A fermenting lactobacillus mucus phage lytic enzyme lysLFP01 and a preparation method and application thereof

Abstract

The present application belongs to the field of fermentation engineering and genetic engineering technology, and particularly relates to a fermentation Lactobacillus muciadis bacteriophage lytic enzyme LysLFP01, a preparation method and application thereof. The present application provides a fermentation Lactobacillus muciadis bacteriophage lytic enzyme LysLFP01, and provides a method for preparing the fermentation Lactobacillus muciadis bacteriophage lytic enzyme LysLFP01 by using a prokaryotic expression system. It is found that the fermentation Lactobacillus muciadis bacteriophage lytic enzyme LysLFP01 has a wider host spectrum than the bacteriophage from which it is derived, and can lyse various gram-positive bacteria including fermentation Lactobacillus muciadis, Streptococcus thermophilus and Staphylococcus aureus. Not only does the fermentation Lactobacillus muciadis bacteriophage lytic enzyme LysLFP01 have an obvious clearing effect on the biofilm of gram-positive bacteria, but also has a good inhibitory effect on gram-positive bacteria.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of fermentation engineering and genetic engineering, and particularly relates to a fermentation Lactobacillus muciadis bacteriophage lytic enzyme LysLFP01 and a preparation method and application thereof. BACKGROUND

[0002] Bacteriophage is a kind of virus capable of infecting microorganisms such as bacteria, and it can quickly lyse host bacteria through a binary lysis system of "perforin-lytic enzyme". Therefore, bacteriophage and its derived lytic enzyme can be used as an alternative antibacterial agent to antibiotics. Bacteriophage has high specificity, and its lysis spectrum is generally narrow. Moreover, because the host bacteria can quickly evolve resistance to the surface receptor, the application of bacteriophage is limited.

[0003] Bacteriophage lytic enzyme is a kind of cell wall hydrolytic enzyme expressed by double-stranded DNA bacteriophage in the late stage of infection of host cells. The lytic enzyme has the following advantages in "killing" host bacteria: first, compared with the bacteriophage from which the lytic enzyme is derived, the lytic enzyme has a wider lysis spectrum, and bacteria are difficult to develop resistance to the lytic enzyme; second, the lytic enzyme has a fast lysis rate, and has strong peptidoglycan degrading activity, and can immediately destroy the cell wall structure of the host bacteria after contacting with the host bacteria, thereby quickly killing the bacteria; third, the lytic enzyme has a synergistic antibacterial effect when used in combination with other antibacterial agents such as antibiotics; fourth, compared with bacteriophage and other antibacterial agents, the lytic enzyme is safer, more stable in nature, and easier to control. At present, many studies have confirmed that the lytic enzyme has great bactericidal potential. In view of the many advantages of the lytic enzyme, it has high development value in the fields of agriculture, food safety, medical health and the like.

[0004] Biofilm is a large number of bacterial aggregates formed by bacteria adhering to different material interfaces in order to adapt to the environment. Biofilm provides a protective lifestyle for bacteria, and is difficult to remove once formed. At present, some chemical cleaning agents that can efficiently remove biofilm have appeared on the market. However, although the chemical cleaning agents can efficiently remove biofilm, they can cause certain harm to the human body and have an impact on the environment and equipment. Although it is pointed out in the prior art that bacteriophage and bacteriophage lytic enzyme have a removing effect on biofilm, however, the existing bacteriophage lytic enzyme has poor environmental tolerance, a narrow host spectrum and low activity. SUMMARY

[0005] The present application aims to provide a fermentation Lactobacillus muciadis bacteriophage lytic enzyme LysLFP01 and a preparation method and application thereof. The fermentation Lactobacillus muciadis bacteriophage lytic enzyme LysLFP01 has good environmental tolerance and a wide host spectrum, not only has an obvious removing effect on biofilm of gram-positive bacteria, but also has a good inhibitory effect on gram-positive bacteria.

[0006] The application provides a fermenting Lactobacillus muciadis bacteriophage lytic enzyme LysLFP01, and the amino acid sequence of the fermenting Lactobacillus muciadis bacteriophage lytic enzyme LysLFP01 is shown as SEQ ID No. 1.

[0007] The application also provides a gene for encoding the fermenting Lactobacillus muciadis bacteriophage lytic enzyme LysLFP01, and the nucleotide sequence of the gene is shown as SEQ ID No. 2.

[0008] The application also provides a preparation method of the fermenting Lactobacillus muciadis bacteriophage lytic enzyme LysLFP01, and the preparation method comprises the following steps: preparing the fermenting Lactobacillus muciadis bacteriophage lytic enzyme LysLFP01 by using a prokaryotic expression system.

[0009] Preferably, the step of preparing the fermenting Lactobacillus muciadis bacteriophage lytic enzyme LysLFP01 by using a prokaryotic expression system comprises the following steps:

[0010] connecting the gene for encoding the fermenting Lactobacillus muciadis bacteriophage lytic enzyme LysLFP01 to a prokaryotic expression vector to obtain a recombinant expression vector;

[0011] transforming the recombinant expression vector into Escherichia coli to obtain a recombinant engineering bacterium;

[0012] inducing expression of the recombinant engineering bacterium to obtain an inclusion body;

[0013] renaturing the inclusion body, purifying the inclusion body after renaturation, and obtaining the fermenting Lactobacillus muciadis bacteriophage lytic enzyme LysLFP01.

[0014] Preferably, the gene is a coding gene of an amino acid sequence after removing a signal peptide of the fermenting Lactobacillus muciadis bacteriophage lytic enzyme LysLFP01;

[0015] the starting vector of the recombinant expression vector comprises a pET30a plasmid; and the gene is inserted between NdeI and Xho I enzyme cutting sites of the pET30a plasmid.

[0016] Preferably, a nucleotide sequence for coding a 6×His tag is connected to the gene, and the nucleotide sequence for coding the 6×His tag is located upstream of a stop codon and connected with the stop codon.

[0017] The application also provides a recombinant expression vector or an engineering bacterium, and the recombinant expression vector comprises a gene for encoding the fermenting Lactobacillus muciadis bacteriophage lytic enzyme LysLFP01 in the above technical solution and a starting vector.

[0018] The engineering bacteria comprise a gene encoding the phage lytic enzyme LysLFP01 of the L. fermentum described in the above technical solution or the recombinant expression vector.

[0019] The application also provides the application of the phage lytic enzyme LysLFP01 of the L. fermentum, the gene, the phage lytic enzyme LysLFP01 prepared by the preparation method, or the recombinant expression vector or the engineering bacteria in removing the biofilm of gram-positive bacteria and / or inhibiting gram-positive bacteria.

[0020] Preferably, the gram-positive bacteria comprise one or more of L. fermentum, Streptococcus thermophilus, Staphylococcus aureus, Lactobacillus plantarum, Lactobacillus delbrueckii bulgaricus, Lactobacillus kefiri, Lactobacillus vini, Lactobacillus paracasei and Lactobacillus pentosus.

[0021] The application also provides a L. fermentum IMAU32646, and the preservation number of the L. fermentum IMAU32646 is CGMCC No.30285.

[0022] Beneficial effects:

[0023] The application provides a phage lytic enzyme LysLFP01 of L. fermentum, and provides a coding gene of the phage lytic enzyme LysLFP01 of L. fermentum, wherein the amino acid sequence of the phage lytic enzyme LysLFP01 of L. fermentum is shown as SEQ ID No.1; and the nucleotide sequence of the coding gene is shown as SEQ ID No.2.

[0024] On this basis, the application also provides a method for preparing the phage lytic enzyme LysLFP01 of L. fermentum by using a prokaryotic expression system, and it is found that the phage lytic enzyme LysLFP01 has a wider host spectrum than the phage from which it is derived, and can lyse a plurality of gram-positive bacteria including L. fermentum, Streptococcus thermophilus and Staphylococcus aureus; not only has an obvious effect on removing the biofilm of gram-positive bacteria, but also has a good inhibitory effect on gram-positive bacteria.

[0025] Biological material preservation information

[0026] The fermenting Limosilactobacillus fermentum IMAU32646 provided by the present application was preserved in the China General Microbiological Culture Collection Center on April 9, 2024, the preservation unit is abbreviated as CGMCC, the address of the preservation unit is No. 1, Beichen West Road, Haidian District, Beijing, China, and the preservation number is CGMCC No. 30285. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below.

[0028] Figure 1 The signal peptide prediction analysis was performed on the fermenting Limosilactobacillus fermentum bacteriophage lytic enzyme LysLFP01 amino acid sequence in Example 1.

[0029] Figure 2 The SDS-PAGE result figure of the prokaryotic expression identification of the fermenting Limosilactobacillus fermentum bacteriophage lytic enzyme LysLFP01 in Example 1 is shown, wherein lane M is a protein molecular weight standard; lane 1 is a plasmid pET30a (empty vector) induction; lane 2 is uninduced; lane 3 is after induction, lane 4 is supernatant after induction and crushing, and lane 5 is precipitate after induction and crushing.

[0030] Figure 3 The fermenting Limosilactobacillus fermentum bacteriophage lytic enzyme LysLFP01 protein purification SDS-PAGE analysis result figure in Example 1 is shown, wherein lane M is a protein molecular weight standard; lane 1 is a crushing after treatment sample, lane 2 is a flow-out liquid, and lanes 3-5 are eluents.

[0031] Figure 4 The determination result of the lytic activity of the fermenting Limosilactobacillus fermentum bacteriophage lytic enzyme LysLFP01 in Example 2 at different concentrations is shown.

[0032] Figure 5 The effect of pH value on the lytic activity of the fermenting Limosilactobacillus fermentum bacteriophage lytic enzyme LysLFP01 in Example 4 is shown.

[0033] Figure 6 The effect of temperature on the lytic activity of the fermenting Limosilactobacillus fermentum bacteriophage lytic enzyme LysLFP01 in Example 4 is shown.

[0034] Figure 7 The effect of the fermenting Limosilactobacillus fermentum bacteriophage lytic enzyme LysLFP01 on mature biofilm in Example 5 is shown.

[0035] Figure 8For the Example 6, the observation of the elimination of biofilm by the fermenting Lactobacillus muciadis bacteriophage lytic enzyme LysLFP01 by scanning electron microscope;

[0036] Figure 9 For the Example 7, the inhibition of bacteria in raw milk by the fermenting Lactobacillus muciadis bacteriophage lytic enzyme LysLFP01;

[0037] Figure 10 For the Example 4, the alignment of the 16S rRNA sequence of the strain IMAU32646 with the NCBI nt database. DETAILED DESCRIPTION

[0038] The present application provides a fermenting Lactobacillus muciadis bacteriophage lytic enzyme LysLFP01, the amino acid sequence of which is shown as SEQ ID No. 1. The amino acid sequence shown as SEQ ID No. 1 in the present application is specifically MQKMKMIKRGAASVAIALLLLPMS DTAHATTAKHYGVDWSKYQGNAGKWGYDRDDFSISQIGGYYNGYFVPQTTYGTQVANTIALNKRAHTYIYAQFSGTAQADQMLDYYLPRVQTPKGSIVMLDVESGNPDTDSVLYALKRVQDSGFTAVLYGYRSFLVNHIDLASIAKQYPLALAEYKDYNVTTEPDYNYFPSFNNVQLFQFTSTYVAGGLDGDVDFTGITENGYKGGNAQKPKTTTPAITTGKQLHQDTHNYTVKSGDTLSTIASRYGMTVNALVILNGIQNANLIYPGQTLRVADSGTGSTVTNKATTPITSTGTQDYTVRYGDTLSGIASRYGTSISALASLNGIGNPNRIYPGQVLKLSGGSSTRSYTVRSGDTLSGIASRLGTSWTSLKAKNGLANANLIYPGQTLYY.

[0039]

[0040] The application further provides a preparation method of the fermenting Lactobacillus muciadis bacteriophage lytic enzyme LysLFP01, which comprises: preparing the fermenting Lactobacillus muciadis bacteriophage lytic enzyme LysLFP01 by using a prokaryotic expression system.

[0041] Preferably, the step of preparing the fermenting Lactobacillus muciadis bacteriophage lytic enzyme LysLFP01 by using a prokaryotic expression system comprises the following steps:

[0042] connecting a gene encoding the fermenting Lactobacillus muciadis bacteriophage lytic enzyme LysLFP01 to a prokaryotic expression vector to obtain a recombinant expression vector;

[0043] transforming the recombinant expression vector into E. coli to obtain a recombinant engineering bacterium;

[0044] inducing expression of the recombinant engineering bacterium to obtain inclusion bodies;

[0045] renaturing the inclusion bodies, purifying the renatured inclusion bodies, and obtaining the fermenting Lactobacillus muciadis bacteriophage lytic enzyme LysLFP01.

[0046] The application connects a gene encoding the fermenting Lactobacillus muciadis bacteriophage lytic enzyme LysLFP01 to a prokaryotic expression vector to obtain a recombinant expression vector. The gene encoding the fermenting Lactobacillus muciadis bacteriophage lytic enzyme LysLFP01 is preferably a coding gene of an amino acid sequence after removing a signal peptide of the fermenting Lactobacillus muciadis bacteriophage lytic enzyme LysLFP01, so as to facilitate prokaryotic expression of the fermenting Lactobacillus muciadis bacteriophage lytic enzyme LysLFP01, and more preferably a nucleotide sequence removing a coding sequence of a signal peptide based on the nucleotide sequence shown in SEQ ID No. 1, and the amino acid sequence of the signal peptide is specifically 5'-MQKMKMIKRGAASVAIALLLLPMSDTAHA-3'(SEQ ID No. 3). The prokaryotic expression vector preferably comprises a pET30a plasmid, and the gene is preferably inserted between Nde I and Xho I enzyme cutting sites of the pET30a plasmid. When the prokaryotic expression vector is constructed, a nucleotide sequence encoding a 6×His tag is preferably connected to the gene, so as to be used for subsequent purification of the fermenting Lactobacillus muciadis bacteriophage lytic enzyme LysLFP01; the nucleotide sequence encoding the 6×His tag is located upstream of a stop codon and connected to the stop codon; and the amino acid sequence encoding the 6×His tag is specifically HHHHHH (SEQ ID No. 4). The construction process of the recombinant expression vector is not particularly limited in the application, and the conventional steps in the art can be used for construction.

[0047] After obtaining the recombinant expression vector, the present application transforms the recombinant expression vector into E. coli to obtain a recombinant engineering bacteria. The present application does not have special limitations on the step of transforming the recombinant expression vector into E. coli, and a conventional step in the art can be used.

[0048] After obtaining the recombinant engineering bacteria, the present application induces expression of the recombinant engineering bacteria to obtain induced bacteria. The present application preferably induces expression of the recombinant engineering bacteria using IPTG. The final concentration of the IPTG in the bacterial solution of the recombinant engineering bacteria is preferably 0.2 mM. The OD value of the bacterial solution of the recombinant engineering bacteria is preferably 0.6-0.8. The present application does not have special limitations on the specific steps of inducing expression using IPTG, and a conventional step of IPTG induction in the art can be used. 600

[0049] After obtaining the induced bacteria, the present application preferably resuspends, ultrasonically breaks and centrifugates the induced bacteria to obtain the inclusion bodies in the bacterial precipitate. The present application preferably resuspends the induced bacteria using PBS buffer, and the steps of ultrasonic breaking and centrifugation do not have special limitations, and a conventional step of ultrasonic breaking and centrifugation in the art can be used.

[0050] After obtaining the bacterial precipitate, the present application resuspends the bacterial precipitate in a lysis solution, ultrasonically breaks and centrifugates, and collects the precipitate as the inclusion bodies. The lysis solution of the present application preferably includes the following components at the following concentrations: 20 mM Tris-HCl, 1 mM PMSF and bacteria protease inhibitor cocktail, and the pH value is 8.0. The power of the ultrasonic breaking of the present application is preferably 400 W, 4 s per run, 8 s intermittently, and preferably 20 min in total. The temperature of the centrifugation of the present application is preferably 4°C, the rotation speed is preferably 10,000 r / min, and the time is preferably 20 min.

[0051] After obtaining the inclusion bodies, the present application preferably washes the inclusion bodies using an inclusion body washing solution, and dissolves the washed inclusion bodies using a dissolution buffer to obtain dissolved inclusion bodies. The inclusion body washing solution of the present application preferably includes the following components: 20 mM Tris, 1 mM EDTA, 2 M urea, 1 M NaCl, 1% Triton X-100, and the pH value is 8.0; the number of washing is preferably 3 times. The dissolution buffer of the present application preferably includes the following components: 20 mM Tris, 5 mM DTT, 0.15 M NaCl and 8 M urea, and the pH value is preferably 8.0.

[0052] ​After obtaining the dissolved inclusion body, the present application preferably centrifuges the dissolved inclusion body after overnight standing, and collects the supernatant. The temperature of the overnight standing is preferably 4℃, and the temperature of the centrifugation is preferably room temperature, the rotation speed is preferably 10000r / min, and the time is preferably 15min.

[0053] After obtaining the supernatant, the present application preferably adds the supernatant dropwise to a dialysis buffer for dissolution, and packs the obtained protein solution into a dialysis bag for dialysis overnight, and collects the effluent. The present application preferably dilutes the supernatant into the dialysis buffer in a gradient manner, and with stirring; the dialysis buffer preferably comprises the following components: 20mM Tris-HCl, 0.15M NaCl, and pH value is 8.0.

[0054] After obtaining the effluent, the present application preferably purifies the effluent to obtain the LysLFP01 bacteriophage lyase of Fermentative Lactamucor. The specific type of the purification of the present application is not particularly limited, and the corresponding purification is performed according to the type of the purification tag used, such as in an embodiment of the present application, the 6×His tag added when constructing the recombinant expression vector is selected to purify by a Ni column.

[0055] The present application also provides a recombinant expression vector or an engineering bacterium, wherein the recombinant expression vector comprises a gene encoding the LysLFP01 bacteriophage lyase of Fermentative Lactamucor in the above technical solution and a starting vector; and the engineering bacterium comprises the gene encoding the LysLFP01 bacteriophage lyase of Fermentative Lactamucor in the above technical solution or the recombinant expression vector. The prokaryotic expression vector of the present application preferably comprises a pET30a plasmid; and the gene is preferably inserted between the Nde I and Xho I enzyme cutting sites of the pET30a plasmid. The initial strain of the engineering bacterium of the present application preferably comprises Escherichia coli. The specific steps for constructing the recombinant expression vector or the engineering bacterium of the present application are not particularly limited, and the construction steps of the conventional recombinant expression vector or engineering bacterium in the art can be used.

[0056] The application also provides the application of the bacteriophage lytic enzyme LysLFP01 of Lactobacillus fermentum, the gene, the bacteriophage lytic enzyme LysLFP01 prepared by the preparation method, or the recombinant expression vector or the engineering bacteria in removing the biofilm of gram-positive bacteria and / or inhibiting gram-positive bacteria, and more preferably in removing the biofilm of gram-positive bacteria and inhibiting gram-positive bacteria. The inhibition of gram-positive bacteria preferably includes the inhibition of gram-positive bacteria in dairy products. The gram-positive bacteria preferably include, but are not limited to, one or more of one or more of Lactobacillus fermentum, Streptococcus thermophilus, Staphylococcus aureus, Lactobacillus plantarum, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus kefiri, Lactobacillus kefiranofaciens, Paracaseicola caseica and Lactobacillus pentosus, and further preferably Lactobacillus fermentum, Streptococcus thermophilus and Staphylococcus aureus; and the Lactobacillus fermentum preferably includes, but is not limited to, Lactobacillus fermentum IMAU32646.

[0057] The application finds that the bacteriophage lytic enzyme LysLFP01 of Lactobacillus fermentum has a wider host spectrum than the bacteriophage from which it is derived, and can lyse various gram-positive bacteria including Lactobacillus fermentum, Streptococcus thermophilus and Staphylococcus aureus; not only has an obvious effect on removing the biofilm of gram-positive bacteria, but also has a good inhibitory effect on gram-positive bacteria.

[0058] The application also provides Lactobacillus fermentum IMAU32646, and the preservation number of the Lactobacillus fermentum IMAU32646 is CGMCC No.30285. The Lactobacillus fermentum IMAU32646 is preferably isolated from an acid milk sample in Tianshan Township, Zhaosu County, Yili Kazakh Autonomous Prefecture, Xinjiang.

[0059] In order to further illustrate the application, the technical solutions provided by the application are described in detail below in combination with the drawings and examples, but they should not be understood as limiting the protection scope of the application.

[0060] Example 1

[0061] A preparation method of a bacteriophage lytic enzyme LysLFP01, and the steps are as follows:

[0062] The bacteriophage lytic enzyme gene is from a Lactobacillus fermentum bacteriophage LFP01, and after sequencing the whole genome, the result is uploaded to the NCBI database, and the accession number is OR048821. The original coding sequence of the bacteriophage lytic enzyme LysLFP01 is obtained by blast comparison, as shown in SEQ ID No.1. The signal peptide of the amino acid sequence is analyzed, as shown in SEQ ID No.2. Figure 1As shown, 1-29aa is a signal peptide sequence, and truncating the signal peptide makes it more conducive to prokaryotic expression in host bacteria;

[0063] The construction of the recombinant expression vector pET30a-LysLFP01 by artificial synthesis of the target gene is as follows:

[0064] 1) After truncating the 1-29aa signal peptide sequence, the nucleotide sequence encoding the 6xHis tag is connected to the C-terminal of the gene encoding the truncated sequence, and a stop codon taa is added. Then the target gene is artificially synthesized and cloned into the Nde I and Xho I enzyme cutting sites of the pET30a plasmid to obtain the recombinant expression vector pET30a-LysLFP01.

[0065] 2) The recombinant plasmid pET30a-LysLFP01 is transformed into the competent E. coli BL21 (DE3) by heat shock method, and the positive transformant of the recombinant strain is screened and identified to obtain the recombinant expression E. coli strain BL21 (DE3)-pET30a-LysLFP01 containing the lytic enzyme gene, which is as follows:

[0066] The competent cells BL21 (DE3) taken out from -80℃ are quickly placed in the prepared ice box, and after melting, 1 μL of the recombinant plasmid is added to 100 μL of the competent cells, which are shaken gently and uniformly, and placed on ice for 30 min. The competent cells BL21 (DE3) after ice bath are placed in the prepared 42℃ water bath for heat shock for 90 s, then quickly placed in ice for 5 min, and finally 600 μL of LB medium is added to the inner part in the clean bench, and cultured in a shaker at 37℃, 200 rpm for 2 h. The competent cells BL21 (DE3) after centrifugation are resuspended with a small amount of culture medium, and then uniformly coated on the LB plate containing 50 μg / mL Kan with a coating rod, and cultured in an incubator at 37℃ for overnight. The colonies with good growth on the resistant plate are picked out and placed in sterilized liquid medium containing Kan, and cultured at 37℃, 200 rpm for 2 h. The amplified bacterial liquid is sent to a sequencing company for sequencing.

[0067] 3) IPTG induces the expression of the recombinant bacterial fusion protein, which is as follows:

[0068] The identified positive transformant monoclonal is inoculated in a 3 mL LB medium containing 50 μg / mL Kan at 37℃, 200 r / min for overnight. The next day, it is inoculated in 30 mL of LB culture medium containing 50 μg / mL Kan at 1:100, and cultured at 37℃, 200 r / min until the OD 600The OD600 value was 0.6-0.8. 1 mL of the culture was removed and centrifuged at 10,000 r / min for 2 min at room temperature, and the supernatant was discarded. The bacterial pellet was resuspended with 100 μL of 1x loading buffer. IPTG was added to the remaining culture to a final concentration of 0.2 mM, and the culture was shaken at 15°C and 200 r / min overnight to induce expression of the fusion protein.

[0069] 1 mL of the culture was removed and centrifuged at 10,000 r / min for 2 min at room temperature, and the supernatant was discarded. The bacterial pellet was resuspended with 100 μL of 1x loading buffer. The remaining culture was centrifuged at 4,000 r / min for 10 min, and the supernatant was discarded. The bacterial pellet was resuspended with PBS. After ultrasonic disruption of the resuspension, the supernatant and the precipitate were resuspended with loading buffer, respectively. Detection and analysis were performed by 12% SDS-PAGE, and the results are shown in Fig. 2. As shown in Fig. 2, the protein was expressed in the form of inclusion bodies in the precipitate. Figure 2

[0070] 4) The inclusion body protein produced was renatured, as follows:

[0071] The bacterial pellet was resuspended in 20 mL of lysis buffer (20 mM Tris-HCl, 1 mM PMSF and bacterial protease inhibitor cocktail, pH 8.0), and ultrasonically disrupted (power 400 W, working for 4 s, intermittent for 8 s, for a total of 20 min). The ultrasonically disrupted cell lysate was centrifuged at 10,000 r / min for 20 min at 4°C, and the precipitate was collected. The inclusion bodies were washed with inclusion body washing buffer (20 mM Tris, 1 mM EDTA, 2 M urea, 1 M NaCl, 1% Triton X-100, pH 8.0) for 3 times. The inclusion bodies were dissolved in a certain proportion of dissolution buffer (20 mM Tris, 5 mM DTT, 0.15 M NaCl, 8 M urea, pH 8.0) and placed at 4°C overnight; centrifuged at 10,000 r / min for 15 min at room temperature. The supernatant was collected. The above solution was added dropwise into 20 mM Tris-HCl, 0.15 M NaCl, pH 8.0 buffer, and gradually diluted by a factor of 2 with slow stirring. The protein solution was loaded into a dialysis bag and dialyzed in 20 mM Tris-HCl, 0.15 M NaCl, pH 8.0 solution overnight.

[0072] 5) The L. mesenteroides bacteriophage lysozyme produced was purified by a Ni column, as follows:

[0073] ​Using low pressure chromatography system, the supernatant solution was loaded into Ni-IDA Binding-Buffer pre-equilibrated Ni-IDA-Sepharose Cl-6B affinity chromatography column at a flow rate of 0.5 mL / min. The flow-through was washed with Ni-IDA Binding-Buffer at a flow rate of 0.5 mL / min until the OD 280 value reached baseline. The flow-through was washed with Ni-IDA Washing-Buffer (20 mM Tris-HCl, 20 mM imidazole, 0.15 M NaCl, 8 M urea, pH 8.0) at a flow rate of 1 mL / min until the OD 280 value reached baseline. The target protein was eluted with Ni-IDA Elution-Buffer (20 mM Tris-HCl, 250 mM imidazole, 0.15 M NaCl, 8 M urea, pH 8.0) at a flow rate of 1 mL / min, and the flow-through was collected. The collected protein solution was added into a dialysis bag and dialyzed into renaturation buffer overnight. After renaturation, the dialyzed solution was stored in PBS. 12% SDS-PAGE analysis was performed, and the results are shown in Figure 3 .

[0074] From the above results, it can be concluded that the bacteriophage lyase LysLFP01 was successfully prepared by prokaryotic expression.

[0075] Example 2

[0076] The bacteriostatic activity of the bacteriophage lyase LysLFP01 in Example 1 was verified by turbidity experiment, as follows:

[0077] The Lactobacillus muciadigestrus was cultured to the early logarithmic phase, and the bacterial cells were collected by centrifugation at 10,000 r / min for 2 min and resuspended in an equal volume of PBS buffer. 100 μL was added to a 96-well plate, and the bacteriophage lyase LysLFP01 prepared in Example 1 was diluted to different concentrations with PBS, and then added to the 96-well plate containing the cell suspension. The OD 600 value was read every 15 min at 37°C in an enzyme marker, and the change in OD 600 value within 135 min was recorded. The results are shown in Figure 4 .

[0078] From the above results, it can be concluded that the bacteriophage lyase LysLFP01 was successfully prepared by prokaryotic expression. Figure 4

[0079] Example 3

[0080] The determination of the lytic spectrum of the bacteriophage lyase LysLFP01 is as follows: ​

[0081] Fermenting Lactobacillus muci, Lactobacillus plantarum, Lactobacillus delbrueckii bulgaricus, Lactobacillus kefiri, Lactobacillus kefiranofaciens, Lactobacillus paracasei, Lactobacillus pentosus, Streptococcus thermophilus, Staphylococcus aureus, Escherichia coli and Salmonella were selected as detection bacteria, a total of 97 strains, and the lysis spectrum of the lytic enzyme was studied by Oxford cup method.

[0082] Two sterilized Oxford cups were placed vertically on the solidified lower layer medium (MRS solid medium for Lactobacillus, M17 solid medium for Streptococcus thermophilus, and NB solid medium for Staphylococcus aureus, Escherichia coli and Salmonella), ensuring that there was no gap between the cup bottom and the medium, then 20 μL of bacteria culture liquid grown in the logarithmic phase was mixed with semi-solid MRS agar medium and poured into the upper layer. After solidification, the Oxford cup was removed, 100 μL of phage lytic enzyme LysLFP01 prepared in Example 1 was injected into the cup hole, and PBS buffer was added as a control, and incubated at 37°C. The presence or absence of a bacteriostatic ring was observed. The determination results are shown in Table 1.

[0083] Table 1 Lysis spectrum of phage lytic enzyme LysLFP01 prepared in Example 1

[0084]

[0085] Note: + represents that the strain can be lysed by the lytic enzyme; - represents that the strain cannot be lysed by the lytic enzyme. IMAU numbers are all laboratory preserved strains, Staphylococcus aureus ATCC 12600 was purchased from American Type Culture Collection, Salmonella CICC 10982 was purchased from China Center of Industrial Culture Collection, and Escherichia coli CMCC 44103 was purchased from National Center for Medical Culture Collection.

[0086] Compared with its source phage LFP01 (the lysis experiment of phage LFP01 is disclosed in the article, DOI number as follows: doi.org / 10.1016 / j.fbio.2024.103604), phage lytic enzyme LysLFP01 can lyse all Gram-positive bacteria covered in the tested bacteria. But it cannot lyse Gram-negative bacteria such as Salmonella and Escherichia coli.

[0087] Example 4

[0088] The effects of pH value and temperature on the lysis activity of phage lytic enzyme LysLFP01 are as follows:

[0089] Figure 10 As can be seen, the 16S rRNA sequence of strain IMAU32646 has 100% similarity to Limosilactobacillus fermentum. Strain IMAU32646 was identified as Limosilactobacillus fermentum, and its accession number at the China General Microbiological Culture Collection Center is CGMCC No. 30285.

[0090] Fermenting Lactobacillus IMAU32646 cultured to the early logarithmic phase was centrifuged at 10000 rpm for 2 min, the supernatant was discarded, and the cells were resuspended in 100 μL PBS buffer to OD200. 600 The initial concentration was 0.5, and then added to a 96-well plate. The phage lysin LysLFP01 prepared in Example 1 was diluted with an equal volume of PBS buffer (pH 2–11) to control the concentration of LysLFP01 to 250 μg / mL, and then added to a 96-well plate containing bacterial cells. The initial OD was measured. 600 The optimal reaction pH value was the one that resulted in the greatest decrease in bacterial turbidity after incubation at 37°C for 2 hours, as shown in the results. Figure 5 As shown.

[0091] Depend on Figure 5 It can be seen that the enzyme has high activity at pH 5-7, and loses its activity at pH 2.

[0092] Similarly, 100 μL of phage lysin LysLFP01 at a concentration of 250 μg / mL was incubated at 4, 25, 37, 50, 63, 72, 90, and 100 °C for 30 min, respectively, and then added to a 96-well plate containing 100 μL of bacterial cells. The plate was incubated at 37 °C for 2 h, and the OD was measured. 600 The optimal reaction temperature was the one where bacterial turbidity decreased the most. Results are as follows: Figure 6 As shown, the lyase exhibits a wide temperature range, showing good activity after treatment at 4–37℃ for 30 min. Activity begins to decrease above 37℃. The effect of untreated lyase on bacterial turbidity serves as a control. OD... 600 The ratio of the difference to the difference in the control group is used to measure the relative activity of the lysin.

[0093] Example 5

[0094] The effects of the phage lysin LysLFP01 prepared in Example 1 on mature biofilms are as follows:

[0095] Place the PVC 96-well plate in the biosafety cabinet in advance and irradiate it with ultraviolet light for 30 minutes.

[0096] Cultured to the end-log phase (OD) 600Staphylococcus aureus (using Staphylococcus aureus ATCC 12600) with a 2% inoculation amount into new 5 mL NB liquid medium, 200 μL of bacterial culture was added to each well. 200 μL of sterile NB medium was used as a negative control. After 48 h of incubation in a constant temperature biochemical incubator at 37°C, the unadhered culture was centrifuged and discarded, washed with PBS buffer for 3 times, 200 μL of phage lytic enzyme LysLFP01 solution (concentrations were 500, 250, 125, 62.5 μg / mL respectively, solvent was PBS buffer with pH value of 7.4) was added, and the reaction was carried out in a constant temperature incubator at 37°C for 1 h. 200 μL of bacterial culture without phage lytic enzyme LysLFP01 was used as a positive control.

[0097] After the incubation was completed, the liquid was discarded and washed, and the formed biofilm was stained with crystal violet: first, 200 μL of methanol was used for fixation for 30 min, the methanol was discarded and dried at room temperature; then 200 μL of 1% crystal violet was used for staining for 30 min; after washing and drying, 200 μL of 33% glacial acetic acid solution was added, and the absorbance was measured at 595 nm by an enzyme-labeled instrument after standing at room temperature for 10 min. The results are shown in Figure 7

[0098] From the above results, it can be concluded that the phage lytic enzyme LysLFP01 has a significant effect on the removal of Staphylococcus aureus biofilm. Figure 7 Example 6.

[0099] The removal effect of the phage lytic enzyme LysLFP01 prepared in Example 1 on the biofilm was observed by scanning electron microscope, as follows:

[0100] Referring to the method in Example 5, the biofilm was cultured in vitro by using a 24-well plate microtiter plate method.

[0101] 200 μL of Staphylococcus aureus ATCC 12600 bacterial solution was added to the wells of a 24-well microtiter plate, and sterile 14 mm diameter cell climbing sheets were added to each well in advance. After 48 h of standing culture, the unadhered culture was discarded, and the biofilm was treated with phage lytic enzyme LysLFP01 with a final concentration of 250 μg / mL and PBS for 1 h. After the liquid was discarded, the wells were washed with PBS for 3 times. Then 2.5% glutaraldehyde was used for fixation, and ethanol was used for dehydration, and then scanning electron microscope was used for observation, and the results are shown in

[0102] Figure 8 From the above results, it can be concluded that the lytic enzyme has a significant effect on the removal of Staphylococcus aureus biofilm.

[0103] Figure 8 From the above results, it can be concluded that the phage lytic enzyme LysLFP01 has a significant effect on the removal of Staphylococcus aureus biofilm. ​​​

[0104] Example 7

[0105] The effect of the bacteriophage lytic enzyme LysLFP01 prepared in Example 1 on bacteria in raw milk is as follows:

[0106] The raw milk was purchased from the fresh milk selling place of Inner Mongolia Agricultural University, and the total number of colonies in the raw milk was preliminarily determined by using PCA medium.

[0107] The raw milk added with the bacteriophage lytic enzyme LysLFP01 (the raw milk and the bacteriophage lytic enzyme LysLFP01 solution (the solvent is PBS buffer with a pH value of 7.4) were mixed at a volume ratio of 3:1, and the concentration of the bacteriophage lytic enzyme LysLFP01 solution was 0.5 mg / mL) was used as the test group, and the raw milk without the bacteriophage lytic enzyme LysLFP01 was used as the control group for the antibacterial experiment. After being fully mixed, they were stored at different temperatures (4, 25 and 37℃), and sampling was performed at 1, 4, 10 and 24 h for counting.

[0108] The results are shown in Table 1. Figure 9 As shown in Table 1, the effect of the bacteriophage lytic enzyme LysLFP01 on bacteria in raw milk is different at different temperatures. When the temperature is 25℃ and 37℃, the growth rate of the blank control group is not much different, but the total number of colonies in the control group is more at 37℃, which may be related to the fact that 37℃ is more suitable for the growth of bacteria in milk. The total number of colonies in the 25℃ experimental group is reduced more than that in the 37℃ experimental group, and the bacteriophage lytic enzyme LysLFP01 is added to reduce 0.3 log after 4 h. When the temperature is 4℃, the total number of colonies in the control group changes little, and only increases by 0.48 log after being placed for 24 h. The total number of colonies in the experimental group decreases most at 10 h, and decreases by 0.92 log. Therefore, the bacteriophage lytic enzyme LysLFP01 has the best antibacterial effect on raw milk at 4℃.

[0109] In summary, the bacteriophage lytic enzyme LysLFP01 in the present application has good environmental tolerance, has a wider host spectrum than the source bacteriophage, and can lyse a variety of gram-positive bacteria. The lytic enzyme not only has a significant clearing effect on the biofilm formed by Staphylococcus aureus, but also has a significant inhibitory effect on bacteria in raw milk.

[0110] Although the above examples have made a detailed description of the present application, it is only a part of the examples of the present application, but not all the examples, and other examples can be obtained under the premise of no creativity according to the present examples, which all belong to the protection scope of the present application.

Claims

1. Use of a bacteriophage lytic enzyme LysLFP01 of Lactobacillus fermentum in inhibiting gram-positive bacteria for non-therapeutic purposes. The preparation steps of the fermenting Lactobacillus muciadis bacteriophage lysing enzyme LysLFP01 include: The gene encoding the bacteriophage lytic enzyme LysLFP01 of Lactobacillus fermentum is expressed by using a prokaryotic expression system to obtain the bacteriophage lytic enzyme LysLFP01 of Lactobacillus fermentum; the amino acid sequence of the bacteriophage lytic enzyme LysLFP01 of Lactobacillus fermentum is shown in SEQ ID No.

1. The gram-positive bacteria include one or more of Lactobacillus fermentum, Streptococcus thermophilus, Staphylococcus aureus, Lactobacillus plantarum, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus kefiri, Lactobacillus kefiranofaciens, Lactobacillus paracasei and Lactobacillus pentosus.

2. Use of a bacteriophage lytic enzyme LysLFP01 of Lactobacillus fermentum in removing Staphylococcus aureus biofilm for non-therapeutic purposes. The preparation steps of the fermenting Lactobacillus muciadis bacteriophage lysing enzyme LysLFP01 include: The gene encoding the bacteriophage lytic enzyme LysLFP01 of Lactobacillus fermentum is expressed by using a prokaryotic expression system to obtain the bacteriophage lytic enzyme LysLFP01 of Lactobacillus fermentum; the amino acid sequence of the bacteriophage lytic enzyme LysLFP01 of Lactobacillus fermentum is shown in SEQ ID No.

1. The concentration of the bacteriophage lytic enzyme LysLFP01 is greater than 125 μg / mL.

3. Use according to claim 1 or 2, characterized in that, The nucleotide sequence of the gene encoding the bacteriophage lytic enzyme LysLFP01 of Lactobacillus fermentum is shown in SEQ ID No.

2.

4. Use according to claim 1 or 2, characterized in that, The step of obtaining the bacteriophage lytic enzyme LysLFP01 of Lactobacillus fermentum includes: connecting the gene encoding the bacteriophage lytic enzyme LysLFP01 of Lactobacillus fermentum to a prokaryotic expression vector to obtain a recombinant expression vector; The recombinant expression vector is transformed into Escherichia coli to obtain a recombinant engineering bacterium; The recombinant engineering bacterium is induced to express to obtain an inclusion body; The inclusion body is renatured, and the renatured inclusion body is purified to obtain the bacteriophage lytic enzyme LysLFP01 of Lactobacillus fermentum.

5. Use according to claim 4, characterized in that, The gene is a coding gene of an amino acid sequence after removing a signal peptide of the bacteriophage lytic enzyme LysLFP01 of Lactobacillus fermentum; The starting vector of the recombinant expression vector includes a pET30a plasmid; the gene is inserted between Nde I and Xho I enzyme cutting sites of the pET30a plasmid.

6. Use according to claim 5, characterized in that, The gene is connected with a nucleotide sequence encoding a 6×His tag, and the nucleotide sequence encoding the 6×His tag is located upstream of a stop codon and connected with the stop codon.

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