and Xho I to obtain the recombinant plasmid pET-28a-DNASE1; then the recombinant plasmid pET-28a-DNASE1 is introduced into the competent cells of Escherichia coli BL21 by heat shock method to obtain the engineering bacteria BL21-pET-28a-DNASE1 containing wild-type deoxyribonuclease, which is stored at -80℃.

[0036] 2. The DNASE1 mutant gene is obtained by three rounds of iterative saturation mutation and superior strain screening, and the specific method is as follows:

[0037] The first round: taking the recombinant plasmid pET-28a-DNASE1 as a template, F69 and R69 as primers, and the 69th alanine in the amino acid sequence is mutated into the remaining 19 kinds of amino acids by site-directed saturation mutation PCR, and then the Escherichia coli is transformed, LB solid medium is coated and cultured, and the superior strain is screened to obtain the strain BL21-pET-28a-DNASE1-A69H.

[0038] The screening method of the superior strain: the mutant single colony is selected from the LB solid medium, inoculated into a 96-well plate containing 500 μL of LB liquid medium, cultured at 37℃, 220 rpm for 15h, inoculated into a 96-well plate containing 500 μL of LB liquid medium at a 2% (v / v) inoculation amount, cultured at 37℃, 220 rpm for 3h, and then induced to culture at 20℃ for 15h after adding IPTG with a final concentration of 0.1 mmol / L; the enzyme activity is detected respectively.

[0039] The enzyme activity detection condition: 40 mM Tris-HCl (pH 8.0), 10 mM MgSO4, 1 mM CaCl2, 1 μg pBR322 plasmid DNA.

[0040] The definition of enzyme activity: at 37℃, the amount of enzyme required to completely degrade 1 μg of pBR322 plasmid DNA within 10 minutes is defined as 1 unit of activity.

[0041] The second round: taking the recombinant plasmid extracted from the strain BL21-pET-28a-DNASE1-A69H as a template, F132 and R132 as primers, and the 132th serine in the amino acid sequence is mutated into the remaining 19 kinds of amino acids by site-directed saturation mutation PCR, and then the Escherichia coli is transformed, LB solid medium is coated and cultured, and the superior strain is screened according to the method of the first round to obtain the strain BL21-pET-28a-DNASE1-A69H-S132R.

[0042] The third round: using the recombinant plasmid extracted from the strain BL21-pET-28a-DNASE1-A69H-S132R as a template, using F215 and R215 as primers, mutating the glutamine at the 215th amino acid of the amino acid sequence to the remaining 19 kinds of amino acids by saturated mutation PCR, then transforming E. coli, coating LB solid medium and culturing, and screening the dominant strain by the method described in the first round, to obtain the strain BL21-pET-28a-DNASE1-A69H-S132R-Q215C.

[0043] The mutation system (25 μL) is shown in the following table.

[0044]

[0045]

[0046] PCR conditions: 95°C pre-denaturation for 5 min, 30 cycles of 90°C for 30 s, 62°C for 30 s, 72°C for 7 min, and finally 72°C for 5 min.

[0047] The recombinant plasmid was extracted from the strain BL21-pET-28a-DNASE1-A69H-S132R-Q215C, and positive verification was performed by DNA agarose gel electrophoresis, and the results are shown in Figure 2 It can be seen from Figure 2 that the target band appears in the gel electrophoresis map and the band is single, indicating that the deoxyribonuclease gene mutation is successful and the expression is successful.

[0048] The specific mutation upstream primers and downstream primers are shown in Table 1.

[0049] Table 1

[0050] Primer name Primer sequence (5'-3') F69 agccacctggtgnnkgtggggaagc R69 ttccccacknncaccaggtggctg F132 aacgacagcttcnnkcgggagcc R132 ggctcccgknngaagctgtcgttc F215 agctccaccttcnnktggctgattc R215 aatcagccaknngaaggtggagctcg

[0051] Example 3, screening of enzyme mutants

[0052] 1. Preparation of enzyme mutants: Taking deoxyribonuclease mutant A69H-S132R-Q215C as an example, the strain BL21-pET-28a-DNASE1-A69H-S132R-Q215C was inoculated into LB liquid medium containing 50 mg / L kanamycin, and cultured for 12 h. Then, the culture was inoculated into fresh LB liquid medium containing 50 mg / L kanamycin at a ratio of 2% (v / v), and cultured for 4 h. Then, 0.1 mmol / L IPTG was added, and the culture was induced at 20°C for 12 h to obtain a fermentation broth. The fermentation broth was centrifuged to obtain wet bacteria, which were ultrasonically broken in phosphate buffer, and then centrifuged to obtain a supernatant, which was the deoxyribonuclease mutant A69H-S132R-Q215C.

[0053] Wild-type deoxyribonuclease DNASE1, deoxyribonuclease mutant A69H, deoxyribonuclease mutant A69R, deoxyribonuclease mutant A69Q, deoxyribonuclease mutant A69H-S132K, deoxyribonuclease mutant A69H-S132R, deoxyribonuclease mutant A69H-S132W, deoxyribonuclease mutant A69H-S132R-Q215A, deoxyribonuclease mutant A69H-S132R-Q215M, and deoxyribonuclease mutant A69H-S132R-Q215Y were prepared in the same manner.

[0054] 2. The enzyme activities of wild-type deoxyribonuclease DNASE1, deoxyribonuclease mutant A69H, deoxyribonuclease mutant A69R, deoxyribonuclease mutant A69Q, deoxyribonuclease mutant A69H-S132K, deoxyribonuclease mutant A69H-S132R, deoxyribonuclease mutant A69H-S132W, deoxyribonuclease mutant A69H-S132R-Q215A, deoxyribonuclease mutant A69H-S132R-Q215C, deoxyribonuclease mutant A69H-S132R-Q215M, and deoxyribonuclease mutant A69H-S132R-Q215Y were determined according to the method described in Example 2, and the results are shown in Table 2.

[0055] Table 2

[0056] Strain number Enzyme activity (U / mL) Enzyme activity improvement rate (%) BL21-pET-28a-DNASE1 1.3 x 10 6 ]]> - BL21-pET-28a-DNASE1-A69H 2.0 x 10 6 ]] 53.8 BL21-pET-28a-DNASE1-A69R 1.6 x 10 6 ]]> 23.1 BL21-pET-28a-DNASE1-A69Q 1.4 x 10 6 ]]> 7.7 BL21-pET-28a-DNASE1-A69H-S132K 2.1 x 10 6 ]]> 61.5 BL21-pET-28a-DNASE1-A69H-S132R 2.7 x 10 6 ]] 107.7 BL21-pET-28a-DNASE1-A69H-S132W 2.1 x 10 6 ]] 61.5 BL21-pET-28a-DNASE1-A69H-S132R-Q215A 2.9 x 10 6 ]] 123.1 BL21-pET-28a-DNASE1-A69H-S132R-Q215C 4.1 x 10 6 ]]> 215.4 BL21-pET-28a-DNASE1-A69H-S132R-Q215M 2.6 x 10 6 ]]> 100 BL21-pET-28a-DNASE1-A69H-S132R-Q215Y 3.8 x 10 6 ]]> 192.3

[0057] As shown in Table 2, the enzyme activities of the deoxyribonuclease mutants were higher than that of the wild-type deoxyribonuclease, and the enzyme activity of deoxyribonuclease mutant A69H-S132R-Q215C reached 4.1 x 10 6 U / mL, which was 2.15 times higher than that of the wild-type deoxyribonuclease.

[0058] Example 4: Preparation of PDRN using the deoxyribonuclease mutant A69H-S132R-Q215C

[0059] A method for preparing PDRN using the deoxyribonuclease mutant A69H-S132R-Q215C includes the following steps:

[0060] (1) Take out the frozen salmon milt, thaw it in a water bath at 25°C, rinse it with purified water, and then crush it with a pulverizer to obtain a paste-like salmon milt.

[0061] (2) At 50℃, add 1000mL of purified water to 100g of the paste-like fish roe from step (1) and stir until homogeneous. Adjust the pH to 8.0 with 1mol / L sodium hydroxide solution and then add 0.3g of deoxyribonuclease mutant.

[0062] A69H-S132R-Q215C was enzymatically hydrolyzed at 50℃ for 60 min. After hydrolysis, 8 g of urea and 40 g of sodium sulfate were added, and the mixture was stirred and incubated for 1 h to obtain the hydrolysate. 95% ethanol was added to the hydrolysate until the ethanol concentration in the mixture was above 50%, causing PDRN to precipitate. The mixture was allowed to stand and separate into layers, the supernatant was discarded, and the precipitate was retained. 75% ethanol (3 times the volume of the precipitate) was added, and the mixture was stirred and washed. The mixture was allowed to stand and separate into layers, the supernatant was discarded, and the precipitate was retained. 85% ethanol (3 times the volume of the precipitate) was added, and the mixture was stirred and washed. The mixture was allowed to stand and separate into layers, the supernatant was discarded, and the precipitate was retained. 95% ethanol (4 times the volume of the precipitate) was added to dehydrate the precipitate for more than 2 h. The mixture was then placed in a vacuum oven and dried at 45℃ for 4 h to obtain PDRN.

[0063] The PDRN prepared in this embodiment was a white granule or powder, with a yield of 15.2%, a purity of 99.1%, a protein content of 0.5%, an endotoxin content of 0.4 EU / mg, and a molecular weight of 1.05 million Daltons. Figure 3 Middle lane 2).

[0064] Comparative Example 1

[0065] The paste-like fish milt was prepared using wild-type deoxyribonuclease DNASE1 according to the method (1) described in Example 4, as follows:

[0066] At 50℃, 1000 mL purified water is added to 100 g of the paste-like fish white, and stirred until uniform. After adjusting the pH to 8.0 using a 1 mol / L sodium hydroxide solution, 0.3 g of wild-type deoxyribonuclease DNASE1 is added, and the enzyme is allowed to react for 60 min at 50℃. After the enzyme reaction, 8 g of urea and 40 g of sodium sulfate are added, and the mixture is stirred and incubated for 1 h to obtain an enzyme reaction solution. 95% ethanol is added to the enzyme reaction solution until the ethanol concentration in the mixture is greater than 50%, and PDRN is precipitated. After standing and layering, the supernatant is discarded, and the precipitate is retained. 3 times the volume of 75% ethanol is added to the precipitate, and the mixture is stirred and washed. After standing and layering, the supernatant is discarded, and the precipitate is retained. 3 times the volume of 85% ethanol is added to the precipitate, and the mixture is stirred and washed. After standing and layering, the supernatant is discarded, and the precipitate is retained. 4 times the volume of 95% ethanol is added to the precipitate, and the mixture is stirred and dehydrated for 2 h or more. The mixture is placed in a vacuum oven, and dried at 45℃ for 4 h to obtain PDRN.

[0067] The PDRN prepared in the present comparative example has a yellow flaky or filamentous solid appearance, a yield of 10.9%, a content of 92.3%, a protein content of 0.8%, an endotoxin of 0.9 EU / mg, and a molecular weight of 4.2 million Daltons. Figure 3 Lane 1).

[0068] As can be seen from the data comparison, the method for extracting PDRN using the deoxyribonuclease mutant A69H-S132R-Q215C in Example 4 has a higher extraction rate and DNA content than Comparative Example 1, and the molecular weight of the prepared PDRN is also smaller than that of Comparative Example 1. Compared with the wild-type enzyme, each aspect is significantly improved. At the same time, the method can also avoid the Maillard reaction that easily occurs at high temperatures, and overcome the problems of complicated two-step enzyme reaction steps, long production cycle, and easy microbial contamination.

Claims

1. A deoxyribonuclease mutant, characterized in that, The amino acid sequence is shown as SEQ ID NO. 1, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO.

2.

2. A recombinant vector, characterized in that, The coding gene of the deoxyribonuclease mutant of claim 1 is inserted into a plasmid vector.

3. The recombinant vector of claim 2, wherein, The plasmid vector is pET-28a plasmid.

4. A recombinant bacterial strain, characterized in that, The recombinant vector of claim 2 is transformed into a host strain.

5. The recombinant vector of claim 4, wherein, The host strain is Escherichia coli.

6. Use of the deoxyribonuclease mutant of claim 1 in the preparation of PDRN.

7. Use according to claim 6, wherein The specific steps of the use are as follows: (1) The salmon white is thawed in water bath at 20-30℃, rinsed with purified water, and then crushed by a crusher to obtain a paste-like fish white; (2) At 35-60℃, purified water is added to the paste-like fish white of step (1), stirred uniformly, and then the pH is adjusted to 7.0-8.5, followed by the addition of the deoxyribonuclease mutant of claim 1, and enzymatic hydrolysis for 18-90 min; after the enzymatic hydrolysis, urea and sodium sulfate are continuously added at 35-60℃, and stirred and incubated for 0.5-1.5 h to obtain an enzymatic hydrolysate; after alcohol precipitation, washing, and drying, PDRN is obtained.

8. Use according to claim 7, wherein the compound is ###0002### In step (2), the mass-volume ratio of the paste-like fish white and purified water is 1:10, g / mL; the mass ratio of the paste-like fish white and the deoxyribonuclease mutant is 1:(0.01-0.08); the mass ratio of the paste-like fish white and urea is 1:(0.03-0.07); and the mass ratio of the paste-like fish white and sodium sulfate is 1:(0.2-0.6).

Images