A method for surface immobilization of bacteriophage lytic enzymes that infect gram-negative bacteria

Abstract

The present application relates to a kind of methods for surface immobilization of bacteriophage lytic enzyme that infects gram-negative bacteria, the present application utilizes bacteriophage lytic enzyme LysSSE1 that can infect G-bacteria, by molecular cloning technology, amino acid type connector and / or affinity ligand SiAP1 / SiAP2 screened from database are introduced from the perspective of molecular biology, construct recombinant lytic enzyme containing peptide chain, then incubated with NaCl and Tween 20, 100 rpm oscillation 30-60 min.The method of the present application successfully realizes the stable and efficient sterilization of lytic enzyme in external environment, the immobilized enzyme containing amino acid chain obtained not only maintains the activity of original lytic enzyme, but also improves its resistance to environmental change and repeated sterilization ability, solves the problem of difficult addition of chemical type connector and environmental unfriendliness etc.It has important practical significance for effectively controlling the surface attachment pollution of G-bacteria in environment.

Description

Technical Field

[0001] This invention relates to a method for surface immobilization of bacteriophage lysins that infect Gram-negative bacteria, belonging to the field of biotechnology. Background Technology

[0002] Bacterial contamination is primarily transmitted through drinking water and food, posing a serious threat to human health and the environment. Traditional antibacterial technologies lack specificity, and long-term use easily leads to bacterial resistance. Currently, the main reason why pathogenic bacterial contamination is difficult to control is the emergence of multidrug-resistant bacteria and the increasing resistance of microorganisms to antibiotics and other treatments. Therefore, the development of novel antibacterial methods is urgently needed. Lyases, a type of bacterial cell wall hydrolytic enzyme encoded by bacteriophages, can efficiently recognize and degrade peptidoglycan, the main component of bacterial cell walls, specifically killing bacteria. Compared with traditional drugs, they are environmentally friendly and less likely to induce bacterial resistance, possessing public health strategic value in reducing and lowering resistance. Bacteriophage lyases have been identified as potential candidates for the specific treatment of bacterial infections in subjects, thus making them a potential candidate for sterilization and inhibition of bacterial surface adhesion. However, as proteins, lyases have poor stability in external applications and are difficult to withstand complex and changing external environments, which limits their bactericidal applications. Enzyme immobilization technology can effectively improve enzyme stability, but it restricts the enzyme's freedom of movement. When acting on the peptidoglycan of the target bacterial cell wall, it cannot flexibly adjust its conformation, which easily leads to the immobilized enzyme having lower activity than the free enzyme.

[0003] Studies have shown that inserting flexible connectors between enzymes and vectors can ensure the enzyme's freedom of movement and help maintain the conformation of the native enzyme. Current reports on lyase immobilization technology mainly focus on phage lyases targeting Gram-positive (G+) bacteria, and most are achieved through covalent immobilization or embedding with chemical connectors. However, pathogenic bacteria in the environment, such as Escherichia coli and Shigella, are Gram-negative (G-). Furthermore, the addition of chemical connectors is subject to stringent conditions and is environmentally unfriendly, typically requiring multiple enzyme purifications before and after insertion. In addition, most chemical connectors lack selectivity at enzyme attachment sites, easily leading to confusion between protein structure and surface attachment sites.

[0004] Therefore, finding a suitable and efficient method for immobilizing phage lysins to control Gram-negative bacterial contamination is of great significance. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for surface immobilization of bacteriophage lysins that infect Gram-negative bacteria. Invention Overview

[0006] This invention utilizes the phage lyase LysSSE1, which can infect Gram-negative bacteria, and employs molecular cloning technology to introduce amino acid linkers and / or affinity ligands SiAP1 / SiAP2 screened from databases from a molecular biology perspective, constructing a recombinant lyase containing peptide chains to achieve surface immobilization of the lyase. This solves the problems of difficulty in incorporating chemical linkers and environmental unfriendliness. The lyase LysSSE1 used in this invention has a modular structure and has been proven to have activity against both Gram-negative and Gram-positive bacteria; all raw materials used are safe and pollution-free. The enzyme immobilization method of this invention maximizes the enzyme activity of the lyase during external sterilization and improves its bactericidal stability and long-lasting effect, effectively controlling surface contamination from Gram-negative bacteria in the environment. A method for surface immobilization of bacteriophage lysins infecting Gram-negative bacteria, comprising the following steps:

[0007] (1) Construct a recombinant plasmid containing a lyase gene based on molecular cloning technology, and then use PCR and molecular cloning to introduce an amino acid chain and / or affinity ligand SiAP1 / SiAP2 to construct a single peptide chain or a double peptide chain recombinant lyase. (2) Mix the recombinant lyase of the single peptide chain or dipeptide chain constructed in step (1) with NaCl and Tween 20, incubate with silica carrier, and wash with sterile PBS after incubation to complete surface fixation.

[0008] According to a preferred embodiment of the present invention, in step (1), the lysin is phage lysin LysSSE1, which is encoded by Shigella phage and has the activity of killing Shigella, Escherichia coli and Staphylococcus aureus.

[0009] According to a preferred embodiment of the present invention, in step (1), the vector plasmid of the lyase is pGS21a, and the recombinant plasmid containing the lyase gene is pGS21a-LysSSE1 constructed based on molecular cloning technology.

[0010] According to a preferred embodiment of the present invention, in step (1), the introduced amino acid chain is NL (AGYGKAGGTVTPTPNT) or GSA (GSAGSAAGSG).

[0011] The affinity ligands of the present invention have silica affinity functions and are used to construct recombinant lyases containing different amino acid chains and affinity ligands.

[0012] According to a preferred embodiment of the present invention, in step (1), when the amino acid chain is NL, the recombinant lyase containing a single peptide chain is LysSSE1-NL, and the recombinant lyase containing a double peptide chain is LysSSE1-NL-SiAP1 or LysSSE1-NL-SiAP2.

[0013] Most preferably, in step (1), the recombinant lyase constructed is LysSSE1-NL-SiAP1 or LysSSE1-NL-SiAP2.

[0014] According to a preferred embodiment of the present invention, in step (1), when the amino acid chain is GSA, the recombinant lyase containing a single peptide chain is constructed as LysSSE1-GSA, and the recombinant lyase containing a double peptide chain is constructed as LysSSE1-GSA-SiAP1 or LysSSE1-GSA-SiAP2.

[0015] Most preferably, in step (1), the recombinant lyase constructed is LysSSE1-GSA-SiAP1 or LysSSE1-GSA-SiAP2.

[0016] According to a preferred embodiment of the present invention, in step (2), the concentration of NaCl in the mixture of NaCl and Tween 20 is 400 mM.

[0017] According to a preferred embodiment of the present invention, in step (2), the concentration of Tween 20 in the mixture of NaCl and Tween 20 is 0.3-0.8%.

[0018] Most preferably, in step (2), the concentration of Tween 20 in the mixture of NaCl and Tween 20 is 0.3% (w / v).

[0019] According to a preferred embodiment of the present invention, in step (2), the silica carrier is three 24×24mm glass slides.

[0020] According to a preferred embodiment of the present invention, in step (2), the incubation is performed by oscillating at 100 rpm for 30-60 min. This invention utilizes phage lyase, a biomaterial that specifically kills Gram-negative bacteria, to construct recombinant lyases containing single or double peptide chains through gene cloning, and successfully immobilizes them onto a silica surface. All materials and methods are safe, pollution-free, and environmentally friendly.

[0021] This invention utilizes multiple amino acid chains as connectors for lyase immobilization. Their insertion does not affect the protein structure and action site, and can maintain the degree of freedom of movement of the immobilized lyase during sterilization, thus ensuring the bactericidal activity of the immobilized lyase.

[0022] This invention utilizes molecular biology techniques to construct an immobilized lyase, which, while maintaining the bactericidal activity of the free enzyme, further improves the bactericidal stability and resistance to environmental changes in the in vitro environment, and also enhances its repeated bactericidal performance. The resulting immobilized lyase has the advantages of stable and long-lasting bactericidal effects, and this technology can be extended to other object surfaces besides silica, which is of great value for the large-scale use of lyases. Attached Figure Description

[0023] Figure 1 This is a simplified structural diagram of the various recombinant lyases constructed according to the present invention. His represents the affinity tag, NL and GSA represent different amino acid-linked peptides, and SiAP represents the silica affinity ligand.

[0024] The amino acid sequence of the phage lysin LysSSE1 (NCBI accession number: QBZ70927.1) is as follows: MNIFEMLRNDEGLRLTLYKDTEGFWTIGIGHLVTKNPSLAVAKAELDRMIGRKCNGTITLDEAEKLFNEDVDKAVRGILGNAKLKPVYDSLDAVRRCALVNMVFQMGVAGVAGFTNSLRMLQQKRWDEAAVNLAQSKWYRQTPNRAKRVISTFKTGTWKEYI. Figure 2 The enzyme activity of the recombinant lyase in Experiment Example 1 was characterized based on absorbance. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto.

[0026] In this embodiment, recombinant lyases containing amino acid linker peptides and silica affinity ligands were constructed using gene cloning technology. The lyases were then immobilized onto a silica surface via covalent affinity bonding to construct immobilized lyases. The effects of environmental factors on the bactericidal performance of the immobilized lyases were analyzed, and the immobilized lyase with the best performance was obtained.

[0027] PCR and molecular cloning are existing technologies.

[0028] SDS-PAGE and Western Blot analysis are existing technologies. Example 1

[0029] The method for surface immobilization of bacteriophage lysins infecting Gram-negative bacteria comprises the following steps: (1) Based on molecular cloning technology, a recombinant plasmid containing a lyase gene was constructed. The vector plasmid of the lyase was pGS21a, and the recombinant plasmid containing the lyase gene was pGS21a-LysSSE1. Then, NL amino acid chains and / or affinity ligands SiAP1 / SiAP2 were introduced using PCR and molecular cloning. Recombinant lyases LysSSE1-NL, LysSSE1-NL-SiAP1, and LysSSE1-NL-SiAP2 were constructed, and the constructed recombinant lyases LysSSE1-SiAP1 and LysSSE1-SiAP2 were used as controls. (2) The different recombinant lyases and the mixture of NaCl and Tween 20 were mixed evenly and incubated with the silica carrier. After incubation, the mixture was washed with sterile PBS to complete the surface fixation. The NaCl concentration in the NaCl and Tween 20 mixture was 400 mM, and the Tween 20 concentration was 0.3% (w / v). The incubation conditions were 2 h, 100 rpm, and room temperature. After incubation, the mixture was washed twice with an equal volume of sterile PBS at 100 rpm for 5 min.

[0030] Recombinant lyases were induced to express in vitro through competent cell transformation, IPTG induction, and ultrasonic disruption. The expression and solubility of the target protein were then identified using SDS-PAGE and Western blotting. The protein concentration was determined using a protein concentration assay kit. E. coli The five recombinant lyases from step (1) of Example 1 were expressed in BL21 Star (DE3), and the induction conditions were optimized using 37℃ / 15℃ and 0.2 mM / 1.0 mMIPTG. The induced recombinant bacteria were sonicated at 60W with 5s intervals and 5s working intervals until a clear light column was formed. SDS-PAGE and Western Blot analyses were then performed to verify the target bands. The obtained recombinant lyases were purified using a Ni-NTA purification kit.

[0031] Shigella bacteria were activated and cultured in LB medium to the logarithmic growth phase. After centrifugation at 8000 rpm for 15 min at 4°C, the bacterial pellet was collected and resuspended in PBS to OD 600 = 1.0. The pellet was then added to 96-well plates, and different purified recombinant lyases were added to each well. An equal volume of PBS was added to the control group. The mixtures were incubated at 37°C and 120 rpm for 2 h, and the OD 600 was measured using a microplate reader to calculate enzyme activity. The experiment was repeated three times. The enzyme activity of the recombinant lyases, characterized by absorbance, is as follows: Figure 2 As shown, the error bars represent the standard deviation. 1. Bactericidal activity test:

[0032] The enzyme-immobilized slide from step (2) of Example 1 was incubated together with a suspension of logarithmic-phase Shigella cells, and the activity of the lysin immobilized on the silica surface was measured.

[0033] Similarly, the procedure was the same as described in Experimental Example 1. The slides were resuspended in PBS and adjusted to an OD value of 1.0. Using immobilized slides without added enzyme as a control group, the slides were incubated at 100 rpm for 2 hours at room temperature. The OD600 of the free solution was then measured. Enzyme leaching analysis was performed on slides immobilized with enzyme but not treated with cell suspension. Compared to the free enzyme, the activities of LysSSE1-SiAP1 and LysSSE1-SiAP2 immobilized enzymes were significantly reduced, but the activity reduction of immobilized enzymes containing the NL chain was not significant. LysSSE1-NL-SiAP1 showed the best bactericidal activity (approximately 50%), followed by LysSSE1-NL-SiAP2.

[0034] Acid and alkali resistance test: The acid and alkali resistance of free recombinant enzymes and immobilized recombinant enzymes were determined separately. Bacterial precipitates were resuspended in sodium phosphate buffer at different pH values ​​(4.1, 5.4, 6.3, 7.4, 8.2, 9.1, 10.2, 11.3) to an OD value of approximately 1.0, and then transferred to 96-well plates. The acid and alkali resistance of different recombinant enzymes in step (2) of Example 1 was determined. The results showed that the acid and alkali resistance of the immobilized lyase containing NL was significantly enhanced, with the order being LysSSE1-NL-SiAP2 > LysSSE1-NL-SiAP1 > LysSSE1-SiAP2 > LysSSE1-SiAP1 > LysSSE1-NL. 3. Thermal stability test: Thermal stability was assessed by heating the enzymes at 60°C for 50 min, then cooling them to room temperature before measuring their bactericidal activity. Unheated enzymes served as a control group. Results showed that LysSSE1-NL-SiAP2 exhibited the strongest heat resistance, followed by LysSSE1-NL-SiAP1. The bactericidal activity of LysSSE1-SiAP2 and LysSSE1-SiAP1 decreased by approximately 53% after heating at high temperatures. 4. Environmental resistance test: Surfactants of 0%, 0.2%, 0.4%, 0.6%, 0.8%, and 1.0% AES were used, and the secondary structure changes of the enzyme in the wavelength range of 190-280 nm were measured by circular dichroism spectroscopy. Because the anions in the surfactants strongly interfered with the interaction between SiAP1 and silica, leading to severe leaching of the enzyme in the presence of AES and affecting the immobilization process, only the bactericidal performance of free LysSSE1 under AES was measured. The results showed that the secondary structure of LysSSE1 changed but did not denature, and the effect of AES on its structure did not increase with increasing AES concentration, consistent with the enzyme activity determination in the presence of AES; this provides reference data for evaluating the recombinant enzyme's resistance to environmental interference.

[0035] 5. Bactericidal repeatability test of immobilized enzymes: After the first enzyme sterilization experiment, the slides were washed twice with sterile PBS at 100 rpm for 5 min each time, and then incubated with sterile PBS and bacterial suspension for 2 h. This was repeated five times. LysSSE1-NL-SiAP1 showed the best repeatable bactericidal activity, with only about 5% reduction in original performance after 3 sterilization cycles.

[0036] In summary, the NL chain plays an important role in the immobilization of lyases. The obtained LysSSE1-NL-SiAP1 and LysSSE1-NL-SiAP2 immobilized lyases are superior to immobilized enzymes without NL peptide chains in terms of bactericidal activity, stability and reusability, and can effectively control surface contamination of Gram-negative bacteria in the environment. Example 2

[0037] The method for surface immobilization of bacteriophage lysins capable of infecting Gram-negative bacteria is the same as described in Example 1, except that: Recombinant lyases LysSSE1-GSA, LysSSE1-GSA-SiAP1, and LysSSE1-GSA-SiAP2 were constructed by replacing the NL amino acid chain with the GSA amino acid chain (GSAGSAAGSG). The constructed recombinant lyases LysSSE1-SiAP1 and LysSSE1-SiAP2 served as controls. Experimental comparisons revealed that the immobilized lyase LysSSE1-GSA-SiAP1 exhibited the highest bactericidal activity, the strongest acid and alkali resistance, and the best reusability. LysSSE1-GSA-SiAP1 and LysSSE1-GSA-SiAP2 showed similar high-temperature resistance, with their bactericidal activity decreasing by approximately 54% after high-temperature treatment, but still outperforming LysSSE1-SiAP1 and LysSSE1-SiAP2. Therefore, the added GSA amino acid chain also contributes to the bactericidal performance of the immobilized lyase.

Claims

1. A method for surface immobilization of bacteriophage lysins infecting Gram-negative bacteria, comprising the following steps: (1) Construct a recombinant plasmid containing a lyase gene based on molecular cloning technology, and then use PCR and molecular cloning to introduce an amino acid chain and / or affinity ligand SiAP1 / SiAP2 to construct a single peptide chain or a double peptide chain recombinant lyase. (2) Mix the recombinant lyase of the single peptide chain or dipeptide chain constructed in step (1) with NaCl and Tween 20, incubate with silica carrier, and wash with sterile PBS after incubation to complete surface fixation.

2. The method according to claim 1, characterized in that, In step (1), the lysin is phage lysin LysSSE1, which is encoded by Shigella phage and has the activity of killing Shigella, Escherichia coli and Staphylococcus aureus.

3. The method according to claim 1, characterized in that, In step (1), the vector plasmid of the lyase is pGS21a, and the recombinant plasmid containing the lyase gene is constructed based on molecular cloning technology as pGS21a-LysSSE1.

4. The method according to claim 1, characterized in that, In step (1), the introduced amino acid chain is NL (AGYGKAGGTVTPTPNT) or GSA (GSAGSAAGSG).

5. The method according to claim 4, characterized in that, In step (1), when the amino acid chain is NL, the recombinant lyase containing a single peptide chain is constructed as LysSSE1-NL, and the recombinant lyase containing a double peptide chain is constructed as LysSSE1-NL-SiAP1 or LysSSE1-NL-SiAP2.

6. The method according to claim 5, characterized in that, In step (1), the recombinant lyase constructed is LysSSE1-NL-SiAP1 or LysSSE1-NL-SiAP2.

7. The method according to claim 4, characterized in that, In step (1), when the amino acid chain is GSA, the recombinant lyase containing a single peptide chain is constructed as LysSSE1-GSA, and the recombinant lyase containing a double peptide chain is constructed as LysSSE1-GSA-SiAP1 or LysSSE1-GSA-SiAP2.

8. The method according to claim 7, characterized in that, In step (1), the recombinant lyase constructed is LysSSE1-GSA-SiAP1 or LysSSE1-GSA-SiAP2.

9. The method according to claim 1, characterized in that, In step (2), the concentration of NaCl in the mixture of NaCl and Tween 20 is 400 mM, and the concentration of Tween 20 in the mixture of NaCl and Tween 20 is 0.3-0.8%.

10. The method according to claim 1, characterized in that, In step (2), the silica support consists of three 24×24mm glass slides, and the incubation is performed by shaking at 100 rpm for 30-60 min.

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