CN114107271B - Heat-resistant and nutrient-resistant salmonella broad spectrum lyase with in-vitro cleavage activity, and preparation and application thereof - Google Patents

Abstract

The invention discloses a heat-resistant and nutrient-resistant salmonella broad-spectrum lyase with in-vitro cleavage activity, which is salmonella phage lyase and is named XFII, wherein the amino acid sequence of the enzyme is shown as SEQ ID NO. 1; the gene encoding the enzyme is named XFII, and the nucleotide sequence of the gene is shown as SEQ ID NO. 2. The invention also discloses application of the lyase in preparing biological bactericides with wide bactericidal spectrum and application of the lyase in cracking pathogenic bacteria under eutrophication conditions. Meanwhile, the chitosan-lyase XFII bactericide compound composition capable of spontaneously cracking gram-negative bacteria from outside the body is provided, and the bactericide is expected to replace antibiotics to inhibit the proliferation of harmful pathogenic bacteria. The lyase provided by the invention has the advantages of wide cleavage spectrum, high activity, high yield, high heat resistance, rich nutrition resistance, capability of spontaneously cleaving gram-negative bacteria without pretreatment of host bacteria, and the like, and has potential application value for sterilizing environment, food surface and in-vivo environment.

Description

Heat-resistant and nutrient-resistant salmonella broad spectrum lyase with in-vitro cleavage activity, and preparation and application thereof

Technical Field

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

Background

Salmonella (Salmonella) is a common gram-negative food-borne pathogenic bacterium. Symptoms such as vomiting, diarrhea and fever can appear when salmonella is infected, and non-typhoid salmonella infection symptoms such as acute gastroenteritis or severe infectious diseases such as typhoid fever are caused. Salmonella is widely distributed in the intestines of humans and animals, is transmitted to the environment, feed, water source and food through animal carrying and excretion, is transmitted to humans through poultry, meat, milk, eggs and the like, and can cause cross contamination in the food processing and transportation processes.

Currently there are 3 main approaches to treat bacterial infections, antibiotic treatment first, but due to the overuse of antibiotics in humans and veterinary drugs, resistant bacterial infections are pandemic, and phages and phage lytic enzymes may be novel antibacterial agents replacing antibiotics. Salmonella lyase has been studied less, and more than 20 have been reported. Examples of the more mature salmonella phage lytic enzymes include LysSE24, SPN9CC, lysSP1, lysloref 22, lysSTP4, SPN1S, lysSTG, lys68, lysWL59, lysWL60, and the like. The stability of free enzyme is poor, the free enzyme needs to resist high temperature during the use, transportation, storage and preparation processes, and the temperature rise can lead to the inactivation of the enzyme, so that a plurality of high-temperature resistant enzyme preparations are developed, and the problem of poor thermal stability of the enzyme preparations can be fundamentally solved. During the application process of the lyase, the surface of the food is rich in nutrition, and the in-vivo environment also contains serum and various nutrients. It is reported that most of the lytic enzymes have significantly reduced or lost killing activity under the eutrophic conditions of LB medium, serum and the like, except that gram positive bacteria staphylococcus aureus phage lytic enzyme CF301 can have the bactericidal activity promoting effect in serum. And so far no report has been made about the bactericidal activity of salmonella lyase in eutrophic conditions. Therefore, the development of the lyase which can be active under various eutrophic conditions has important significance and application value. In addition, besides being applied to environmental sterilization, the lyase has potential application value in the aspects of food sterilization and in-vivo sterilization, and the application range is also enlarged. The enhanced activity and functionality of lytic enzymes in human blood, serum and plasma can provide new therapeutic agents and improved antibacterial methods.

While the research of the lyase against gram-positive bacteria has been carried out satisfactorily, the research of the lyase against gram-negative bacteria has faced a great difficulty. 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 contact of the lyase and the peptidoglycan, so that the sterilization effect cannot be achieved. However, with continued attempts and efforts by researchers, the problems faced by gram-negative bacterial lyases are expected to be overcome or resolved by: (1) identifying a natural lyase that recognizes a membrane-penetrating agent; (2) Synergistic action of lyase and outer membrane penetrating agent such as EDTA, citric acid, malic acid, cationic polypeptide, bacteriostat, etc.; (3) Fusion expression of the lyase and a polypeptide capable of penetrating the outer membrane is performed. However, the compounding of the lyase and the bacteriostat chitosan is not reported to solve the problems. The compatibility of the pathogenic bacteria capable of being spontaneously cracked from the outside of the body enables the pathogenic bacteria to be used as a food additive, and has potential application for coping with food pollution. Can be used as a livestock feed additive to improve the immunity of livestock and increase the capability of the livestock to resist the infection of exogenous pathogenic bacteria, and has potential application value in the livestock and poultry raising industry.

Currently, a molecular cloning method is generally adopted to prepare phage lyase and a prokaryotic expression system is utilized to express recombinant protein, and the method is applicable to phage capable of determining genes encoding the phage lyase. However, in the induction expression process of phage lyase, a certain killing effect is generated on escherichia coli due to the generation of the lyase, so that the expression strain generates growth defects, and the expression quantity of the lyase is low and unstable. Through searching, the gene construction expression vector of the obtained lyase is transferred into the competence of engineering expression strain C43 (DE 3) pLysS, and the competence C43 (DE 3) pLysS is utilized to be different from BL21 in that the gene contains at least one unknown mutation, and the unknown mutation obtains the capability of efficiently expressing toxic proteins. This mutation site is involved in the cell death pathway when the toxic protein is expressed by E.coli. Recombinant expression strains are obtained through screening, and strain and enzyme products thereof of gram-negative phage lyase with the advantages of wide expression splitting spectrum, high activity, high yield, high heat resistance, serum resistance, capability of spontaneously splitting gram-negative bacteria without pretreatment of host bacteria and the like are obtained through IPTG induction.

Disclosure of Invention

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

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

The lyase XFII has good heat resistance, still retains higher activity after heat treatment for 2 hours at 50-80 ℃, can be stored for more than 175 days at 4 ℃, and hardly loses activity after repeated freeze thawing 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 invention provides a gene for encoding the heat-resistant and nutrient-resistant salmonella wide-spectrum lyase with in-vitro cleavage activity, which is characterized in that: the gene is named XFII, and the nucleotide sequence of the gene is shown as SEQ ID NO. 2.

The preparation method of the heat-resistant and nutrient-resistant salmonella broad-spectrum lyase with in-vitro cleavage activity comprises the steps of constructing and expressing a heat-resistant and nutrient-resistant salmonella broad-spectrum lyase engineering strain with in-vitro cleavage activity, IPTG induction, and preparation and purification of a lyase; the method is characterized by comprising the following steps of:

(1) Co-culturing salmonella phage and salmonella to obtain a lysate containing salmonella phage, purifying the phage lysate and extracting salmonella phage genome DNA; wherein the salmonella phage 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 XFII, and the nucleotide sequence of the gene is shown as SEQ ID NO. 2; wherein, the nucleotide sequence of the primer pair is as follows:

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

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

(3) The amplified lyase gene XFII is connected with an expression vector pET-29b (+) after double enzyme digestion by utilizing restriction enzymes XhoI and HindIII, and a recombinant plasmid connected with the lyase gene XFII is obtained, and the plasmid is named pET-29b-XFII;

(4) Transferring the purified pET-29b-XFII plasmid into engineering expression strain C43 (DE 3) pLysS competence by a heat shock conversion method, obtaining recombinant expression strain by screening, and naming the recombinant expression strain as C43-pET29b-XFII;

(5) Activating recombinant expression strain C43-pET29b-XFII and transforming itShaking culture at 37deg.C and 220rpm to OD in LB medium ₆₀₀ =0.6-0.8, isopropyl-beta-D-thiogalactopyranoside (IPTG) is added and transferred to 16 ℃ and induced to express for 4-5 hours under the condition of shaking culture at 100rpm, so as to obtain a bacterial fermentation broth capable of expressing lyase;

(6) Centrifuging the obtained fermentation liquor at 6800-7000rpm and 4 ℃, collecting precipitated thalli, re-suspending the thalli by using 50mM sodium phosphate and 300mM sodium chloride buffer solution with pH=7.4, performing ultrasonic crushing, centrifuging the crushed thalli for 20min at 12000rpm, and collecting supernatant as a target crude enzyme solution of the lyase;

(7) Purifying and separating the crude enzyme liquid by a nickel column affinity chromatographic column, and finally obtaining the target protein which is salmonella phage lyase and named XFII.

The application of the lyase XFII in preparing biological bactericides with wide bactericidal spectrum.

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

The application of the lyase XFII in the pathogen cracking under the eutrophic condition.

Wherein: the eutrophication condition refers to rabbit serum or human serum albumin or LB culture medium, or complex culture medium simulating environment, food and animal body components; the pathogenic bacteria are pathogenic escherichia coli, salmonella, acinetobacter baumannii, klebsiella pneumoniae and staphylococcus aureus. The application of the lyase XFII in the process of lysing pathogenic bacteria under the eutrophication condition enables the lyase XFII to have potential application in the aspects of environment disinfection, food sterilization and in-vivo environment 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, is obtained by taking C43 (DE 3) pLysS as an original strain, transferring into an expression plasmid pET-29b-XFII containing a nucleotide sequence gene XFII shown in SEQ ID NO.2, and screening.

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

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

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

(2) The lyase provided by the invention has good thermal stability. Most of the currently reported lyase is normal-temperature type lyase, and has higher activity at normal temperature, and the lyase has catalytic activity in the range of 4-80 ℃ and highest catalytic activity in the range of 4-70 ℃, so that the lyase is applicable to sterilization in a higher-temperature environment.

(3) The lyase coded by the phage lyase sequence provided by the invention has good catalytic activity under the eutrophic condition. One challenge with the use of gram-negative lyases is their significantly reduced or lost bactericidal activity in the presence of nutrient rich conditions such as LB medium and serum. Under the background, the lyase XFII provided by the invention can maintain the bactericidal activity under the eutrophic conditions such as LB culture medium, rabbit serum and the like, and can promote the lytic activity of 10-50% of rabbit serum when the reaction system is added, so that the activity is improved by about 136%.

(4) The salmonella lyase XFII provided by the invention belongs to broad spectrum lyase. The lyase XFII has good bactericidal effect on various gram-negative bacteria, wherein the lyase has strong bactericidal effect on escherichia coli DH5 alpha, escherichia coli JM109 and multiple environmental separation pathogenic escherichia coli, acinetobacter baumannii AB1 and klebsiella pneumoniae K3, has good bactericidal effect on multiple environmental separation pathogenic salmonella, and even has certain bactericidal effect on gram-positive bacteria staphylococcus aureus, thus indicating that the lyase has a wider bactericidal spectrum.

(5) Importantly, the bactericide prepared by compounding and combining the lyase XFII and the chitosan provided by the invention can spontaneously generate a lysis effect from outside the body under the condition that gram-negative pathogenic bacteria are not required to be pretreated by EDTA, so that the barrier of adding the outer membrane breaker EDTA with cytotoxicity additionally is overcome. The compound combination can spontaneously crack pathogenic bacteria from outside the body, so that the compound combination can be used as a food additive and has potential application for coping 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, can increase 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 showing electrophoresis of the XFII product of the thermostable lyase gene of the present invention.

Wherein: lane 1 is Marker, lanes 2, 3 are the gene product XFII, which is 489bp in size.

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

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

FIG. 3A shows the cleavage curves of the cleavage enzymes XFII and LysSE24 for EDTA-pretreated E.coli JM109, and FIG. 3B shows the relative enzyme activities remaining after treatment of the cleavage enzyme XFII according to the invention at 4-80℃for 2h.

FIG. 4A shows the cleavage activity of the present invention when the cleavage enzyme is stored at 4℃and FIG. 4B shows the cleavage activity of the present invention when the cleavage enzyme is stored at ordinary temperature, and FIG. 4℃ shows the cleavage activity of the present invention after repeated freeze thawing 5 times at-20 ℃.

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

FIG. 6 shows the cleavage activity of the cleavage enzymes XFII and 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 the cleavage enzyme XFII in combination with chitosan in different ratios.

Detailed Description

The present invention will be described in detail with reference to the following drawings and examples. The following examples are only preferred embodiments of the present invention, and it should be noted that the following descriptions are merely for explaining the present invention, and are not limiting in any way, and any simple modification, equivalent variation and modification of the embodiments according to the technical principles of the present invention are within the scope of the technical solutions of the present invention.

In the examples below, the viral genomic DNA/RNA extraction kit involved was purchased from Beijing Tiangen Biochemical technology Co. Other materials, plasmids, strains, reagents, etc., used, unless otherwise specified, are commercially available.

Example 1: extraction of salmonella phage XFII-1 genome

1) Obtaining pure culture solution of salmonella phage XFII-1.

Salmonella single colonies (designated XFII) were picked from the solid medium and inoculated into 15mL LB medium and cultured with shaking at 37℃for 6-8h.

The salmonella phage plaque (marked as XFII) is picked up to obtain the salmonella phage, and the phage is named as XFII-1; 500 mu L of salmonella phage XFII-1 is inoculated into the salmonella culture medium, shaking culture is carried out for 4-6 hours at 37 ℃ to obtain a lysate containing the salmonella phage XFII-1, then the lysate is centrifuged at 8000rpm for 10min, and a filter membrane with 0.22 mu m is used for filtering to obtain a purified phage lysate without residual salmonella, and the purified phage lysate is subsequently used for genome extraction.

2) Phage XFII-1 genomic DNA extraction.

Extracting salmonella phage genome DNA from the obtained purified phage lysate by using a viral genome DNA/RNA extraction kit according to the method described in the specification of the use of the kit, and finally obtaining an extracted product, namely the genome DNA of the salmonella phage XFII-1, and storing the obtained product at the temperature of minus 20 ℃ for later use.

Example 2: functional alignment of the lyase genes in NCBI library

The extracted genome DNA of salmonella phage XFII-1 is sent to a biological company for whole genome sequencing, genome is subjected to gene prediction by a RAST tool, and the ORF sequence predicted by the sample is converted into an amino acid sequence by a transceq program in EMBOSS, wherein the amino acid sequence is shown as SEQ ID NO. 1. The predicted amino acid sequence with the function of lysing the cell wall is compared with public data, and homology comparison is carried out by uploading NCBI library, so that the amino acid sequence (SEQ ID No. 1) of the lyase provided by the invention has higher similarity with a plurality of salmonella phage lyase comprising lyase LysSP1, lysSE24 and the like, but no report of research and application of the lyase with 100% similarity is found at present.

The salmonella phage lyase gene is obtained through sequence annotation and alignment 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 XFII.

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

1. Amplification of the lyase Gene XFII

Primers P1 and P2 were designed according to the nucleotide sequence of XFII gene (SEQ ID No. 2), double restriction sites XhoI, hindIII and a protective base were added to the 5' end, the 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, the PCR product was identified by electrophoresis in 1.5% agarose, the fragment size was 489bp, the band was single and bright, and the gel electrophoresis result was 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 a PCR reaction system is as follows:

the PCR reaction procedure is as follows:

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

The amplified XFII gene product and empty vector pET-29b (+) were subjected to double digestion with restriction enzymes XhoI and HindIII according to the following digestion reaction system, and DNA bands with correct sizes were recovered.

Enzyme digestion reaction system:

and respectively taking 4 mu L of XFII gene fragments 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 DNAligase Solution I ligase, uniformly mixing, and then placing at 16 ℃ for overnight connection, and converting a connection product into DH5 alpha.

The transformants were picked for PCR verification and the target fragments on the plasmids were sequenced to determine if their sequences were completely correct. The recombinant plasmid identified as correct was designated pET-29b-XFII.

3. Construction of E.coli C43 expression Strain containing target Gene XFII

The correctly sequenced plasmid pET-29b-XFII was transferred into E.coli C43 (DE 3) pLysS competent.

mu.L of plasmid pET-29b-XFII was added to 20. Mu.L of competent C43 (DE 3) pLysS and mixed well and then left on ice for 30min. And (5) carrying out heat shock for 90sec in a water bath at 42 ℃ after taking out, and rapidly taking out the ice bath for 2min. Then, 900. Mu.L of LB medium was added thereto, and the culture was incubated at 37℃for 1 hour with shaking at 220 rpm. The bacterial liquid is coated on LB solid medium containing 50 mug/mL kanamycin, and cultured for 8-12h at 37 ℃. Single colonies were randomly picked on plates and inoculated into 5mL of LB medium containing 50. Mu.g/mL kanamycin, and cultured at 37℃with shaking at 220rpm for 10-16h. Thus, a C43 expression strain containing a target gene XFII is obtained, named as C43-pET29b-XFII, and is placed in a refrigerator at-80 ℃ to store glycerol strains for later use.

LysSE24 is a recently reported lyase with higher thermostability, and the invention compares the bactericidal activity and part of the properties of the two lyases.

The amino acid sequence of LysSE24 lyase is shown as SEQ ID NO. 3. The amino acid sequence of the lyase LysSE24 was aligned with the amino acid sequence of the lyase XFII, and the similarity of the lyase LysSE24 to the present invention was found to be 98%.

In the same way, the engineering strain construction method is that pET29b-XFII plasmid is firstly used for constructing pET29b-LysSE24 plasmid vector through site-directed mutagenesis, then plasmid pET29b-LysSE24 is used for heat shock conversion of engineering strain C43 (DE 3) pLysS to obtain recombinant strain which can express lyase LysSE24, named C43-pET29b-LysSE24, and the recombinant strain is placed in a refrigerator at-80 ℃ to store glycerol strain for standby.

Example 4: preparation and purification of recombinant protein lyase XFII

1. Induction and preparation of recombinant proteins

The C43 expression strains of C43-pET29b-XFII and C43-pET29b-LysSE24 obtained in example 3 were activated in 5mL LB medium, respectively, and transferred into 50mL Erlenmeyer flasks at 37℃and 220rpm to OD ₆₀₀ =0.6-0.8, 1mM IPTG was added to 16 ℃, and incubated for 4h in a shaker at 100 rpm. The cells were collected by centrifugation at 7000rpm, 1mL of the supernatant of the fermentation broth was taken out for use, the cell pellet was suspended with 50mM sodium phosphate (pH=7.4) and 300mM sodium chloride buffer, and then sonicated, the disrupted cell broth was centrifuged at 12000rpm for 20 minutes, the supernatant of the disrupted broth was collected, and the disrupted cell pellet was resuspended with 2mL of buffer. Respectively collecting 50 μl of fermentation broth supernatant, crushing solution precipitate, adding 12.5 μl of 5×loading BThe buffer was bathed in boiling water for 10min, and then SDS-PAGE was performed to detect the expression of the lyase, which showed that most of the lyase was present in the supernatant of the disruption solution, and the protein yield was over 100mg/L.

2. Extended preparation and purification of recombinant proteins

The preserved strains C43-pET29b-XFII and C43-pET29b-LysSE24 were inoculated into 5mL of LB medium containing 50. Mu.g/mL kanamycin, cultured overnight at 37℃under shaking at 220rpm, added to fresh 300mL of LB medium containing 50. Mu.g/mL kanamycin in an inoculum size of 1% by volume the following day, and cultured to OD at 37 ℃ ₆₀₀ =0.6-0.8, and the expression of the lyase was induced by adding 1mM iptg,16 ℃, in a shaker at 100 rpm. The cells were collected by centrifugation at 7000rpm for 10min, resuspended in 30mL of 20mM imidazole solution, sonicated until the imidazole solution containing cells was clear, centrifuged at 12000rpm for 20min, and the supernatant of the disruption solution containing crude enzyme solution was collected and suction-filtered for use.

And (3) eluting 20% alcohol in the nickel column affinity chromatography column stored at the temperature of 4 ℃ until the alcohol is completely eluted. 10 column volumes of 500mM NaCl,20mM Tris-HCl,20mM imidazole were added to completion. And (3) loading all the supernatant of the crushed liquid containing the crude enzyme liquid after suction filtration to a column until all the solution flows out. Adding 10mL 500mM NaCl,20mM Tris-HCl,50mM imidazole and eluting the hetero protein at pH 7.4; 2mL,5mL and 7mL of buffer solutions with imidazole concentrations of 50mM, 100mM and 200mM, respectively, were collected and stored at 4 ℃.

The collected buffers containing the target protein were taken out in an amount of 50. Mu.L, and 12.5. Mu.L of 5×loading Buffer was added thereto, followed by 10min in a boiling water bath and centrifugation at 12000rpm for 5min. SDS-PAGE gel is used to detect the purity of recombinant protein lyase XFII, and the experimental results are shown in FIG. 2. Wherein FIG. 2A is a map of the lyase XFII and FIG. 2B is a map of the lyase LysSE 24.

Finally, the concentration of the lyase XFII was measured using the Bradford kit and adjusted to the same concentration by dilution. The method for detecting the relevant cleavage activity and specific experimental procedures are described in example 5.

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

1. Bactericidal effect of lyase XFII on Escherichia coli JM109

The host bacterium E.coli JM109 was activated with 5mL of LB medium, transferred into a 50mL Erlenmeyer flask, and cultured at 37℃to OD ₆₀₀ =0.6-0.8. Centrifugation at 7000rpm for 10min, the supernatant was discarded and incubated with 10mL of 20mM Tris-HCl (pH=8.0) and 100mM EDTA buffer at 37℃for 5min. Centrifugation at 7000rpm for 10min to remove EDTA, resuspending the cells in the above buffer, washing twice, and finally adjusting to OD with buffer ₆₀₀ About 1.0. Purified lyase XFII was adjusted to a protein concentration of 0.104mg/mL, and 1. Mu.L (20-25 nM) of lyase XFII and 199. Mu.L of EDTA-pretreated E.coli JM109 were added to a 96-well plate. The test was set up in triplicate with 1 μl sodium phosphate buffer as negative control. Immediately placing the ELISA plate in an ELISA reader, detecting at 37deg.C for 30min, and taking cleavage time as abscissa, ΔOD ₆₀₀ On the ordinate, a lysis curve was obtained.

The bactericidal activity of the lyase XFII is detected by using a turbidity method, and the bacterial liquid is found to be clarified rapidly under the action of the lyase XFII, and OD is found to be within 5min ₆₀₀ I.e., from 0.8 to 0.2, the result of which is shown in fig. 3A. The experiment shows that the expressed lyase XFII has good bactericidal activity.

The purified enzyme of LysSE24 was adjusted to the same protein concentration (0.104 mg/mL) as XFII, the bactericidal activity was compared, the bactericidal activity of LysSE24 was examined by the same method, and the bactericidal activity of LysSE24 was compared with that of LysSE XFII, and as a result, as shown in fig. 3A, the activity of LysSE XFII of the present invention was 193% higher than that of LysSE 24. The method shows that the use cost and the industrial production cost of the lyase XFII can be greatly reduced in the application process.

The active site of lyase XFII was found by conserved sequence analysis to be: glu18, pro29, thr35. Whereas the lyase LysSE24 was obtained by cleaving the XFII mutation sites N-40-Y, A-97-V, A-159-D, the Swiss-model simulated three-dimensional structure revealed that sites 40, 97 and 159 were all located on the surface of the protein, and that the steric hindrance of 3 amino acids after mutation was greater than that of the original amino acids, and this was probably one reason for affecting LysSE24 enzyme activity.

2. Sterilization profile detection of lyase XFII

The results of the sterilization spectrum measurement of the lyase XFII using the sterilization detection method in the first section of example 5, respectively selecting Escherichia coli DH5 alpha, escherichia coli JM109, escherichia coli C43 (DE 3) and a plurality of environmental isolation pathogenic Escherichia coli, acinetobacter baumannii standard strain, a plurality of environmental isolation salmonella, klebsiella pneumoniae and Staphylococcus aureus, showed that the lyase XFII had a very remarkable sterilization effect on these indicator bacteria on the premise of EDTA pretreatment of the indicator bacteria, and further compared the sterilization effect of the lyase XFII on these indicator bacteria, and are shown in Table 1. Meanwhile, the lyase XFII has good bactericidal effect on various gram-negative bacteria, wherein the lyase XFII has strong bactericidal effect on escherichia coli DH5 alpha, escherichia coli JM109 and multiple environmental separation pathogenic escherichia coli, acinetobacter baumannii AB1 and klebsiella pneumoniae K3, has good bactericidal effect on multiple environmental separation pathogenic salmonella, and even has certain bactericidal effect on gram-positive bacteria staphylococcus aureus, so that the lyase XFII belongs to a broad-spectrum lyase.

Table 1: sterilization profile of lyase XFII

Note that: ++++: clear by lysis, OD ₆₀₀ About 0.6 drop; +++: cleavage of the clarified OD ₆₀₀ About 0.5 drop; ++: cleavage is clearer, OD ₆₀₀ About 0.3 drop; +: cracking is turbid, insufficient OD ₆₀₀ About 0.2 drop, -: does not crack.

Example 6: thermal stability detection of lyase XFII and analysis of partial enzymatic Properties (storability, pH value)

1. Thermal stability and low-temperature storability detection of lyase XFII

20. Mu.L of purified lyase XFII was placed at 4℃respectively,Treating in a water bath at 20deg.C, 30deg.C, 37deg.C, 45deg.C, 50deg.C, 55deg.C, 60deg.C, 65deg.C, 70deg.C, 75deg.C and 80deg.C for 2 hr. The treated sample was taken out for 30 minutes in ice bath, and the bactericidal activity was measured as in the first section of example 5, and the relative bactericidal activity was calculated using untreated lyase as a control, relative lytic activity=lytic activity of treated enzyme/lytic activity of untreated enzyme×100%, wherein bactericidal activity=Δod ₆₀₀ /Δt. As shown in FIG. 3B, the bactericidal activity of the lyase XFII treated for 2 hours at the temperature below 70 ℃ is similar to that of the lyase not treated, but the bactericidal activity of the lyase treated for 2 hours at the temperature of 80 ℃ still has 54.82%, which proves that the stability is good, and the protease XFII has unique advantages 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.ΔOD within 5min on storage date ₆₀₀ On the ordinate, a lysis curve was obtained over the date of storage. As a result, as shown in FIG. 4A, the bactericidal activity was still significant after 175 days of storage at 4℃and the longer time data was not shown, and the bactericidal activity after storage of the lyase XFII at normal temperature was examined by the same method. As a result, as shown in FIG. 4B, the enzyme activity was hardly decreased within 15 days.

The purified lyase XFII is repeatedly frozen and thawed at-20 ℃ for 2 hours each time, taken out and thawed for 1 hour, and 20 mu L of the purified lyase XFII is taken out according to the requirement to detect the bactericidal activity. As shown in FIG. 4C, the sterilizing activity of the lyase XFII after repeated freeze thawing for 5 times at-20 ℃ is still more remarkable, and more data are not shown, and all the results prove that the lyase XFII is relatively stable and is suitable for long-term storage.

2. Thermal resistance analysis of lyase XFII, optimal temperature and optimal pH detection

A reaction system was prepared as in the first part of example 5, and the host JM109 was uniformly conditioned to OD ₆₀₀ Approximately 1.0, then the systems were placed at 4℃at 10℃at 20℃at 30℃at 37℃at 40℃at 45℃at 50℃at 60℃for 5 minutes, the centrifuge tube was removed, and the test was repeated with the same volume of sodium phosphate buffer as negative control. Immediately placing in an enzyme labeling instrument, and measuring the residual OD ₆₀₀ . ΔOD within 5min with reaction temperature on the abscissa ₆₀₀ The sterilization curve of the optimal reaction temperature is obtained on the ordinate. As shown in FIG. 5A, the results show that the cleavage enzyme XFII has good cleavage activity in the range of 4-60 ℃, and particularly has the highest catalytic activity at 37 ℃.

Escherichia coli JM109 cells were resuspended in universal buffer to adjust pH between 3-12, and then subjected to the method of the first part of example 5 at an optimum temperature (37 ℃) according to OD before and after the reaction ₆₀₀ The change in (2) determines the optimum pH, and the result is 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 culture medium

1. Cleavage Activity of lyase XFII in LB Medium

EDTA-treated E.coli JM109 was washed and then resuspended in LB medium, and the bactericidal activity was measured as in the first part of example 5. As a result, as shown in FIG. 6, the bactericidal activity of the lyase XFII was slightly inhibited in LB medium, while the bactericidal activity of the lyase LysSE24 against E.coli JM109 in LB medium was examined by the same method, as a result, as shown in FIG. 6, 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 buffer solutions, the effectiveness of the lyases tends to decrease under bacterial growth supporting conditions, which limits their broader effectiveness, especially for in vitro applications. The lyase XFII of the invention can show bactericidal activity under the eutrophic condition of LB culture medium, which becomes one of the unique advantages and has wider application value.

2. Cleavage Activity of lyase XFII in serum Medium

After adjusting to OD as in the first part of example 5 ₆₀₀ 0%, 10%, 20%, 30%, 40%, 50% rabbit serum is added into a reaction system of Escherichia coli JM109 (Escherichia coli) of approximately 1.0, and 50% rabbit serum and sodium phosphate buffer are used for preventingThe OD was measured as in the first part of example 5, with the addition of lyase as a negative control ₆₀₀ Is a variation of (c). As shown in FIG. 7A, the cleavage enzyme XFII was able to promote the cleavage of E.coli JM109 when up to 50% of rabbit serum was added. In contrast, when the cleavage enzyme LysSE24 was subjected to serum resistance as in the first section of example 5, the cleavage enzyme LysSE24 also exhibited bactericidal activity in serum, which was comparable to that in Tris-HCl buffer, without exhibiting significant characteristics of promoting bactericidal activity in serum, as shown in FIG. 7B.

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

The host bacterium E.coli JM109 was activated with 5mL of LB medium, transferred into a 50mL Erlenmeyer flask, and cultured at 37℃to OD ₆₀₀ =0.6-0.8. Centrifugation at 7000rpm for 10min, discarding supernatant, washing the cells once with 10mL of 20mM Tris-HCl (pH=8.0), and finally adjusting to OD with buffer ₆₀₀ About 1.0. Purified lyase XFII was adjusted to a protein concentration of 0.54mg/mL and 6mg/mL chitosan was formulated with 1% acetic acid, attempting to mix the lyase and chitosan in varying proportions (1:1, 1:2, 2:1, 3:1, 1:3, 4:1, 1:4, V/V) to form a bactericide formulation. In vitro bactericidal activity assays were performed on each group of different ratio mixes in 96-well plates.

At the time of each group test, 30 mu L of bactericide was added to each well of a 96-well plate to lyse E.coli JM109. The test was performed in triplicate with Tris-HCl buffer, lyase XFII, chitosan solution as negative control. Immediately placing the ELISA plate in an ELISA reader, detecting at 37deg.C for 30min, and taking cleavage time as abscissa, ΔOD ₆₀₀ On the ordinate, a lysis curve was obtained. As shown in FIG. 8, the Escherichia coli JM109 which is not pretreated by EDTA has a certain cracking effect under the action of the bactericide compound combination, and the combined bactericidal effects of different formula ratios are different, so that the effect is most obvious when the chitosan/lyase is 1:3, and the OD is the same as that of the Escherichia coli JM109 which is not pretreated by EDTA ₆₀₀ Can and OD within 5min ₆₀₀ I.e. from 0.9 to 0.55. In the case of acting on non-pretreated JM109 with only lyase XFII, it is necessary to use EDTA, chloroform and lemon as the external membrane of gram-negative bacteriaThe acid and other outer membrane damaging agents have good cracking effect, so when the lyase XFII is singly acted, the OD of JM109 escherichia coli ₆₀₀ The trend was shown to be horizontal and neither Tris-HCl buffer of the lyase XFII nor chitosan itself produced a lysis effect on E.coli JM109. Meanwhile, experiments also prove that the compound combination of the lyase XFII and the chitosan has good in-vitro cracking effect on the escherichia coli DH5 alpha, so that the escherichia coli DH5 alpha has broad spectrum.

The formula of the complex combination of the XFII and Chitosan, which breaks through the outer membrane of gram-negative bacteria and spontaneously cracks pathogenic bacteria from outside the body, has never been reported, chitosan (Chitosan) is also called deacetylated chitin, and is obtained by deacetylation of chitin (Chitosan) widely existing in nature, and the excellent properties of the natural polymer, such as biological functionality, compatibility, blood compatibility, safety, microbial degradability and the like, are widely focused by various industries, accord with national food additive use standard GB-2760, and have made great progress in application research in various fields of medicine, food, chemical industry, cosmetics, water treatment, gold biochemical engineering, biomedical engineering and the like. Meanwhile, since chitosan is positively charged, it is hypothesized that chitosan can interact with the surface of negatively charged cells to destroy membrane permeability, and thus has a synergistic effect with lyase XFII. The compatibility of the pathogenic bacteria capable of being spontaneously cracked from the outside of the body enables the pathogenic bacteria to be used as a food additive, and has potential application for coping with food pollution. Meanwhile, the compound feed additive can be used as a feed additive for livestock and poultry, can improve the immunity of the livestock and poultry, can increase 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 raising industry.

Sequence list

<110> university of Shandong

<120> heat-resistant and nutrient-resistant salmonella broad spectrum lyase with in-vitro cleavage 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 of XFII Gene encoding lyase

<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 (2)

1. The application of the heat-resistant and nutrient-resistant salmonella wide-spectrum lyase with in-vitro cracking activity in preparing a pathogen preparation under the nutrient-rich condition is characterized in that: the lyase is salmonella phage lyase, named XFII, the amino acid sequence of the lyase is shown as SEQ ID NO.1, the gene encoding the lyase is named XFII, and the nucleotide sequence of the gene is shown as SEQ ID NO. 2; the eutrophic condition refers to rabbit serum or LB culture medium; the pathogenic bacteria are pathogenic escherichia coli, salmonella, acinetobacter baumannii, klebsiella pneumoniae and staphylococcus aureus.

2. A broad spectrum bactericide of compound combination is characterized in that: the bactericide is prepared from heat-resistant and nutrient-resistant salmonella enterica broad-spectrum lyase with in-vitro cleavage activity and chitosan, wherein the amino acid sequence of the heat-resistant and nutrient-resistant salmonella broad-spectrum lyase is shown as SEQ ID NO. 1; wherein the mixing volume ratio of the lyase to the chitosan is as follows: 6mg/mL chitosan and 0.54mg/mL lyase were 1:3.

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