A bacteriophage-derived staphylococcus aureus antibacterial peptide enzyme lysozyme mutant with high thermal stability and its preparation method and application

Abstract

The application relates to the technical field of biotechnology, and particularly discloses a bacteriophage-derived lysostaphin mutant with high thermal stability and an application and a preparation method thereof. The mutant is obtained by using a PROSS online prediction tool to design the stability of wild-type antimicrobial peptidase lysostaphin (APL), and nine mutation schemes (design 1-design 9) are obtained. The thermal stability of the mutant APL (design 9) is significantly improved, the optimum temperature is increased to 55 DEG C, and the half-life at 70 DEG C and 80 DEG C is 57+ / -4 min and 21+ / -1 min, respectively, which is 1.4 times and 2.1 times that of the wild type. The preparation method comprises gene synthesis, vector construction, recombinant expression and purification. The mutant can be used for the prevention and treatment of staphylococcus aureus infection, veterinary antibiotic substitutes, food preservatives and compound disinfectants, and is suitable for high-temperature processing environment and has industrialization advantages.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a phage-derived Staphylococcus aureus antimicrobial peptidase lysozyme mutant with high thermal stability, its preparation method, and its application. Background Technology

[0002] Staphylococcus aureus is a common zoonotic pathogen that can cause various diseases, including skin and soft tissue infections, pneumonia, and sepsis. With the spread of methicillin-resistant Staphylococcus aureus (MRSA) and other drug-resistant strains, clinical treatment is becoming increasingly challenging. Antimicrobial peptidase lysostaphin (APL), derived from Staphylococcus simulans, is a bacteriophage-derived endolysin that specifically recognizes and hydrolyzes the peptidoglycan cross-linked structure in the Staphylococcus aureus cell wall. It possesses advantages such as high bactericidal efficiency, strong targeting, and low likelihood of inducing bacterial resistance, and is considered a potential candidate molecule to replace traditional antibiotics.

[0003] However, wild-type APL exhibits poor thermal stability, with an optimal reaction temperature of only 37°C. Enzyme activity declines rapidly above 40°C and is almost completely inactivated at 70°C and above. For example, the half-life of wild-type APL is approximately 41±3 min at 70°C and only 10±1 min at 80°C. This deficiency severely limits its application in high-temperature processing scenarios (such as food pasteurization, medical device thermal sterilization, and high-temperature feed pelleting). Current technologies for endosomalin stability modification largely rely on directed evolution or random mutation, which involves a large screening workload, long cycles, and low success rates, making it difficult to efficiently obtain stable mutants that meet the demands of industrial high-temperature processing.

[0004] To address the aforementioned issues, it is necessary to develop a highly thermally stable APL mutant through rational design strategies to expand its application value in high-temperature environments and reduce reliance on cold chain in production, storage, and transportation processes. Summary of the Invention

[0005] This invention first provides a phage-derived Staphylococcus aureus antimicrobial peptidase lysozyme mutant with high thermal stability. The mutant was designed from wild-type APL (sequence shown in SEQ ID NO: 1) using the PROSS stability prediction tool. Its thermal stability is higher than that of wild-type APL, and its half-life is not less than 40 min at 70°C.

[0006] In some embodiments, the mutant is APL (design9), with an optimal temperature of 55°C, a half-life of 57±4 min at 70°C, and a half-life of 21±1 min at 80°C.

[0007] In some embodiments, the mutant amino acid sequence is shown in SEQ ID NO: 11.

[0008] The present invention also provides a nucleic acid molecule encoding the above-mentioned mutant, the sequence of which is obtained by site-directed mutagenesis based on the wild-type APL gene.

[0009] The present invention also provides a recombinant expression vector comprising the above-mentioned nucleic acid molecule, wherein the vector is pET21a(+) and carries a histidine tag.

[0010] The present invention also provides a host cell, which is a prokaryotic cell transformed into the above-mentioned vector, preferably Escherichia coli BL21(DE3).

[0011] The present invention also provides a method for preparing the above-mentioned APL mutant, comprising the following steps:

[0012] (1) Synthesize the mutant gene and clone it into the expression vector;

[0013] (2) Transform the vector into the host cell for induction;

[0014] (3) Disrupt cells and purify proteins using Ni-NTA affinity chromatography.

[0015] In some embodiments, expression was induced overnight at 16°C using 0.1 mM IPTG, and purification was performed by elution with a buffer containing 250 mM imidazole.

[0016] Finally, this invention provides the application of the above-mentioned APL mutant in the preparation of antibacterial drugs, food preservatives or disinfectants, for inhibiting or killing Staphylococcus aureus.

[0017] In some embodiments, the application includes use in combination with vancomycin or gentamicin to synergistically enhance the antibacterial effect.

[0018] Compared with the prior art, the present invention has at least the following beneficial effects:

[0019] (1) Significantly improved thermal stability: The optimal temperature of mutant APL (design9) is increased to 55℃, and the half-life at 70℃ and 80℃ is extended to 1.4 times and 2.1 times that of wild type, respectively, making it suitable for high-temperature environments.

[0020] (2) Industrialization advantages: Enhanced stability reduces cold chain transportation and storage costs, and facilitates application in medical device disinfection and food processing.

[0021] (3) Broad application prospects: It can be used as an antibiotic substitute in veterinary drugs and food preservatives, or combined with traditional antibiotics to enhance the effect and delay the development of drug resistance.

[0022] (4) Rational design efficiency: The PROSS tool accurately predicts and avoids the blindness of random mutations, thus improving R&D efficiency. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the sequence alignment between APL (Uniprot ID: Q2FYD8) and the template protein (PDB ID: 4LXC).

[0024] Figure 2 This is a superimposed image of the three-dimensional structures of APL (Uniprot ID: Q2FYD8) and the template protein (PDB ID: 4LXC).

[0025] Figure 3 The distribution of RMSD values ​​for APL (Uniprot ID: Q2FYD8) and the template protein (PDB ID: 4LXC).

[0026] Figure 4 A table showing the nine mutation schemes predicted by PROSS.

[0027] Figure 5 This is a diagram showing the specific mutation sites and mutated amino acids.

[0028] Figure 6 This is a graph showing the results of SDS-PAGE. Note: W: whole cell; S: supernatant; P: precipitate; U: uninduced. Detailed Implementation

[0029] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0030] Example 1: Structural analysis of Staphylococcus aureus endolysin APL

[0031] To obtain highly stable Staphylococcus aureus ( Staphylococcus aureus The endolysin APL mutant was first analyzed in this study. S.a The crystal structure of the unresolved strain (Uniprot ID: Q2FYD8) is not yet resolved. We use the resolved crystal structure of the mimic staphylococcus (…). Staphylococcus simulans The APL crystal structure (PDB ID: 4LXC) was used as the research template. Sequence alignment results showed significant differences between the two. like Figure 1-3 As shown in the figure, the crystal structure is used directly for subsequent design.

[0032] I. Predicting APL stability sites online using PROSS

[0033] PROSS provides nine possible schemes to improve APL stability (design_1 - design_9), with the number of mutated amino acids ranging from 2 to 11, and mutation rates ranging from 0.81% to 4.47% (e.g., Figure 4 (As shown). Specific mutation sites and mutated amino acids are as follows: Figure 5 As shown, we constructed a mutation library using nine designed mutation schemes.

[0034] II. Construction of Mutant Libraries (des 1 – des 9)

[0035] The APL gene sequence was obtained as follows:

[0036] ATGGCTGCAACACATGAACATTCAGCACAATGGTTGAATAATTACAAAAAAGGATATGGTTACGGTCCTTATCCATTAGGTATAAATGGCGGTATGCACTACGGAGTTGATTTTTTTATGAATATTGGAACACCAGTAAAAGCTATTTCAAGCGGAAAAATAGTTGAAGCTGGTTGGAGTAATTACGGAGGAGGTAATCAAATAGGTCTTATTGAAAATGATGGAGTGCATAGACAATGGTATATGCATCTAAGTAAATATAATGTTAAAGTAGGAGATTATGTCAAAGCTGGTCAAATAATCGGTTGGTCTGGAAGCACTGGTTATTCTACAGCACCACATTTACACTTCCAAAGAATGGTTAATTCATTTTCAAATTCAACTGCCCAAGATCCAATGCCTTTCTTAAAGAGCGCAGGATATGGAAAAGCAGGTGGTACAGTAACTCCAACGCCGAATACAGGTTGGAAAACAAACAAATATGGCACACTATATAAATCAGAGTCAGCTAGCTTCACACCTAATACAGATATAATAACAAGAACGACTGGTCCATTTAGAAGCATGCCGCAGTCAGGAGTCTTAAAAGCAGGTCAAACAATTCATTATGATGAAGTGATGAAACAAGACGGTCATGTTTGGGTAGGTTATACAGGTAACAGTGGCCAACGTATTTACTTGCCTGTAAGAACATGGAATAAATCTACTAATACTTTAGGTGTTCTTTGGGGAACTATAAAGTGA (SEQ ID No.1).

[0037] The amino acid sequence is as follows (the first amino acid M is numbered starting from 247).

[0038] >4LXC_1|Chains A, B, C, D|Lysostaphin|Staphylococcus simulans (1286)

[0039] MAATHEHSAQWLNNYKKGYGYGPYPLGINGGMHYGVDFFMNIGTPVKAISSGKIVEAGWSNYGGGNQIGLIENDGVHRQWYMHLSKYNVKVGDYVKAGQIIGWSGSTGYSTAPHLHFQRMVNSFSNSTAQDPMPFLKSAGYGKAGGTVTPTPNTGWKTNKYGTLYKSESASFTPNTDIITRTTGPFRSMPQSGVLKAGQTIHYDEVMKQDGHVWVGYTGNSGQRIYLPVRTWNKSTNTLGVLWGTIKLEHHHHHH(SEQ ID No.2);

[0040] >des_1

[0041] MAATHEHSAQWLNNYKKGYGYGPYPLGINGGMHYGVDFFMNIGTPVKAISSGKVVEAGWSNYGGGNQIGLIENDGVHRQWYMHLSKYNVKVGDYVKAGQIIGWSGSTGYSTGPHLHFQRMVNSFSNSTAQDPMPFLKSAGYGKAGGTVTPTPNTGWKTNKYGTLYKSESASFTPNTDIITRTTGPFRSMPQSGVLKAGQTIHYDEVMKQDGHVWVGYTGNSGQRIYLPVRTWNKSTNTLGVLWGTIKLEHHHHHH(SEQ ID No.3);

[0042] >des _2

[0043] MAATHEHSAQWLNNYKKGYGYGPYPLGINGGMHYGVDFFMNIGTPVKAISSGKVVEAGWSNYGGGNQIGLIENDGVHRQWYMHLSKYNVKVGDYVKTGQIIGWSGSTGYSTGPHLHFQRMVNSFSNSTAQDPMPFLKSAGYGKAGGTVTPTPNTGWKTNKYGTLYKSESASFTPNTDIITRTTGPFRSMPQSGVLKAGQTIHYDEVMKQDGHVWVGYTGNSGQRIYLPVRTWNKSTNTLGVLWGTIKLEHHHHHH(SEQ ID No.4);

[0044] >des _3

[0045] MAATHEHSAQWLNNYKKGYGYGPYPLGINGGMHYGVDFFMNIGTPVKAISSGKVVEAGWSNYGGGNQIGLIENDGVHRQWYMHLSKYNVKVGDYVKTGQIIGWSGSTGYSTGPHLHFQRMVNSFSNSTAQDPMPFLKSAGYGKAGGTVTPTPNTGWKTNKYGTLYKSESASFTPNTDIITRTTGPFRSMPQAGVLKAGQTIHYDEVMKQDGHVWVGYTGNSGQRIYLPVRTWNKSTNTLGVLWGTIKLEHHHHHH(SEQ ID No.5);

[0046] >des _4

[0047] MAATHEHSAQWLNNYKKGYGYGPYPLGINGGMHYGVDFFMNIGTPVKAISSGKVLEAGWSNYGGGNQIGLIENDGVHRQWYMHLSKYNVKVGDYVKTGQIIGWSGSTGYSTGPHLHFQRMVNSFSNSTAQDPMPFLKSAGYGKAGGTVTPTPNTGWKTNKYGTLYKSESASFTPNTDIITRTTGPFRSMPQAGVLKAGQTIHYDEVMKQDGHVWVGYTGNSGQRIYLPVRTWNKSTNTLGVLWGTIKLEHHHHHH(SEQ ID No.6);

[0048] >des _5

[0049] MAATHEHSAQWLNNYKKGYGYGPYPLPINGGMHYGVDFFMNIGTPVKAISSGKVLEAGWSNYGGGNQIGLIENDGVHRQWYMHLSKYNVKVGDYVKTGQIIGWSGSTGYSTGPHLHFQRMVNSFSNSTAQDPMPFLKSAGYGKAGGTVTPTPNTGWKTNKYGTLYKSESASFTPNTDIITRTTGPFRSMPQAGVLKAGQTIHYDEVMKQDGHVWVGYTGNSGQRIYLPVRTWNKSTNTLGVLWGTIKLEHHHHHH(SEQ ID No.7);

[0050] >des _6

[0051] MAATHEHSAQWLNNYKKGYGYGPYPLPINGGMHYGVDFFMNIGTPVKAISSGKVLEAGWSNYGGGNQIGLIENDGVHRQWYMHLSKYNVKVGDYVKTGQIIGWSGSTGYSTGPHLHFQRMVGSFSNSTAQDPMPFLKSAGYGKAGGTVTPTPNTGWKTNKYGTLYKSESASFTPNTDIITRTTGPFRSMPQAGVLKAGQTIHYDEVMKQDGHVWVGYTGNSGQRIYLPVRTWNKSTNTLGVLWGTIKLEHHHHHH(SEQ ID No.8);

[0052] >des _7

[0053] MAATHEHSAQWLNNYKKGYGYGPYPLPINGGMHYGVDFFMNIGTPVKAISSGKVLEAGWSNYGGGNQIGLQENDGVHRQWYMHLSKYNVKVGDYVKTGQIIGWSGSTGYSTGPHLHFQRMVGSFSNSTAQDPMPFLKSAGYGKAGGTVTPTPNTGWKTNKYGTLYKSESASFTPNTDIITRTTGPFRSMPQAGVLKAGQTIHYDEVMKQDGHVWVGYTGNSGQRIYLPVRTWNKSTNTLGVLWGTIKLEHHHHHH(SEQ ID No.9);

[0054] >des _8

[0055] MAATHEHSAAWLNNYKKGYGYGPYPLPINGGMHYGVDFFMNIGTPVKAISSGKVVEAGWSNYGGGNQIGLKENDGVHRQWYMHLSKYNVKVGDYVKTGQIIGWSGSTGYSTGPHLHFQRMVGSFSNSTAQDPMPFLKSAGYGKAGGTVTPTPNTGWKTNKYGTLYKSESASFTPNTDIITRTTGPFRSMPQAGVLKAGQTIHYDEVMKQDGHVWVGYTGNSGQRIYLPIRTWNKSTNTLGVLWGTIKLEHHHHHH(SEQ ID No.10)

[0056] >des _9

[0057] MAATHEHSAAWLNNYKKGYGYGPYPLPINGGMHYGVDFFMNIGTPVKAISSGKVVEAGWSNYGGGNQIGLKENDGVHRQWYMHLSKYNVKVGDYVKTGQIIGWSGSTGASTGPHLHFQRMVGSFSNSTA QDPMPFLKSAGYGKAGGTVTPTPNTGWKTNKYGTLYKSESASFTPNTDIITRTTGPFRSMPQAGVLKAGQTIHYDEVMKQDGHVWVGYTGSSGQRIYLPIRTWNKSTNTLGVLWGTIKLEHHHHHH (SEQ ID No.11);

[0058] The above mutants were synthesized by Beijing Qingke Biotechnology Co., Ltd. and constructed in the pET21a(+) vector, and named pET21a(+)-des 1, pET21a(+)-des 2, pET21a(+)-des 3, pET21a(+)-des 4, pET21a(+)-des 5, pET21a(+)-des 6, pET21a(+)-des 7, pET21a(+)-des 8, and pET21a(+)-des 9, respectively.

[0059] III. Large-scale acquisition of plasmid pET21a(+)-des1-9

[0060] LB medium: 10 g peptone / L, 5 g yeast extract / L, 10 g sodium chloride / L, with 1.5% agar powder added to the solid medium. The steps for transforming the plasmid into *E. coli* DH5α strain are as follows: Add 100 ng of plasmid to *E. coli* DH5α competent cells, incubate on ice for 30 minutes, then heat shock at 42 ℃ for 30 seconds, immediately incubate on ice for 2 minutes, add 0.5 mL of LB medium for recovery for 45 minutes, centrifuge at 3000-6000 rpm for 1 minute to collect the cells, and the remaining... The plating was spread on Amp-resistant plates and incubated overnight at 37 °C. The next day, single clones were picked from the plates and inoculated into 4 mL test tubes (with Amp antibiotic added), and incubated at 37 °C and 200 rpm for 8-12 hours. Plasmids were extracted using a plasmid extraction kit (TIANGEN, catalog number DP103).

[0061] IV. Recombinant Escherichia coli E. coli Construction of BL21(DE3) / pET21a(+)-des1-9

[0062] The steps for transforming plasmid pET21a(+)-des1-9 into *E. coli* BL21(DE3) strain are as follows: Add 100 ng of plasmid to *E. coli* BL21(DE3) competent cells, incubate on ice for 30 minutes, then heat shock at 42 °C for 45 seconds, immediately incubate on ice for 2 minutes, add 0.5 mL of LB medium for recovery for 1 hour, centrifuge at 3000-6000 rpm for 1 minute to collect cells, and the remaining... Spread on Amp-resistant plates and incubate overnight at 37°C.

[0063] Example 2: Induced expression of recombinant bacteria

[0064] LB medium: 10 g peptone / L, 5 g yeast extract / L, 10 g sodium chloride / L, solid medium with 1.5% agar powder added. Lysis buffer: 25 mM Tris-HCl, 200 mM NaCl, 10% glycerol, pH 8.0.

[0065] Single clones were picked from the plate and placed in 4 mL of... The ampicillin was cultured overnight at 37°C and 220 rpm in LB broth to obtain the primary seed culture. The next day, the seed culture in the test tubes was inoculated at a ratio of 1:100 into 50 mL of LB broth and... In a shake flask containing ampicillin, the cells were cultured at 37 °C and 200 rpm until the logarithmic growth phase. Then, 0.1 mM isopropyl-β-D-thiogalactoside (IPTG) was added, and the cells were induced overnight at 16 °C to express APL. The cells were collected by centrifugation at 3000-6000 rpm for 15 minutes, and an appropriate amount of Lysis buffer was added to adjust the OD... 600 The bacterial cells were ultrasonically disrupted for 15 minutes at 30% power, with a working time of 1 second and an interval of 3 seconds. The disrupted cells were then centrifuged at 12,000 rpm for 1 hour. The supernatant and precipitate samples were collected separately, and APL protein expression was detected by SDS-PAGE.

[0066] Example 3: Purification of recombinant protein APL

[0067] The APL protein was purified using Ni-NTA Sepharose 6FF (Sangon Biotech), and the steps are as follows:

[0068] Add 1 mL of Ni beads (Roche Diagnostics Shanghai Co., Ltd.) to a gravity column. First, wash the Ni beads with 10 column volumes of ddH2O, then equilibrate them with 50 column volumes of lysis buffer. Mix the supernatant from the previous high-speed centrifugation with the Ni beads in a 50 mL centrifuge tube and place it in a 4°C rotary mixer in a chromatography cabinet. Rotate at low speed for 2 hours to allow the His-tagged target protein to bind to the Ni beads. After 2 hours, pour the liquid into the gravity column. At this point, the liquid flowing through the gravity column contains unbound contaminating proteins. Continue washing the Ni beads with approximately 500 mL of lysis buffer to further remove contaminating proteins. Continuously monitor the residual protein in the flow-through using Bradford assay until no obvious blue color is observed after adding 100 μL of Bradford assay to 100 μL of flow-through. Near the end of the washing process, add lysis buffer containing 250 mM imidazole to the gravity column to elute the target protein, and collect the flow-through. This is the target protein. The theoretical size of APL is 45 kDa, and the SDS-PAGE results are as follows: Figure 6 As shown, the display is consistent with the expected size.

[0069] Example 4: Determination of enzyme thermostability

[0070] I. Thermal stability determination

[0071] The purified wild-type APL and the nine mutants predicted by PROSS (design 1-9) were incubated at 60℃-80℃ for different times, and then immediately placed on ice to measure the residual enzyme activity.

[0072] Table 1: Comparison of thermal stability between wild-type APL and PROSS predicted mutants

[0073]

[0074] The optimal temperature of APL (design 9) has been increased to 55°C, and its thermal stability has been significantly enhanced. The half-life at 70°C and 80°C has been extended to 1.4 times and 2.1 times that of the wild type, respectively.

[0075] Application Example 1: Prevention and treatment of Staphylococcus aureus infection

[0076] The purified APL (design 9) protein from Example 3 was diluted to 0.5 mg / mL with sterile phosphate-buffered saline (PBS, pH 7.4) for in vitro antibacterial experiments or therapeutic studies in animal infection models. Experimental results showed that APL (design 9) maintained high antibacterial activity at 55°C, making it suitable for medical device disinfection and food surface sterilization in high-temperature processing environments. This endolysin mutant can be applied to the control of Staphylococcus aureus contamination in hospitals, food processing plants, and other locations through spraying, coating, or immersion.

[0077] Application Example 2: Development of Alternatives to Veterinary Antibiotics

[0078] APL (design 9) is used as the active ingredient and mixed with suitable pharmaceutically acceptable excipients (such as mannitol, trehalose, gelatin, etc.) to prepare lyophilized powder injections or topical ointments for the treatment of animal diseases such as mastitis in dairy cows and skin infections in pets caused by Staphylococcus aureus. Due to its high thermal stability, this formulation can be transported and stored at room temperature, reducing cold chain costs and offering significant advantages for industrialization.

[0079] Application Example 3: Functional food additives or preservatives

[0080] APL (design 9) can be added to dairy products, meat products, or ready-to-eat foods in appropriate proportions as a natural antibacterial ingredient to inhibit the growth of Staphylococcus aureus and extend the shelf life of the food. It remains active at 70°C and is suitable for heat treatment processes such as pasteurization, exhibiting good processing adaptability.

[0081] Application Example 4: Compound application of veterinary drugs or disinfectant products

[0082] When APL (design 9) is used in combination with other antimicrobial peptides or traditional antibiotics (such as vancomycin and gentamicin), in vitro experiments show a synergistic antibacterial effect, which can reduce the dosage of single antibiotics and slow down the development of drug resistance. This compound formulation can be used to prepare novel veterinary drugs, disinfectant sprays, or medical dressing coatings.

[0083] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A phage-derived Staphylococcus aureus antimicrobial peptidase lysozyme mutant with high thermal stability, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID NO:

11.

2. A nucleic acid molecule encoding the mutant of claim 1.

3. A recombinant expression vector comprising the nucleic acid molecule of claim 2, wherein the vector is pET21a(+) and carries a histidine tag.

4. A host cell, which is a prokaryotic cell transferred into the vector of claim 3.

5. The host cell as described in claim 4, characterized in that, The host cell was Escherichia coli BL21(DE3).

6. A method for preparing the mutant of claim 1, comprising the following steps: (1) Synthesize the mutant gene and clone it into the expression vector; (2) Transform the vector into host cells to induce expression; (3) Disrupt cells and purify proteins using Ni-NTA affinity chromatography.

7. The method as described in claim 6, characterized in that, Expression was induced overnight at 16°C using 0.1 mM IPTG, and purification was performed by elution with buffer containing 250 mM imidazole.

8. The use of the mutant of claim 1 in the preparation of antibacterial drugs, food preservatives or disinfectants, for inhibiting or killing Staphylococcus aureus.

9. The application as described in claim 8, characterized in that, The application includes use in combination with vancomycin or gentamicin to synergistically enhance the antibacterial effect.

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