Endolysin sw16, its engineering and use

Abstract

The application discloses an endolysin SW16 and engineering and application thereof, and belongs to the field of genetic engineering. A new phage endolysin is mined from sewage metagenomic sequencing data, and a new endolysin SW16 is found. The new endolysin after engineering can efficiently lyse pseudomonas aeruginosa. The discovery of the new endolysin not only has important theoretical research significance, but also provides a new selection for clinical treatment of multi-drug resistant bacteria.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and relates to endosomalin SW16, its engineering modification, and applications. More specifically, this invention relates to an endosomal protein containing a target, and to the enzymatic activity verification of a mined natural endosomalin by expressing the endosomalin. Furthermore, this invention also relates to a method for inhibiting the growth of *Pseudomonas aeruginosa* using an engineered natural endosomalin. Background Technology

[0002] Currently, the global overuse and abuse of antibiotics has led to the emergence and spread of increasingly new drug-resistant bacteria, posing a significant threat to public health. [1] Statistics show that infections caused by multidrug-resistant (MDR) pathogens claim approximately 700,000 lives worldwide each year, and this number is expected to increase in the coming years. [2] Therefore, in the fight against infection, the development of innovative antibacterial drugs is imperative. [3] According to statistics from the US NIH (https: / / clinicaltrials.gov / ) and the WHO International Clinical Trials Platform (ICTRP) clinical trial database (https: / / www.who.int / clinical-trials-registry-platform), there are currently 80 antimicrobial drugs undergoing clinical trials, including 46 antimicrobial drugs and 34 non-antimicrobial drugs, encompassing antibodies, microbiome modulators, immunomodulators, phages, and phage endolysins. Phage endolysins (lysins) are peptidoglycan (PG) degrading enzymes encoded by phages. They lyse the host bacterial cell membrane at the end of the phage cycle and have become a promising new class of antimicrobials. Among these unconventional treatment options, phage endolysins possess the following key advantages. [4] (1) Specificity and effective activity: It can specifically kill multidrug-resistant bacteria without disrupting the normal human microecological community. (2) Low risk of drug resistance: Due to its reliance on a relatively conserved peptidoglycan structure, endolysins have a low probability of developing drug resistance. (3) Synergistic effect: When used in combination with other endolysins or antibiotics, endolysins can produce a synergistic effect on pathogenic bacteria that colonize mucosal surfaces and form biofilms. They are particularly effective against Gram-positive bacteria. [5] To date, three drugs related to phage lysins have entered clinical trials for Gram-positive bacteria, including Staphylococcus aureus and Clostridium difficile. [6-8]:SAL200, CF-301, and LMN-201. Conversely, the development of lysozyme inhibitors targeting Gram-negative bacteria has lagged behind. The outer membrane (OM) of the cell wall of Gram-negative bacteria forms a barrier to lysozyme entry and degradation of the PG layer, rendering lysozyme therapy ineffective against Gram-negative bacteria. [9] To overcome this obstacle, researchers have used feasible methods to facilitate the penetration of endosomal into the outer cell membrane, including co-administration with chemical agents called outer membrane permeabilizers (OMPs), which can disrupt the outer cell membrane, such as ethylenediaminetetraacetic acid (EDTA) or citric acid, or modification of endosomal through protein engineering, including fusing natural endosomal with membrane-penetrating peptides. This could be a promising strategy against Gram-negative bacteria.

[10] .

[0003] To expand the potential pool of endosomal candidates for clinical application, the field still needs to discover more effective endosomal drugs. 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 an endocytoxin SW16.

[0005] Another object of the present invention is to provide an engineered modified endosomalin KWK-SW16.

[0006] Another object of the present invention is to provide applications of the above-mentioned SW16 or KWK-SW16.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] An endolysin SW16, the amino acid sequence of which is shown in SEQ ID NO: 1, or a similar sequence of which is obtained by substitution, insertion or deletion of one or more amino acids but still has the same or similar function, or a sequence that is more than 95% similar to the amino acid sequence of SEQ ID NO: 1 and still has the same or similar function.

[0009] The aforementioned biomaterials related to endolysin SW16 are any one or more combinations of the following biomaterials:

[0010] (1) A nucleic acid molecule encoding the endolysin SW16;

[0011] (2) An expression cassette containing the nucleic acid molecules described in (1);

[0012] (3) A recombinant expression vector containing the nucleic acid molecules described in (1);

[0013] (4) A recombinant expression vector containing the expression cassette described in (2);

[0014] (5) Recombinant microorganisms containing the nucleic acid molecules described in (1);

[0015] (6) Recombinant microorganisms containing the expression cassette described in (2);

[0016] (7) Recombinant microorganisms containing the recombinant expression vector described in (3) or (4).

[0017] Furthermore, the nucleic acid molecule described in (1) is the gene sequence encoding endosomalin SW16, as shown in SEQ ID NO: 3, or a similar sequence as shown in SEQ ID NO: 3 that still has the same or similar function obtained by base insertion, deletion, or substitution.

[0018] Furthermore, the starting vector for the recombinant expression vector described in (3) and (4) is a pET series vector, etc.; preferably a pET32a(+) vector.

[0019] Furthermore, the host microorganisms corresponding to the recombinant microorganisms mentioned in (5), (6), and (7) are selected from prokaryotes, yeast, or higher eukaryotic cells; the prokaryotes include bacteria of the genera *Escherichia*, *Bacillus*, *Salmonella*, *Pseudomonas*, and *Streptomyces*. More specifically, the prokaryotes are *Escherichia*, preferably *Escherichia coli*, specifically *Escherichia coli* BL21(DE3).

[0020] An engineered endolysin KWK-SW16, the amino acid sequence of which is shown in SEQ ID NO: 2, or a similar sequence as shown in SEQ ID NO: 2 obtained by substitution, insertion or deletion of one or more amino acids, still having the same or similar function, or a sequence that has more than 95% similarity to the amino acid sequence shown in SEQ ID NO: 2 and still has the same or similar function.

[0021] The biomaterials related to the above-mentioned engineering modification of endosomalin KWK-SW16 are any one or more combinations of the following biomaterials:

[0022] (a) The nucleic acid molecule encoding the engineered endolysin KWK-SW16;

[0023] (b) An expression cassette containing the nucleic acid molecules described in (a);

[0024] (c) A recombinant expression vector containing the nucleic acid molecules described in (a);

[0025] (d) A recombinant expression vector containing the expression cassette described in (b);

[0026] (e) Recombinant microorganisms containing the nucleic acid molecules described in (a);

[0027] (f) Recombinant microorganisms containing the expression cassette described in (b);

[0028] (g) Recombinant microorganisms containing the recombinant expression vector described in (c) or (d).

[0029] Furthermore, the nucleic acid molecule described in (a) is a gene sequence encoding an engineered endosomalin KWK-SW16, as shown in SEQ ID NO: 4, or a similar sequence as shown in SEQ ID NO: 4 that still has the same or similar function obtained by base insertion, deletion, or substitution.

[0030] Furthermore, the starting vector for the recombinant expression vector described in (c) and (d) is a pET series vector, etc.; preferably, it is a pET32a(+) vector.

[0031] Furthermore, the host microorganisms corresponding to the recombinant microorganisms described in (e), (f), and (g) are selected from prokaryotes, yeast, or higher eukaryotic cells; the prokaryotes include bacteria of the genera *Escherichia*, *Bacillus*, *Salmonella*, *Pseudomonas*, and *Streptomyces*. More specifically, the prokaryotes are *Escherichia*, preferably *Escherichia coli*, specifically *Escherichia coli* BL21(DE3).

[0032] The application of the above-mentioned biomaterials related to endolysin SW16 or the biomaterials related to engineered endolysin KWK-SW16 in the preparation of endolysin SW16 or engineered endolysin KWK-SW16.

[0033] A method for preparing endolysin SW16 or engineered endolysin KWK-SW16 includes the following steps: culturing recombinant microorganisms to obtain endolysin SW16 or engineered endolysin KWK-SW16 from the recombinant microorganisms.

[0034] Furthermore, a method for preparing engineered endolysin KWK-SW16 includes the following steps:

[0035] Using genetic engineering methods, a short peptide was linked to the N-terminus of endolysin SW16 to form engineered endolysin KWK-SW16. The gene sequence was transformed into a host microorganism to obtain a recombinant microorganism, which was then cultured to prepare engineered endolysin KWK-SW16.

[0036] The applications of the aforementioned endolysin SW16 and its biomaterials, or engineered endolysin KWK-SW16 and its biomaterials, are any one or more combinations of the following applications:

[0037] ① Application in the preparation of products resistant to Gram-negative bacteria;

[0038] ② Application in the preparation of products for treating Gram-negative bacterial infections;

[0039] ③ Application in the preparation of products resistant to Gram-positive bacteria;

[0040] ④ Application in the preparation of products for treating Gram-positive bacterial infections.

[0041] Furthermore, the Gram-negative bacteria mentioned in ① and ② include, but are not limited to, at least one of Pseudomonas aeruginosa, Acinetobacter baumannii, and Salmonella typhimurium.

[0042] Furthermore, the Gram-positive bacteria mentioned in ③ and ④ include, but are not limited to, Listeria monocytogenes.

[0043] In one embodiment, the engineered endosomalin KWK-SW16 of the present invention can lyse Pseudomonas aeruginosa, and its ability to lyse Pseudomonas aeruginosa is significantly improved compared with the control.

[0044] The present invention has the following advantages and effects compared with the prior art:

[0045] This invention mines novel phage endolysins from wastewater metagenomic sequencing data, discovering a novel endolysin, SW16. This engineered novel endolysin can efficiently lyse *Pseudomonas aeruginosa*. The discovery of this novel endolysin not only has significant theoretical research value but also provides a new option for the clinical treatment of multidrug-resistant bacteria. Attached Figure Description

[0046] Figure 1 The expression and purification strategy based on endolysin candidate proteins and the structure of the pET32a-POI-His-tag expression vector used are shown; where POI is SW16.

[0047] Figure 2The image shows the SDS-PAGE analysis results of the expression and purification of the SW16 endosomal candidate protein using the His-tag method. A: SDS-PAGE analysis results of the lysate supernatant (ES) and lysate precipitate (EP) after expression; B: SDS-PAGE analysis results of the purified SW16 endosomal protein after ES purification using a nickel column, with BSA values ​​of 0.03125, 0.0625, 0.25, and 0.5 mg / mL for BSA 1-4, respectively.

[0048] Figure 3 The diagram shows the saturation curve results of endosomalase activity detection analysis and endosomal candidate proteins.

[0049] Figure 4 The image shows the SDS-PAGE analysis results of the expression and purification of KWK-SW16 engineered endosomal protein using the His-tag method. A: SDS-PAGE analysis results of the lysate supernatant (ES) and lysate precipitate (EP) after expression. B: SDS-PAGE analysis results of the purified KWK-SW16 engineered endosomal protein after nickel column purification, with BSA values ​​of 0.03125, 0.0625, 0.25, and 0.5 mg / mL for BSA 1-4, respectively.

[0050] Figure 5 The graph shows the results of the minimum inhibitory concentration (MIC) determination of the KWK-SW16 engineered endosomal candidate protein.

[0051] Figure 6 The activity verification of KWK-SW16 engineered endosomal is shown; where A: the effect of concentration on engineered endosomal; B: the effect of temperature on engineered endosomal; C: the effect of pH on engineered endosomal.

[0052] Figure 7 The fragmentation spectrum of KWK-SW16 engineered endosomal is shown. Detailed Implementation

[0053] 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.

[0054] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0055] Example

[0056] To make the technical solutions and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below. It should be understood that the embodiments should not be construed as limiting, and those skilled in the art can make further adjustments to the embodiments based on the principles of the present invention.

[0057] The strains *Escherichia coli* DH5α and BL21(DE3) were obtained from TransGen Biotech, Beijing. DNA sequencing was performed by Sangon Biotech, Guangzhou, China. Restriction endonucleases and DNA polymerases were purchased from New England Biolabs (Beverly, USA). DNA purification, gel recovery, genomic DNA extraction, and plasmid amplification kits were purchased from Tiangen Biotech, Ltd. *Pseudomonas aeruginosa* PAO1 was a gift from Professor Hu Fuquan of the Third Military Medical University. Oligonucleotides used for cloning were synthesized by Sangon Biotech, Guangzhou, China.

[0058] Example 1. Obtaining the gene encoding endolysin and analyzing its protein sequence.

[0059] We performed data mining on wastewater metagenomic sequencing data. The wastewater (SW) dataset was obtained from the EBI database (https: / / www.ebi.ac.uk / ena / ), metagenomic project number ERP015409. We ultimately identified one endolysin candidate protein, designated SW16. The amino acid sequence of SW16 endolysin is shown in SEQ ID NO: 1.

[0060] The phage in vivo lysozyme protein sequences showed an endolysozyme similarity of 60-90%. This study further annotated the sequences with domains using Interproscan software and the Pfam database. Specific sequence information is shown in Table 1.

[0061] Table 1. Sequence information of endorphin SW16 in this embodiment.

[0062]

[0063] Example 2: Construction of an endosomal protein expression vector

[0064] The SW16 endosomalin gene in this embodiment was synthesized by Ribobio Biotechnology Co., Ltd. (Guangzhou, China). Its DNA sequence was codon-optimized in Escherichia coli, and the codon-optimized nucleotide sequence of E. coli is shown in SEQ ID NO: 3.

[0065] To construct the expression vector pET32a-POI-His-tag (where POI is SW16), the sequence between the NdeⅠ and XhoⅠ restriction enzyme sites of the pET32a(+) vector was replaced with the aforementioned SW16 endosomalin gene (a histidine tag (His-tag) was added to the 3' end of the SW16 endosomalin gene to facilitate the purification of the target protein), thus constructing the recombinant expression vector pET32a-SW16-His-tag. This was completed by Ribobio Biotechnology Co., Ltd. (Guangzhou, China). The plasmid construction diagram can be found in [link to diagram]. Figure 1 .

[0066] Example 3: Expression and purification of endosomal protein

[0067] Test bacteria: recombinant bacteria BL21(DE3)-pET32a-SW16-His-tag and blank control bacteria BL21(DE3)-pET32a. The recombinant bacteria were prepared by transforming the recombinant expression vector pET32a-SW16-His-tag from Example 2 into *Escherichia coli* BL21(DE3); the blank control bacteria were prepared by transforming the pET32a(+) vector into *Escherichia coli* BL21(DE3).

[0068] 1. Inoculate a single colony of the *E. coli* to be tested into LB liquid medium containing 100 μg / mL ampicillin resistance and incubate overnight (12-16 h) at 37°C with shaking at 220 rpm. Transfer the overnight culture 1:50 to LB liquid medium containing 100 μg / mL ampicillin resistance and incubate at 37°C with shaking at 220 rpm until OD (dose eluent) is reached. 600 When the value reaches 0.6-0.8, pre-cool the bacterial culture and shaker to 16℃ for 30 min, add IPTG to a final concentration of 0.2 mM for induction expression, induce expression at 16℃ and 220 rpm for 24 h, and collect the bacterial cells by centrifugation at 4℃ and 4,000 rpm for 20 min.

[0069] 2. After completing step 1, disrupt the bacterial cells: resuspend the collected bacterial cells in Binding Buffer to 20 OD. 600 Cells were disrupted using an ultrasonic disruptor;

[0070] 3. Purification of endosomal proteins:

[0071] (1) Nickel column purification method: After ultrasonic disruption, centrifuge at 15,000g for 30 min at 4℃ to separate the lysis supernatant (ES) and lysis precipitate (EP). ES and EP samples were prepared for SDS-PAGE detection. SDS-PAGE results are shown below. Figure 2 As shown in A. From Figure 2As shown in Table A, comparison with the pET32a(+) empty vector expression band indicates that SW16 protein can be expressed and is mostly expressed in the supernatant (ES), with the band size consistent with the theoretical molecular mass of 17.8 kDa (see Table 4). The ES was filtered through a 0.22 μm filter and added to a HisPurNi-NTA centrifuge column. The column was incubated on a shaker at 4°C and 100 rpm for 30 min to ensure specific binding of the target protein to the column. The column was then centrifuged at 4°C and 700 g for 2 min, and the flow-through was collected. A gradient elution method was then used, sequentially increasing the imidazole concentration with 10%, 30%, 50%, 80%, and 100% elution buffer, and collecting the eluent at each concentration.

[0072] 4. Concentration and dialysis: After purification, the protein was concentrated using a 10kDa ultrafiltration tube to a small volume (1-2mL). Then, it was dialyzed using a 10kDa dialysis bag. The buffer was replaced with 10mM PBS (pH 7.4), and the protein was dialyzed overnight at 4°C. After dialysis, a small amount of sample was collected for sample preparation.

[0073] 5. All samples were analyzed by SDS-PAGE. ImageJ was used for protein grayscale calculation. A BSA protein standard concentration sample was used as a reference to construct a BSA standard protein concentration curve to determine the concentration of the obtained candidate endolysins. The SDS-PAGE results are shown below. Figure 2 As shown in B. From Figure 2 B indicates that the SW16 protein can be successfully purified using a nickel column purification method with a purity of 99%.

[0074] Example 4: Detection of Endolysin Protein Activity

[0075] The method for preparing the Pseudomonas aeruginosa outer membrane permeability cell substrate used in this embodiment is based on the method described by Yoyeon and Lim et al. [11,12] Method. The positive control used in this example was egg white lysozyme (HEWL, Sigma-Aldrich, L6876, CAS: 12650-88-3).

[0076] 1. Single colonies of *Pseudomonas aeruginosa* strain PAO1 were cultured overnight (12-16 h) at 37°C with shaking at 220 rpm in antibiotic-free MHB liquid medium. The overnight culture was then transferred 1:50 to fresh antibiotic-free MHB liquid medium and grown to the logarithmic growth phase, i.e., OD100. 600Centrifuge at 0.5-0.6°C, 4°C, 4,000g for 15 min. After centrifugation, collect the cells and wash once with 20 mM Tris-HCl (pH 8.0). Incubate at 25°C for 5 min with a buffer containing 20 mM Tris-HCl (pH 8.0) and 100 mM EDTA. Finally, wash three times with 20 mM Tris-HCl, centrifuge to remove residual EDTA, and resuspend the cell pellet in protein buffer and 10 mM PBS (pH 7.4) to OD. 600 The concentration was 0.8-1.2, and the substrate was placed on ice. The preparation of the cell substrate with permeable outer membrane was completed.

[0077] 2. Add 20 μL of purified 10 μg / mL endosomal protein to a 96-well plate. Add 180 μL of outer membrane permeabilized cell substrate to each well. Incubate at 25°C. The blank control is 20 μL of 10 mM PBS (pH 7.4), and the positive control is 20 μL. Place the plate in a preheated microplate reader and continuously measure the OD. 600 The experiment lasted 30 minutes, and all experiments were repeated three times independently.

[0078] 3. Quantification of endolysin activity: Add 20 μL of candidate proteins at different dilutions (the specific concentration gradient should be adjusted according to the activity of the candidate endolysins) to a 96-well plate. Add 180 μL of outer membrane permeable cell substrate to each protein, and incubate at 25°C. Use 20 μL of 10 mM PBS (pH 7.4) as a blank control. In a multi-functional microplate reader, OD is continuously measured. 600 The total duration was 30 minutes. All experiments were repeated independently three times.

[0079] 4. Enzyme activity calculations were based on data from Briers et al.

[13] The saturation curves of endosomal proteins at different concentrations were calculated using the method described above. Figure 3 The enzyme activity was finally calculated in units / mg. The experiment showed that the enzyme activity of SW16 was 9.7 times that of the positive control (HEWL), as shown in Table 2.

[0080] Table 2. Results of endolysin protein activity detection in this embodiment.

[0081]

[0082] Example 5: Engineering Modification of Endolysin Protein

[0083] This embodiment involves an engineering modification experiment of SW16 endosomalin.

[0084] KWK (KWKLFKKI) short peptide is derived from the first 8 amino acids of cecropin A (CepA).

[14] According to reports, KWK's antibacterial activity is negligible.

[14] KWK short peptides were synthesized by Sangon Biotech (Ningbo, China) using the Fmoc solid-phase chemical method.

[0085] 1. Construction of an engineered endosomal plasmid: An expression plasmid for the engineered endosomal protein SW16 was constructed using the pET32a-KWK-AG Linker-SW16-His-tag expression vector. The KWK peptide was fused to the N-terminus of the endosomal sequence via PCR (the amino acid sequence of KWK-SW16 is shown in SEQ ID NO: 2, and the nucleotide sequence is shown in SEQ ID NO: 4). Two primers containing the peptide coding sequence were synthesized by Shanghai Sangon Biotech Co., Ltd., and the oligonucleotide primers are shown in Table 3. Two target protein DNA fragments, AmpR and KWK-lacI, were amplified from the plasmid pET32a-SW16-His-tag constructed in Example 2 and assembled using the Gibson assembly method.

[0086] 2. Expression and purification: The nickel column purification method described in Example 3 was followed. The purification results of natural and engineered endosomalin are shown in Table 4. Referring to Example 3, the SDS-PAGE detection results are as follows: Figure 4 As shown. From Figure 4 This indicates that the KWK-SW16 protein can be expressed, and the band size is consistent with the theoretical molecular mass of 19.2 kDa (see Table 4).

[0087] Table 3 Primer information for the vector used in this embodiment.

[0088]

[0089] Note: The bold underlined part is the KWK sequence, and the underlined part is the AG Linker sequence.

[0090] Table 4. Statistics on the purification results of natural and engineered endolysins used in this embodiment.

[0091]

[0092] Example 6: Antibacterial Activity Test of Engineering Modified Endolysin

[0093] In this embodiment, the engineered KWK-SW16 strain from Example 5 was selected for bacterial inhibition experiments. Pseudomonas aeruginosa PAO1 was used as the test strain in this study.

[0094] Pseudomonas aeruginosa PAO1 strain was streaked onto antibiotic-free MHB plates for resuscitation. Single colonies were picked and incubated overnight (12-16 h) at 37°C with shaking at 220 rpm in antibiotic-free liquid medium. The overnight culture was then transferred 1:50 to fresh antibiotic-free liquid medium and grown to the logarithmic growth phase (OD2). 600 =0.5-0.6), centrifuged at 4℃, 4,000g for 15 min, and the culture medium was discarded. The cells were washed once with 10mM PBS (pH 7.4) and resuspended to OD. 600 The concentration was approximately 0.5, so the test bacterial suspension was diluted to 1×10⁻⁵. 5 CFU / mL. Add 100 μL of different concentrations of endolysin to 100 μL of test bacterial suspension, for a total volume of 200 μL. The bacterial control consisted of 100 μL of PBS buffer and 100 μL of test bacterial suspension, while the blank control was 200 μL of PBS buffer. Incubate the mixture at 37°C for 1 h. Spread 100 μL of the mixture evenly onto a plate and incubate overnight at 37°C. Record the colony count. All experiments were independently repeated three times.

[0095] The endolysin was incubated with logarithmic-phase PAO1 cells as described above, and colony counts were performed using the plating method to further characterize the antibacterial activity of the endolysin. Figure 5 As shown, engineered endosomalin exhibits higher antibacterial activity than the unmodified natural endosomalin, and this activity increases with increasing concentration. In the SW16 series, engineered endosomalin demonstrates even higher antibacterial activity at high concentrations.

[0096] Example 7: Enzymatic Characterization of Engineering-Modified Endolysin

[0097] 1. Effect of concentration on endolysin: Pseudomonas aeruginosa PAO1 cells in the logarithmic growth phase were collected (4℃, 4,000g, 15min), washed once with PBS (pH 7.4), and resuspended at OD. 600 The concentration was approximately 0.5, so the test bacterial suspension was diluted to 1×10⁻⁵. 5 CFU / mL; 100 μL of KWK-SW16 with final concentrations ranging from 1 to 128 μg / mL was added to 100 μL of the test bacterial suspension (total volume 200 μL), and incubated at 37°C for 1 h. 100 μL of the mixture was then evenly spread onto a plate and incubated overnight at 37°C. The colony count was recorded. All experiments were independently replicated three times. IC50 was calculated using https: / / www.aatbio.com / tools / ic50-calculator. The calculated IC50 for the engineered endolysin KWK-SW16 was 34.6 μg / mL. Figure 6 As shown in Figure A.

[0098] 2. Effect of temperature on endolysins: Pseudomonas aeruginosa PAO1 cells in the logarithmic growth phase were collected (4℃, 4,000g, 15min), washed once with PBS (pH 7.4), and resuspended at OD. 600 The concentration was approximately 0.5, so the test bacterial suspension was diluted to 1×10⁻⁵. 5 CFU / mL; 100 μL of KWK-SW16, with a final concentration of 100 μg / mL, was incubated for 30 min at different temperatures (4-70℃). Then, it was added to 100 μL of the test bacterial suspension (total volume 200 μL) and incubated at 37℃ for 1 h. The relative activity of each treatment group (4-70℃) relative to the control group (37℃) was determined, and all treatments were performed in triplicate. The results of the effect of different temperatures on the antibacterial activity of KWK-SW16 are as follows: Figure 6 As shown in B. From Figure 6 B indicates that the optimal temperature range for KWK-SW16 protein is 4-42℃, with enzyme activity exceeding 80%. Furthermore, it maintains over 60% antibacterial activity within the 4-70℃ temperature range, demonstrating the stability of KWK-SW16 protein at different temperatures.

[0099] 3. Effect of pH on endolysins: Pseudomonas aeruginosa PAO1 cells in the logarithmic growth phase were collected (4℃, 4,000g, 15min), washed once with PBS (pH 7.4), and resuspended at OD. 600 The concentration was approximately 0.5, so the test bacterial suspension was diluted to 1×10⁻⁵. 5 CFU / mL; 100 μL of KWK-SW16 to a final concentration of 100 μg / mL was added to 100 μL of bacterial suspension at different pH values ​​(total volume 200 μL), and incubated at 37℃ for 1 h. The relative activity of each treatment group (pH 4-10) to the control group (pH 7.4) was determined, and all experiments were independently repeated three times. The results of the effect of different pH values ​​on the antibacterial activity of KWK-SW16 are as follows. Figure 6 As shown in C. From Figure 6 C indicates that the KWK-SW16 protein exhibits an antibacterial rate of over 80% at pH 4-10, demonstrating the stability of the KWK-SW16 protein at different pH values.

[0100] Example 8: Determination of the fragmentation spectrum of endosomalin in engineering modification

[0101] All test strains used in this embodiment were obtained from our laboratory. A total of 10 Gram-negative bacteria and 3 Gram-positive bacteria were tested, as shown in Table 5. Among them, 3 strains of *Pseudomonas aeruginosa* (PALWL1.001, PALWL1.002, and PALWL1.003) were isolated and cultured in our laboratory, as described in the references.

[15] It is publicly available in China.

[0102] Table 5. Test strains used in this example.

[0103]

[0104] We used the engineered high-activity endolysin candidate KWK-SW16 from Example 5 as the research object, and conducted preliminary analysis of its domains and three-dimensional structure. KWK-SW16 lysis spectrum determination was performed. 500 μg / mL of KWK-SW16 was mixed with an equal volume of different logarithmic-phase cells and incubated for 1 h. The antibacterial activity of the endolysin was evaluated by counting the number of residual colonies after treatment. The IC50 of KWK-SW16 against PAO1 was calculated using https: / / www.aatbio.com / tools / ic50-calculator / , and was 34.6 μg / mL. Furthermore, the lysis spectrum of KWK-SW16 was tested... Figure 7 As shown, KWK-SW16 exhibited lytic activity against all tested Gram-negative bacterial strains (10 / 10). KWK-SW16 also showed high antibacterial activity against Gram-positive bacteria (Listeria monocytogenes CICC 21634). It holds promise for the treatment of drug-resistant infections caused by Pseudomonas aeruginosa, Acinetobacter spp., Salmonella typhimurium, and Listeria monocytogenes.

[0105] References:

[0106] [1]ANTIMICROBIAL RESISTANCE C.Global burden of bacterialantimicrobial resistance in 2019:a systematic analysis[J].Lancet,2022,399(10325):629-655.

[0107] [2]GHOSH C,SARKAR P,ISSA R,et al.Alternatives to ConventionalAntibiotics in the Era of Antimicrobial Resistance[J].Trends Microbiol,2019,27(4):323-38.

[0108] [3]BUTLER M S,GIGANTE V,SATI H,et al.Analysis of the ClinicalPipeline of Treatments for Drug-Resistant Bacterial Infections:DespiteProgress,More Action Is Needed[J].Antimicrob Agents Chemother,2022,66(3):e0199121.

[0109] [4]VAZQUEZ R,BLANCO-GANAN S,RUIZ S,et al.Mining of Gram-NegativeSurface-Active Enzybiotic Candidates by Sequence-Based Calculation ofPhysicochemical Properties[J].Front Microbiol,2021,12:660403.

[0110] [5]GRISHIN A V,KARYAGINA A S,VASINA D V,et al.Resistance topeptidoglycan-degrading enzymes[J].Critical Reviews in Microbiology,2020,46(6):703-726.

[0111] [6]ALSHRARI A S,HUDU S A,ELMIGDADI F,et al.The Urgent Threat ofClostridioides difficile Infection:A Glimpse of the Drugs of the Future,withRelated Patents and Prospects[J].Biomedicines,2023,11(2):426.

[0112] [7]SCHUCH R,KHAN B K,RAZ A,et al.Bacteriophage Lysin CF-301,a PotentAntistaphylococcal Biofilm Agent [J].Antimicrob Agents Chemother,2017,61(7):10-1128.

[0113] [8]HO M K Y,ZHANG P,CHEN X,et al.Bacteriophage endolysins againstgram-positive bacteria,an overview on the clinical development and recentadvances on the delivery and formulation strategies[J].Crit Rev Microbiol,2021,1-24.

[0114] [9]VAZQUEZ R,SEOANE-BLANCO M,RIVERO-BUCETA V,et al.MonomodularPseudomonas aeruginosa phage JG004 lysozyme(Pae87)contains a bacterialsurface-active antimicrobial peptide-like region and a possible substrate-binding subdomain[J].Acta Crystallogr D Struct Biol,2022,78(Pt 4):435-454.

[0115]

[10] GERSTMANS H,GRIMON D,GUTIERREZ D,et al.A VersaTile-drivenplatform for rapid hit-to-lead development of engineered lysins[J].Sci Adv,2020,6(23):eaaz1136.

[0116]

[11] CHA Y,SON B,RYU S.Effective removal of staphylococcal biofilms onvarious food contact surfaces by Staphylococcus aureus phage endolysinLysCSA13[J].Food Microbiol,2019,84:103245.

[0117]

[12] LIM J A,SHIN H,KANG D H,et al.Characterization of endolysin froma Salmonella Typhimurium-infecting bacteriophage SPN1S[J].Res Microbiol,2012,163(3):233-241.

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[13] BRIERS Y,LAVIGNE R,VOLCKAERT G,et al.A standardized approach foraccurate quantification of murein hydrolase activity in high-throughputassays[J].J Biochem Biophys Methods,2007,70(3):531-533.

[0119]

[14] CHEN X,LIU M,ZHANG P,et al.Membrane-Permeable AntibacterialEnzyme against Multidrug-Resistant Acinetobacter baumannii[J].ACS Infect Dis,2021,7(8):2192-204.

[0120]

[15] GUO Y,CHEN P,LIN Z,et al.Characterization of Two Pseudomonasaeruginosa Viruses vB_PaeM_SCUT-S1 and vB_PaeM_SCUT-S2[J].Viruses,2019,11(4):318.

[0121] 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. An endolysin SW16 or an engineered endolysin KWK-SW16, characterized in that: The amino acid sequence of the endolysin SW16 is shown in SEQ ID NO: 1; The amino acid sequence of the engineered endolysin KWK-SW16 is shown in SEQ ID NO:

2.

2. The biomaterials related to endolysin SW16 or engineered endolysin KWK-SW16 as described in claim 1, characterized in that: The biomaterials related to endolysin SW16 are any one or more combinations of the following biomaterials: (1) The nucleic acid molecule encoding the endolysin SW16; (2) An expression cassette containing the nucleic acid molecules described in (1); (3) A recombinant expression vector containing the nucleic acid molecules described in (1); (4) A recombinant expression vector containing the expression cassette described in (2); (5) Recombinant microorganisms containing the nucleic acid molecules described in (1); (6) Recombinant microorganisms containing the expression cassette described in (2); (7) Recombinant microorganisms containing the recombinant expression vector described in (3) or (4); The biomaterials related to the engineering modification of endolysin KWK-SW16 are any one or more combinations of the following biomaterials: (a) The nucleic acid molecule encoding the engineered endosomalin KWK-SW16; (b) An expression cassette containing the nucleic acid molecule described in (a); (c) A recombinant expression vector containing the nucleic acid molecules described in (a); (d) A recombinant expression vector containing the expression cassette described in (b); (e) Recombinant microorganisms containing the nucleic acid molecules described in (a); (f) Recombinant microorganisms containing the expression cassette described in (b); (g) Recombinant microorganisms containing the recombinant expression vector described in (c) or (d).

3. The biomaterial according to claim 2, characterized in that: (1) The nucleic acid molecule described is the gene sequence encoding endosomalin SW16, as shown in SEQ ID NO: 3; The nucleic acid molecule described in (a) is the gene sequence encoding the engineered endosomalin KWK-SW16, as shown in SEQ ID NO:

4.

4. The biomaterial according to claim 2 or 3, characterized in that: The starting vector for the recombinant expression vectors described in (3), (4), (c), and (d) is a pET series vector; The host microorganisms corresponding to the recombinant microorganisms mentioned in (5), (6), (7), (e), (f), and (g) are selected from prokaryotes, yeast, or higher eukaryotic cells; the prokaryotes include Escherichia coli (Escherichia coli). Escherichia ), Bacillus spp. ( Bacillus Salmonella ( Salmonella ), Pseudomonas spp. Pseudomonas ) or Streptomyces ( Streptomyces ) bacteria.

5. The biomaterial according to claim 4, characterized in that: The starting vector for the recombinant expression vectors described in (3), (4), (c), and (d) is the pET32a(+) vector; The host microorganisms corresponding to the recombinant microorganisms mentioned in (5), (6), (7), (e), (f), and (g) are selected from *Escherichia coli* (Escherichia coli). E. coli ).

6. The biomaterial according to claim 5, characterized in that: The host microorganisms corresponding to the recombinant microorganisms mentioned in (5), (6), (7), (e), (f), and (g) are selected from Escherichia coli BL21 (DE3).

7. The use of the biomaterial according to any one of claims 2 to 6 in the preparation of endosomalin SW16 or engineered endosomalin KWK-SW16.

8. A method for preparing endolysin SW16 or engineered endolysin KWK-SW16, characterized in that: The method includes the following steps: culturing the recombinant microorganism as described in any one of claims 2 to 6, and obtaining endolysin SW16 or engineered endolysin KWK-SW16 from the recombinant microorganism.

9. The application of the engineered endosomalin KWK-SW16 according to claim 1, or the biomaterials related to the engineered endosomalin KWK-SW16 according to any one of claims 2 to 6, characterized in that: For any one or more of the following applications: ① In the preparation of anti-Pseudomonas aeruginosa ( Pseudomonas aeruginosa Acinetobacter baumannii ( Acinetobacter baumannii ) and Salmonella typhimurium ( Salmonella typhimurium Application in at least one of the following products; ② Use in the preparation of drugs for treating at least one infection of Pseudomonas aeruginosa, Acinetobacter baumannii and Salmonella typhimurium; ③ In the preparation of anti-Listeria monocytogenes ( Listeria monocytogenes Applications in products; ④ Its application in the preparation of drugs for treating Listeria monocytogenes infection.

10. The use of the endosomalin SW16 of claim 1, or the biomaterial related to endosomalin SW16 of any one of claims 2 to 6, in the preparation of a product containing Pseudomonas aeruginosa that resists outer membrane permeability.

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