Peptide fusion antibiotic enzyme for splitting gram-negative bacteria independent of outer membrane penetrant and application of peptide fusion antibiotic enzyme

Abstract

The invention relates to the technical field of antibiotic enzymes, and provides a peptide fusion antibiotic enzyme, the peptide fusion antibiotic enzyme comprises a catalytic domain LysS23, a cell-penetrating peptide and a connecting peptide, the catalytic domain LysS23 comprises an amino acid sequence represented by SEQ ID NO: 1, the cell-penetrating peptide comprises an amino acid sequence represented by SEQ ID NO: 2, and the connecting peptide comprises an amino acid sequence represented by SEQ ID NO: 3. Or an amino acid sequence which is obtained by substitution / deletion / addition of one or more amino acid residues on the amino acid sequence as shown in SEQ ID NO.1 and has more than 98% of identity is obtained by substitution / deletion / addition of one or more amino acid residues on the amino acid sequence as shown in SEQ ID NO.1. The catalytic domain LysS23 in the peptide fusion antibiotic enzyme has peptidoglycan lysis activity, the cell-penetrating peptide exerts a cell-penetrating function and has an outer membrane destruction effect, the cell-penetrating peptide acts on an extracellular membrane and helps the catalytic domain LysS23 to smoothly reach the peptidoglycan position of a cell wall so as to quickly exert the lysis effect, and the connecting peptide maintains the integrity of the structures and functions of the cell-penetrating peptide and the catalytic domain. Meanwhile, the antibacterial activity of the peptide fusion antibiotic enzyme is ensured. The peptide fusion antibiotic enzyme provided by the invention can directly act on common gram-negative bacteria without depending on the addition of a chemical outer membrane penetrant, exerts a good antibacterial effect, and has a very good application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of anti-enzyme, in particular to a peptide fusion anti-enzyme for lysis of gram-negative bacteria independent of outer membrane permeabilizers and application thereof. BACKGROUND

[0002] The current era is facing a serious situation of antimicrobial resistance (AMR), and it is urgent to explore alternative and sustainable antimicrobial agents, especially the demand for new antimicrobial agents targeting gram-negative bacteria. Enzybiotics, a mixed word of enzyme and antibiotic, was introduced by Dr. Vincent Fischetti's team at Rockefeller University in New York in 2001, which refers to a special antibacterial protein with the function of lysis of peptidoglycan. It is also called peptidoglycan hydrolase (PGH), which was first defined as a bacteriophage lyase with antibacterial ability by degrading bacterial cell wall, and now it is generally also included in bacteriocin, endolysin and lysozyme. Enzybiotics can quickly and specifically lyse peptidoglycan in cell wall to release cell contents and kill bacteria. Enzybiotics protein provides a very promising way for the development of new antimicrobial agents. Enzybiotics has multiple favorable properties, including high efficiency and rapid bactericidal activity, substrate specificity for microorganisms, reducing the possibility of inducing drug resistance, easy protein editing and modification, compatibility with other antimicrobial agents, and antimicrobial activity unaffected in the presence of antibodies. Given these characteristics and advantages, enzybiotics has attracted more and more attention and has great potential to become a powerful weapon against antibiotic resistance.

[0003] While exogenously applied lysobactins have shown antimicrobial potential against Gram-positive pathogens as peptidoglycan is readily accessible, their efficacy against Gram-negative bacteria is affected by the outer membrane barrier. To overcome this challenge, one effective approach is to utilize chemical outer membrane permeabilizers (such as EDTA / organic acids) to facilitate the penetration of lysobactins through the outer membrane to reach peptidoglycan. However, the practical application of this approach is limited. For example, EDTA is not suitable for systemic use in humans or agriculture due to its strong anticoagulant properties and environmental persistence, and thus its application is mainly limited to topical use or food preservation applications. Meanwhile, the differences in the composition of the outer membrane and lipopolysaccharide (LPS) of different bacteria can result in different sensitivities of bacteria to organic acid damage. In order to more effectively solve this problem, protein engineering technology is introduced into the study of lysobactins. Researchers successfully fused peptides with outer membrane penetration function, referred to as cell-penetrating peptides (OMPs), to lysobactins, enabling them to penetrate the outer membrane and reach the peptidoglycan portion, where they cut the peptidoglycan to kill bacteria. While many cell-penetrating peptides exhibit cytotoxicity, studies have shown that this can be due to non-specific damage to mammalian cell membranes caused by the aggregation of cell-penetrating peptide molecules with each other, but fortunately, the cytotoxicity of lysobactins fused with cell-penetrating peptides is significantly reduced, indicating that lysobactins fused with cell-penetrating peptides have good therapeutic potential.

[0004] The antimicrobial activity of lysobactin-peptide fusions is influenced by a variety of factors, such as the length and sequence of the connecting peptide, the fusion position (N- or C-terminal), and the specific OMP and lysobactin used. Therefore, constructing a peptide-fused lysobactin that is active against Gram-negative pathogenic bacteria and does not rely on the addition of outer membrane permeabilizers to exert high antibacterial activity is of great significance in curbing the spread and harm of drug-resistant Gram-negative pathogenic bacteria. SUMMARY

[0005] To overcome the above technical problems, the present application provides a peptide-fused lysobactin that exerts high antibacterial activity against Gram-negative pathogenic bacteria without relying on the addition of outer membrane permeabilizers, in order to improve the limited application of existing lysobactins and meet people's needs.

[0006] In a first aspect, the present application provides a peptide-fused lysobactin, which comprises a catalytic domain LysS23, a cell-penetrating peptide, and a connecting peptide Linker connecting the catalytic domain and the cell-penetrating peptide, wherein the catalytic domain LysS23 comprises the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having 98% or more identity to the amino acid sequence shown in SEQ ID NO. 1 by substitution / deletion / addition of one or more amino acid residues, preferably an amino acid sequence having 98.5% or more, 99% or more, 99.5% or more, 99.8% or more identity.

[0007] In the peptide fusion antibacterial enzyme provided in the present application, the catalytic domain LysS23 is from the lytic enzyme of Salmonella phage vB_SenS_ER23, and the amino acid sequence is as shown below.

[0008] MSNRNISNNGIKFTAAFEGFRGTAYKATKNEKYFTIGYGSYGPHVKEGQKITEGQGLLLLHK DMAKAVAAVDAVAHPSLNQSQFDAVCDLVYNAGAGVIAASTGTGQALRKGDVATLRNKLT QFHYQNGKSLLGLRRRAAGRVALFDGMLWQQAEAVGREAK (SEQ ID NO: 1).

[0009] The catalytic domain LysS23 in the peptide fusion antibacterial enzyme has peptidoglycan cleavage activity, the cell-penetrating peptide has a cell-penetrating function, has an outer membrane damaging effect, and helps the catalytic domain LysS23 to reach the peptidoglycan position of the cell wall while acting on the outer membrane, so as to quickly exert the cleavage effect. The connecting peptide maintains the structural and functional integrity of the cell-penetrating peptide and the catalytic domain, and ensures the antibacterial activity of the peptide fusion antibacterial enzyme. Therefore, the peptide fusion antibacterial enzyme of the present application does not depend on the addition of a chemical outer membrane permeabilizer (such as EDTA / organic acid), directly acts on common gram-negative bacteria, and has good antibacterial effect.

[0010] In some embodiments, the connecting peptide comprises the amino acid sequence shown in SEQ ID NO: 2.

[0011] In the present application, the connecting peptide is preferably the amino acid sequence of GGGGSGGGGS (SEQ ID NO: 2), which can realize the stable connection of the cell-penetrating peptide and the catalytic domain LysS23, and maintain the stability of the tertiary protein structure of the peptide fusion antibacterial enzyme.

[0012] In some embodiments, the cell-penetrating peptide is located at the N-terminus or C-terminus of the catalytic domain.

[0013] In the present application, the structure of the peptide fusion antibacterial enzyme can be cell-penetrating peptide- connecting peptide Linker-catalytic domain LysS23, or catalytic domain LysS23-connecting peptide Linker-cell-penetrating peptide, regardless of whether the cell-penetrating peptide is connected at the N-terminus or C-terminus of the catalytic domain, the peptide fusion antibacterial enzyme can exert very high antibacterial activity without the addition of a chemical outer membrane permeabilizer.

[0014] In some embodiments, the cell-penetrating peptide is selected from any one of the amino acid sequences shown in SEQ ID NO: 3-7.

[0015] In the present application, the catalytic domain is modified by using an antibacterial peptide known in the art as a transmembrane peptide, but not any antibacterial peptide connected with the catalytic domain LysS23 can maintain the stability of the tertiary spatial structure of the peptide fusion antibacterial enzyme, higher and broader spectrum antibacterial activity, and smooth folding and secretory expression. The preferred five transmembrane peptides are connected with the N-terminus or C-terminus of the catalytic domain LysS23 through a connecting peptide Linker, which not only can realize the stability of the spatial structure of the protein, but also can maintain the hydrophobicity of lysozyme and higher and broader spectrum antibacterial activity, and finally can be smoothly expressed and secreted through a prokaryotic expression system or a eukaryotic expression system.

[0016] In some embodiments, the amino acid sequence of the peptide fusion antibacterial enzyme is shown in SEQ ID NO: 8-17.

[0017] In the present application, the synthetic peptide fusion antibacterial enzyme has a completely new protein structure and physical and chemical properties. Specifically, the number of amino acids is 192, the molecular weight is about 20.5KD, the isoelectric point is about 10.0, it is a hydrophilic and stable protein, easy to express, and has good temperature and pH stability.

[0018] In the second aspect, the present application also provides the use of the peptide fusion antibacterial enzyme of the first aspect in the preparation of food, medicine, health care product, feed or feed additive for preventing or treating gram-negative bacterial infection.

[0019] In the present application, the synthetic peptide fusion antibacterial enzyme can exert high antibacterial activity against gram-negative pathogenic bacteria without relying on the addition of outer membrane permeabilizers, can effectively curb the spread of drug-resistant gram-negative pathogenic bacteria, and has good application prospect in the preparation of food, medicine, health care product, feed or feed additive for preventing or treating gram-negative bacterial infection.

[0020] In the third aspect, the present application also provides a pharmaceutical composition for preventing or treating gram-negative bacterial infection, wherein the effective component of the pharmaceutical composition comprises the peptide fusion antibacterial enzyme of the first aspect of the present application.

[0021] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or additive.

[0022] In some embodiments, the dosage form of the pharmaceutical composition is injection, oral agent or external agent, the content of the peptide fusion antibacterial enzyme in the injection is 0.01-500 mg / branch, the content of the peptide fusion antibacterial enzyme in the oral agent is 0.01-500 mg / branch, and the content of the peptide fusion antibacterial enzyme in the external agent is 0.001-1000 mg / branch.

[0023] The pharmaceutical composition containing the peptide fusion antibacterial enzyme described above can be used for preventing and treating infections of gram-negative bacteria, and sterilizing and disinfecting instruments and physical sites. The content of the peptide fusion antibacterial enzyme is controlled within the range described above, so that the pharmaceutical composition can be suitable for most scenarios.

[0024] Compared with the prior art, the present application has the following technical effects: 1) The present application modifies the catalytic domain LysS23, i.e. connects a transmembrane peptide to the N- or C-terminus of the catalytic domain LysS23, to form a peptide fusion antibacterial enzyme with a completely new protein structure. The peptide fusion antibacterial enzyme can directly act on common gram-negative bacteria without the addition of a chemical outer membrane permeabilizer (such as EDTA / organic acid), and has good antibacterial effect.

[0025] 2) The peptide fusion antibacterial enzyme of the present application has stronger antibacterial activity. The MIC value of the peptide fusion antibacterial enzyme of the present application for Klebsiella pneumoniae and Acinetobacter baumannii is equal to or lower than 0.78 μM, while the MIC value of the non-fusion antibacterial enzyme LysS23 for Klebsiella pneumoniae and Acinetobacter baumannii is equal to or higher than 3.66 μM. The peptide fusion antibacterial enzyme of the present application has a wider antibacterial spectrum, and can effectively kill common gram-negative bacteria such as Escherichia coli, Salmonella, Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii, with MIC values equal to or lower than 0.78 μM. The non-fusion antibacterial enzyme LysS23 has no bactericidal effect on Escherichia coli and Salmonella.

[0026] 3) The peptide fusion antibacterial enzyme of the present application has lower hemolytic activity and higher safety, and the cytotoxicity of the transmembrane peptide is significantly reduced after fusion with the antibacterial enzyme. The concentration of the peptide fusion antibacterial enzyme that does not produce hemolytic effect on sheep whole blood is greater than 0.17 mM, while the concentration of the transmembrane peptide that does not produce hemolytic effect on sheep whole blood is less than 0.06 mM.

[0027] 4) The peptide fusion antibacterial enzyme of the present application can tolerate high salt environment. The activity of the peptide fusion antibacterial enzyme remains more than 90% under the condition of up to 600 mM NaCl. At the same time, the peptide fusion antibacterial enzyme of the present application also has high activity under complex conditions such as serum and body fluid. The activity of the peptide fusion antibacterial enzyme is basically not affected in the presence of 10% and 20% different concentrations of serum. Therefore, the peptide fusion antibacterial enzyme of the present application can adapt to more complex environments. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1Figure 3 is a tertiary structure model and electropherogram of LysS23-BQ. Wherein, left: AlphaFold tertiary structure model of LysS23-BQ, dark blue: LysS23, black: (GGGGS)2, green: BQ; right: Purified collection peak electropherogram of LysS23-BQ, using 15% SDS-PAGE gel, the first lane on the left is Marker, the second lane is the elution peak of LysS23-BQ fermentation broth after cation chromatography purification, the molecular weight of LysS23-BQ protein is 20.54 KDa.

[0029] Figure 2 Figure 6 is a determination diagram of the effect of salt ion concentration on the enzyme activity of peptide fusion penicillinase.

[0030] Figure 3 Figure 7 is a determination diagram of the effect of serum concentration on the enzyme activity of peptide fusion penicillinase. DETAILED DESCRIPTION

[0031] The present application will be further described below in conjunction with specific examples, which should be understood as merely illustrating the present application but not limiting the scope of the present application. The experimental methods in the following examples, if not specified, are generally carried out according to the conventional conditions, the conditions described in the laboratory manual or the conditions suggested by the manufacturer. EXAMPLE

[0032] A total of 5 different sequences of transmembrane peptides were selected, and the specific amino acid sequences are shown in Table 1.

[0033] Table 1: Name and amino acid sequence of transmembrane peptide Meanwhile, according to the different connection modes between the catalytic domain LysS23 and different transmembrane proteins (connected to the N-terminus or C-terminus), the specific structure of the peptide fusion penicillinase synthesized in the examples is shown in Table 2.

[0034] Table 2: Name and amino acid sequence of peptide fusion penicillinase Number Name SEQ ID NO Number Name SEQ ID NO 1 LysS23-BQ 8 2 BQ-LysS23 9 3 LysS23-K17 10 4 K17-LysS23 11 5 LysS23-L10 12 6 L10-LysS23 13 7 LysS23-K6 14 8 K6-LysS23 15 9 LysS23-K11 16 10 K11-LysS23 17 Wherein, “-” represents a connecting peptide.

[0035] Example 1: Escherichia coli expression system According to the codon bias of E. coli, the nucleic acid sequences of the peptide fusion lysozyme numbered 1-10 in Table 2 were artificially designed and synthesized. The recombinant vector of pET-28α-peptide fusion lysozyme numbered 1-10 was obtained by ligating the nucleic acid sequences into the pET-28α vector through the restriction enzyme sites Nco I and Hird III. The recombinant vector was then transformed into the E. coli expression host BL(DE3) by the heat transformation method, and the final recombinant lysozyme E. coli expression strain was obtained by screening on LB plates containing 50 μg / mL Kan.

[0036] The basic steps of recombinant lysozyme expression are as follows: the monoclonal strain was streaked on LB plates with the addition of 50 μg / mL kan and incubated at 37°C overnight. A loopful of the strain was picked from the plate and inoculated into a 250 mL conical flask containing 50 mL of fresh LB medium, which was incubated at 37°C, 200 rpm overnight as the top tank fermentation seed liquid. The fermentation medium was inoculated at a rate of 2%, and the fermentation parameters were 37°C, 300 rpm, and aeration amount of 2 L / min for 2-6 h of pre-culture to make the OD600 in the range of 0.8-1.2. Under the conditions, the final concentrations of 1% penetration enhancer and 0.5% lactose were added, and the fermentation parameters were changed to 24°C, 150 rpm, and aeration amount of 1 L / min for overnight induction of protein synthesis and secretion.

[0037] The fermentation supernatant was purified by two-step purification of cation exchange and / or hydrophobic chromatography. After purification, the peptide fusion lysozyme numbered 1-10 was obtained and stored at -20°C for standby use.

[0038] Example 2 Pichia pastoris expression system According to the codon bias of Pichia pastoris, the nucleic acid sequences encoding the peptide fusion lysozyme numbered 1-10 were artificially designed and synthesized. The enzyme cleavage sites Xho I (CTCGAG) and Xba I (TCTAGA) were introduced at both ends, and TAA and TAG were the stop codons to obtain the recombinant plasmid pGAPZαA-peptide fusion lysozyme numbered 1-10. The yeast strain SMD1168 was made competent according to the method in the manual of Invitrogen Company, and 10 μg of the enzyme cleavage product was used to transform the competent yeast strain SMD1168 using a Bio-Rad electroporator under the conditions of 25 μF and 1500 V according to the manual. The transformation product was plated on YPDS plates with Zeocin resistance at concentrations of 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, and 500 μg / mL, and incubated at 30°C for 2-4 days until single colonies grew. The highest concentration of Zeocin-resistant bacterial cells that grew were picked with a toothpick, shaken overnight, and the bacterial cells were collected by centrifugation for PCR identification. The engineered bacteria with integrated target genes were screened, and the positive yeast strain was obtained.

[0039] The positive yeast strains screened were inoculated into 100 mL of BMGY medium and cultured at 30 °C and 250 r / min until the OD600 reached 2-6. The bacterial cells were collected by centrifugation and resuspended in 500 mL of BMMY medium for induced expression. The culture was incubated at 28 °C and 250 r / min for 96 h, during which 0.5% (V / V) of methanol was added every 24 h. After 96 h of induced expression, the culture supernatant was collected by centrifugation at 10,000 r / min for 5 min.

[0040] The fermentation supernatant was purified by two steps of cation exchange and / or hydrophobic chromatography. After purification, the peptide fusion antifungin with sample numbers 1-10 was obtained and stored at -20 °C for future use.

[0041] Comparative Example According to the expression method of Example 1 or Example 2, peptide fusion antifungins containing other different types of transmembrane peptides (as shown in Table 3) were expressed, as shown in Table 4.

[0042] Table 3 Amino acid sequences of different types of transmembrane peptides Table 4 Synthesized peptide fusion antifungins Number Name SEQ ID NO Number Name SEQ ID NO 11 LysS23-L180 22 12 L180-LysS23 23 13 LysS23-L618 24 14 L618-LysS23 25 15 LysS23-L108 26 16 L108-LysS23 27 17 LysS23-MPG 28 18 MPG-LysS23 29 wherein “-” represents a connecting peptide.

[0043] Example 1 Structural characterization of peptide fusion antifungin proteins The physical and chemical parameters of the peptide fusion antifungins numbered 1-18 in the examples and comparative examples were characterized, and the determination method used the ProtParam online bioinformatics analysis tool (https: / / web.expasy.org / protparam / ). The determination results are shown in Table 5.

[0044] Table 5 Physical and chemical parameters of peptide fusion antifungins Note: Generally, a protein with an instability index greater than or equal to 40 is considered unstable.

[0045] As can be seen from Table 5, the peptide fusion antifungins numbered 1-10 and 15-18 can form stable protein tertiary structures, among which the protein tertiary structure of the peptide fusion antifungin numbered 1 is as shown in Figure 1 The peptide fusion antifungins numbered 11-14 did not form stable protein tertiary structures, which shows that not all common transmembrane peptides connected to antifungin LysS23 can obtain the peptide fusion antifungin with a stable protein tertiary structure according to the present application.

[0046] Example 2 Minimum inhibitory concentration (MIC) determination The minimum inhibitory concentration (MIC) of the peptide fusion penicillinase against different bacteria was determined by 96-well plate micro two-fold dilution method. The bacteria species were Acinetobacter baumannii 34AB (strain 1), Acinetobacter baumannii AB17H194 (strain 2), Klebsiella pneumoniae SWJQ2966Rt (strain 1), Klebsiella pneumoniae SWJQ2990Rt (strain 2), Salmonella typhi 9079 (strain 1), Salmonella typhi 17137 (strain 1), Pseudomonas aeruginosa CMCC (B) 10104, and Escherichia coli CMCC (B) 44102.

[0047] After the bacteria were cultured to the exponential growth phase, the nutrient broth medium was adjusted to OD600 of 0.5, and then diluted 100 times with the nutrient broth. The sample nutrient broth medium was diluted by two-fold gradient to obtain sample solutions with different concentrations. Each well was added with 50 μl of the medium (or containing EDTA, so that the final concentration was 0.5 mM), 20 μl of the bacterial suspension, and 50 μl of the sample solution with different concentrations (the final concentration was 0.09-100 μM), and then incubated at 37°C for 18-24 hours. The bacterial medium mixture without the penicillinase was used as the positive control. The minimum concentration completely inhibiting the growth of the bacteria was determined as the MIC value by visual observation. The determination results are shown in Table 6.

[0048] Table 6 Determination of the minimum inhibitory concentration (MIC / μM) As can be seen from the determination results in Table 6, the peptide fusion penicillinase has stronger antibacterial activity. Without adding the outer membrane permeabilizer, the MIC value of the peptide fusion penicillinase against Klebsiella pneumoniae and Acinetobacter baumannii is equal to or lower than 0.78 μM, while the MIC value of the non-fusion penicillinase LysS23 against Klebsiella pneumoniae and Acinetobacter baumannii is equal to or higher than 3.66 μM. The peptide fusion penicillinase has a wider antibacterial spectrum. Without adding the outer membrane permeabilizer, the peptide fusion penicillinase can effectively kill common gram-negative bacteria such as Escherichia coli, Salmonella, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii, and the MIC value is equal to or lower than 0.78 μM. The non-fusion penicillinase LysS23 has no bactericidal effect on Escherichia coli and Salmonella. The MIC value, antibacterial spectrum, and antibacterial spectrum of the peptide fusion penicillinase numbered 15-18 are equivalent to those of the non-fusion penicillinase LysS23. The antibacterial spectrum is not increased, and the individual MIC value is even higher. The antibacterial activity is not improved. It can be seen that not all common membrane penetrating peptides connected with the penicillinase LysS23 can obtain the peptide fusion penicillinase of the present application, so that the antibacterial activity of the recombinant is stronger.

[0049] In addition, the MIC value of the transmembrane peptide for Acinetobacter baumannii, Klebsiella pneumoniae, Salmonella typhi, Pseudomonas aeruginosa and Escherichia coli was the lowest at 5.44 μM, i.e. the concentration of the transmembrane peptide for exerting the antibacterial effect was at least 5.44 μM, while the average MIC value of the peptide fusion antibacterial enzyme was 0.78 μM, and the transmembrane peptide and lyase in the peptide fusion antibacterial enzyme were combined at a ratio of 1:1, i.e. 0.78 μM of the peptide fusion antibacterial enzyme contained 0.78 μM of the transmembrane peptide, which was far from the MIC value 5.44 μM of the transmembrane peptide, thus indicating that the peptide fusion antibacterial enzyme did not exert the antibacterial effect by the transmembrane peptide. The MIC value (average 0.78 μM) of the peptide fusion antibacterial enzyme was equivalent to or lower than the MIC value (0.91 μM) of the non-fusion antibacterial enzyme LysS23 in the presence of EDTA, and it can be seen that the catalytic domain LysS23 of the peptide fusion antibacterial enzyme exerted the main antibacterial effect, and the antibacterial effect of the catalytic domain LysS23 was obviously enhanced with the help of the transmembrane peptide, reaching the antibacterial level when the non-fusion antibacterial enzyme LysS23 was mixed with the outer membrane permeabilizer EDTA.

[0050] Example 3 Hemolysis assay The hemolytic activity of the antibacterial enzyme was determined by detecting the release of hemoglobin (OD value at 414 nm) in a fresh 4% red blood cell suspension. Defibrillated sheep blood was washed with PBS (PBS: 35 mM phosphate buffer, 150 mM NaCl, pH 7.0), and 100 μl of an 8% human red blood cell suspension was taken in a 96-well plate, 100 μl of the antibacterial enzyme solution was added to each well, and incubated at 37°C for 1 hour, centrifuged at 1500 rpm for 5 minutes, and 100 μl of the supernatant was transferred to a new 96-well plate, and the absorption at 414 nm was detected by an enzyme marker. The negative control used PBS, and the positive control used 0.1% Triton X-100.

[0051] The result analysis calculation formula was: hemolysis rate % = (OD experiment - OD negative) / (OD positive - OD negative) x 100. The judgment standard was: hemolysis rate less than 5% was no hemolytic effect, and hemolysis rate greater than 5% was hemolytic effect.

[0052] The determination results are shown in Table 7.

[0053] Table 7 Hemolysis assay results As can be seen from Table 7, the peptide fusion lysozyme of the present application has lower hemolysis and is safer, and the cell toxicity of the penetratin is significantly reduced after the penetratin is fused with the lysozyme. The concentration of the peptide fusion lysozyme that does not cause hemolysis to the sheep whole blood is greater than 0.17 mM, while the concentration of the penetratin that does not cause hemolysis to the sheep whole blood is less than 0.06 mM. The hemolysis of the peptide fusion lysozyme numbered 15 and 16 is improved compared with the penetratin BQ, but the hemolysis is increased compared with the non-fusion protein LysS23, and the hemolysis is still detected at the determined concentration. The hemolysis of the peptide fusion lysozyme numbered 17 and 18 does not change compared with the penetratin BQ, and the concentration that does not cause hemolysis is less than 0.06 mM. It can be seen that not all common penetratins connected with the lysozyme LysS23 can obtain the peptide fusion lysozyme of the present application, which has very low hemolysis.

[0054] Effect Example 4 Influence of salt ion concentration on enzyme activity The peptide fusion lysozymes numbered 1, 3, 6, 9 and 10 were respectively resuspended in 20 mM Tris-HCl pH 7.5 buffer containing 0 mM, 75 mM, 150 mM, 300 mM and 600 mM NaCl, so that the final concentration of the sample peptide fusion lysozyme was 0.6 mg / mL. After incubation at 37°C for 30 min, the peptidoglycan cleavage activity was respectively determined. The determination results are shown in Table 8. Figure 2 As can be seen from the column chart, the peptide fusion lysozyme of the present application can tolerate high salt environment, and the activity of the peptide fusion lysozyme is maintained at more than 90% under the condition of up to 600 mM NaCl.

[0055] Effect Example 5 Influence of serum concentration on enzyme activity The peptide fusion lysozymes numbered 1, 3, 6, 9 and 10 were respectively resuspended in 20 mM Tris-HCl pH 7.5 150 mM NaCl buffer containing 0%, 1%, 5%, 10% and 20% human serum, so that the final concentration of the sample peptide fusion lysozyme was 0.6 mg / mL to obtain the experimental samples. After incubation at 37°C for 30 min, the peptidoglycan cleavage activity was respectively determined. The determination results are shown in Table 9. Figure 3 As can be seen from the column chart, the activity of the peptide fusion lysozyme is basically not affected in the presence of different concentrations of serum of 1%, 5%, 10% and 20%.

[0056] Effect Example 6 The peptide fusion lysozymes numbered 1-10 prepared in the examples of the present application can be used as active ingredients to prepare biological antibacterial preparations. Herein, only as an example, the peptide fusion lysozyme LysS23-BQ numbered 1 is used to prepare 100 ml of a biological antibacterial preparation.

[0057] 1. Formulation: LysS23-BQ of Example 2 0.02 g; glycine 2 g; glycerol 4 g; potassium dihydrogen phosphate 0.16 g; sodium phosphate dibasic 0.12 g; sodium chloride 0.6 g; and the rest is made up to 100 ml with distilled water.

[0058] 2. Preparation procedure (1) Dissolve potassium dihydrogen phosphate and sodium phosphate dibasic in distilled water to obtain solution A; (2) According to the formulation, weigh the required LysS23-BQ and glycine, and add them to part of solution A, stir and dissolve to obtain solution B; (3) Weigh sodium chloride and glycerol according to the ratio, dissolve them in distilled water to obtain solution C; (4) Mix solution B and solution C, and stir until uniform to obtain the finished product.

[0059] The product is directly used as a disinfectant in plastic or glass bottles; or it can be prepared into a disinfectant spray by being filled into a spray bottle with a hand pump; or it can be used as an additive liquid for wet wipes and be absorbed in non-woven fabric to make sanitary wet wipes; or it can be mixed with a thickening agent to make a gel; or it can be mixed with a stabilizer and excipient to make a powder; or it can be mixed with other dressings to make tablets or capsules.

[0060] Example 7 The biological antibacterial preparation containing the peptide fusion enzyme LysS23-BQ can be used for skin disinfection and sterilization, 1-2 times per day.

[0061] I. Apparatus 1. Test bacteria: Escherichia coli 6th generation (provided by the Disinfection Testing Center of the Military Medical Academy) and Pseudomonas aeruginosa 6th generation (provided by the Disinfection Testing Center of the Military Medical Academy).

[0062] 2. Sample: the biological antibacterial preparation of Example 6.

[0063] 3. Neutralizing agent: 2% histidine, 8% lecithin, and 10% Tween-80 in D / E broth.

[0064] 4. PBS: 0.03 mol / L phosphate buffer.

[0065] 5. GNP-9160 type water-resistant constant temperature incubator (Shanghai Jinghong Experimental Equipment Co., Ltd.).

[0066] 6. Culture medium: ordinary nutrient agar.

[0067] 7. Mechanical stopwatch (type 504).

[0068] 8. Adjustable constant temperature water bath (CBN28-3).

[0069] II. Method 1. Test basis: Hygienic Standard for Disposable Hygienic Products GB15979-2002.

[0070] 2. Neutralizer identification test: test strains: Escherichia coli, Pseudomonas aeruginosa.

[0071] 3. Quantitative bactericidal test: sample stock solution.

[0072] 4. Test temperature 20℃, test repeated 3 times.

[0073] III. Results 1. Test bacteria: Escherichia coli, the neutralizer identification test results are shown in Table 8.

[0074] Table 8 Neutralizer identification test results 2. Test bacteria: Pseudomonas aeruginosa, the neutralizer identification test results are shown in Table 9.

[0075] Table 9 Neutralizer identification test results It is determined that the biological antibacterial preparation containing the peptide fusion antibiotic LysS23-BQ has the bactericidal results on the experimental bacteria as shown in Table 10.

[0076] Table 10 Bactericidal effect on experimental bacteria According to the test results in Tables 8-10, the neutralizer identification error rates for Escherichia coli are 12.63%, 10.34% and 11.66% respectively, which meet the requirements. The neutralizer identification error rates for Pseudomonas aeruginosa are 10.97%, 11.76% and 12.88% respectively, which meet the requirements. The bactericidal test results show that the biological antibacterial preparation containing the peptide fusion antibiotic LysS23-BQ has a bactericidal result greater than 90% on Escherichia coli and Pseudomonas aeruginosa for 2 min, 5 min, 10 min and 20 min, reaching the antibacterial level and meeting the hygienic standard for disposable hygienic products.

[0077] This specific embodiment is only an explanation of the present application, which is not a limitation of the present application. Those skilled in the art can make modifications to the present embodiment without creative contribution after reading the present specification, but as long as it is within the scope of the claims of the present application, it is protected by the patent law.

Claims

1. A peptide fusion antibacterial enzyme, characterized in that, The peptide fusion antibiotic enzyme comprises a catalytic domain LysS23, a cell-penetrating peptide, and a connecting peptide connecting the catalytic domain and the cell-penetrating peptide, wherein the catalytic domain LysS23 comprises an amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having 98% or more identity by substitution / deletion / addition of one or more amino acid residues to the amino acid sequence shown in SEQ ID NO.

1.

2. The pepti-botics of claim 1, wherein, The connecting peptide comprises an amino acid sequence shown in SEQ ID NO:

2.

3. The pepti-botics of claim 1, wherein, The cell-penetrating peptide is connected to the N-terminus or C-terminus of the catalytic domain through the connecting peptide.

4. The pepti-botics of claim 1, wherein, The cell-penetrating peptide is selected from any one of the amino acid sequences shown in SEQ ID NO: 3-7.

5. The peptidic fusion enzyme according to any one of claims 1 to 4, characterized in that, The amino acid sequence of the peptide fusion antibiotic enzyme is shown in SEQ ID NO: 8-17.

6. Use of the peptide fusion antibiotic enzyme according to any one of claims 1-5 in the preparation of a food, a drug, a health food, a feed, or a feed additive for preventing or treating a Gram-negative bacterial infection.

7. Use according to claim 6, characterized in that, The Gram-negative bacteria include, but are not limited to, Escherichia coli, Salmonella, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii.

8. A pharmaceutical composition for preventing or treating Gram-negative bacterial infection, characterized by, The pharmaceutical composition comprises the peptide fusion antibiotic enzyme according to any one of claims 1-5 as an effective ingredient.

9. The pharmaceutical composition of claim 8, wherein, The pharmaceutical composition further comprises a pharmaceutically acceptable carrier or additive.

10. The pharmaceutical composition of claim 9, wherein, The pharmaceutical composition is in the form of an injection, an oral agent, or a topical agent.

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