Cell wall degrading enzymes with antibacterial activity

Abstract

The present invention relates to a series of cell wall degrading enzymes with antibacterial activity for effectively controlling Staphylococcus aureus, and the cell wall degrading enzymes with antibacterial activity of the present invention are a series of lyases with new amino acid sequences. The cell wall degrading enzymes with antibacterial activity of the present invention can be used as agents capable of effectively inhibiting Staphylococcus aureus, and can be applied to the disinfection of Staphylococcus aureus in the environment and the treatment of diseases caused by Staphylococcus aureus infection, etc.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a series of cell wall degrading enzymes that effectively control Staphylococcus aureus, including their applications and production methods. Background Technology

[0002] Staphylococcus aureus is one of the most important pathogens in humans, causing a variety of diseases, such as skin, soft tissue, wound, bone and blood infections, toxic shock syndrome, and food poisoning. [McCaig LF et al. Emerg Infect Dis 2006;12:1715–1723. Laureano AC et al. Clin Dermatol 2014;32:711–714. Brüssow HEnviron Microbiol 2016;18:2089–2102.]. It is one of the most common causes of hospital-acquired infections, and this bacterium has become a serious threat to hospitals. Furthermore, the emergence and increase of antibiotic-resistant bacteria in the clinical setting is worrying, especially methicillin-resistant bacteria. Recent data from the World Health Organization indicate that methicillin-resistant Staphylococcus aureus (MRSA) strains account for more than 20% of all infection cases in WHO regions, but in some countries, this figure reaches 80% (WHO. 2014. Antimicrobial resistance, global report on surveillance 2014. WHO, Geneva, Switzerland).

[0003] Furthermore, Staphylococcus aureus is one of the major pathogens of foodborne illnesses, producing enterotoxins in humans (Le Loir Y et al. 2003. Genet Mol Res 2:63–76.). In 2014, a major foodborne illness outbreak occurred in the European Union, with 7.5% of food contaminated with Staphylococcus aureus (EFSA, ECDC. 2016. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2014. EFSA J 13:4329.). Similarly, methicillin-resistant Staphylococcus aureus (MRSA) infection is a common problem in animal husbandry, as livestock can not only be infected with the bacteria but also transmit it (Normanno G et al. 2015. Food Microbiol 51:51–56.). It is well known that the overuse of antibiotics in food, animal husbandry, and other fields has increased the emergence of multidrug-resistant bacteria (MDR), contributing to the current global health crisis. In response to this problem, some countries have restricted the use of antibiotics in livestock farming (Maron DF, et al. 2013. Global Health 9:48.).

[0004] With the rapid emergence of resistance to classic antibiotics, there is an urgent need for alternative treatments. In recent years, bacteriophages and cell wall lysins extracted from bacteriophages have emerged as an alternative approach to overcome this problem.

[0005] Bacteriophages are viruses that infect only bacteria during their life cycle. In most cases, the lysing life cycle ends with the death of the bacterial cell, making bacteriophages natural killers of bacteria. Lysis can occur through two mechanisms: first, bacteriophages with single-stranded genomes encode lysis factors that inhibit bacterial peptidoglycan biosynthesis; second, in double-stranded DNA (dsDNA) bacteriophages, the release of bacteriophage progeny is mediated by two proteins, holin and endolysin, responsible for cell membrane rupture. Once the viral particle matures inside the bacterial cell, the hollow protein holin forms a pore in the inner cell membrane, allowing the endolysin to enter the cell wall. Subsequently, endolysin degrades peptidoglycan, causing permeolysis. Furthermore, bacteriophages require additional proteins, called transmembrane proteins, to help break down the outer membrane of Gram-negative host cells when infecting them. (Catalao MJ et al. 2013. FEMS Microbiol Rev 37:554–571)

[0006] In addition, virus particle-associated peptidoglycan hydrolases (VAPGHs) are structural components of viral particles. They participate in the initial steps of infection by slightly degrading peptidoglycan, allowing bacteriophage genetic material to enter bacterial cells. Some researchers also refer to them as phage-virus particle tail-associated cell wall degrading enzymes (TAME) (Paul VD, et al., 2011;11: 226.), believing that all phage particles contain TAME associated with the tail structure, which facilitates the injection of phage DNA by causing local degradation of the cell wall.

[0007] Gram-positive dsDNA phage-encoded lysin proteins (endolysin and VAPGHs) share a common characteristic: a modular structure composed of different functional domains. On the one hand, this structure endows the lysin proteins with significant substrate specificity; on the other hand, this modular structure facilitates the design of new proteins with enhanced antibacterial activity based on existing ones (Oliveira H et al. 2013. J Virol 87:4558–4570.). Most staphylococcal phage endolysins possess one or two N-terminal catalytic domains and a C-terminal cell wall-binding domain (CBD); similarly modular VAPGHs also consist of one or two catalytic domains but lack the CBD domain; moreover, no signal peptide or transmembrane domain has been found in staphylococcal phage endolysins (Rodríguez-Rubio L et al. 2013. CritRev Microbiol 39:427–434.).

[0008] To understand the catalytic mechanism of phage lysis proteins, studying the structure of peptidoglycans in target bacteria is crucial. Vollmer W et al. pointed out that peptidoglycans consist of linear glycan chains crosslinked with short peptides. These chains are composed of alternating N-acetylglucosamine (GIcNAc) and N-acetylmuramic acid (MurNAc) residues linked by β-1,4 glycosidic bonds. In Staphylococcus aureus, the D-lactyl group of each MurNAc residue is replaced by a peptidyl stem, with the stem structure being L-Ala-Glu-Lys-D-Ala (Vollmer W et al. 2008. FEMS Microbiol Rev 32:259–286.). Researchers have classified bacteriophage lysis proteins based on their catalytic active sites: N-acetylmuramidase (also known as lysozymes or muramidases); endo-β-N-acetylglucosaminidases (also known as glucosaminidases); N-acetylmuraxnyl-L-alanineamidase; transglycosylases; and endopeptidases. Endopeptidases are further divided into L-alanyl-D-glutamate endopeptidase and interpeptide bridge-specific endopeptidases. Glucosamine glycosylases, lysozymes, and transglycosylases act on the glycan portion, while endopeptidases are responsible for cleaving peptide bridges, and amidases degrade the amide bond between sugars and peptides. Staphylococcal phage lysins rarely contain transglycosylases. The main catalytic domains are the phage lysozyme domain M23 (LYSO-M23), the peptidase domain M23 (PET-M23), the amidase 2 domain (AMI-2), the amidase 3 domain (AMI-3), and the cysteine ​​and histidine-dependent amidohydrolase / peptidase (CHAP). Among them, CHAP is the most common domain (>74%) (Oliveira et al. 2013. J Virol 87:4558-4570.).

[0009] The CBD region of Staphylococcus aureus phage lysozymes typically contains SH3-associated domains, most commonly SH3_5 and SH3b (13,15). The SH3b domain has been shown to bind to peptidoglycan peptide bridges (Grundling A, et al., 2006. J Bacteriol 188:2463–2472.). However, some staphylococcal phage lysozymes have CBDs that do not share homology with SH3b (Daniel A, et al., 2010. AntimicrobAgents Chemother 54:1603–1612.), such as lysozymes derived from phages phiNM3, phi13, and MW1. Daniel et al. suggested that the phiNM3 CBD may bind to cell wall-associated carbohydrates rather than peptide bridges. Recently, a novel CBD was discovered in the endolysin of phage SA97 (LysSA97), which has only 19% homology with other staphylococcal endolysins stored in the database (Chang Y et al. 2017. Appl Microbiol Biotechnol 101:147–158.).

[0010] Most phage lysozymes exhibit high specificity for the genus or species they infect, a significant advantage over classic broad-spectrum antibiotics. However, the molecular-level interactions between these proteins and their substrates are not fully understood, and it remains unclear which molecular fragment determines specificity. Therefore, researchers are working to produce novel chimeric proteins by combining the domains of different lysin proteins. Interestingly, the design of chimeric proteins has shown promising results in improving lysin proteins. For example, the Lysk-based chimeric protein PRF-119, obtained by fusing the CHAP domain of Lysk lysozyme with the SH3b domain of lysozyme, exhibits excellent activity and a minimum inhibitory concentration (MIC) against both Staphylococcus aureus and methicillin-resistant Staphylococcus aureus. 90The concentration was 0.391 μg / mL. Similarly, combining the CHAP domain of Ply187 endosomalase with the CBD (SH3b) region of Lysk endosomalase showed a 10-fold increase in specificity compared to the single CHAP domain (Mao J, et al. 2013. FEMS Microbiol Lett 342:30–36.). Similar chimeric proteins include ClyH (derived from the CHAP domain of Ply187 endosomalase and the CBD of phiNM3 endosomalase), ClyF (derived from the CHAP domain of Ply187 endosomalase and the CBD of Plyss2 endosomalase), and HydH5_SH3b (derived from the CHAP domain of HydH5 and the SH3b domain of lysozyme) (US9868943B2), all of which showed higher antibacterial activity against Staphylococcus aureus than before the combination. From these data, we can conclude that combining different functional domains is a powerful tool to increase the activity and specificity of phage lysis proteins.

[0011] In summary, both types of phage lysins, endolysins and VAPGHs, possess the potential to degrade peptidoglycan and can serve as useful antibacterial agents when added exogenously. Currently, endolysins are the most extensively studied, with existing endolysins being improved through combinations of different functional modules. Research on the functional domains of VAPGHs is relatively limited. However, the development of novel antibacterial phage lysin proteins requires a systematic exploration of naturally occurring lysin proteins. Besides modularly combining and designing new proteins, analyzing protein structures that mimic individual functional domains is extremely helpful for the targeted design of mutants with altered activity or substrate specificity. Summary of the Invention

[0012] Against this backdrop, this invention, by exploring novel phage lysin proteins, altering their length, or mutating the CHAP functional domain of VAPGH tail lysins, and combining them with CBD regions from different sources, yields a series of cell wall lysins with diverse properties. This increases the diversity of existing cell wall lysins, broadens their application scenarios, and provides a foundation for obtaining more and better lysins. Therefore, the purpose of this invention is to provide cell wall lysins with good control effects against Staphylococcus aureus as antibiotic alternatives.

[0013] This invention first provides a cell wall degrading enzyme with antibacterial activity, characterized in that it is obtained by fusing a peptidoglycan lyase or its cell wall lysing domain with a cell wall binding domain, wherein the amino acid sequence of the peptidoglycan lyase is shown in SEQ ID NO:1, and the amino acid sequence of its cell wall lysing domain is shown in SEQ ID NO:5, or a combination of mutations A36V, G66S, P88N, Q89A and A129Q based on SEQ ID NO:1, or a combination of mutations R69K, P88N, Q89A, Y113W and A129Q based on SEQ ID NO:1, or a combination of mutations A36V, R69K, A76S, I79V, P88A and Y113W based on SEQ ID NO:1, or a single-point mutation V36N based on SEQ ID NO:5.

[0014] Preferably, the C-terminal cell wall-binding domain of the lysozyme is derived from staphylococcal bacteriophage; more preferably, its amino acid sequence is shown in SEQ ID NO:8.

[0015] More preferably, the amino acids of the cell wall degrading enzyme with antibacterial activity are shown in SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, and SEQ ID NO. 14, respectively.

[0016] The present invention thereby provides the encoding gene of the cell wall degrading enzyme with antibacterial activity.

[0017] Further, we provide an expression vector for the encoded gene and recombinant cells.

[0018] The present invention further provides an antibacterial agent with the aforementioned cell wall degrading enzyme having antibacterial activity as the active ingredient.

[0019] Preferably, it is used to inhibit Staphylococcus aureus. Alternatively, a disinfectant containing the aforementioned cell wall-degrading enzyme with antibacterial activity as an active ingredient can be used to disinfect Staphylococcus aureus in the environment.

[0020] The present invention also provides the use of the aforementioned cell wall degrading enzyme with antibacterial activity in the preparation of medicaments for the prevention or treatment of diseases caused by Staphylococcus aureus infection.

[0021] The present invention further provides the application of the aforementioned cell wall degrading enzyme with antibacterial activity in the preparation of drugs or skin care products for the prevention or treatment of skin infections.

[0022] The present invention also provides the application of the aforementioned cell wall degrading enzyme with antibacterial activity in the preparation of skin care products for the prevention or treatment of skin infections.

[0023] Preferably, the target of prevention or treatment is a mammal, more specifically a human, primate, bird, cattle, horse, goat, cat, sheep, rodent, dog, pig or poultry.

[0024] This invention analyzes the functional domains and related homologous proteins of bacteriophage genomes (VAPGHs), predicts mutations through structural simulation analysis, and combines these with CBD from different sources to obtain a series of chimeric lyase proteins with antibacterial activity. The chimeric proteins provided by this invention exhibit either stronger antibacterial activity compared to the CHAP1 domain alone, or their corresponding mutants also exhibit strong antibacterial activity; or they demonstrate good thermostability; or they exhibit substrate specificity; or they show high inhibition rates under different pH conditions, thus possessing significant practical value. Attached Figure Description

[0025] Figure 1 The figure shows the experimental results of the CHAP1 protein crude enzyme solution's antibacterial effect against Staphylococcus aureus in Example 1. In the figure, B represents the Buffer control, and 1, 2, and 3 represent three parallel experiments of the CHAP1 protein crude enzyme solution.

[0026] Figure 2 Example 2: SDS-PAGE electrophoresis results of different chimeric proteins after purification. In the figure, 1-6 represent proteins SA2, SA2-M1, SA1, SA1-M1, SA1-M2, and SA1-M3, respectively.

[0027] Figure 3 Figure 3 shows the experimental results of testing the antibacterial effect of different chimeric protein crude enzyme solutions on Staphylococcus aureus.

[0028] Figure 4 Figure 4 shows the experimental results of MIC testing of different chimeric proteins in Example 4.

[0029] Figure 5 Example 5 shows the experimental results of the antibacterial effect test of different chimeric proteins against Staphylococcus aureus and Staphylococcus epidermidis. In the figure, A represents the antibacterial effect against Staphylococcus aureus, and B represents the antibacterial effect against Staphylococcus epidermidis.

[0030] Figure 6 Example 6: Comparison of enzyme activity of chimeric proteins under different pH conditions. Detailed Implementation

[0031] The invention is further illustrated below with reference to specific embodiments. Unless otherwise specified, the reagents and instruments used in the following embodiments are all commercially available products.

[0032] Example 1: Analysis of the VAPGHs lysin encoding gene to verify its antibacterial activity.

[0033] Bacteriophage KSAP7 exhibits inhibitory activity against Staphylococcus aureus. Analysis of the KSAP7 genome revealed the presence of the endolysin lyase and peptidoglycan lyases VAPGHs. VAPGHs also possess the potential to degrade peptidoglycan and could serve as a useful antibacterial agent when added exogenously; however, research on this topic is limited, and their activity when used alone has not been systematically investigated. This invention analyzed the amino acid sequence of the peptidoglycan lyase VAPGHs, altered its length to obtain the amino acid sequence of the CHAP domain, and named it CHAP1 (amino acid sequence as shown in SEQ ID NO:1).

[0034] First, the CHAP1 amino acid sequence was optimized according to the E. coli codon, and the DNA sequence was integrated into the pET30a expression vector between NdeI and XhoI. The resulting vector was then transformed into the BL21(DE3) expression strain.

[0035] The above-mentioned expression strain was transferred to LB medium for activation and cultured at 37°C. When the OD600 reached approximately 0.8, IPTG was added to a final concentration of 0.5 mM, and the culture was incubated overnight at 16°C and 200 rpm. The overnight culture was centrifuged at 10,000 rpm to collect the cells, and the cells were resuspended in a buffer solution of 20 mM Tris-HCl, 150 mM NaCl, and 10 mM CaCl2 (pH 7.0). The cells were then disrupted using an ultrasonic cell disruptor, and the cell disruption solution was centrifuged at 10,000 rpm for at least 30 min. The supernatant was collected as the crude protein enzyme solution.

[0036] The crude protein enzyme solution was used as the test object to determine its antibacterial effect against Staphylococcus aureus strain GIM 1.481. The test was conducted according to section 5.1.1 of the WS / T 650-2019 standard for quantitative suspension inhibition, as follows: Staphylococcus aureus culture medium incubated overnight at 37°C was diluted 1000-fold with a buffer solution of 20 mM Tris-HCl, 150 mM NaCl, 10 mM CaCl2, and pH 7.0 (hereinafter referred to as Buffer). This buffer was then added to a 50 μg / mL protein solution at a concentration of 2% (v / v), and incubated at 37°C for 1 h. The solution was then diluted 10-fold with the same buffer solution and plated (LB medium). For the negative control, the Staphylococcus aureus culture medium was diluted to the appropriate gradient with the buffer solution and plated. The growth of Staphylococcus aureus was observed and compared. The experimental results are shown in Table 1. The negative control plate showed an appropriate growth rate of 10 μg / mL Staphylococcus aureus. 3The number of colonies on the plates treated with the crude CHAP1 protein enzyme solution did not decrease significantly, and the results of the three parallel experiments were consistent, indicating that CHAP1 protein has no significant antibacterial activity against Staphylococcus aureus. The corresponding plate inhibition effect is shown in the figure below. Figure 1 As shown.

[0037] sample Number of colonies (cells) negative control <![CDATA[>10 3 ]]> CHAP1 Parallel Experiment 1 <![CDATA[>10 3 ]]> CHAP1 Parallel Experiment 2 <![CDATA[>10 3 ]]> CHAP1 Parallel Experiment 3 <![CDATA[>10 3 ]]>

[0038] Example 2: Analysis of CHAP1-related homologous proteins and construction of different chimeric proteins with antibacterial activity.

[0039] CHAP1 was found to be inactive. Therefore, NCBI-Blast sequence alignment was performed based on the CHAP1 amino acid sequence. The results showed that the homology of each amino acid sequence was over 90% compared to other naturally occurring CHAP domains, indicating strong regional conservation, low genetic diversity, and the function of most naturally occurring CHAP domains remained unproven. To investigate the functional domains of CHAP, CHAP2 (amino acid sequence shown in SEQ ID NO: 5), a CHAP domain derived from the Staphylococcus phage vB_SsapH-Golestan-105-M genome with less than 90% homology to CHAP1, was selected for functional studies. Simultaneously, based on the amino acid sequence variation patterns of other confirmed functional CHAP homologs, site-directed mutagenesis was performed on CHAP1 and CHAP2 using structural simulation analysis to obtain new CHAP amino acid sequences. CHAP1 alone was ineffective. Different combinations of the CHAP catalytic domain and CBD can enhance specificity and catalytic activity through synergistic effects. This invention also combines CHAP1, CHAP2, and related mutants with CBD from different sources to construct new chimeric proteins. Among them, the relevant mutations include CHAP1 mutant 1, which is a combination mutation of A36V, G66S, P88N, Q89A and A129Q on the basis of CHAP1 (amino acid sequence as shown in SEQ ID NO: 2); CHAP1 mutant 2, which is a combination mutation of R69K, P88N, Q89A, Y113W and A129Q on the basis of CHAP1 (amino acid sequence as shown in SEQ ID NO: 3); CHAP1 mutant 3, which is a combination mutation of A36V, R69K, A76S, I79V, P88A and Y113W on the basis of CHAP1 (amino acid sequence as shown in SEQ ID NO: 4); and CHAP2 mutant 1, which is a single-point mutation of V36N on the basis of CHAP2 (amino acid sequence as shown in SEQ ID NO: 6).

[0040] First, the CHAP1 amino acid sequence was combined with the CBD amino acid sequence derived from LysP108 lysozyme (amino acid sequence as shown in SEQ ID NO:7), and the CHAP2 amino acid sequence was combined with the CBD amino acid sequence derived from lysostaphin lysozyme (amino acid sequence as shown in SEQ ID NO:8) to synthesize the whole gene, named SA1 and SA2 respectively (amino acid sequences shown in SEQ ID NO:9 and SEQ ID NO:13 respectively); according to E. coli codon optimization, the DNA sequence was integrated between the pET30a expression vector NdeI and XhoI. Using the DNA sequence of SA1 as a template, primers were designed to construct three CHAP1 mutants, which were then integrated into the target vector pET30a via one-step recombination and named SA1_M1, SA1_M2, and SA1_M3, respectively. SA1_M1 was designed with five mutation sites based on the SA1 amino acid sequence: A36V, G66S, P88N, Q89A, and A129Q (amino acid sequences as shown in SEQ ID NO:10). SA1_M2 was designed with five other mutation site combinations: R69K, P88N, Q89A, Y113W, and A129Q (amino acid sequences as shown in SEQ ID NO:11). SA1_M3 was designed with six mutation sites: A36V, R69K, A76S, I79V, P88A, and Y113W (amino acid sequences as shown in SEQ ID NO:12). Using the SA2 DNA sequence as a template, primers were designed to construct a CHAP2 mutant, which was then integrated into the target vector pET30a via one-step recombination and named SA2_M1. This mutant has a mutation site V36N (amino acid sequence as shown in SEQ ID NO:14) in the SA2 amino acid sequence. Specific information is shown in Table 2. The above vector was then transformed into the BL21(DE3) expression strain for protein expression.

[0041] coding CHAP catalytic domain CBD mutation site Serial Number SA1 CHAP1 LysP108 endosomal enzyme CBD wild type SEQ ID NO:9 SA1-M1 CHAP1 mutant 1 LysP108 endosomal enzyme CBD A36V, G66S, P88N, Q89A, A129Q SEQ ID NO:10 SA1-M2 CHAP1 mutant 2 LysP108 endosomal enzyme CBD R69K, P88N, Q89A, Y113W, A129Q SEQ ID NO:11 SA1-M3 CHAP1 mutant 3 LysP108 endosomal enzyme CBD A36V, R69K, A76S, I79V, P88A, Y113W SEQ ID NO:12 SA2 CHAP2 lysostaphin lysostaphin CBD wild type SEQ ID NO:13 SA2-M1 CHAP2 mutant 1 lysostaphin lysostaphin CBD V36N SEQ ID NO:14

[0042] Example 3: Preparation of lyase chimeric protein

[0043] The expression strain from Example 2 was transferred to LB medium for activation and cultured at 37°C until it grew to OD. 600The concentration was approximately 0.8. IPTG was added to a final concentration of 0.5 mM, and the mixture was incubated overnight at 16°C and 200 rpm. The overnight culture was centrifuged at 10,000 rpm to collect the cells. The cells were resuspended in a buffer solution of 20 mM Tris-HCl, 150 mM NaCl, and 10 mM CaCl2 (pH 7.0). The cells were then disrupted using an ultrasonic cell disruptor, and the cell disruption buffer was centrifuged at 10,000 rpm for at least 30 minutes. The supernatant was collected as the crude protein enzyme solution. The purified protein was then obtained by His-Tag affinity chromatography. The protein concentration was determined spectrophotometrically, and the purity and size of the protein were detected by SDS-PAGE (polyacrylamide gel electrophoresis). Figure 2 The results showed that each chimeric protein band after purification was single, and the correct size was around 28 kDa, with an estimated purity of >95%.

[0044] Example 4: Crude enzyme solution activity determination

[0045] The protein supernatant prepared in Example 3 above was used as the test object to determine the expression level and antibacterial activity of different chimeric proteins, excluding inactive mutants. Staphylococcus aureus GIM 1.481 was used as the antibacterial test strain. The quantitative suspension antibacterial experiment was performed according to WS / T650-2019 5.1.1, as follows: First, the Staphylococcus aureus culture broth incubated overnight at 37°C on LB medium was diluted 1000-fold with 20 mM Tris-HCl, 150 mM NaCl, 10 mM CaCl2, and pH 7.0 buffer (hereinafter referred to as Buffer). Then, it was added to 50 μg / mL protein solution at a dosage of 2% (v / v), incubated at 37°C for 1 h, and then diluted 10-fold with the above buffer for plate culture (LB medium). For the negative control, the Staphylococcus aureus culture broth was diluted to the appropriate gradient with buffer and plate cultured. The growth of Staphylococcus aureus was observed and compared.

[0046] The experimental results are shown in Table 3. The appropriate amount of Staphylococcus aureus on the negative control plate reached 10. 3 The number of Staphylococcus aureus on the plates treated with the chimeric protein and the corresponding mutant crude enzyme solution was significantly reduced by about 2-3 orders of magnitude (Table 3), indicating that the new chimeric protein and the corresponding mutant obtained in this invention have antibacterial activity against Staphylococcus aureus, and the crude enzyme solution has an inhibition rate of over 99%. The corresponding plate inhibition effect is shown in the figure below. Figure 3 As shown.

[0047] sample Number of colonies (cells) negative control <![CDATA[>10 3 ]]> SA1 0 SA1-M1 4 SA1-M2 0 SA1-M3 0 SA2 34 SA2-M1 0

[0048] Example 5: MIC test of each chimeric protein

[0049] The minimum inhibitory concentration (MIC) of each protein was determined using the microdilution method (Wiegand-Nature Protocol 2008) in 96-well plates. Proteins were diluted separately with MH(B) medium and added to MH(B) medium at 10 μL. 6 A 1:1 mixture of CFU / ml Staphylococcus aureus culture was prepared to achieve a final protein concentration of 128 μg / ml to 0.25 μg / ml. MH(B) medium was used as a blank control, with a bacterial count of 5 × 10⁻⁶. 5 cfu / ml. After incubating the wells at 37°C for 18 hours, the optical density was measured at a wavelength of 600 nm. The lowest protein concentration at which no bacterial growth occurred was recorded as the MIC value of the protein.

[0050] The results show that ( Figure 4 When the concentration of SA1 protein (amino acid sequence as shown in SEQ ID NO:9) is greater than or equal to 2 μg / mL, no bacterial growth occurs; when the concentration is less than 2 μg / mL, bacterial growth occurs to varying degrees. 2 μg / mL is the minimum inhibitory concentration (MIC) of this protein. The MIC of the mutant protein SA1-M2 (amino acid sequence as shown in SEQ ID NO:11) remains unchanged, while the MICs of mutants SA1-M1 (amino acid sequence as shown in SEQ ID NO:10) and SA1-M3 (amino acid sequence as shown in SEQ ID NO:11) are 0.52 μg / mL, a four-fold increase. This indicates that the combined mutations of A36V, G66S, P88N, Q89A, A129Q and A36V, R69K, A76S, I79V, P88A, Y113W help to enhance the activity of the chimeric protein and lower the MIC. Similarly, the combined mutations of R69K, P88N, Q89A, Y113W, A129Q... Although it did not lower the minimum inhibitory concentration, it also did not reduce the activity of the chimeric protein;

[0051] Compared to the chimeric protein SA1, the wild-type SA2 (amino acid sequence as shown in SEQ ID NO:13) has a higher minimum inhibitory concentration (MIC), with cell growth only occurring at a concentration ≥128 μg / mL, which is 128 μg / mL. The mutant SA2-M1 (amino acid sequence as shown in SEQ ID NO:14) shows a significantly higher MIC than the wild-type SA2, increasing it by 64 times to 2 μg / mL, indicating that the V36N mutation site has the ability to enhance the activity of the chimeric protein. Although there is currently no difference in MIC between the mutant SA2-M1 and the wild-type SA1, based on the above results, the V36N mutation site has the potential to further enhance the activity of SA1 and related mutants, and lower the MIC.

[0052] sample MIC (ug / mL) SA1 2 SA1-M1 0.5 SA1-M2 2 SA1-M3 0.5 SA2 128 SA2-M1 2

[0053] Example 5: Determination of thermal stability data of chimeric proteins

[0054] The chimeric protein was treated at 100℃, 80℃, and 55℃ for 5 min to assess its thermal stability. The initial protein concentration was 50 ug / mL. After high-temperature treatment, the sample was centrifuged and the decrease in optical density was measured by photometry to assess the remaining protein concentration. The proportion of remaining protein was calculated. The specific results are shown in Table 5.

[0055] The results show that the chimeric protein SA2 (amino acid sequence as shown in SEQ ID NO:13) exhibits good thermal stability, retaining 45% of the protein residue after treatment at 100℃ for 5 min. In contrast, the chimeric protein SA1 (amino acid sequence as shown in SEQ ID NO:9) has relatively poor thermal stability, with over 80% of the protein inactivating and precipitating after treatment at temperatures above 55℃ for 5 min. Other SA1 mutants show varying degrees of improved thermal stability compared to wild-type SA1, especially the SA1-M2 (amino acid sequence as shown in SEQ ID NO:11) mutant, which shows a more than 4-fold improvement after treatment at different temperatures, indicating that different combinations of mutations are beneficial for improving thermal stability. The wild-type chimeric protein SA2 mutant also maintains high thermal stability, retaining 67% of the protein residue after treatment at 100℃ for 5 min, which is 1.5 times the residue of SA2 under the same conditions. The V36N mutation site helps to further improve thermal stability.

[0056] Table 5. Thermal stability data of chimeric proteins

[0057]

[0058] Example 6: Chimeric protein specificity assessment

[0059] Antibacterial specificity is an important criterion for evaluating the application scenarios of lysed proteins. Following the quantitative suspension antibacterial assay method in WS / T 650-2019 5.1.1, Staphylococcus aureus GIM1.481 and Staphylococcus epidermidis ATCC12228 were tested. The specific method is as follows: First, the Staphylococcus aureus and Staphylococcus epidermidis cultures incubated overnight at 37°C on LB medium were diluted 1000-fold with 20 mM Tris-HCl, 150 mM NaCl, 10 mM CaCl2, and pH 7.0 buffer. This solution was then added to a 50 μg / mL protein solution at a concentration of 2% (v / v) and incubated at 37°C for 1 h. Afterward, each culture was diluted 10-fold with the same buffer and plated (LB medium). For negative controls, the Staphylococcus aureus and Staphylococcus epidermidis cultures were diluted to the appropriate gradients with buffer and plated. The growth of Staphylococcus aureus and Staphylococcus epidermidis was observed and compared.

[0060] Experimental results: As shown in Table 6, the chimeric protein and its corresponding mutants showed an inhibition rate of over 99% against Staphylococcus aureus, with only a small number of colonies growing or no growth at all on the corresponding plates. The wild-type chimeric proteins SA1 (amino acid sequence as shown in SEQ ID NO:9) and SA2 (amino acid sequence as shown in SEQ ID NO:13) showed some antibacterial activity against Staphylococcus epidermidis, but their activity against Staphylococcus aureus was relatively poor, exhibiting a certain degree of substrate specificity. Compared with the control, the number of colonies decreased by only one order of magnitude, and the inhibition rate was less than 90%. The corresponding mutants showed a significantly reduced antibacterial effect against Staphylococcus epidermidis, with no change in the number of colonies by an order of magnitude, and a low inhibition rate.

[0061] The results indicate that different site combinations of mutations or single-point mutations of V36N all enhanced the substrate specificity of the chimeric protein to some extent, resulting in the chimeric protein exhibiting antibacterial activity only against Staphylococcus aureus within a certain concentration range. The corresponding plate antibacterial effect experimental results are shown in the figure below. Figure 5 As shown.

[0062] Table 6. Number of Staphylococcus aureus and Staphylococcus epidermidis colonies in plates

[0063]

[0064] Example 7: Comparison of enzyme activity of chimeric proteins under different pH conditions

[0065] Enzymatic activity of chimeric proteins under different pH conditions is also an important standard for evaluating protein application scenarios. First, various chimeric proteins were prepared into protein solutions with different pH values, with a protein concentration of 50 μg / mL. The pH values ​​were 4.5, 5.5, 6.5, 7.5, 8.0, 9.0, and 10.0. Following the quantitative suspension antibacterial experiment of WS / T 650-2019 5.1.1, the antibacterial activity of Staphylococcus aureus GIM1.481 was tested. 20 mM Tris-HCl, 150 mM NaCl, and 10 mM CaCl2 buffers at different pH values ​​were used as blank controls. The number of Staphylococcus aureus colonies growing on the blank control plates (A) and the number of colonies growing on plates at different pH values ​​(B) were counted and compared to calculate the inhibition rate, i.e., inhibition rate = (AB) / A*100%. The results are shown in [Figure number missing]. Figure 6 .

[0066] The results showed that, through comparison of enzyme activity data of chimeric proteins under different pH conditions, the chimeric protein SA1 (amino acid sequence as shown in SEQ ID NO:9) exhibited a high antibacterial rate of over 95% under various pH conditions. Its three corresponding mutants (amino acid sequences as shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12) showed reduced antibacterial rates under neutral or slightly alkaline pH conditions, reaching as low as 70%-80%. However, under slightly acidic pH conditions, they still exhibited high antibacterial activity, with an inhibition rate of over 90%. Although the combined mutations of SA1 showed reduced antibacterial activity under neutral or slightly alkaline conditions, they still possessed the potential to adapt to strongly acidic environments. Compared to wild-type SA1, the chimeric protein SA2 (amino acid sequence as shown in SEQ ID NO:13) showed high antibacterial efficiency of over 98% under weakly acidic, weakly alkaline, or neutral conditions, but the antibacterial efficiency decreased significantly to 77% when the pH was adjusted to 10. The mutant SA2-M1 (amino acid sequence as shown in SEQ ID NO:14) corresponding to SA2 showed strong antibacterial activity under different pH conditions, with an inhibition rate of over 98.5%. Under pH 10 conditions, the inhibition rate was more than 1.2 times higher than that of SA2 (77%), indicating that the V36N mutation site enhanced the antibacterial activity of the mutant SA2-M1 under alkaline conditions, which is a positive mutation. <110> Baikui Rui (Shenzhen) Biotechnology Co., Ltd. <120> Cell wall degrading enzymes with antibacterial activity <160> 14 <210> 1 <211> 141 <212> PRT <213> Artificial sequence <400> 1 MTLASLEKYNGKLPKHDPNFVQPGNRHYKYQCTWYAYNRRGELGIPVPLWGDAADWIGSAKSAGYGVGRTPKQGACVIWQRGAPGGSPQYGHVAFVEKVLDGGASIFISEHNYATPNGYGTRTIDMSSAIGKGAQFIYDKG141 <210> 2 <211> 141 <212> PRT <213> artificial sequence <400> 2 MTLASLEKYNGKLPKHDPNFVQPGNRHYKYQCTWYVYNRRGELGIPVPLWGDAADWIGSAKSAGYSVGRTPKQGACVIWQRGAPGGSNAYGHVAFVEKVLDGGASIFISEHNYATPNGYTRTIDMSSQIGKGAQFIYDKG141 <210> 3 <211> 141 <212> PRT <213> artificial sequence <400> 3 MTLASLEKYNGKLPKHDPNFVQPGNRHYKYQCTWYAYNRRGELGIPVPLWGDAADWIGSAKSAGYGVGKTPKQGACVIWQRGAPGGSNAYGHVAFVEKVLDGGASIFISEHNWATPNGYGTRTIDMSSQIGKGAQFIYDKG141 <210> 4 <211> 141 <212> PRT <213> artificial sequence <400> 4 MTLASLEKYNGKLPKHDPNFVQPGNRHYKYQCTWYVYNRRGELGIPVPLWGDAADWIGSAKSAGYGVGKTPKQGSCVVWQRGAPGGSAQYGHVAFVEKVLDGGASIFISEHNWATPNGYGTRTIDMSSAIGKGAQFIYDKG141 <210> 5 <211> 141 <212> PRT <213>Artificial Sequence <400>5 MSLDSLKKYNGKLPKHDPSFVQPGNRHYKYQCTWYVYNRRGQLGIPVPLWGDAADWIGGAKGAGYGVGKTPKQGSCVVWQRGVQGGSAQYGHVAFVEKVLDGGKKIFISEHNWATPNGYGTRTIDMSSAIGKNAQFIYDKK 141 <210>6 <211>141 <212>PRT <213>Artificial Sequence <400>6 MSLDSLKKYNGKLPKHDPSFVQPGNRHYKYQCTWYNYNRRGQLGIPVPLWGDAADWIGGAKGAGYGVGKTPKQGSCVVWQRGVQGGSAQYGHVAFVEKVLDGGKKIFISEHNWATPNGYGTRTIDMSSAIGKNAQFIYDKK 141 <210>7 <211>111 <212>PRT <213>Artificial Sequence <400>7 KTSSASTPATRPVTGSWKKNQYGTWYKPENATFVNGNQPIVTRIGSPFLNAPVGGNLPAGATIVYDEVCIQAGHIWIGYNAYNGNRVYCPVRTCQGVPPNHIPGVAWGVFK111 <210>8 <211>97 <212>PRT <213>Artificial Sequence <400>8 TPNTGWKTNKYGTLYKSESASFTPNTDIITRTTGPFRSMPQSGVLKAGQTIHYDEVMKQDGHVWVGYTGNSGQRIYLPVRTWNKSTNTLGVLWGTIK 97 <210>9 <211>252 <212>PRT <213>Artificial Sequence <400>9 MTLASLEKYNGKLPKHDPNFVQPGNRHYKYQCTWYAYNRRGELGIPVPLWGDAADWIGSAKSAGYGVGRTPKQGACVIWQRGAPGGSPQYGHVAFVEKVLDGGASIFISEHNYATPNGYGTRTIDMSSAIGKGAQFIYDKGKTSSASTPATRPVTGSWKKNQYGTWYKPENATFVNGNQPIVTRIGSPFLNAPVGGNLPAGATIVYDEVCIQAGHIWIGYNAYNGNRVYCPVRTCQGVPPNHIPGVAWGVFK 252 <210>10 <211>252 <212>PRT <213>Artificial Sequence <400>10 MTLASLEKYNGKLPKHDPNFVQPGNRHYKYQCTWYVYNRRGELGIPVPLWGDAADWIGSAKSAGYSVGRTPKQGACVIWQRGAPGGSNAYGHVAFVEKVLDGGASIFISEHNYATPNGYGTRTIDMSSQIGKGAQFIYDKGKTSSASTPATRPVTGSWKKNQYGTWYKPENATFVNGNQPIVTRIGSPFLNAPVGGNLPAGATIVYDEVCIQAGHIWIGYNAYNGNRVYCPVRTCQGVPPNHIPGVAWGVFK252 <210>11 <211>252 <212>PRT <213>Artificial Sequence <400>11 Mtlaslekyngklpkhdpnfvqpgnrhykyqctwyaynrrgelgipvplwgdaadwigsaksagygvgktpkqgacviwqrgapggsnayghvafvekvldggasifisehnwatpngygtrtidmssqigkgaqfiydkgktssastpatrpvtgswkknqygtwykpenatfvngnqpivtrigspflnapvggnlpagativydevciqaghiwigynayngnrvycpvrtcqgvppnhipgvawgvfk 252 <210>12 <211>252 <212>PRT <213>Artificial Sequence <400>12 MTLASLEKYNGKLPKHDPNFVQPGNRHYKYQCTWYVYNRRGELGIPVPLWGDAADWIGSAKSAGYGVGKTPKQGSCVVWQRGAPGGSAQYGHVAFVEKVLDGGASIFISEHNWATPNGYGTRTIDMSSAIGKGAQFIYDKGKTSSASTPATRPVTGSWKKNQYGTWYKPENATFVNGNQPIVTRIGSPFLNAPVGGNLPAGATIVYDEVCIQAGHIWIGYNAYNGNRVYCPVRTCQGVPPNHIPGVAWGVFK 252 <210>13 <211>240 <212>PRT <213>Artificial Sequence <400>13 MSLDSLKKYNGKLPKHDPSFVQPGNRHYKYQCTWYVYNRRGQLGIPVPLWGDAADWIGGAKGAGYGVGKTPKQGSCVVWQRGVQGGSAQYGHVAFVEKVLDGGKKIFISEHNWATPNGYGTRTIDMSSAIGKNAQFIYDKKLETPNTGWKTNKYGTLYKSESASFTPNTDIITRTTGPFRSMPQSGVLKAGQTIHYDEVMKQDGHVWVGYTGNSGQRIYLPVRTWNKSTNTLGVLWGTIK 240 <210>14 <211>240 <212>PRT <213>Artificial Sequence <400>14 MSLDSLKKYNGKLPKHDPSFVQPGNRHYKYQCTWYNYNRRGQLGIPVPLWGDAADWIGGAKGAGYGVGKTPKQGSCVVWQRGVQGGSAQYGHVAFVEKVLDGGKKIFISEHNWATPNGYGTRTIDMSSAIGKNAQFIYDKKLETPNTGWKTNKYGTLYKSESASFTPNTDIITRTTGPFRSMPQSGVLKAGQTIHYDEVMKQDGHVWVGYTGNSGQRIYLPVRTWNKSTNTLGVLWGTIK 240

Claims

1. A cell wall degrading enzyme with antibacterial activity, characterized in that, Its amino acid sequence is as shown in any one of SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.13, SEQ ID No.

14.

2. A gene encoding the cell wall degrading enzyme with antibacterial activity as claimed in claim 1.

3. An expression vector containing the gene as claimed in claim 2.

4. A recombinant cell containing the gene as claimed in claim 2.

5. The antibacterial agent with the cell wall degrading enzyme having antibacterial activity as the active ingredient according to claim 1, characterized in that The antibacterial agent is used to inhibit Staphylococcus aureus.

6. Use of the cell wall degrading enzyme with antibacterial activity as claimed in claim 1 in the preparation of a medicament for inhibiting Staphylococcus aureus.

7. Use of the cell wall degrading enzyme with antibacterial activity as described in claim 1 in the preparation of a medicament or skin care product for preventing or treating skin infections, characterized in that, The skin infection refers to Staphylococcus aureus infection.

8. The application according to claim 7, characterized in that, The subject for prevention or treatment is a mammal.

9. The application according to claim 8, wherein The mammal is a primate, cattle, horse, goat, cat, sheep, rodent, dog, pig or poultry.

10. The application according to claim 8, wherein, The mammal is a human.

Images