Gram-negative bacterium lyase and application thereof

Abstract

The invention discloses gram-negative bacterium lyase and application thereof, and relates to the technical field of biological medicine. The gram-negative bacterium lyase is a lyase lysin of which the amino acid sequence is as shown in SEQ ID NO.1 or a lyase lysin-Mix of which the amino acid sequence is as shown in SEQ ID NO.3. The gram-negative bacterium lyase is a lyase lysin of which the amino acid sequence is as shown in SEQ ID NO.1. According to the invention, a novel lyase lysin is excavated from a bacteriophage, and the lyase lysin is subjected to bioinformatics analysis and modification, so that a novel lyase, namely lysin-Mix, is prepared. A lyase activity detection experiment shows that the modified new lyase can penetrate through the outer membrane of gram-negative bacteria and has good cracking activity. The invention provides technical support for developing novel antibacterial drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, in particular to Gram-negative bacteria lysing enzyme and application thereof. Background Art

[0002] Escherichia coli (Escherichia coli) is a common Gram-negative bacterium that inhabits the intestines of humans and animals. In recent years, the overuse of antibiotics has led to a growing global problem of drug resistance. As the most common multidrug-resistant bacterium, E. coli poses a serious threat to public health and the livestock industry. Multidrug-resistant E. coli not only spreads among animals but also spreads to humans through the food chain, increasing the difficulty of treating complicated infections. Furthermore, once E. coli enters the human body, it can become a reservoir for E. coli resistance, exacerbating the resistance environment. The increasing problem of bacterial resistance urges the search for new alternatives to antibiotics. Compared to traditional broad-spectrum antibiotics, bacteriophages possess a natural ability to lyse bacteria, exhibit high specificity, enhance efficacy, and minimize side effects. These phages offer a natural basis for developing antimicrobial agents and are therefore considered one of the most promising and viable solutions to combat bacterial antibiotic resistance.

[0003] Endolysins, also known as endolysins, are peptidoglycan hydrolases encoded by bacteriophages. Phage lytic enzymes typically differ in their targeting of Gram-positive and Gram-negative bacteria, primarily due to differences in their domain structure. Lytic enzymes from Gram-positive phages possess a dual-domain structure, consisting of an N-terminal catalytic domain (CD) and a C-terminal cell wall-binding domain (CBD). CD lytic enzymes possess multiple enzymatic activity domains, each targeting a different cell wall component. These domains include: Muramidase (lysozyme), which cleaves glycosidic bonds (β-1,4 glycosidic bonds) in peptidoglycan; N-acetylmuramidase, which cleaves the bond between N-acetylmuramic acid and N-acetylglucosamine; and N-acetylglucosaminidase, which cleaves the bond between N-acetylglucosamine. Glycylglycine endopeptidase (GEPE) cleaves the peptide bonds connecting glycine bridges in peptidoglycan. The CD is responsible for hydrolyzing the bacterial peptidoglycan layer, while the CBD ensures the lytic enzyme specifically binds to the bacterial cell wall. Gram-positive bacterial lytic enzymes, through their dual-domain structure, effectively hydrolyze the bacterial peptidoglycan layer, thereby killing the bacteria. However, Gram-negative bacterial lytic enzymes typically have only a single catalytic domain and lack a binding domain. Most Gram-negative bacterial lytic enzymes are unable to penetrate the peptidoglycan layer to kill bacteria. This is because Gram-negative bacteria have an outer membrane composed of lipopolysaccharides, phospholipids, outer membrane proteins, and lipoproteins. Consequently, Gram-negative bacterial lytic enzymes require the aid of an outer membrane permeabilizer to penetrate the outer membrane and hydrolyze the peptidoglycan layer to kill the bacteria.

[0004] Lytic enzymes are a new strategy for combating drug-resistant bacterial infections. Compared to bacteriophages themselves, they possess advantages such as highly effective antibacterial activity, low resistance to bacterial resistance, rapid onset of action, efficient biofilm penetration, no damage to beneficial bacteria and biota, and ease of production and modification. Studies have revealed that the three-dimensional structures of various lytic enzymes have been resolved using structural biology techniques (such as X-ray crystallography and cryo-electron microscopy), revealing their catalytic mechanisms and structural characteristics, providing a theoretical basis for further optimization and modification of lytic enzymes. On this basis, genetic engineering can be used to construct and express lytic enzymes with multiple, highly effective antibacterial activities. These enzymes exhibit significant antibacterial effects against a variety of Gram-positive bacteria in vitro and clinically demonstrate significant efficacy against bacterial infections, particularly in the treatment of infections caused by antibiotic-resistant strains. The application of lytic enzymes in the food industry and agriculture has also garnered widespread attention, with broad application prospects in preventing food contamination, extending food shelf life, and controlling plant diseases. Therefore, lytic enzymes are expected to achieve greater breakthroughs in clinical treatment.

[0005] However, there are still many technical problems in the current research on lytic enzyme preparations. The poor solubility and insufficient stability of lytic enzymes limit their clinical application. The presence of the outer membrane of Gram-negative bacteria increases the difficulty of the action of lytic enzymes. Lytic enzymes have shown potential to combat multidrug-resistant bacteria, but the development of bacterial resistance to lytic enzymes is still a problem that needs to be monitored and solved. In response to this, the present invention, based on the accumulation of phage resources in the early stage, mines novel lytic enzymes and transforms them through genetic modification methods to enable them to penetrate the outer membrane of Gram-negative bacteria and improve lysis efficiency, thereby providing basic data and experimental basis for the development of new antibacterial drugs. Summary of the Invention

[0006] The purpose of the present invention is to provide a Gram-negative bacteria lytic enzyme and its application to solve the problems existing in the above-mentioned prior art. The Gram-negative bacteria lytic enzyme provided by the present invention can penetrate the outer membrane of Gram-negative bacteria and has good lytic activity.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a Gram-negative bacteria lysing enzyme. The Gram-negative bacteria lysing enzyme is a lysin having an amino acid sequence as shown in SEQ ID NO.1 or a lysin-Mix having an amino acid sequence as shown in SEQ ID NO.3.

[0009] The present invention also provides a gene encoding the above-mentioned Gram-negative bacterial lytic enzyme. When the Gram-negative bacterial lytic enzyme is the lysin lytic enzyme, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2;

[0010] When the Gram-negative bacteria lytic enzyme is the lytic enzyme lysin-Mix, the nucleotide sequence of the encoding gene is shown as SEQ ID NO.4.

[0011] The present invention also provides a recombinant vector comprising the above-mentioned encoding gene.

[0012] The present invention also provides a recombinant host cell, comprising the above-mentioned recombinant vector.

[0013] The present invention also provides the use of the above-mentioned encoding gene, recombinant vector or recombinant host cell in the preparation of the above-mentioned Gram-negative bacteria lytic enzyme.

[0014] The present invention also provides use of the Gram-negative bacteria lytic enzyme in preparing drugs for resisting Gram-negative bacteria.

[0015] The present invention also provides a drug for resisting Gram-negative bacteria, wherein the active ingredient includes the Gram-negative bacteria lytic enzyme.

[0016] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0017] The present invention also provides use of the Gram-negative bacteria lysing enzyme in the preparation of an environmental disinfectant, wherein the environmental disinfectant has the function of lysing Gram-negative bacteria.

[0018] The present invention also provides an environmental disinfectant, the active ingredient of which includes the above-mentioned Gram-negative bacteria lytic enzyme.

[0019] The present invention discloses the following technical effects:

[0020] This study identified a novel lysin lytic enzyme from a bacteriophage and conducted bioinformatics analysis and modification of this enzyme, resulting in a novel lysin-Mix lytic enzyme. Lysin-Mix is ​​derived by adding KRKRKFFVAIIP to the C-terminus of lysin lytic enzyme. Lysin activity assays revealed that the modified enzyme can penetrate the outer membrane of Gram-negative bacteria and exhibits excellent lytic activity. This study provides technical support for the development of novel antibacterial drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 This is a phylogenetic tree analysis of lysin. KFJDMLJL00007 Endolysin in the figure represents lysin.

[0023] Figure 2 The primary structure diagram of lysin;

[0024] Figure 3 The hydrophilicity arrangement diagram of amino acids in lysin;

[0025] Figure 4 This is the protein secondary structure diagram of lysin;

[0026] Figure 5 This is the predicted structural domain of lysin;

[0027] Figure 6 This is a diagram of the transmembrane domain analysis of the lysin enzyme;

[0028] Figure 7This is the signal peptide analysis diagram of lysin;

[0029] Figure 8 This is the predicted three-dimensional structure of lysin;

[0030] Figure 9 Schematic diagram of the conserved and non-conserved amino acids of lysin;

[0031] Figure 10 This is the three-dimensional distribution diagram of the hydrophilic and hydrophobic amino acids of the lysin enzyme; hydrophobic amino acids are yellow and hydrophilic amino acids are blue;

[0032] Figure 11 This is the three-dimensional distribution diagram of positive and negative charged amino acids in the lysin enzyme; positively charged amino acids are yellow, and negatively charged amino acids are red;

[0033] Figure 12 Electropherogram for enzyme digestion verification;

[0034] Figure 13 The SDS gel electrophoresis diagram of the expressed protein;

[0035] Figure 14 This is the result of the lytic enzyme activity verification experiment. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0038] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0039] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0040] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0041] Example 1

[0042] 1. Experimental Materials

[0043] 1.1 Main experimental reagents (see Table 1)

[0044] Table 1 Main experimental reagents

[0045]

[0046] 1.2 Main experimental instruments (see Table 2)

[0047] Table 2 Main experimental instruments

[0048]

[0049] 1.3 Partial experimental reagent formula

[0050] Binding Buffer: Weigh the drug using an analytical balance and adjust the volume to 1 L with ddH2O. The concentrations of the drug in the solution are: Tris-base 20 mmol, NaCl 30 mmol, imidazole 10 mmol. Adjust the pH to 7.8 with HCl and NaOH.

[0051] Elution Buffer: Weigh the drug using an analytical balance and dilute to 1 L with ddH2O. The concentrations of the drug in the solution are: Tris-base 20 mmol, NaCl 30 mmol, imidazole 60 mmol. Adjust the pH to 7.8 with HCl and NaOH.

[0052] 2. Experimental methods

[0053] 2.1 Discovery of novel lytic enzymes

[0054] The present invention mines a new type of lytic enzyme from the phage resources isolated in the early stage and names it as lysin.

[0055] The amino acid sequence of the lysin enzyme is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2.

[0056] SEQ ID NO.1:

[0057] MVSKVQFNPRSRTDAIFVHCSATKPEMDIGVETIRMWHKQQGWLDVGYHFIIKRDGT VEEGRPVNVVGSHVKDWNSRSVGVCLVGGIDAKGEFEANFTPAQMNSLRNKLADLKVLY PQAEIKAHHDVAPKACPSFDLQRWLTTNELVTSDRG.

[0058] SEQ ID NO.2:

[0059] ATGGTAAGTAAGGTACAGTTCAACCCACGGTCCCGGACTGACGCTATCTTCGTTCACTGTTCGGCTACCAAGCCAGATGGACATCGGGGTAGAGACCATCCGTATGTGGCACAAGCAGCAAGGCTGGCTGGACGTAGGATACCACTTTATCATCAAGCGAGATGGCACTGTGG AAGAGGGTCGCCCGGTCAATGTCGTAGGGTCACACGTTAAGGACTGGAACTCACGGTCTGTAGGCGTCTGCCTTGTAGGTGGAATTGACGCTAAGGGCGAGTTTGAAGCTAACTTCACTCCGGCCCAGATGAACTCACTGCGCAACAAGCTGGCTGACCTGAAGGTCCTGTATCC TCAGGCAGAAATCAAAGCACACCATGACGTAGCACCAAAGGCGTGTCCAAGTTTCGACTTGCAACGCTGGCTGACTACCAACGAACTGGTCACTTCCGACCGAGGCTAA.

[0060] 2.2 Modification and expression of lyase

[0061] 2.2.1 Modification of lyase

[0062] Bioinformatics analysis revealed that adding a positively charged or hydrophobic amino acid to the C-terminus of the amino acid sequence allows the lyase to penetrate the outer membrane of Gram-negative bacteria without the need for penetrating agents such as EDTA. Therefore, the present invention uses the lysin lyase as a basis to modify the lysin lyase: lysin-Mix (the amino acid sequence is shown in SEQ ID NO. 3, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO. 4). The lysin lyase is obtained by adding KRKRKFFVAIIP to the C-terminus of the lysin lyase. This sequence is a polypeptide containing cationic and hydrophobic amino acids.

[0063] SEQ ID NO.3:

[0064] MVSKVQFNPRSRTDAIFVHCSATKPEMDIGVETIRMWHKQQGWLDVGYHFIIKRDGT VEEGRPVNVVGSHVKDWNSRSVGVCLVGGIDAKGEFEANFTPAQMNSLRNKLADLKVLY PQAEIKAHHDVAPKACPSFDLQRWLTTNELVTSDRGKRKRKFFVAIIP.

[0065] SEQ ID NO.4:

[0066] ATGGTGAGTAAAGTCCAGTTTAACCCGCGTAGCCGTACCGATGCCATCTTCGTTCACTGCAGCGCGACCAAACCGGAAATGGATATCGGTGTTGAAACCATTCGTATGTGGCACAAACAGCAGGGTTGGCTGGATGTTGGTTACCACTTCATCATCAAACGTGATGGTACCGTTGAAGAAGGTCGTCCGGTTAACGTTGTTGGTTCTCACGTTAAAGATTGGAACTCTCGTTCTGTTGGTGTTTGCCT GGTTGGTGGTATCGATGCGAAAGGTGAATTTGAAGCGAACTTCACCCCGGCCGAGATGAACAGCCTGCGTAACAAACTGGCGGATCTGAAAGTTCTGTATCGCAGGGCAAATCAAAGCGCACCACGATGTTGCGCCGAAAGCGTGCCCGTCTTTCGATCTGCAGCGTTGGCTGACCACCAACGAACTGGTTACCTCTGATCGTGGTAAACGTAAACGTAAATTCTTCGTTGCGATCATCCCGTAA.

[0067] 2.2.2 Expression of Lysase

[0068] (1) The lytic enzyme coding gene (SEQ ID NO. 2 or SEQ ID NO. 4) was connected to the pET28a(+) vector to obtain the recombinant plasmid, which was sent to a biological company for sequencing. The correct positive clone was selected and stored at -20°C for future use.

[0069] (2) Prokaryotic expression of proteins

[0070] Use the heat shock method to transform the recombinant plasmid into Rosetta (DE3) competent cells: Take the competent cells out from -80℃ and quickly insert them into ice. After 5 minutes, wait for the bacterial block to melt, add the target plasmid, and gently mix it by hand at the bottom of the EP tube (avoid using a gun to suck it up), and let it stand on ice for 25 minutes. Heat shock in a 42℃ water bath for 45 seconds, quickly put it back on ice and let it stand for 2 minutes. Shaking will reduce the transformation efficiency. Add 700μL of sterile culture medium (2YT or LB) without antibiotics to the centrifuge tube, mix well, and recover at 37℃, 200rpm for 60 minutes. Centrifuge at 5000rpm for one minute to collect the bacteria, retain about 100μL of supernatant, gently blow to resuspend the bacterial block and spread it on 2YT (or LB) medium containing the corresponding antibiotics. Place the plate upside down in a 37℃ incubator for overnight culture, and then perform the following operations:

[0071] 1) Micro-inoculation: Prepare 1-3 mL of liquid LB containing the appropriate antibiotic in a breathable test tube or breathable centrifuge tube, inoculate a fresh single colony containing the target plasmid, and shake overnight at 37°C, 200 rpm for 15 hours.

[0072] 2) Large shake inoculation: Inoculate 2% of the small shake solution from the first step into 50 mL of LB medium containing the corresponding antibiotics, shake at 37°C, 220 rpm until the OD 600 The value is 0.5-0.8.

[0073] 3) Blank control sampling: Before adding the inducer IPTG, sample 1 mL of bacterial solution into a 1.5 mL centrifuge tube, centrifuge at 12,000 rpm for 10 minutes, discard the supernatant, and store the precipitate at -20°C until use.

[0074] 4) Adding inducer: Add IPTG to the flask in step (3) to a final concentration of 1 mM and continue shaking at 37°C, 220 rpm for 4 h.

[0075] 5) Harvest the bacteria by centrifugation: Remove the flask from the shaker and centrifuge at 5000 g for 10 minutes at 4°C. Discard the supernatant and store the pellet at -20°C.

[0076] 6) SDS-PAGE analysis of protein expression.

[0077] (3) Identification of protein expression products

[0078] 1) Use 12% precast gel, add 5×SDS loading buffer in proportion to the bacterial sample, denature in a metal bath at 100℃ for 10 minutes, and load the sample.

[0079] 2) First, secure the electrophoresis plate with the electrophoresis clamp and pour in 1× electrophoresis buffer (Tris-Gly). Check that the clamp is secure. After loading the sample, add 1× electrophoresis buffer until the sample wells are covered. Check the power supply polarity and, after confirming that they are correct, turn on the power supply. Use 70V to quickly run the sample through the stacking gel. Once the blue indicator band of bromophenol blue is flush with the separating gel, adjust the voltage to 110V. At this point, determine the position of the target band based on the position of the marker and determine the stopping voltage.

[0080] 3) Remove the plate after electrophoresis and slowly cut the gel using a gel cutter. Place the gel in a running box and add Coomassie Brilliant Blue R250 staining solution until the gel is completely submerged. Stain the gel on a shaker for 30 minutes. Discard the staining solution, gently rinse the box with tap water, and slowly destain with destaining solution. Change the destaining solution every 30 minutes. After the second change, destain overnight.

[0081] 4) Take a photo of the clean decolorized gel and save it.

[0082] (4) Protein expression and fragmentation

[0083] According to the above identification results, strains with better expression levels were selected for expanded culture. The specific experimental operations are the same as those for the prokaryotic expression of the protein above and will not be repeated here. Ultrasonic disruption was used for bacterial disruption. The specific operations were as follows: 500 mL of culture medium was used for expanded culture. After culture, the culture was centrifuged at 4200 rpm for 20 minutes, the supernatant was removed, the bacteria were resuspended with PBS buffer, and the cells were thoroughly disrupted using an ultrasonic disruptor. Clean the ultrasonic disruptor in advance, adjust the parameters to 20 kHz, work for 3 seconds, stop for 2 seconds, and continue for 30 minutes for cell disruption. The homogenate after disruption was centrifuged at 4°C and 12000 rpm for 15 minutes, the supernatant was filtered with a 0.22 μm filter, and stored at 4°C for later use.

[0084] (5) Protein purification and concentration

[0085] Before the experiment, use 20% ethanol and UP water to Clean the system (20% ethanol: Systemwash, B1 wash, A1 wash; UP water: System wash, B1 wash, A1 wash; Bypass; flush the entire system at a flow rate of 5 mL / min for 2 minutes). Then install the Ni NTABeads 6FF chromatography column, taking care to avoid the generation of bubbles and inspect each interface for leaks to ensure the flow path is sealed. Equilibrate the column with 20% ethanol, UP water, 0.5 mol / L NaOH, UP water, 100% Elution Buffer, and 100% Lysis Buffer, respectively, at a flow rate of 3 mL / min. Once equilibration is complete, load the sample and adjust the system flow rate to 1.5 mL / min. After loading, purify the target protein using gradient elution. First, elute with 100% Lysis Buffer until the UV signal drops to 50 mAU. Then, adjust the elution buffer ratio to 2% B, 5% B, 10% B, and 15% B for elution, using a gradient of 5-8 column volumes each, until the UV signal stabilizes. Next, elute with 50% B to obtain the target protein. Finally, rinse the medium with 100% B, terminate the run, and collect samples from each stage for SDS-PAGE verification. Upon completion of the purification process, clean the column and system. Follow the above cleaning procedure in reverse order. Desalt and concentrate the sample eluted with 50% B using a 10K ultrafiltration tube using Lysis Buffer at 4500g at 4°C for 40 min until the imidazole concentration drops below 1 mmol / L and the protein volume is less than 4 mL. Store at 4°C until further use.

[0086] (6) Protein BCA quantification

[0087] BCA is an alkaline water-soluble complex with stable properties. In the alkaline environment it provides, proteins can convert the Cu in the BCA reagent into 2+ Reduction to Cu + , Cu + This is then chelated with the BCA reagent to produce a blue-purple complex with a strong absorption peak at 562nm. By measuring the absorbance and combining it with a standard curve, the protein concentration can be determined. This assay is highly sensitive, simple to operate, and minimally affected by interfering substances such as detergents. Based on this, this experiment used the BCA assay to quantify the final collected target protein, following the instructions for the BCA protein quantification kit.

[0088] 2.3 Lyase antibacterial ability test

[0089] E. coli EIII4 (lysin-derived phage host bacteria) in the logarithmic growth phase was washed three times with sterile saline and then diluted to 10 3 CFU / mL, aliquot 150 μL into four tubes. Add 150 μL of unmodified lysin and modified lysin-Mix lyase at a concentration of 1.5 μg / μL to two tubes, respectively. Add 150 μL of phage as a positive control to one tube, and 150 μL of saline to the remaining tube as a blank control. Mix thoroughly and incubate at 37°C for 1 hour. Then, take 100 μL of the bacterial solution from each tube and evenly spread it on LB solid medium. Plate three replicates and incubate at 37°C for 10 hours before counting the colonies.

[0090] 3. Experimental results and analysis

[0091] 3.1 Experimental Results

[0092] 3.1.1 Physicochemical properties and modification of new phage lytic enzymes

[0093] 1) MEGA phylogenetic tree analysis

[0094] The target protein was analyzed by NCBI Blast analysis, and the 50 sequences with the smallest E values ​​(E values ​​less than 10 5 ) to conduct MEGA evolutionary tree analysis ( Figure 1 ). The analysis found that the closest related proteins had a similarity of 94.6%.

[0095] 2) Primary structure analysis

[0096] The online software ExPasy was used to analyze the isoelectric point, molecular weight, amino acid composition and other primary structures of the lyase. Figure 2 ), and obtain the hydrophilicity analysis of this protein ( Figure 3 ).

[0097] 3) Secondary structure analysis

[0098] The secondary structure of the protein amino acid sequence was analyzed using the online software SOPMA, and it was found that α-helix accounted for 27.63%, extended chain accounted for 15.79%, and irregular coil accounted for 56.58% ( Figure 4 ).

[0099] 4) Transmembrane region and structural domain prediction

[0100] Analysis of the lytic enzyme domain using the online software SMART revealed that it possesses two domains, PGRP and Ami_2, with a high degree of overlap. PGRP is an animal peptidoglycan recognition protein homologous to the Gram-negative phage, bacteriophage T3. The Ami-2 domain possesses zinc-dependent MurNAc-L-alanine amidase activity. It can be assumed that this lytic enzyme only has a CD (peptidoglycan hydrolysis domain), which is consistent with the characteristics of Gram-negative bacteria ( Figure 5 ); Secondary structure analysis was performed using the online software TMHMM, which revealed that the lytic enzyme had no transmembrane domain and signal peptide ( Figure 6 、 Figure 7 ).

[0101] 5) Homology modeling and three-dimensional structure prediction

[0102] The online software SWISS-MODEL was used for homology modeling and tertiary structure prediction. The online server TheConsurf Server was used for analysis and annotation of the conserved and non-conserved amino acids of the natural protein to obtain a three-dimensional structure distribution map ( Figure 8-Figure 9 ).

[0103] 6) Prediction of the three-dimensional distribution of positive and negative charge amino acids and hydrophilic and hydrophobic amino acids

[0104] The obtained PDB was analyzed and edited using PyMOL software to obtain the three-dimensional distribution of the hydrophilicity of amino acids ( Figure 10 ), the three-dimensional distribution of positive and negative amino acids ( Figure 11 ).

[0105] 3.1.2 Modification and expression of lyase

[0106] 1) Enzyme digestion and enzyme ligation

[0107] The target gene was connected to the pET-28a plasmid, such as Figure 12 As shown, the 5000 bp band is the pET-28a plasmid, and the 500 bp band is the target gene.

[0108] 2) Rosetta (DE3) expression competent cell transformation

[0109] The plasmid of the positive clone lysin-Mix was extracted and transformed into Rosetta (DE3) expression competent cell. A single colony was picked and expanded for colony PCR to verify the positive clone. Lysin-Mix was the positive clone.

[0110] 3) Induce Rosetta (DE3) to express competent expression of lytic enzyme

[0111] The positive clones were electrophoresed by SDS-page. Figure 13As shown, the lysin-Mix clone successfully expressed the lytic enzyme, with a clear protein band at around 25KDa, while the protein of lysin-Mix was 23KDa. Therefore, it can be preliminarily determined that the lytic enzyme was successfully expressed.

[0112] 3.1.3 Lyase activity test

[0113] like Figure 14 As shown, compared with the blank control group of normal saline and the unmodified lysin, the number of colonies generated by the addition of lysin was significantly reduced, which was equivalent to the lysis effect of the phage from which it was derived, indicating that the modified lysin-Mix has lysis activity, and the activity is significantly higher than that of the unmodified lysin.

[0114] 3.2 Results Analysis

[0115] 3.2.1 Physicochemical properties and modification of new phage lytic enzymes

[0116] The newly discovered lyase, MurNAc-L-alanine amidase, is a Gram-negative lyase. It lacks an autonomously traversable outer membrane structure and cannot directly penetrate the cell outer membrane for lysis, making it feasible and necessary to modify it. Bioinformatics analysis also revealed that lysin is a lysin from Escherichia coli phage. NCBI analysis revealed that lysin is homologous to 50 proteins with high similarity. However, phylogenetic tree analysis revealed that the closest relative is 94.6% similar, suggesting that it is a novel lysin. Furthermore, no studies have been conducted on the function and activity of its most similar homologous protein. Therefore, lysin is a novel lysin worthy of study in antibacterial applications.

[0117] 3.2.2 Modification and expression of lyase

[0118] This study successfully discovered and modified a novel lysin lysin lyase. By amplifying the target gene and determining the ligation conditions, a lysin-Mix recombinant plasmid was constructed. The antibacterial activity of lysin-Mix was preliminarily verified. The study also found that by adding a peptide containing cationic and hydrophobic amino acids to the C-terminus of the lysin lyase, it was able to autonomously cross the bacterial outer membrane, achieving a highly effective antibacterial effect without the need for an outer membrane permeabilizer.

[0119] In summary, the present invention provides basic data and experimental basis for the development of Gram-negative bacteria lytic enzyme preparations, and provides important technical support for the solution of using lytic enzymes to combat bacterial resistance.

[0120] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A Gram-negative bacterial lytic enzyme, characterized in that The Gram-negative bacteria lysing enzyme is a lysin enzyme having an amino acid sequence as shown in SEQ ID NO.1 or a lysin enzyme lysin-Mix having an amino acid sequence as shown in SEQ ID NO.

3.

2. A gene encoding a Gram-negative bacterial lytic enzyme according to claim 1, characterized in that: When the Gram-negative bacterial lytic enzyme is the lytic enzyme lysin, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2; When the Gram-negative bacteria lytic enzyme is the lytic enzyme lysin-Mix, the nucleotide sequence of the encoding gene is shown as SEQ ID NO.

4.

3. A recombinant vector, characterized in that Comprising the coding gene according to claim 2.

4. A recombinant host cell, characterized in that Comprising the recombinant vector according to claim 3.

5. Use of the encoding gene according to claim 2, the recombinant vector according to claim 3 or the recombinant host cell according to claim 4 in preparing the Gram-negative bacteria lytic enzyme according to claim 1.

6. Use of the Gram-negative bacteria lytic enzyme according to claim 1 in the preparation of drugs against Gram-negative bacteria.

7. A drug for use against Gram-negative bacteria, characterized in that: The active ingredient comprises the Gram-negative bacteria lytic enzyme according to claim 1.

8. The drug according to claim 7, characterized in that The drug also includes pharmaceutically acceptable excipients.

9. Use of the Gram-negative bacteria lytic enzyme according to claim 1 in the preparation of an environmental disinfectant, characterized in that: The environmental disinfectant has the function of lysing Gram-negative bacteria.

10. An environmental disinfectant, characterized in that The active ingredient comprises the Gram-negative bacteria lytic enzyme according to claim 1.

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