A bacteriophage lytic enzyme mutant and its application

Abstract

The present invention discloses a phage lytic enzyme mutant and its application. The present invention constructs a large number of point mutations for En6-1 protein, and screens out a phage lytic enzyme mutant 18# with improved enzyme activity and stronger bactericidal activity against Acinetobacter baumannii. The in vitro minimum inhibitory concentration of phage lytic enzyme mutant 18# against Acinetobacter baumannii is 2.0 μg / mL, which can achieve efficient killing of Acinetobacter baumannii.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and in particular relates to a bacteriophage lytic enzyme mutant and application thereof. Background Art

[0002] Since the advent of penicillin, many antimicrobial drugs have been developed and widely used to combat pathogen infections, but many bacteria have become resistant to these drugs. The declining efficacy of antibiotics against pathogens has made it urgent to find potential alternative antibiotics (Tacconelli et al. 2018). Antibiotic resistance is a major threat to global public health. Unfortunately, the emergence of drug-resistant pathogens is growing rapidly, and the world is moving towards a post-antibiotic era. Acinetobacter baumannii is one of the most important pathogens in clinical practice, and the treatment of drug-resistant isolates has caused serious problems. Acinetobacter baumannii is a Gram-negative bacterium that can cause a range of infections, including wounds, skin and urinary tract infections, pneumonia, and bacteremia (V. Manchanda et al, 2019). In the 2024 World Health Organization (WHO) list of priority pathogens for antibiotic-resistant bacteria, carbapenem-resistant Acinetobacter baumannii has been listed as the top priority.

[0003] Phage lytic enzymes are antimicrobial proteins from bacteriophages. They are released in the late stage of phage infection of host cells and can destroy the peptidoglycan layer in the bacterial cell wall to achieve the purpose of bacterial lysis. According to the different chemical bonds in the peptidoglycan layer of the bacterial cell wall that they act on, they can be divided into endopeptidases, amidases, and glycosidases. Phage lytic enzymes often exhibit a modular structure, usually consisting of a catalytic domain and a cell wall binding domain. However, in the outer layer of the cell wall of Gram-negative bacteria, the peptidoglycan layer is covered by a cell wall outer membrane, which makes them more resistant to the action of phage lytic enzymes than Gram-positive bacteria (Vermassen et al. 2019).

[0004] LysECP26 is a phage lytic enzyme from Escherichia coli phage (Dowon Park, 2020). In previous studies, Chinese patent CN117625658A disclosed a phage lytic enzyme recombinant protein En6-1, which added a cell wall binding domain SP10 (renamed En6-1) to the C-terminus of LysECP26 lytic enzyme, improving its activity against Acinetobacter baumannii. However, the antibacterial effect of the recombinant protein En6-1 on Acinetobacter baumannii was limited. The minimum inhibitory concentration of the recombinant protein En6-1 on Acinetobacter baumannii in vitro was 3.9 μg / mL, which could not achieve efficient killing of Acinetobacter baumannii in the environment. Summary of the invention

[0005] In order to overcome the above-mentioned deficiencies in the prior art, further improve the specific activity of phage lytic enzymes and reduce the inhibitory concentration of phage lytic enzymes against Acinetobacter baumannii, the purpose of the present invention is to provide a phage lytic enzyme mutant with improved enzyme activity and stronger bactericidal activity against Acinetobacter baumannii and its application. The present invention selects the 9th, 63rd, 90th and 130th amino acids in the En6-1 protein for random mutation, and after screening, obtains a phage lytic enzyme mutant 18# with improved enzyme activity, which has better bactericidal activity against Acinetobacter baumannii than the En6-1 protein.

[0006] The purpose of the present invention is achieved by at least one of the following technical solutions.

[0007] The first aspect of the present invention provides a bacteriophage lytic enzyme mutant, wherein the bacteriophage lytic enzyme mutant comprises one of the following features:

[0008] 1) a protein having an amino acid sequence as shown in SEQ ID NO: 6;

[0009] 2) A protein having a sequence identity of 90% or more to SEQ ID NO: 6, and the amino acid sequences at positions 9, 63, 90 and 130 are identical to those of SEQ ID NO: 6.

[0010] Further, the protein in 2) specifically refers to a protein obtained by replacing, deleting or inserting one or more amino acids in the amino acid sequence shown in SEQ ID No: 6, or by adding one or more amino acids at the N-terminus and / or C-terminus, and having the function of the protein shown in the amino acid sequence of SEQ ID NO: 6, and the amino acid sequence in 2) may have a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% with SEQ ID NO: 6. The amino acid sequences at positions 9, 63, 90 and 130 are the relative positions of the four amino acid mutation sites in the protein. For example, when an amino acid is added to the N-terminus, the amino acid sequences at positions 9, 63, 90 and 130 are adjusted to the amino acid sequences at positions 10, 64, 91 and 131 respectively.

[0011] In the present invention, the protein with the amino acid sequence shown in SEQ ID NO: 6 is named as bacteriophage lytic enzyme mutant 18#.

[0012] The second aspect of the present invention is to provide a polynucleotide encoding the aforementioned bacteriophage lytic enzyme mutant.

[0013] Further, the polynucleotide comprises a nucleic acid fragment whose nucleotide sequence is shown in SEQ ID NO: 5; or, the polynucleotide has a sequence identity of 90% or more with SEQ ID NO: 5, and the polynucleotide comprises nucleic acid fragments at positions 25-27, 187-189, 268-270 and 388-390 that are identical to SEQ ID NO: 5.

[0014] The third aspect of the present invention is a primer pair, which is used to amplify the aforementioned polynucleotide.

[0015] Furthermore, the nucleotide sequence of the upstream primer of the primer pair is shown as SEQ ID NO:3, and the nucleotide sequence of the downstream primer of the primer pair is shown as SEQ ID NO:4.

[0016] The fourth aspect of the present invention is to provide a nucleic acid construct, which comprises the aforementioned polynucleotide and a backbone plasmid.

[0017] Furthermore, the backbone plasmid is pET28a(+).

[0018] The fifth aspect of the present invention is to provide a cell, which comprises the aforementioned nucleic acid construct or the aforementioned polynucleotide.

[0019] Furthermore, the cell is selected from Escherichia coli.

[0020] Furthermore, the cell is selected from Escherichia coli DH5α strain or Escherichia coli BL21 strain.

[0021] The sixth aspect of the present invention is to provide a method for preparing the aforementioned phage lytic enzyme mutant, comprising the following steps: (i) culturing the aforementioned cells; (ii) collecting the culture containing the phage lytic enzyme mutant; and (iii) isolating the phage lytic enzyme mutant from the culture.

[0022] The seventh aspect of the present invention is to provide the use of the aforementioned bacteriophage lytic enzyme mutant in the preparation of products against Acinetobacter baumannii, wherein the products include antibacterial agents, drugs, disinfectants or cleaning agents.

[0023] The eighth aspect of the present invention is to provide an antibacterial agent or a drug, the active ingredient of which comprises the aforementioned bacteriophage lytic enzyme mutant.

[0024] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0025] The present invention constructs a large number of point mutations for the En6-1 protein, constructs a random saturation mutation library of the 9th, 63rd, 90th and 130th amino acids in the En6-1 amino acid sequence, prepares a series of En6-1 protein mutants with point mutation sites, and screens out phage lytic enzyme mutants with improved enzyme activity and stronger bactericidal activity against Acinetobacter baumannii, which provides a reference for the molecular modification of other proteins.

[0026] The specific activity of the phage lytic enzyme mutant 18# screened by the present invention has a significantly higher bactericidal activity against Acinetobacter baumannii than that of the En6-1 protein. As shown in the examples of the present invention, at the same protein concentration, the bactericidal rate of the phage lytic enzyme mutant 18# against Acinetobacter baumannii is 99.5%, while the bactericidal rate of the En6-1 protein against Acinetobacter baumannii is only 60.4%; the in vitro minimum inhibitory concentration of the phage lytic enzyme mutant 18# against Acinetobacter baumannii is 2.0 μg / mL, while the in vitro minimum inhibitory concentration of the En6-1 protein sample against Acinetobacter baumannii is 4.0 μg / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a picture of the E. coli BL21 transformants containing the random saturation mutation library in Example 1 cultured on a lactose-induced screening plate at 37° C. for 24 hours.

[0028] Figure 2 This is a bar graph showing the bactericidal rates of the phage lytic enzyme mutant 18# protein sample and the parent protein sample against Acinetobacter baumannii in Example 4.

[0029] Figure 3 This is a bar graph of the in vitro minimum inhibitory concentration of the phage lytic enzyme mutant 18# protein sample and the parent protein sample against Acinetobacter baumannii in Example 5.

[0030] Among them, 18# refers to the phage lytic enzyme mutant 18#, and the parent refers to the En6-1 protein. DETAILED DESCRIPTION

[0031] The present invention provides a bacteriophage lytic enzyme mutant, wherein the bacteriophage lytic enzyme mutant comprises one or more of the following characteristics:

[0032] 1) a protein having an amino acid sequence as shown in SEQ ID NO: 6;

[0033] 2) A protein having a sequence identity of 90% or more to SEQ ID NO: 6 and having the function of the bacteriophage lytic enzyme mutant defined in 1).

[0034] Further, the protein in 2) specifically refers to a protein obtained by replacing, deleting or inserting one or more (usually 1-20, preferably 1-10, and even more preferably 1-8, 1-5, 1-3 or 1-2) amino acids in the amino acid sequence shown in SEQ ID No: 6, or adding one or more (usually within 20, preferably within 10, and even more preferably within 5) amino acids at the N-terminus and / or C-terminus, and having the function of the protein shown in the amino acid sequence of SEQ ID NO: 6. The amino acid sequences at positions 9, 63, 90 and 130 are the relative positions of the four amino acid mutation sites in the protein. For example, when an amino acid is added to the N-terminus, the amino acid sequences at positions 9, 63, 90 and 130 are adjusted to the amino acid sequences at positions 10, 64, 91 and 131 respectively. The amino acid sequence in 2) may have a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more than 99% with SEQ ID NO:6.

[0035] In some specific embodiments, the phage lytic enzyme mutant may further comprise a secretion tag, which may be a peptide segment that affects the protein, such as increasing protein secretion, fluorescent labeling, increasing protein solubility and expression.

[0036] In some embodiments, the bacteriophage lytic enzyme mutant comprises a histidine tag. A histidine tag or polyhistidine tag is a sequence of 2 to 20 histidine residues attached to a bacteriophage lytic enzyme mutant. The histidine tag can be used to purify the protein by a chromatography method using a nickel-based chromatography medium.

[0037] In some embodiments, the bacteriophage lytic enzyme mutant comprises a fluorescent protein tag. In one embodiment, the bacteriophage lytic enzyme mutant comprises green fluorescent protein (GFP) and / or red fluorescent protein (RFP).

[0038] In some embodiments, the phage lytic enzyme mutant further comprises a protease cleavage site. The protease cleavage site can be used to cleave the recombinantly produced phage lytic enzyme mutant to remove a portion of the protein. The removable portion of the polypeptide includes a secretion tag, a histidine tag, a fluorescent protein tag and / or a beta-lactamase. Proteases include endoproteases, exoproteases, serine proteases, cysteine ​​proteases, threonine proteases, aspartic proteases, glutamic proteases and metalloproteases.

[0039] The present invention also provides a polynucleotide encoding the aforementioned bacteriophage lytic enzyme mutant.

[0040] In some specific embodiments, the polynucleotide comprises a nucleic acid fragment having a nucleotide sequence as shown in SEQ ID NO:5.

[0041] In some specific embodiments, the polynucleotide may also be a nucleic acid fragment having a sequence identity of 90% or more to SEQ ID NO:5, and having the function of the nucleotide sequence defined by SEQ ID NO:5.

[0042] Furthermore, the nucleic acid fragment specifically refers to: a nucleic acid fragment obtained by replacing, deleting or adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5 or 1-3) nucleotides of the nucleic acid sequence as shown in SEQ ID NO: 5, or adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5 or 1-3) nucleotides at the 5' end and / or 3' end, and having the function of a nucleic acid fragment with a nucleotide sequence as shown in SEQ ID NO: 5, and the nucleotide sequence in the nucleic acid fragment may have a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more with SEQ ID NO: 5.

[0043] The polynucleotide sequences of the present invention can be used to express or produce recombinant bacteriophage lytic enzyme mutants by conventional recombinant DNA techniques. Generally, the following steps are involved:

[0044] (1) transforming or transducing a suitable host cell with the polynucleotide (or variant) encoding the bacteriophage lytic enzyme mutant of the present invention, or with a recombinant expression vector containing the polynucleotide;

[0045] (2) Host cells cultured in a suitable culture medium;

[0046] (3) Isolate and purify proteins from culture medium or cells.

[0047] The present invention also provides a primer pair, which is used to amplify the aforementioned polynucleotide.

[0048] In some specific embodiments, the nucleotide sequence of the upstream primer of the primer pair is shown as SEQ ID NO:3; the nucleotide sequence of the downstream primer of the primer pair is shown as SEQ ID NO:4.

[0049] The present invention also provides a nucleic acid construct, which comprises the aforementioned polynucleotide and a backbone plasmid.

[0050] In some specific embodiments, the backbone plasmid is selected from pET expression vector, pCW expression vector, pUC expression vector, pAO815, pPIC9, pPIC9K, pPIC3.5, pPIC3.5K, pPICZαA, pPICZαB, pPICZαC, pGAPZαA, pGAPZαB, pGAPZαC, pPICZ A, pPICZ B, pPICZ C, pGAPZ A, pGAPZ B or pGAPZ C. Preferably, the backbone plasmid is selected from pET expression vector. In one embodiment, the backbone plasmid is pET28a (+).

[0051] In the present application, a backbone plasmid is a circular DNA molecule or a linear DNA molecule that can autonomously replicate and express an inserted target gene in a cell. The backbone plasmid may contain regulatory sequences such as promoters, replicons, transcription and translation start and stop codons. The backbone plasmid is usually connected to the target gene to form a complete expression vector that can express a specific output in a cell.

[0052] The present invention also provides a cell, comprising the aforementioned nucleic acid construct or the aforementioned polynucleotide.

[0053] In some embodiments, the cell is selected from bacteria or yeast cells. More specifically, the bacteria is selected from Escherichia coli, Lactobacillus, Bacillus licheniformis, Bacillus subtilis or Streptomyces; the yeast cell is selected from Saccharomyces cerevisiae, Candida, Torulopsis, Rhodotorula, Hansenula, Pichia pastoris or Kluyveromyces. Preferably, the cell is selected from Escherichia coli. In a specific embodiment, the cell is selected from Escherichia coli DH5α strain or Escherichia coli BL21 strain.

[0054] The present invention also provides a method for preparing the aforementioned phage lytic enzyme mutant, comprising the following steps: (i) culturing the aforementioned cells; (ii) collecting the culture containing the phage lytic enzyme mutant; and (iii) isolating the phage lytic enzyme mutant from the culture.

[0055] The present invention also provides the use of the aforementioned bacteriophage lytic enzyme mutant in the preparation of products against Acinetobacter baumannii, wherein the products include antibacterial agents, drugs, disinfectants or cleaning agents.

[0056] The present invention also provides an antibacterial agent or a medicine, wherein the active ingredient of the antibacterial agent or the medicine comprises the aforementioned bacteriophage lytic enzyme mutant.

[0057] As used herein, the term "homology" or "identity" or "similarity" refers to the sequence similarity between two proteins or between two nucleic acid molecules. Homology can be determined by comparing corresponding positions in different proteins or nucleic acid molecules. When the same position in the sequence of the compared molecules is occupied by the same base or amino acid in the different sequences, then the molecules are homologous at that position. The degree of homology between sequences is determined as a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence should have less than 20% homology to one of the sequences disclosed herein.

[0058] A polynucleotide or polynucleotide region (or protein or protein region) has a certain percentage of sequence homology (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%) with another polynucleotide or polynucleotide region (or protein or protein region) means that when compared, the percentage of bases (or amino acids) in the two compared sequences are the same. The comparison and percentage homology or sequence identity can be determined using software programs and methods known in the art.

[0059] In the present invention, the terms "polynucleotide" and "oligonucleotide" are used interchangeably, and they refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or their analogs. "Polynucleotide" refers to both double-stranded and single-stranded polynucleotide molecules. Unless otherwise specified or required, any embodiment of the polynucleotide disclosed in the present invention includes any of its double-stranded form and two complementary single-stranded forms known or predicted to constitute the double-stranded form.

[0060] In the present invention, the terms "peptide", "polypeptide" and "protein" are used interchangeably in the present invention and refer to a polymeric form of amino acids of any length, which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones.

[0061] As used herein, if it is necessary to represent a protein with an amino acid sequence as shown in SEQ ID NO: 6, it will be labeled as "bacteriophage lytic enzyme mutant 18#", "bacteriophage lytic enzyme mutant 18# protein" or "18#".

[0062] As used herein, where reference is made to the En6-1 protein, it will be designated as the "parent protein" or "parent".

[0063] The specific implementation of the present invention is further described below in conjunction with the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that if there are processes that are not particularly described in detail below, they can be implemented or understood by those skilled in the art with reference to the prior art. If the manufacturer of the reagents or instruments used is not indicated, they are deemed to be conventional products that can be purchased commercially.

[0064] The sequence information of the amino acid sequence or nucleotide sequence used in this application is as follows:

[0065] SEQ ID NO: 1 En6-1 protein nucleotide sequence:

[0066] ATGGCTAAACTGGATAAAAACGTGCAGCGCAAAGCGTTTCTGGATATGATTGCGTATAGCGAAGGCACCGATAACGGCCGTCAGCCGACCAAAGATCGCGGCTATGATGTGATTGTGGGCGGCAAACTGTTTAGCGACTATAGCAAACATCCGGGCGTGTATGTGAAACTGAACGCGAAACTGACGAGCAGCGCGGCGGGCCGCTATCAGATTCTGGAAAAATTTGCGAAACACTATATGGAACAGCTGGGCCTGCCGGATTTTGGCCCGGCGAGCCAAGATAAAATTGCGCTGCAGCTGATTCGCGAATGCAAAGCGCTGACCGATATTGATGAAGGCCGCATTCATGAAGCGATTCGCAAATGCCGCAGCCGCTGGGCGAGCCTGCCGGGCGCGGGCTATGGTCAGCATGAACAGAAAATTGAAAAACTGATTGAAGTGTTTAAAAAAGCGGGCGGCATTGTGCTGGATGGATCCAATAGTACCTCTAATAGTAGCACCAATTCAGGCTCTACCGGTAAAGTGAGTCTGCCGAATCGTGTGATTCGCGTGACCAAACCGATTGTTCATGGTAGCGATGTGCTGGCAATTCAGAAAGCACTGTCAAGCCTGTATTTTTATCCGGAAAAAGGTGCCAAAGATAATGGTTCCGATAGCTATTATGGTCCGAAAACCGCCAATGCCGTTAAACGCTTTCAGTCTGTTAATGGCTTAGTTGCAGATGGCGTGTATGGCCCGAAAACCCGCGCAGCCATTCTGAAAAAACTGTAA

[0067] SEQ ID NO: 2 Amino acid sequence of En6-1 protein

[0068] MAKLDKNVQRKAFLDMIAYSEGTDNGRQPTKDRGYDVIVGGKLFSDYSKHPGVYVKLNAKLTSSAAGRYQILEKFAKHYMEQLGLPDFGPASQDKIALQLIRECKALTDIDEGRIHEAIRKCRSRWAS LPGAGYGQHEQKIEKLIEVFKKAGGIVLDGSNSTSNSSTNSGSTGKVSLPNRVIRVTKPIVHGSDVLAIQKALSSLYFYPEKGAKDNGSDSYYGPKTANAVKRFQSVNGLVADGVYGPKTRAAILKKL

[0069] SEQ ID NO: 3 Upstream primer T7 nucleotide sequence:

[0070] TAATACGACTCACTATAGGG

[0071] SEQ ID NO: 4 Downstream primer T7t nucleotide sequence:

[0072] TGCTAGTTATTGCTCAGCGG

[0073] SEQ ID NO: 5 Phage lytic enzyme mutant 18# nucleotide sequence:

[0074] ATGGCTAAACTGGATAAAAACGTGCGGCGCAAAGCGTTTCTGGATATGATTGCGTATAGCGAAGGCACCGATAACGGCCGTCAGCCGACCAAAGATCGCGGCTATGATGTGATTGTGGGCGGCAAACTGTTTAGCGACTATAGCAAACATCCGGGCGTGTATGTGAAACTGAACGCGAAACTGACGAGGAGCGCGGCGGGCCGCTATCAGATTCTGGAAAAATTTGCGAAACACTATATGGAACAGCTGGGCCTGCCGGATTTTGGCCTGGCGAGCCAAGATAAAATTGCGCTGCAGCTGATTCGCGAATGCAAAGCGCTGACCGATATTGATGAAGGCCGCATTCATGAAGCGATTCGCAAATGCCGCAGCCGCTGGGCGAGCCTGGCGGGCGCGGGCTATGGTCAGCATGAACAGAAAATTGAAAAACTGATTGAAGTGTTTAAAAAAGCGGGCGGCATTGTGCTGGATGGATCCAATAGTACCTCTAATAGTAGCACCAATTCAGGCTCTACCGGTAAAGTGAGTCTGCCGAATCGTGTGATTCGCGTGACCAAACCGATTGTTCATGGTAGCGATGTGCTGGCAATTCAGAAAGCACTGTCAAGCCTGTATTTTTATCCGGAAAAAGGTGCCAAAGATAATGGTTCCGATAGCTATTATGGTCCGAAAACCGCCAATGCCGTTAAACGCTTTCAGTCTGTTAATGGCTTAGTTGCAGATGGCGTGTATGGCCCGAAAACCCGCGCAGCCATTCTGAAAAAACTGTAA

[0075] SEQ ID NO: 6 Amino acid sequence of phage lysozyme mutant 18#:

[0076] AKLDKNVRRKAFLDMIAYSEGTDNGRQPTKDRGYDVIVGGKLFSDYSKHPGVYVKLNAKLTRSAAGRYQILEKFAKHYMEQLGLPDFGLASQDKIALQLIRECKALTDIDEGRIHEAIRKCRSRWAS LAGAGYGQHEQKIEKLIEVFKKAGGIVLDGSNSTSNSSTNSGSTGKVSLPNRVIRVTKPIVHGSDVLAIQKALSSLYFYPEKGAKDNGSDSYYGPKTANAVKRFQSVNGLVADGVYGPKTRAAILKKL

[0077] Experimental Materials

[0078] 1. Experimental strains and plasmids

[0079] Bacteria strains: Escherichia coli DH5α and Escherichia coli BL21 were purchased from Nanjing Novazon Biotechnology Co., Ltd.; Acinetobacter baumannii ATCC 19606 was purchased from Guangdong Provincial Microbiological Culture Collection Center.

[0080] Plasmid: pET28a-En6-1 was stored in our laboratory.

[0081] 2. Culture medium and screening plates

[0082] LB liquid medium: 0.5% yeast extract, 1% tryptone, 1% NaCl, pH 7.0.

[0083] LB solid medium: Add 2.0% agar to LB liquid medium.

[0084] Lactose induction medium: 2.5% tryptone, 1.25% yeast extract, 0.75% NaCl, 0.6% ammonium chloride, 0.6 lactose.

[0085] Lactose-induced screening plate: Acinetobacter baumannii (ATCC 19606) was cultured overnight in a shaker at 37°C and 200 rpm, and the bacterial solution was collected by centrifugation at 5000 rpm, washed once with sterile water, and fixed to a final concentration of 5 OD / mL. 2× fermentation medium (2.5% tryptone, 1.25% yeast extract, 0.75% NaCl, 0.6% ammonium chloride, 0.6% lactose, 2% agar) and Baumannin bacteria solution were sterilized separately and mixed in a 1:1 ratio, and poured into plates, with each plate containing about 20 mL.

[0086] All culture media and solutions were sterilized at 121°C for 20 min.

[0087] Protein concentration determination method (Lowry method)

[0088] Reagent preparation:

[0089] Preparation of standard solution:

[0090] Take 1 bottle of bovine serum albumin standard and prepare it to 0.2 mg / ml.

[0091] Alkaline copper test solution:

[0092] Liquid A: Take 10 g of sodium hydroxide and 50 g of anhydrous sodium carbonate, add 400 ml of water to dissolve.

[0093] Liquid B: Take 0.5 g of potassium tartrate and add 50 ml of water to dissolve it; take 0.25 g of copper sulfate and add 30 ml of water to dissolve it. Mix the two liquids as Liquid B.

[0094] Before use, combine Solution A and Solution B and add water to 500 ml to obtain the alkaline copper test solution.

[0095] Folin-phenol test solution: Take the phenol reagent stock solution (2 mol / L acid concentration) in the Folin-phenol kit, prepare it before use, and store it away from light.

[0096] Make a standard curve:

[0097] Take 200 μg / mL BSA standard protein solution, add reagents in the order of Table 1, add alkaline copper test solution, mix well, leave at room temperature for 10 min, then add 2 ml of Folin phenol test solution, mix immediately, leave at room temperature for 30 min, and then measure the absorbance at a wavelength of 650 nm, with tube 0 as the blank. Calculate the linear regression equation based on the absorbance corresponding to the concentration of the standard solution and draw a standard curve.

[0098] Table 1 Amount and order of reagent addition to each test tube of the standard curve

[0099]

[0100] The linear regression equation was calculated based on the concentration of the standard solution and its corresponding absorbance.

[0101] The regression curve is A=KC+B, and the correlation coefficient should be greater than 99.5%.

[0102] Where K is the slope of the standard curve; B is the intercept; A is the absorbance; C is the concentration of the standard (mg / mL)

[0103] Sample protein concentration determination:

[0104] Take a sample, dilute it to about 0.2 mg / mL, add 0.25 mL of sample, and determine it in the same way. Calculate the sample protein concentration according to the following formula: Sample protein concentration C0 (mg / mL) = (C 测 ×N) / v

[0105] Where: C0------is the actual protein content of the sample (mg / mL) 测 ------Protein obtained from the standard curve (mg / mL)

[0106] N------is the sample dilution multiple v------the sample volume during measurement (mL)

[0107] 4. Purification of target protein by CMFF

[0108] Buffer preparation:

[0109] Equilibration solution: 20 mM phosphate buffer (pH 7.2).

[0110] Regeneration solution (1M NaCl): Weigh 58.44 g of NaCl and dissolve it, then dilute to 1 L with purified water.

[0111] Eluent 1 (0.4M NaCl): Prepare by mixing 300 mL of equilibration solution with 200 mL of 1M NaCl solution.

[0112] Eluent 2 (0.7M NaCl): Prepare by mixing 150 mL of the equilibration solution with 350 mL of 1M NaCl solution.

[0113] Disinfectant (0.5M NaOH): weigh 20 g of NaOH and dilute to 500 mL with purified water.

[0114] Preservation solution (20% ethanol): Measure 200 mL of ethanol and add purified water to make up to 1 L.

[0115] Column Packing:

[0116] Pour 50 mL of CMFF weakly cationic gel filler (purchased from Si Tuofan) into the chromatography column, let it stand for about 30 min, aspirate the upper suspended gel, add purified water to resuspend, let it stand for 30 min, then tighten the top, adjust the injection flow rate to 5 mL / min, slowly press the filler down, maintain the pressure at 0.2 MPa, stop when the distributor is close to the filler, increase the flow rate, and maintain the pressure at 0.2 MPa for 30 min.

[0117] The fermentation broth containing the target protein was centrifuged at 8 000 rpm and 4 °C for 30 min, and the supernatant was collected and diluted with purified water. The pH of the supernatant was adjusted to 7.0 and the conductivity was adjusted to 7.5 S·m -1 , and obtain the loading solution containing the target protein.

[0118] Table 2 CMFF purification steps for target protein

[0119]

[0120] Example 1 Construction and screening of saturation mutant library

[0121] Using the expression vector pET28a-En6-1 (the preparation method of the expression vector pET28a-En6-1 can refer to Chinese patent CN117625658A) as a template, General Biotech Co., Ltd. was commissioned to perform random saturation mutations on the 9th, 63rd, 90th, and 130th amino acids in the amino acid sequence of En6-1 to construct a random saturation mutation library (En6-1 nucleotide sequence and amino acid sequence are SEQ ID NO: 1 and SEQ ID NO: 2, respectively). The random saturation mutation library was transformed into the Escherichia coli DH5α strain, and all the Escherichia coli transformants grown were mixed and inoculated into LB liquid medium (containing 30 μg / mL kanamycin) and cultured at 37°C, 180 rpm for 4 h. The library plasmid was extracted using a plasmid extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., plasmid extraction kit DP103), and transformed into the E. coli BL21 expression host to obtain the transformation liquid. The transformation liquid was spread on LB solid medium (containing 30 μg / mL kanamycin) and cultured at 37°C for 16 hours to obtain E. coli BL21 transformants containing the random saturation mutation library. Use a sterile toothpick to pick the transformants on the culture medium onto a lactose induction screening plate and culture at 37°C for 24 hours. The transformant with the largest hydrolysis circle was selected and numbered 18# ( Figure 1 The phage lytic enzyme mutant in the 18# transformant was named phage lytic enzyme mutant 18#.

[0122] Example 2 Sequencing analysis of bacteriophage lytic enzyme mutant 18#

[0123] The 18# transformant was inoculated into 5 mL LB liquid medium (containing 30 μg / mL kanamycin), cultured overnight at 37°C and 180 rpm, and genomic DNA was extracted using a bacterial genome extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., Bacterial Genomic DNA Extraction Kit (DP302)). PCR amplification was performed using the genomic DNA of the 18# transformant as a template. The reaction system was: 25 μL 2×Phanta Max Master Mix (Nanjing Novogene Biotechnology Co., Ltd.); 2 μL upstream primer T7 (10 μM); 2 μL downstream primer T7t (10 μM); 2 μL genomic DNA; 19 μL ddH2O. The PCR reaction program was: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 s; 56°C annealing for 15 s; 72°C extension for 1 min; 35 cycles, and finally 72°C extension for 5 min. The PCR product of the phage lytic enzyme in the 18# transformant was obtained. The PCR products were sent to Suzhou Genewise Biotechnology Co., Ltd. for sequencing and analysis.

[0124] T7:TAATACGACTCACTATAGGG (SEQ ID NO: 3)

[0125] T7t: TGCTAGTTATTGCTCAGCGG (SEQ ID NO: 4)

[0126] The sequencing results of the nucleotide sequence encoding the bacteriophage lytic enzyme mutant 18# are shown in SEQ ID NO: 5: The coding sequence of the bacteriophage lytic enzyme in the 18# transformant has 4 base mutations, and the amino acid sequence encoded by it is shown in SEQ ID NO: 6. The sequencing results show that the 9th, 63rd, 90th, and 130th amino acids in its amino acid sequence have mutated from the original glutamine, serine, proline, and proline to arginine, arginine, leucine, and alanine, respectively.

[0127] Example 3 Shake flask fermentation and purification of bacteriophage lytic enzyme mutant 18#

[0128] Inoculate 18# transformant and parent strain (Escherichia coli BL21 strain containing expression vector pET28a-En6-1, preparation method can refer to Chinese patent CN117625658A) into 5 mL LB liquid medium (containing 30 μg / mL kanamycin), 37°C, 180 rpm, culture for 4 h, and then inoculate 1% into 50 mL lactose induction medium. Culture overnight (about 16 h) at 30°C, 200 rpm, lactose induces the expression of target protein. Centrifuge the fermentation broth at 4°C, 12 000 rpm for 10 min, and collect the supernatant. Dilute the fermentation supernatant by one half, adjust the pH to 7.2, and purify the target protein using a CMFF weak cation exchange column. The obtained sample was dialyzed and desalted using a dialysis bag with a molecular weight cutoff of 10 KDa (dialysis buffer was 50 mM Tris, pH 8.0). The protein concentration after dialysis was determined by the Lowry method, and the protein concentration was diluted to 1 mg / mL to obtain 1 mg / mL phage lytic enzyme mutant 18# protein sample and parent protein (En6-1 protein) sample.

[0129] Example 4 Determination of bacteriocidal activity of bacteriophage lytic enzyme mutant 18#

[0130] According to the Disinfection Technical Specifications - 2002 Edition 2.1.1.7.4, the suspension quantitative bactericidal test was carried out. The Acinetobacter baumannii activated overnight on LB solid medium was inoculated into 5 mL LB liquid medium, cultured at 37°C, 180 rpm, and the bacterial concentration was measured with a bacterial turbidity meter after 5 h of incubation. The bacterial concentration was controlled at 1-5×10 8 CFU / mL. Take 500 μL of bacterial solution to a 15 mL centrifuge tube, add 500 μL of 20 mM PBS (pH 7.2) and mix well, and place at room temperature for 5 min. Add 4 mL of the 18# protein sample and the parent protein sample obtained in Example 3, mix quickly and place in a 37°C reaction box. After 1 hour, take 0.5 mL to a 15 mL centrifuge tube, add 4.5 mL of neutralizer (DE neutralization broth (Guangdong Huankai Microbiological Technology Co., Ltd., 022014)), neutralize for 10 min, perform 10-fold gradient dilution, and take 1.0 mL of sample solution for pouring plate method live bacteria count. After culturing in a 37°C incubator for 48 h, count the bacterial colonies, and perform three parallels in each group. Use 20 mM PBS solution as a positive control.

[0131] Bactericidal rate (%) = (number of colonies in the experimental group / number of colonies in the positive control group) × 100%.

[0132] The killing rates of phage lytic enzyme mutant 18# protein sample and parent protein sample against Acinetobacter baumannii are as follows Figure 2As shown in the figure, the killing rate of the phage lytic enzyme mutant 18# protein sample against Acinetobacter baumannii was 99.5%, and the killing rate of the parent protein sample was 60.4%. At the same protein concentration (protein concentration 500 μg / mL), the killing rate of the phage lytic enzyme mutant 18# protein sample against Acinetobacter baumannii increased by 64.7% compared with the parent sample, that is, the specific activity of the phage lytic enzyme mutant 18# was higher than that of the En6-1 protein.

[0133] Example 5 In vitro minimum inhibitory concentration (MIC) determination

[0134] The overnight cultured Acinetobacter baumannii was diluted to 1.0 OD / mL with 2×LB liquid medium, and then diluted to 10 4 CFU / mL, take 100 μL of bacterial solution to a 96-well cell culture plate. The phage lytic enzyme mutant 18# protein sample and the parent protein sample with a protein concentration of 1 mg / mL obtained in Example 3 were diluted twice with 20 mM PBS (pH 7.2), and 100 μL of phage lytic enzyme mutant 18# protein sample and the parent protein sample with different dilutions were taken to a 96-well plate, mixed with the test bacterial solution, and cultured at 37 °C for 48 h. LB liquid culture medium was used as a negative control. Three parallels were set for each well.

[0135] The results of in vitro MIC determination experiments are as follows Figure 3 As shown: The MIC of the phage lytic enzyme mutant 18# protein sample against Acinetobacter baumannii is 2.0 μg / mL. The MIC of the parent protein sample against Acinetobacter baumannii is 4.0 μg / mL. The bactericidal rate of the phage lytic enzyme mutant 18# mutant protein sample against Acinetobacter baumannii increased by 100% compared with the parent sample.

[0136] The above embodiments are only preferred implementation modes of the present invention and are only used to explain the present invention rather than to limit the present invention. Any changes, substitutions, modifications, etc. made by those skilled in the art without departing from the spirit of the present invention should fall within the scope of protection of the present invention.

Claims

1. A bacteriophage lytic enzyme mutant, characterized in that: The bacteriophage lytic enzyme mutant is a protein with an amino acid sequence as shown in SEQ ID NO:

6.

2. A polynucleotide, characterized in that The polynucleotide encodes the bacteriophage lytic enzyme mutant according to claim 1; The polynucleotide is a nucleic acid fragment whose nucleotide sequence is shown in SEQ ID NO:

5.

3. A nucleic acid construct, characterized in that The nucleic acid construct comprises the polynucleotide according to claim 2 and a backbone plasmid.

4. The nucleic acid construct according to claim 3, characterized in that The backbone plasmid is pET28a(+).

5. A cell, characterized in that The cell comprises the polynucleotide of claim 2 or the nucleic acid construct of any one of claims 3-4.

6. The cell according to claim 5, characterized in that The cell is selected from Escherichia coli, and contains the polynucleotide according to claim 2 or the nucleic acid construct according to any one of claims 3-4.

7. The method for preparing a bacteriophage lytic enzyme mutant according to claim 1, characterized in that: The following steps are involved: (i) culturing the cell according to any one of claims 5 to 6; (ii) collecting a culture containing the bacteriophage lytic enzyme mutant; and (iii) isolating the bacteriophage lytic enzyme mutant from the culture.

8. Use of the bacteriophage lytic enzyme mutant according to claim 1 in the preparation of a product against Acinetobacter baumannii, characterized in that: The products include bacteriostatic agents, drugs, disinfectants or cleaning agents.

9. An antibacterial agent or drug, characterized in that: The active ingredient of the bacteriostatic agent or drug comprises the bacteriophage lytic enzyme mutant according to claim 1.

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