CN114107271A

CN114107271A - Heat-resistant and eutrophic salmonella broad-spectrum lyase with in-vitro cracking activity and preparation and application thereof

Info

Publication number: CN114107271A
Application number: CN202111532742.5A
Authority: CN
Inventors: 卢雪梅; 张书航
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2021-12-15
Filing date: 2021-12-15
Publication date: 2022-03-01
Anticipated expiration: 2041-12-15
Also published as: CN114107271B

Abstract

The invention discloses a heat-resistant eutrophic salmonella broad-spectrum lyase with in vitro lytic activity, which is salmonella phage lyase and is named as XFII, and the amino acid sequence of the salmonella phage lyase is shown in SEQ ID No. 1; the gene coding the enzyme is named as XFII, and the nucleotide sequence is shown in SEQ ID NO. 2. The invention also discloses application of the lyase in preparation of a biological bactericide with a wide bactericidal spectrum and application of the lyase in cracking pathogenic bacteria under a eutrophic condition. Simultaneously, the chitosan-lyase XFII bactericide compound combination capable of spontaneously cracking gram-negative bacteria in vitro is provided, and the bactericide is expected to replace antibiotics to inhibit the proliferation of harmful pathogenic bacteria. The lyase which has the advantages of wide lysis spectrum, high activity, high yield, high heat resistance, rich nutrition resistance, capability of spontaneously lysing gram-negative bacteria without pretreating host bacteria and the like has potential application value in sterilization of environment, food surface and in-vivo environment.

Description

Heat-resistant and eutrophic salmonella broad-spectrum lyase with in-vitro cracking activity and preparation and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a heat-resistant eutrophic salmonella broad-spectrum lyase with in vitro lytic activity, and a preparation method and an application thereof.

Background

Salmonella (Salmonella) is a common gram-negative food-borne pathogen. Salmonella infection causes symptoms such as vomiting, diarrhea, fever, etc., and causes non-typhoid salmonella infection such as acute gastroenteritis, or typhoid fever, etc., as a virulent infectious disease. Salmonella is widely distributed, parasitizes in human and animal intestines, is carried and excreted by animals to be transmitted to the environment, feed, water source and food, and then is transmitted to human beings through poultry, meat, milk, eggs and the like, and cross contamination can occur in the food processing and transportation process.

There are mainly 3 current approaches to the treatment of bacterial infections, antibiotic therapy in the first place, but drug-resistant bacterial infections are prevalent due to the overuse of antibiotics in human and veterinary medicine, and bacteriophages lytic enzymes may be novel antibacterial agents replacing antibiotics. Salmonella lyase studies are rare, and more than 20 are reported at present. More mature Salmonella phage lyase including LysSE24, SPN9CC, LysSP1, LysLorf22, LysSTP4, SPN1S, LysSTG2, Lys68, LysWL59 and LysWL 60. The stability of free enzyme is poor, the free enzyme needs to endure high temperature in the processes of using, transporting, storing and preparing, and the enzyme is inactivated due to the rise of temperature, so that the problem of poor thermal stability of the enzyme preparation can be fundamentally solved by developing some high-temperature-resistant enzyme preparations. The application process of the lyase is characterized in that the food surface is rich in nutrition, and the in vivo environment also contains serum and various nutrients. Reports show that except that the gram-positive bacteria staphylococcus aureus bacteriophage lytic enzyme CF301 can promote bactericidal activity in serum, the killing activity of most lytic enzymes under rich nutrient conditions such as LB culture medium, serum and the like can be obviously reduced or lost. And no bactericidal activity of Salmonella lyase in eutrophic conditions has been reported so far. Therefore, the development of the lyase which can be active under various nutrient-rich conditions has important significance and application value. In addition, besides being applied to environmental sterilization, the lyase also has potential application value in the aspects of food sterilization and in-vivo sterilization, and the application range is enlarged accordingly. The enhanced activity and functionality of lytic enzymes in human blood, serum and plasma may provide novel therapeutic agents and improved antimicrobial methods.

The research of lyase aiming at gram-positive bacteria has been carried out more satisfactorily, while the research of lyase aiming at gram-negative bacteria faces more difficult problems. The main reason is that the action target of the lyase is the peptidoglycan component of the cell wall, and the outer membrane wrapped outside the peptidoglycan layer of the gram-negative bacteria prevents the lyase from contacting with the peptidoglycan and cannot play a role in sterilization. However, through the continuous trial and effort of researchers, the problems faced by gram-negative bacteria lytic enzymes are expected to be overcome or solved by: (1) identifying a native lyase that recognizes its ability to penetrate the outer membrane; (2) the lyase is cooperated with outer membrane penetrant such as EDTA, citric acid, malic acid, cationic polypeptide, bacteriostatic agent, etc.; (3) performing fusion expression on the lyase and a section of polypeptide capable of penetrating the outer membrane, and the like. However, no report is found about the compounding of lyase and chitosan as bacteriostatic agent to solve the above problems. The compatibility of the pathogenic bacteria which can be spontaneously cracked from the outside of the body enables the pathogenic bacteria to be used as food additives and have potential application in dealing with food pollution. Can be used as a livestock and poultry feed additive, can improve the immunity of livestock and poultry, increases the capability of the livestock and poultry to resist the infection of exogenous pathogenic bacteria, and has potential application value in the livestock and poultry breeding industry.

At present, the method of molecular cloning is generally adopted to prepare phage lyase, a prokaryotic expression system is utilized to express recombinant protein, and the method is suitable for determining phage encoding lyase genes. However, in the induction expression process of the phage lyase, the lyase generates a certain killing effect on escherichia coli, so that the expression strain generates growth defects, and the expression amount of the lyase is low and unstable. Through retrieval, an expression vector constructed by using the gene of the obtained lyase is transferred into the competence of an engineering expression strain C43(DE3) pLysS, and the competence difference of the competence C43(DE3) pLysS and BL21 is that the gene contains at least one unknown mutation, and the unknown mutation obtains the capacity of efficiently expressing toxic protein. This mutation site is involved in the cell death pathway when toxic proteins are expressed in E.coli. Recombinant expression strains are obtained by screening, and IPTG is used for inducing the strains to obtain the gram-negative phage lyase strains which have the advantages of wide lysis spectrum, high activity, high yield, high heat resistance, serum resistance, capability of spontaneously lysing gram-negative bacteria without pretreating host bacteria and the like, and enzyme products thereof are rarely reported.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a heat-resistant eutrophic salmonella broad-spectrum lyase with in vitro lytic activity and preparation and application thereof.

The heat-resistant eutrophic salmonella broad-spectrum lyase with in-vitro cracking activity is characterized in that: the lyase is salmonella phage lyase and is named as XFII, and the amino acid sequence of the lyase is shown in SEQ ID NO. 1.

The lyase XFII has good heat resistance, still retains higher activity after being subjected to heat treatment for 2h at 50-80 ℃, can be stored for more than 175 days at 4 ℃, and hardly loses activity after being repeatedly frozen and thawed for 5 times at-20 ℃; the activity is remarkable under the condition of pH 5-11, the optimal reaction pH is 8.0, and the optimal reaction temperature is 37 ℃.

The present invention provides a gene encoding the heat-resistant and eutrophic salmonella broad spectrum lyase having in vitro lytic activity of claim 1, characterized in that: the gene is named as XFII, and the nucleotide sequence of the gene is shown in SEQ ID NO. 2.

The preparation method of the heat-resistant and eutrophic salmonella broad-spectrum lyase with in vitro cracking activity comprises the steps of constructing and expressing a heat-resistant and eutrophic salmonella broad-spectrum lyase engineering strain with in vitro cracking activity, inducing IPTG, and preparing and purifying the lyase; the method is characterized by comprising the following steps:

(1) co-culturing salmonella bacteriophage and salmonella to obtain lysate containing salmonella bacteriophage, purifying the lysate and extracting salmonella bacteriophage genome DNA; wherein the Salmonella bacteriophage is designated XFII-1;

(2) amplifying a gene of salmonella phage lyase by using salmonella phage genome DNA as a template and using a primer pair P1 and P2, wherein the gene is named as XFI, and the nucleotide sequence of the gene is shown as SEQ ID NO. 2; wherein the nucleotide sequences of the primer pairs are as follows:

p1 upstream primer: 5- 'GCGaagcttATGTCAAACCGAAACATCAGTGAC-3',

p2 downstream primer: 5 'TATctcgagCTTAGCAGCGCGCCCTACAGCTTC-3';

(3) the lyase gene XFI obtained by amplification is subjected to double enzyme digestion by using restriction enzymes XhoI and HindIII and then is connected with an expression vector pET-29b (+), so as to obtain a recombinant plasmid connected with the lyase gene XFI, wherein the plasmid is named as pET-29b XFI;

(4) transferring the purified pET-29b XFI plasmid into an engineering expression strain C43(DE3) pLysS competence by a heat shock transformation method, obtaining a recombinant expression strain by screening, and naming the recombinant expression strain as C43 pET29b XFII;

(5) activating the recombinant expression strain C43 pET29b XFII and transferring the strain into LB culture medium, shaking and culturing at 37 ℃ and 220rpm until OD₆₀₀Adding isopropyl beta D thiogalactopyranoside (IPTG) 0.6-0.8, carrying out induction expression for 4-5h under the conditions of shaking table culture at the temperature of 16 ℃ and the rpm of 100, and obtaining thallus fermentation liquor capable of expressing lyase;

(6) centrifuging the obtained fermentation liquor at 6800-7000rpm and 4 ℃, collecting precipitated thalli, resuspending the thalli by using 50mM sodium phosphate with pH of 7.4 and 300mM sodium chloride buffer solution, carrying out ultrasonic crushing, centrifuging the crushed bacterial liquid at 12000rpm for 20min, and collecting supernatant, namely the crude enzyme liquid of the target lyase;

(7) and purifying and separating the crude enzyme solution by a nickel column affinity chromatography column to finally obtain the target protein which is the salmonella phage lyase and is named as XFII.

The lyase XFII is applied to preparation of biological bactericides with wide bactericidal spectrum.

Wherein: the lyase XFII can crack pathogenic escherichia coli, salmonella, acinetobacter baumannii, klebsiella pneumoniae and staphylococcus aureus in gram-negative pathogenic bacteria and gram-positive pathogenic bacteria.

The lyase XFII is applied to cracking pathogenic bacteria under the condition of eutrophication.

Wherein: the eutrophication condition refers to a rabbit serum or human serum albumin or LB culture medium, or a complex culture medium simulating the internal components of environment, food and animals; the pathogenic bacteria are pathogenic Escherichia coli, Salmonella, Acinetobacter baumannii, Klebsiella pneumoniae and Staphylococcus aureus. The application of the lyase XFII in cracking pathogenic bacteria under the condition of rich nutrition makes the lyase XFII have potential application in the aspects of environmental disinfection, food sterilization and in-vivo environmental sterilization.

The invention provides an engineering strain capable of expressing the lyase XFII, which is characterized in that: the engineering strain is named as C43 pET29b XFII; the strain is obtained by taking C43(DE3) pLysS as an original strain, transferring an expression plasmid pET-29b XFII containing a nucleotide sequence gene XFII shown in SEQ ID NO.2, and screening.

The invention provides a compound broad-spectrum bactericide, which is characterized in that: the bactericide contains the lyase XFII and chitosan; wherein the mixing volume ratio of the lyase XFII to the chitosan is preferably as follows: 6mg/mL chitosan 0.54mg/mL lyase XFII 1: 3.

The invention provides a heat-resistant eutrophic salmonella broad-spectrum lyase with in vitro cracking activity, and preparation and application thereof, and the heat-resistant eutrophic salmonella broad-spectrum lyase has the following unique advantages and remarkable effects:

(1) the lyase XFII provided by the invention has high expression quantity and strong enzyme activity in a prokaryotic expression system, can efficiently lyse bacteria, is expected to replace the traditional antibiotic treatment, especially the treatment aiming at multi-drug resistant pathogenic bacteria, and can also be used as a bactericide for sterilizing the environment. The nucleotide sequence disclosed by the invention can be used for constructing a genetic engineering strain for producing the lyase and producing the lyase XFII on a large scale.

(2) The lyase provided by the invention has good thermal stability. Most of the currently reported lyases are normal temperature type lyases, have high activity at normal temperature, have catalytic activity within the range of 4-80 ℃, have highest catalytic activity at 4-70 ℃, and are suitable for sterilization in high temperature environment.

(3) The lyase coded by the phage lyase sequence provided by the invention has good catalytic activity under the condition of rich nutrition. One challenge in the use of gram-negative lyases is their significant reduction or loss of bactericidal activity under nutrient-rich conditions such as LB medium and serum. Under the background, the lyase XFII provided by the invention can keep the bactericidal activity under the conditions of LB culture medium, rabbit serum and the like which are rich in nutrition, and when 10-50% of rabbit serum is added into a reaction system, the lyase XFII can promote the lytic activity of the rabbit serum, so that the activity of the rabbit serum is improved by about 136%.

(4) The salmonella lyase XFII provided by the invention belongs to broad-spectrum lyase. The lyase XFII has a good bactericidal effect on various gram-negative bacteria, wherein the bactericidal effect on Escherichia coli DH5 alpha, Escherichia coli JM109 and multiple strains of environment-separated pathogenic Escherichia coli, Acinetobacter baumannii AB1 and Klebsiella pneumoniae K3 is strong, the bactericidal effect on multiple strains of environment-separated pathogenic Salmonella is good, even a certain bactericidal effect on gram-positive bacteria staphylococcus aureus is achieved, and the lyase XFII has a wide bactericidal spectrum.

(5) Importantly, the bactericide prepared by compounding and combining the lyase XFII and the chitosan provided by the invention can spontaneously generate the lytic effect from the outside of the body under the condition that gram-negative pathogenic bacteria are not required to be pretreated by EDTA, thereby overcoming the obstacle of additionally adding the cytotoxic outer membrane damaging agent EDTA. The compound combination can spontaneously crack pathogenic bacteria from the outside of the body, so that the compound combination can be used as a food additive and has potential application in dealing with food pollution. The feed additive can also be used as a feed additive for livestock and poultry, can improve the immunity of the livestock and poultry, increases the capability of the livestock and poultry to resist the infection of exogenous pathogenic bacteria, and has potential application value in the livestock and poultry breeding industry.

Drawings

FIG. 1 is a schematic diagram of the electrophoresis of the product of the heat-resistant lyase gene XFII of the present invention.

Wherein: lane 1 is Marker,

lanes

2 and 3 are the gene product XFII, which is 489bp in size.

FIG. 2 shows a SDS-PAGE of target proteins of lyase XFII and lyase LysSE24 eluted with different concentrations of imidazole, FIG. 2A shows a map of the lyase XFII, and FIG. 2B shows a map of the lyase LysSE 24.

Wherein: m represents a protein Marker, and 1-2 represents target protein eluted by 50mM imidazole; 3-4 represents the target protein eluted by 100mM imidazole; 5-7 represent the target protein eluted with 200mM imidazole.

FIG. 3A shows the lysis curves of the lyase XFII and the lyase LysSE24 on EDTA-pretreated Escherichia coli JM109, and FIG. 3B shows the relative enzyme activities remaining after treatment of the lyase XFII of the present invention at 4-80 ℃ for 2 h.

FIG. 4A shows the lytic activity of the lyase of the present invention stored at 4 ℃, FIG. 4B shows the lytic activity of the lyase of the present invention stored at room temperature, and FIG. 4℃ shows the lytic activity of the lyase of the present invention after repeated freeze-thawing at-20 ℃ for 5 times.

FIG. 5A shows the results of the effect of reaction temperature on the lyase activity, and FIG. 5B shows the results of the effect of reaction pH on the lyase activity.

FIG. 6 shows the lytic activity of the lyase XFII and the lyase LysSE24 in LB medium.

FIG. 7A shows the cleavage activity of the cleavage enzyme XFII in rabbit serum medium and Tris-HCl buffer, and FIG. 7B shows the cleavage activity of the cleavage enzyme LysSE24 in rabbit serum medium and Tris-HCl buffer.

FIG. 8 shows the in vitro spontaneous cleavage activity of different ratios of the combination of lyase XFII and chitosan.

Detailed Description

The present invention will be described in detail with reference to the following detailed drawings and examples. The following examples are only preferred embodiments of the present invention, and it should be noted that the following descriptions are only for explaining the present invention and not for limiting the present invention in any form, and any simple modifications, equivalent changes and modifications made to the embodiments according to the technical spirit of the present invention are within the scope of the technical solution of the present invention.

In the following examples, the viral genomic DNA/RNA extraction kit was purchased from Beijing Tiangen Biochemical technology Ltd. Other materials, plasmids, strains, reagents and the like used, unless otherwise specified, were obtained commercially.

Example 1: extraction of Salmonella phage XFII-1 genome

1) And (3) obtaining a pure culture solution of salmonella bacteriophage XFII-1.

A single colony of Salmonella (designated as XFII) was picked from the solid medium and inoculated into 15mL of LB medium and cultured with shaking at 37 ℃ for 68 hours.

Picking salmonella (marked as XFII) phage plaques to obtain the salmonella phage, wherein the phage is named as XFII-1; inoculating 500 mu L of salmonella phage XFII-1 into the salmonella culture medium, performing shake culture at 37 ℃ for 46 h to obtain lysate containing salmonella phage XFII-1, centrifuging the lysate at 8000rpm for 10min, filtering with a 0.22 mu m filter membrane to obtain purified phage lysate without residual salmonella, and subsequently using the purified phage lysate for genome extraction.

2) Extracting the genome DNA of the bacteriophage XFII-1.

Extracting salmonella phage genome DNA from the obtained purified phage lysate by using a virus genome DNA/RNA extraction kit according to the method described in the instruction manual, wherein the finally extracted product is the genome DNA of the salmonella phage XFII-1, and storing at-20 ℃ for later use.

Example 2: functional comparison of lyase genes in NCBI library

Sending the extracted genome DNA of the salmonella bacteriophage XFII-1 to a biological company for whole genome sequencing, carrying out gene prediction on the genome through an RAST tool, and converting the ORF sequence predicted by the sample into an amino acid sequence through a trans eq program in EMBOSS, wherein the amino acid sequence is shown as SEQ ID NO. 1. The predicted amino acid sequence with the function of cracking cell walls is compared with public data, and the homology comparison is carried out by uploading an NCBI library to find that the amino acid sequence (SEQ ID No.1) of the lyase provided by the invention has higher similarity with the lyases of a plurality of salmonella bacteriophages, including lysSP1, LysSE24 and the like, but no research and application report of the lyase with the similarity of 100 percent is found at present.

The gene of the Salmonella phage lyase is obtained by sequence annotation and comparison analysis, the size of the gene is 489bp, the nucleotide sequence of the gene is shown as SEQ ID NO.2, and the gene is named as XFII.

Example 3: XFII gene cloning, recombinant expression vector and construction of expression strain

1. Amplification of lyase Gene XFII

Primers P1 and P2 were designed based on the nucleotide sequence of XFII gene (SEQ ID No.2), double restriction sites XhoI, HindIII and protective bases were added to the 5' end, amplification of XFII gene was performed by PCR using the Salmonella phage genomic DNA obtained in example 1 as a template according to the following procedure, and the PCR product was identified by electrophoresis in 1.5% agarose, the fragment size was 489bp, the band was single bright, and the gel electrophoresis results are shown in FIG. 1.

Wherein, the nucleotide sequences of the primers P1 and P2 are:

p1 upstream primer: 5- 'GCGaagcttATGTCAAACCGAAACATCAGTGAC-3',

p2 downstream primer: 5 'TATctcgagCTTAGCAGCGCGCCCTACAGCTTC-3'.

The DNA polymerase Phanta Max Super Fidelity carries out PCR amplification, and the PCR reaction system is as follows:

the PCR reaction procedure is as follows:

2. construction of recombinant plasmid pET-29b-XFII

The XFII gene product obtained by amplification and an empty vector pET-29b (+) are subjected to double digestion treatment by restriction enzymes XhoI and HindIII according to the following digestion reaction system, and a DNA band with the correct size is recovered.

An enzyme digestion reaction system:

respectively taking 4 mu L of XFII gene fragment recovered from double-enzyme digested glue and 1 mu L of pET-29b (+) empty plasmid recovered from double-enzyme digested glue, adding 5 mu L of DNA ligand Solution I ligase, uniformly mixing, placing at 16 ℃ for overnight ligation, and converting the ligation product into DH5 alpha.

And selecting transformants for PCR verification, sequencing the target fragment on the plasmid, and determining whether the sequence is completely correct. The correctly identified recombinant plasmid was designated pET-29b XFII.

3. Construction of Escherichia coli C43 expression strain containing target gene XFII

The correctly sequenced plasmid pET-29b XFII was transformed into E.coli C43(DE3) pLysS competence.

mu.L of the plasmid pET-29b XFII was added to 20. mu.L of competent C43(DE3) pLysS, mixed well and then placed on ice for 30 min. Taking out, heat-shocking in 42 deg.C water bath for 90sec, and rapidly taking out ice bath for 2 min. Then, 900. mu.L of LB medium was added thereto, and the mixture was incubated at 37 ℃ for 1 hour with shaking at 220 rpm. The bacterial suspension was spread on LB solid medium containing 50. mu.g/mL kanamycin, and cultured at 37 ℃ for 812 hours. Single colonies were randomly picked from the plate, inoculated into 5mL of LB medium containing 50. mu.g/mL of kanamycin, and cultured at 37 ℃ for 1016 hours with shaking at 220 rpm. Thus, the C43 expression strain containing the target gene XFII is obtained and named as C43 pET29b XFII and is placed in a refrigerator at minus 80 ℃ to store glycerol strains for later use.

The lyase LysSE24 is a lyase with higher thermal stability recently reported, and the bactericidal activity and partial properties of the two lyases are compared in the invention.

The amino acid sequence of LysSE24 lyase is shown in SEQ ID NO. 3. The cleavage enzyme LysSE24 amino acid sequence and cleavage enzyme XFII amino acid sequence alignment, the lyase LysSE24 and the invention of the cleavage enzyme XFII similarity of 98%.

Similarly, the method for constructing the engineering strain comprises the steps of firstly constructing a pET29b LysSE24 plasmid vector by a plasmid of pET29b XFII through a site-directed mutagenesis method, then carrying out heat shock transformation on the plasmid pET29b LysSE24 to the engineering strain C43(DE3) pLysS to obtain a recombinant strain capable of expressing the lyase LysSE24, wherein the recombinant strain is named as C43 pET29b LysSE24 and is placed in a refrigerator at the temperature of-80 ℃ for storage of a glycerol strain for later use.

Example 4: preparation and purification of recombinant protein lyase XFII

1. Induction and preparation of recombinant proteins

The C43-expressing strains C43 pET29b XFII and C43 pET29b LysSE24 obtained in example 3 were activated in 5mL LB medium, respectively, and were shake-cultured at 220rpm in 50mL Erlenmeyer flask 37 ℃ to OD₆₀₀After adding 1mM IPTG to 0.6-0.8, the mixture was transferred to a shaker at 16 ℃ and 100rpm, and cultured for 4 hours. The cells were collected by centrifugation at 7000rpm, 1mL of the supernatant of the fermentation broth was taken out and used, the cell pellet was suspended in 50mM sodium phosphate (pH 7.4) and 300mM sodium chloride buffer and then sonicated, the disrupted bacterial solution was centrifuged at 12000rpm for 20min, the supernatant of the disrupted solution was collected, and the disrupted pellet was resuspended in 2mL of the buffer. 50 mu L of fermentation liquid supernatant, crushing liquid supernatant and crushing liquid sediment are respectively added into 12.5 mu L of 5 XLoading Buffer boiling water bath for 10min, and then SDS-PAGE is carried out to detect the expression condition of the lyase, and the result shows that most of the lyase exists in the crushing liquid supernatant, and the protein yield exceeds 100 mg/L.

2. Extended preparation and purification of recombinant proteins

The preserved strains C43 pET29b XFII and C43 pET29b LysSE24 were respectively inoculated into 5mL LB medium containing 50. mu.g/mL kanamycin, cultured overnight at 37 ℃ with shaking at 220rpm, and the next day, added to fresh 300mL LB medium containing 50. mu.g/mL kanamycin in an inoculum size of 1% by volume, and cultured at 37 ℃ to OD₆₀₀When the concentration was changed to 0.6 to 0.8, 1mM IPTG was added, and the expression of the lyase was induced in a shaker at 16 ℃ and 100 rpm. Centrifuging at 7000rpm for 10min to collect thallus, resuspending with 30mL of 20mM imidazole solution, and ultrasonicatingUntil the imidazole solution containing the thalli is transparent and clear, centrifuging at 12000rpm for 20min, collecting the supernatant of the broken solution containing the crude enzyme solution, and performing suction filtration for later use.

Eluting 20% alcohol in the nickel column affinity chromatography column stored at 4 deg.C until it is completely eluted. 10 column volumes of 500mM NaCl, 20mM Tris-HCl, 20mM imidazole were added to run off. And (4) putting all the crushed liquid supernatant containing the crude enzyme solution after suction filtration on the column until the solution flows out completely. Adding 10mL of 500mM NaCl, 20mM Tris-HCl, 50mM imidazole, and pH7.4 to elute the hybrid protein; the resulting mixture was stored at 4 ℃ in 2mL, 5mL and 7mL of buffers containing 50mM, 100mM and 200mM of imidazole, respectively.

50. mu.L of the collected Buffer solution containing the target protein was taken out, and 12.5. mu.L of 5 Xloading Buffer was added thereto, and the mixture was centrifuged at 12000rpm for 5min in a boiling water bath for 10 min. The purity of the recombinant protein lyase XFII is detected by SDS-PAGE gel map, and the experimental result is shown in figure 2. Wherein FIG. 2A is a map of lyase XFII and FIG. 2B is a map of lyase LysSE 24.

Finally, the concentration of the cleavage enzyme XFII was measured using a Bradford kit, and adjusted to the same concentration by dilution. The method and the specific experimental procedure for the detection of the relevant cleavage activity refer to example 5.

Example 5: antibacterial effect of lyase XFII on Escherichia coli JM109 and bactericidal spectrum analysis of other bacteria

1. Sterilization effect of lyase XFII on Escherichia coli JM109

The host bacterium Escherichia coli JM109 was activated with 5mL of LB medium and transferred to a 50mL conical flask and cultured at 37 ℃ to OD₆₀₀0.6-0.8. The mixture was centrifuged at 7000rpm for 10min, the supernatant was discarded, and the mixture was incubated with 10mL of 20mM Tris-HCl (pH 8.0) and 100mM EDTA buffer at 37 ℃ for 5 min. Centrifugation at 7000rpm for 10min to remove EDTA, cell resuspension in the above buffer and washing twice, and final buffer to OD₆₀₀About 1.0. The purified lyase XFI was adjusted to a protein concentration of 0.104mg/mL, and 1. mu.L (20-25nM) of the lyase XFI and 199. mu.L of EDTA-pretreated E.coli JM109 were added to a 96-well plate. The assay was set up in triplicate, and 1 μ L of sodium phosphate buffer served as a negative control. Immediately placing the ELISA plate in an ELISA reader, and detecting at 37 DEG C30min,. DELTA.OD in lysis time as abscissa₆₀₀As ordinate, a cleavage curve was obtained.

The bactericidal activity of the lyase XFII is detected by a turbidity method, and the bacterial liquid is found to be quickly clarified under the action of the lyase XFII and OD within 5min₆₀₀I.e., from 0.8 to 0.2, the results are shown in fig. 3A. The lyase XFII expressed in the experiment has good bactericidal activity.

As shown in FIG. 3A, the activity of the lyase XFII of the present invention was 193% higher than that of the lyase LysSE24, as a result of adjusting the concentration of the purified enzyme LysSE24 to the same protein as XFII (0.104mg/mL) and comparing the bactericidal activity, the bactericidal activity of the lyase LysSE24 was detected in the same manner, and the bactericidal activity of the lyase LysSE24 was compared with that of the lyase XFII. The lyase XFII can greatly reduce the use cost and the industrial production cost in the application process.

The active site of the lyase XFII was found by conserved sequence analysis to be: glu18, Pro29, Thr 35. The lyase LysSE24 is obtained by mutating points N-40-Y, A-97-V and A-159-D of the lyase XFII, and the three-dimensional structure simulation by Swiss-model shows that the 40 th site, the 97 th site and the 159 th site are all positioned on the surface of the protein, and the steric hindrance of the 3 amino acids after mutation is larger than that of the original amino acid, so the lysSE24 enzymatic activity is suspected to be influenced.

2. Bactericidal spectrum detection of lyase XFII

Coli DH5 alpha, E.coli JM109, E.coli C43(DE3) and a plurality of strains of environmentally isolated pathogenic E.coli, Acinetobacter baumannii standard strains, a plurality of strains of environmentally isolated Salmonella, Klebsiella pneumoniae and Staphylococcus aureus were selected respectively by the sterilization detection method in the first part of example 5, and the bactericidal spectrum of the lyase XFII was determined, and the results showed that the lyase XFII had a very obvious bactericidal effect on indicator bacteria on the premise of EDTA pretreatment of the indicator bacteria, and the bactericidal effects of the lyase XFII on the indicator bacteria were further compared, and the results are shown in Table 1. Meanwhile, the lyase XFII has a good bactericidal effect on various gram-negative bacteria, wherein the lyase XFII has a strong bactericidal effect on Escherichia coli DH5 alpha, Escherichia coli JM109, and a plurality of strains of environmentally separated pathogenic Escherichia coli, Acinetobacter baumannii AB1 and Klebsiella pneumoniae K3, has a good bactericidal effect on a plurality of strains of environmentally separated pathogenic Salmonella, and even has a certain bactericidal effect on gram-positive bacteria Staphylococcus aureus, which indicates that the lyase XFII belongs to broad-spectrum lyase.

Table 1: bactericidal spectrum of lyase XFII

Note: ++++: lysis clear, OD₆₀₀The reduction is about 0.6; +++: clear OD of lysis₆₀₀The reduction is about 0.5; ++: clear lysis, OD₆₀₀The reduction is about 0.3; +: cracking turbid, insufficient, OD₆₀₀Decrease by about 0.2, -: it is not cracked.

Example 6: detection of thermal stability and analysis of partial enzymological Properties (storage, pH value) of the lyase XFII

1. Detection of thermal stability and Low temperature storage Properties of lyase XFII

20 μ L of purified lyase XFII was treated in a water bath at 4 deg.C, 20 deg.C, 30 deg.C, 37 deg.C, 45 deg.C, 50 deg.C, 55 deg.C, 60 deg.C, 65 deg.C, 70 deg.C, 75 deg.C, 80 deg.C for 2 h. The treated samples were taken out of the bath for 30 minutes and the bactericidal activity assay was performed as described in the first part of example 5, with the untreated lyase as a control and the relative bactericidal activity was calculated as the lytic activity of the treated enzyme/the lytic activity of the untreated enzyme x 100%, where bactericidal activity Δ OD₆₀₀And/Δ t. The result is shown in figure 3B, lyase XFII below 70 ℃ for 2h, its bactericidal activity is similar to that of untreated lyase, and 80 ℃ heat treatment for 2h bactericidal activity still has 54.82%, prove that its stability is good, all have its own unique advantage in future storage and transportation.

The purified lyase XFII was stored at 4 ℃ and periodically sampled for residual bactericidal activity as described in the first part of example 5. Delta OD within 5min with storage date as abscissa₆₀₀As ordinate, the lysis curve within the storage date is obtained. The results are shown in fig. 4A, the bactericidal activity was still significant after 175 days of storage at 4 ℃, data not shown for longer periods of time, and the bactericidal activity of lyase XFII after storage at ambient temperature was tested in the same manner. As a result, as shown in FIG. 4B, the enzyme activity was hardly decreased within 15 days.

And (3) repeatedly freezing and thawing the purified lyase XFII at-20 ℃, freezing for 2h each time, taking out and thawing for 1h, and taking out 20 mu L as required to detect the bactericidal activity. The results are shown in fig. 4C, the bactericidal activity of the lyase XFII is still significant after 5 times of repeated freeze-thawing at-20 ℃, and more times of data are not shown, and these results all prove that the lyase XFII is stable and suitable for long-term storage and use.

2. Heat-resistant analysis, optimum temperature and optimum pH detection of lyase XFII

The reaction system was prepared in accordance with the method described in the first part of example 5, and host bacterium JM109 was adjusted to OD₆₀₀Approximately equals to 1.0, then each system is placed at the temperature of 4 ℃, 10 ℃, 20 ℃, 30 ℃, 37 ℃, 40 ℃, 45 ℃, 50 ℃ and 60 ℃ for reaction for 5 minutes, the centrifuge tube is taken out, the test is set to three times, and the same volume of sodium phosphate buffer solution is used as a negative control. Immediately placed in a microplate reader and the remaining OD measured₆₀₀. Delta OD within 5min with the reaction temperature as abscissa₆₀₀The sterilization curve of the optimum reaction temperature is obtained for the ordinate. The results are shown in FIG. 5A, showing that the lyase XFII has good cleavage activity in the range of 4-60 ℃, especially the highest catalytic activity at 37 ℃.

Escherichia coli JM109 cells were resuspended in a universal buffer to adjust pH to 3-12, and then subjected to OD before and after the reaction at the optimum temperature (37 ℃) according to OD before and after the reaction as described in the first part of example 5₆₀₀The optimum pH was determined by the amount of change in XFII, and the results are shown in FIG. 5B, which shows that the catalytic range of the lyase XFII is pH 7-11, and the optimum pH is 8.

Example 7: bactericidal Activity of lyase XFII in Complex Medium

1. Cleavage Activity of the cleavage enzyme XFII in LB Medium

The EDTA-treated Escherichia coli JM109 was washed and then resuspended in LB medium, and bactericidal activity was examined as in the first part of example 5. As a result, as shown in FIG. 6A, the bactericidal activity of the lyase XFII was slightly inhibited in LB medium, whereas the bactericidal activity of the lyase LysSE24 against Escherichia coli JM109 in LB medium was examined in the same manner, and as a result, as shown in FIG. 6B, the bactericidal activity of the lyase LysSE24 was severely inhibited and almost no bactericidal activity was observed.

Although many lyases have strong bactericidal activity in simple buffered solutions, their effectiveness tends to decrease under conditions supported by bacterial growth, which limits their broader effectiveness, particularly in vitro applications. The lyase XFII can show bactericidal activity under the condition of rich nutrition existing in an LB culture medium, and becomes one of unique advantages of the lyase XFII and has wider application value.

2. Lytic Activity of the lyase XFII in serum Medium

Adjustment to OD according to the method in the first part of example 5₆₀₀Coli JM109 reaction system of 1.0, 0%, 10%, 20%, 30%, 40%, 50% rabbit serum was added, and OD was measured by the method described in the first part of example 5 using 50% rabbit serum and sodium phosphate buffer without adding lyase as negative control₆₀₀A change in (c). The results are shown in FIG. 7A, the lyase XFII still promoted its cleavage of Escherichia coli JM109 when up to 50% rabbit serum was added. On the other hand, when the LysSE24 was also assayed for serum resistance as in the first part of example 5, the LysSE24 exhibited bactericidal activity in serum comparable to that in Tris-HCl buffer, but did not exhibit significant serum-activity-promoting properties, as shown in fig. 7B.

Example 8: in-vitro bactericidal activity of lyase XFII and chitosan compound combination

Activation of the sink with 5mL LB MediumEscherichia coli JM109, a main bacterium, was transferred to a 50mL conical flask and cultured at 37 ℃ to OD₆₀₀0.6-0.8. The cells were centrifuged at 7000rpm for 10min, the supernatant was discarded, and the cells were washed once with 10mL of 20mM Tris-HCl (pH 8.0), and finally adjusted to OD with a buffer₆₀₀About 1.0. Purified lyase XFII is adjusted to have a protein concentration of 0.54mg/mL, 1% acetic acid is used for preparing 6mg/mL chitosan, and the lyase and the chitosan are mixed according to different proportions (1:1, 1:2, 2:1, 3:1, 1:3, 4:1, 1:4, V/V) to form a bactericide composite combination. In vitro bactericidal activity tests were performed on each group of different ratio mixes in 96-well plates.

In each group of tests, 30 mu L of bactericide complex combination is added into each hole of a 96-hole plate to crack escherichia coli JM 109. The test was set up in triplicate, Tris-HCl buffer, lyase XFII, chitosan solution as negative controls. Immediately placing the ELISA plate in an ELISA reader, detecting at 37 deg.C for 30min, with lysis time as abscissa and delta OD₆₀₀As ordinate, a cleavage curve was obtained. The results are shown in fig. 8, the escherichia coli JM109 without EDTA pretreatment has a certain lysis effect under the action of the bactericide compound combination, the combined bactericidal effects of different formula ratios are different, the effect is most obvious when the ratio of chitosan to lyase is 1:3, and the OD is OD₆₀₀Can achieve OD within 5min₆₀₀I.e. from 0.9 to 0.55. On the other hand, when only the lyase XFII is used to act on the JM109 which is not pretreated, the good lytic activity must be obtained by the action of an outer membrane-disrupting agent such as EDTA, chloroform or citric acid due to the presence of the outer membrane of gram-negative bacteria, and therefore, when the lyase XFII is used alone, the OD of Escherichia coli of JM109 is observed₆₀₀Shows a trend of levels and neither Tris-HCl buffer of the lyase XFII nor chitosan itself produces a lytic effect on Escherichia coli JM 109. Meanwhile, experiments also prove that the compound combination of the lyase XFII and the chitosan has good in-vitro cracking effect on Escherichia coli DH5 alpha, so that the compound combination has broad spectrum.

The formula of the compound combination of the lyase XFI which breaks through the outer membrane of gram-negative bacteria and spontaneously cracks pathogenic bacteria in vitro and the Chitosan is not reported, Chitosan (Chitosan) is also called deacetylated chitin and is obtained by deacetylating chitin (chitin) widely existing in the nature, and the excellent performances of the natural polymer, such as biological functionality, compatibility, blood compatibility, safety, microbial degradability and the like, are widely concerned by various industries, meet the national food additive use standard GB-2760, and make great progress in the application research in the fields of medicine, food, chemical industry, cosmetics, water treatment, gold biochemistry, biomedical engineering and the like. Meanwhile, chitosan is supposed to have positive charge and can act with the surface of a negatively charged cell to destroy the membrane permeability, so that the chitosan has a synergistic effect with the lyase XFII. The compatibility of the pathogenic bacteria which can be spontaneously cracked from the outside of the body enables the pathogenic bacteria to be used as food additives and have potential application in dealing with food pollution. Meanwhile, the feed additive can be used as a feed additive for livestock and poultry, can improve the immunity of the livestock and poultry, increases the capability of the livestock and poultry to resist the infection of exogenous pathogenic bacteria, and has potential application value in the livestock and poultry breeding industry.

Sequence listing

<110> Shandong university

<120> heat-resistant eutrophic salmonella broad-spectrum lyase with in-vitro cracking activity and preparation and application thereof

<141> 2021-12-09

<160> 1

<210> 1

<211> 162

<212> PRT

<213> Artificial sequence

<221> amino acid sequence of lyase XFII

<222>（1）…（162）

<400> 1

Met Ser Asn Arg Asn Ile Ser Asp Asn Gly Leu His Phe Thr Ala Ala

1 5 10 15

Phe Glu Gly Phe Arg Gly Thr Ala Tyr Arg Ala Thr Pro Ser Glu Lys

20 25 30

Tyr Phe Thr Ile Gly Tyr Gly His Asn Gly Ala Asp Val Lys Glu Gly

35 40 45

Gln Lys Ile Thr Glu Gly Gln Gly Leu Leu Leu Leu His Lys Asp Met

50 55 60

Ala Lys Ala Val Ala Ala Val Asp Ala Val Ala His Pro Ser Leu Asn

65 70 75 80

Gln Ser Gln Phe Asp Ala Val Cys Asp Leu Val Tyr Asn Ala Gly Ala

85 90 95

Gly Ala Ile Ala Val Ser Thr Gly Thr Gly Gln Ala Leu Arg Lys Gly

100 105 110

Asp Ala Ser Thr Leu Arg Asn Lys Leu Thr Gln Phe His Tyr Gln Asn

115 120 125

Gly Lys Ser Leu Leu Gly Leu Arg Arg Arg Ala Ala Gly Arg Val Ala

130 135 140

Leu Phe Asp Gly Met Leu Trp Gln Gln Ala Glu Ala Val Gly Arg Ala

145 150 155 160

Ala Lys

　　162

<210> 2

<211> 489

<212> DNA

<213> Artificial sequence

<221> nucleotide sequence encoding lyase XFII gene

<222>（1）…（489）

<400> 2

atgtcaaacc gaaacatcag tgacaacgga ttacacttca ccgccgcgtt cgaggggttc 60

cgcgggaccg cctaccgcgc gacaccttca gaaaaatact ttactattgg ctacggccac 120

aacggcgcag atgtaaaaga aggtcagaag attaccgaag gccagggtct cctgcttctg 180

cataaagata tggctaaggc cgtagctgct gtagacgccg tagcgcatcc gtctctaaat 240

cagtcacagt tcgacgccgt gtgtgacctg gtgtataacg ctggtgcagg tgcgattgct 300

gtgtcaaccg gaacaggtca ggcgctgcgc aaaggcgatg catctacact gcgtaataag 360

ttaactcagt tccattatca gaacggcaaa tcactcctcg gattgcggcg ccgagctgct 420

ggtcgtgttg cactgttcga cggtatgctg tggcaacagg ccgaagctgt agggcgcgct 480

gctaagtag 489

<210> 3

<211> 162

<212> PRT

<213> Artificial sequence

<221> amino acid sequence of lyase LysSE24

<222>（1）…（162）

<400> 3

Met Ser Asn Arg Asn Ile Ser Asp Asn Gly Leu His Phe Thr Ala Ala

1 5 10 15

Phe Glu Gly Phe Arg Gly Thr Ala Tyr Arg Ala Thr Pro Ser Glu Lys

20 25 30

Tyr Phe Thr Ile Gly Tyr Gly His Tyr Gly Ala Asp Val Lys Glu Gly

35 40 45

Gln Lys Ile Thr Glu Gly Gln Gly Leu Leu Leu Leu His Lys Asp Met

50 55 60

Ala Lys Ala Val Ala Ala Val Asp Ala Val Ala His Pro Ser Leu Asn

65 70 75 80

Gln Ser Gln Phe Asp Ala Val Cys Asp Leu Val Tyr Asn Ala Gly Ala

85 90 95

Gly Val Ile Ala Val Ser Thr Gly Thr Gly Gln Ala Leu Arg Lys Gly

100 105 110

Asp Ala Ser Thr Leu Arg Asn Lys Leu Thr Gln Phe His Tyr Gln Asn

115 120 125

Gly Lys Ser Leu Leu Gly Leu Arg Arg Arg Ala Ala Gly Arg Val Ala

130 135 140

Leu Phe Asp Gly Met Leu Trp Gln Gln Ala Glu Ala Val Gly Arg Asp

145 150 155 160

Ala Lys

　　162

Claims

1. A heat-resistant and eutrophic Salmonella broad-spectrum lyase having in vitro lytic activity, characterized in that: the lyase is salmonella phage lyase and is named as XFII, and the amino acid sequence of the lyase is shown in SEQ ID NO. 1.

2. A gene encoding the heat-resistant, nutrient-tolerant salmonella broadspectrum lyase having in vitro lytic activity of claim 1, wherein: the gene is named as XFII, and the nucleotide sequence of the gene is shown in SEQ ID NO. 2.

3. The preparation method of the heat-resistant and eutrophic salmonella broad-spectrum lyase with in vitro lytic activity as claimed in claim 1, comprising the steps of constructing a heat-resistant and eutrophic salmonella broad-spectrum lyase engineering strain expressing the in vitro lytic activity, IPTG induction, preparation and purification of lyase; the method is characterized by comprising the following steps:

p1 upstream primer: 5- 'GCGaagcttATGTCAAACCGAAACATCAGTGAC-3',

p2 downstream primer: 5'-TATctcgagCTTAGCAGCGCGCCCTACAGCTTC-3', respectively;

(3) the lyase gene XFI obtained by amplification is subjected to double enzyme digestion by using restriction enzymes XhoI and HindIII and then is connected with an expression vector pET-29b (+), so as to obtain a recombinant plasmid connected with the lyase gene XFI, and the plasmid is named as pET-29 b-XFI;

(4) transferring the purified pET-29b-XFII plasmid into an engineering expression strain C43(DE3) pLysS competence by a heat shock transformation method, obtaining a recombinant expression strain by screening, and naming the recombinant expression strain as C43-pET29 b-XFII;

(5) activating the recombinant expression strain C43-pET29b-XFII, transferring the strain into an LB culture medium, and shaking-culturing at 37 ℃ and 220rpm until OD₆₀₀Adding isopropyl-beta-D-thiogalactopyranoside (IPTG) 0.6-0.8, carrying out induced expression for 4-5h under the conditions of 16 ℃ and 100rpm shake culture to obtain thallus fermentation liquor capable of expressing lyase;

4. Use of the lyase of claim 1 for the preparation of a wide bactericidal spectrum of biological fungicides.

5. Use according to claim 4, characterized in that: the lyase can cleave pathogenic escherichia coli, salmonella, acinetobacter baumannii, klebsiella pneumoniae and staphylococcus aureus in gram-negative pathogenic bacteria and gram-positive pathogenic bacteria.

6. Use of the lyase of claim 1 for the lysis of pathogenic bacteria under conditions of eutrophication.

7. Use according to claim 6, characterized in that: the eutrophication condition refers to a rabbit serum or human serum albumin or LB culture medium, or a complex culture medium simulating the internal components of environment, food and animals; the pathogenic bacteria are pathogenic Escherichia coli, Salmonella, Acinetobacter baumannii, Klebsiella pneumoniae and Staphylococcus aureus.

8. An engineered strain capable of expressing the lyase of claim 1, wherein: the engineering strain is named as C43-pET29 b-XFII; the strain is obtained by taking C43(DE3) pLysS as an original strain, transferring an expression plasmid pET-29b-XFII containing a nucleotide sequence gene XFII shown in SEQ ID NO.2, and screening.

9. A compound broad-spectrum bactericide is characterized in that: the bactericide comprises the lyase described in claim 1 and chitosan.

10. The broad spectrum fungicide of the compounded combination according to claim 9, which is characterized in that: the mixing volume ratio of the lyase to the chitosan in the bactericide is as follows: 6mg/mL chitosan: 0.54mg/mL lyase was 1: 3.