CN111876400A

CN111876400A - Normal temperature lyase Sly and polynucleotide for coding same

Info

Publication number: CN111876400A
Application number: CN202010780549.2A
Authority: CN
Inventors: 林连兵; 张瑶; 陆瑶; 张棋麟; 王峰
Original assignee: Kunming University of Science and Technology
Current assignee: Kunming University of Science and Technology
Priority date: 2020-08-06
Filing date: 2020-08-06
Publication date: 2020-11-03
Anticipated expiration: 2040-08-06
Also published as: CN111876400B

Abstract

The invention discloses a normal temperature lyase and a polynucleotide for coding the lyase, wherein the amino acid sequence of the lyase is shown as SEQ ID NO. 1, and the nucleotide sequence of the lyase is shown as SEQ ID NO. 2; the lyase can inhibit the growth of partial gram-positive bacteria and gram-negative bacteria, has catalytic activity within the range of 20-50 ℃, has higher catalytic activity within the range of 30-45 ℃, and can be used for constructing a genetic engineering strain for producing the lyase by a nucleotide sequence.

Description

Normal temperature lyase Sly and polynucleotide for coding same

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a normal-temperature lyase Sly and a nucleotide sequence for encoding the same.

Background

In recent years, due to the unreasonable use of antibiotics, the problems of bacterial multi-drug resistance, drug residues, food safety, imbalance of environmental flora and the like are caused, and the health of human beings and animals is seriously threatened. There is an urgent need to develop novel antibacterial agents, and bacteriophages, which are natural enemies of bacteria, provide new ideas for people to resist bacterial invasion, and more people are trying to treat bacterial infections with bacteriophage lytic enzymes.

The phage lyase (hereinafter referred to as lyase) is a kind of peptidoglycan hydrolase expressed at the end stage of phage infection of a host, and can rapidly degrade peptidoglycan on the cell wall of host bacteria, so that the infected bacteria are cracked and progeny phage particles are released. Phage lytic enzymes can be classified into glucosidases, amidases, endopeptidases and transglycosylases, depending on the site of covalent bond acting on the peptidoglycan. In addition, lytic enzymes have a typical modular structure, including an N-terminal Catalytic Domain (CD) and a C-terminal Cell wall binding domain (CBD). The catalytic domain structure has catalytic activity and can specifically cut off the chemical bond of peptidoglycan on the bacterial cell wall; the binding domain structure can be combined with a specific substrate on the cell wall of host bacteria, and mediates the effective combination and cleavage of the catalytic domain structure and an action target. In addition, Rehman estimates that there are roughly 10 on earth³²There were individual phages present, which is roughly 10 times the number of bacteria. It can be said that there are phages present wherever bacteria are distributed. Therefore, lyase has attracted the attention of many researchers as a widely available antibiotic alternative.

Related studies have shown that lyases have many advantages. First, the specificity of the lytic enzymes is better compared to antibiotics and the spectrum of lysis is expanded compared to phages. Researches show that penicillin-resistant streptococcus pneumoniae strains can be killed by lyase, and have no influence on normal flora of human bodies, while antibiotics kill pathogenic bacteria and damage part of normal flora. Secondly, the action of the lyase is efficient and rapid, and the lyase rapidly breaks bacterial cells at the moment of contacting with bacteria in vitro, so that the turbidity of the bacteria is rapidly reduced. Schuch et al add 2 units (2. mu.g) of Bacillus anthracis gamma phage lyase PlyG to 1.0X 10⁴Streptomycin-resistant bacillus cereus rsv f1 was able to lyse bacteria within 10 s. Finally, there is a synergistic effect, either between the lytic enzymes or between the lytic enzymes and the antibiotic. Jado et al found that the combination of low Dp-1 and low Cpl-1 concentrations allowed mice to survive all over, while Dp-1 or Cpl-1 alone, when used alone, was minimal due to severe bacteremiaAll mice died. Djurkovic et al found that the minimal inhibitory concentration of penicillin against Streptococcus pneumoniae could be reduced by combining the lyase Cpl-1 with gentamicin. When Cpl-1 is used in combination with penicillin, it may also be used to synergistically kill penicillin-resistant strains.

Extensive research shows that the lyase has high efficiency, high stability, potential extensive bactericidal property and safety, can specifically kill bacteria and is not easy to cause the bacteria to generate drug resistance; the lyase has a great development value in the aspect of antibiosis, is expected to become a substitute of antibiotics, and has a wide application prospect.

Disclosure of Invention

The invention aims to provide a normal-temperature lyase Sly which can inhibit the growth of partial gram-positive and gram-negative bacteria, has catalytic activity within the range of 20-50 ℃, and is derived from a bacteriophage SP 26. The amino acid sequence of the normal temperature lyase Sly is shown as SEQ ID NO. 1, or polypeptide, analogue or derivative with at least 90% of homology with the amino acid sequence shown as SEQ ID NO. 1.

Another objective of the invention is to provide a polynucleotide encoding a cold-lyase Sly, the nucleotide sequence of which is shown in SEQ ID NO. 2, or a complementary sequence thereof, or a polynucleotide having at least 80% identity with the nucleotide sequence shown in SEQ ID NO. 2 and a complementary sequence thereof.

The invention has the beneficial effects that:

the lyase has catalytic activity within the range of 20-50 ℃, has the highest catalytic activity at 37 ℃, has higher enzyme activity at the pH value of 6-10, and contains Mg²⁺、Na⁺、K⁺The three ions have an activating effect on the enzyme, and the normal-temperature lyase can efficiently crack bacteria, so that the normal-temperature lyase is expected to replace the traditional antibiotic treatment and can also be used as a bacteriostatic agent for sterilization of the environment; the nucleotide sequence can be used for constructing a genetic engineering strain for producing the lyase.

Drawings

FIG. 1 is a schematic diagram of the electrophoresis of the PCR product of the room temperature lyase Sly gene of the invention, wherein a lane 1 represents a Marker; lane 2 is a negative control; lane 3 is the Sly gene PCR product;

FIG. 2 is a schematic diagram of a double-restriction enzyme map of the recombinant plasmid pET28a-Sly of the present invention, wherein lane 1 represents Marker, and lane 2 is a band of the recombinant plasmid pET28a-Sly which is not subjected to double restriction enzyme; lane 3 shows two bands generated by the recombinant plasmid pET28a-Sly after double digestion;

FIG. 3 is a schematic diagram of detection of protein expression of a normal temperature lyase Sly according to the present invention, wherein a lane 1 is GenStar M221-01 protein Marker; lane 2, pET28a-Sly/Rosetta non-induced whole protein; lane 3 shows pET28a-Sly/Rosetta induced 6h of whole protein at 37 ℃ and 150rpm with lactose (1 g/L); lane 4 shows pET28a-Sly/Rosetta induction of the supernatant for 6h at 37 ℃ and 150rpm with lactose (1 g/L); lane 5 shows pET28a-Sly/Rosetta induced 8h of whole protein at 37 ℃ and 150rpm with lactose (1 g/L); lane 6 shows pET28a-Sly/Rosetta induction of the supernatant at 37 ℃ for 8h with lactose (1 g/L) at 150 rpm; lane 7 shows pET28a-Sly/Rosetta induced 10h of whole protein at 37 ℃ and 150rpm with lactose (1 g/L); lane 8 shows pET28a-Sly/Rosetta induction of the supernatant at 37 ℃ for 10h with lactose (1 g/L) at 150 rpm; lane 9 shows pET28a-Sly/Rosetta induced 12h of whole protein at 37 ℃ and 150rpm with lactose (1 g/L); lane 10 shows pET28a-Sly/Rosetta induction of the supernatant at 37 ℃ for 12h with lactose (1 g/L) at 150 rpm;

FIG. 4 is a schematic diagram showing the detection of the purification result of the normal temperature lyase Sly protein of the invention, wherein 1 is GenStar M221-01 protein Marker, and 2 is a second tube after 500 mM imidazole buffer solution passes through the column after the loading;

FIG. 5 is a schematic diagram showing the effect of different temperatures on the activity of a normal temperature lyase Sly in the present invention;

FIG. 6 is a schematic diagram showing the effect of different pH values on the activity of a normal temperature lyase Sly in the invention;

FIG. 7 is a schematic diagram showing the effect of different metal ions on the activity of a normal temperature lyase Sly in the invention;

FIG. 8 is a schematic diagram showing the results of crystal violet staining of Escherichia coli CMCC (B)44102 and Staphylococcus aureus ATCC6538 by the action of the room temperature lyase Sly of the present invention. Wherein A is the thallus form of Escherichia coli CMCC (B)44102 acted by inactivated lyase; b is the thallus form of Escherichia coli CMCC (B)44102 acted by lyase; c is the thallus form of staphylococcus aureus ATCC6538 with the inactivated lyase; d is the thallus form of staphylococcus aureus ATCC6538 under the action of lyase;

FIG. 9 shows the effect of the room temperature lyase Sly according to the invention on the growth of the strains Escherichia coli CMCC (B)44102, Staphylococcus aureus ATCC6538, Salmonella CMC (B)50094 and Shigella DS26 over time.

Detailed Description

The present invention will be described in further detail with reference to the following examples, but the present invention is not limited thereto, and the method in the present example is carried out in a conventional manner unless otherwise specified, and reagents used in the present example are conventional reagents or reagents prepared in a conventional manner unless otherwise specified.

Example 1: cloning and expression of Normal temperature lyase Sly

1. Amplification of lyase Sly Gene (with genomic DNA of phage SP26 as template)

(1) The primer sequences used for amplification of the normal temperature lyase Sly gene are as follows:

a forward primer: 5' -CATGCCATGGCAATGGCTATTAGCAAAAACATGAAG -3’

Reverse primer: 5' -CGGAATTCTGCCACAGCTCCGCCAGCCTCTTTA -3’

Enzyme cutting site:Ncoi andEcoRI

(2) the amplification system was as follows:

TABLE 1 amplification System Components

；

(3) The amplification conditions were as follows:

the reaction system is mixed evenly, the pre-denaturation is carried out for 10min at 94 ℃, then the denaturation is carried out for 45s at 94 ℃, the annealing is carried out for 45s at 58 ℃, the extension is carried out for 90s at 72 ℃, and after 30 cycles, the extension is carried out for 10min at 72 ℃. After amplification, 5. mu.L of the product was collected and analyzed by electrophoresis on a 1.2% agarose gel (see FIG. 1), and the size of the PCR product was 465 bp.

2. Recovery of PCR product gel of lyase Sly gene

(1) Pouring 1.2% agarose gel in an electrophoresis apparatus;

(2) carrying out sample application electrophoresis on the PCR product to be separated and purified, and stopping electrophoresis at a proper position;

(3) cutting the gel containing the target gene fragment under an ultraviolet lamp, and transferring the gel into a 1.5mL EP tube;

(4) recovering target gene segment with StarPrep fast DNA glue recovering kit and the recovering process is as the instruction.

3. Construction of recombinant plasmid

In order to ligate the desired gene fragment to plasmid pET28a, it is necessary to have cohesive ends, i.e., cleavage sites, in both the desired gene fragment and plasmid pET28 a.

(1) Extraction of plasmid pET28a

Activating strains: dipping a strain preservation solution of a plasmid pET28a frozen at-80 ℃ in an aseptic inoculating loop, inoculating the strain preservation solution to an LB solid plate containing kanamycin (the final concentration is 50 mug/mL) by a continuous line method, and culturing for 12-16 h at 37 ℃;

enrichment of bacteria liquid: picking a single colony by using an inoculating loop, and culturing the single colony in a 5mL LB liquid test tube containing kanamycin (the final concentration is 50 mug/mL) at 37 ℃ at 150rpm for 12-16 h;

extraction of plasmid pET28 a: plasmid pET28a was extracted using StarPrep Rapid plasmid Mini-extraction kit, the extraction procedure was as described.

(2) Target gene fragment and double enzyme digestion of plasmid pET28a

The enzyme digestion system is as follows:

TABLE 2 digestion System Components

Reaction conditions: and (3) carrying out enzyme digestion in a metal bath at 37 ℃ for 3h, and recovering the target gene fragment and the plasmid pET28a after enzyme digestion.

(3) Construction of recombinant plasmid

Constructing a recombinant plasmid by connecting and transforming a target gene fragment with a cohesive end and a plasmid pET28 a;

the connecting system is as follows:

TABLE 3 ligation System Components

Reaction conditions: the recombinant plasmid was recovered after 12 hours of ligation in a metal bath at 16 ℃.

4. Expression of recombinant plasmids

(1) The recombinant plasmid is transformed into Escherichia coli DH5 alpha competent cells

Firstly, transferring 10 mu L of recombinant plasmid into a competent cell of a 100 mu L system;

② after ice-bath for 30min, heat shock is carried out for 90s at 42 ℃, and ice-bath is carried out for 30 min;

③ adding 890 microliter LB liquid culture medium, shaking and culturing for 1h at 37 ℃ and 100 rpm;

putting the cultured liquid culture medium into a centrifuge, and centrifuging for 10min at 10,000 rpm;

fifthly, abandoning the supernatant, leaving about 100 mu L of the supernatant, lightly blowing and uniformly mixing the precipitate, coating the mixture on an LB solid plate containing kanamycin (the final concentration is 50 mu g/mL), and culturing for 12-16 h at 37 ℃;

sixthly, observing single colonies of the flat plate, randomly picking the single colonies by using an inoculating loop, and culturing the single colonies in a 5mL LB liquid test tube containing kanamycin (the final concentration is 50 mu g/mL) at 37 ℃ for 12h at 150 rpm.

(2) Double restriction enzyme verification

Firstly, extracting recombinant plasmids by using a StarPrep rapid plasmid miniextraction kit, wherein the extraction method is carried out according to a specification;

secondly, carrying out double enzyme digestion verification on the extracted recombinant plasmid, wherein the double enzyme digestion system is as follows:

TABLE 4 double restriction enzyme System Components

Reaction conditions are as follows: and (3) carrying out enzyme digestion in a metal bath at 37 ℃ for 3h, recovering the recombinant plasmid after enzyme digestion, and carrying out electrophoresis verification (shown in figure 2).

(3) Transformation of the recombinant plasmid intoE. coli Rosetta Strain

Transferring the correctly verified recombinant plasmid intoE. coli RosettIn the strain a, the method is the same as the method; the expression strain pET28a-Sly/Rosetta can be obtained.

5. Induced expression and purification of recombinant protein

Firstly, inoculating the expression strain pET28a-Sly/Rosetta into 5mL LB liquid test tube containing kanamycin (the final concentration is 50 mu g/mL), and culturing at 37 ℃ and 150rpm until OD is reached₆₀₀The value is 0.6 to 0.8;

② inoculating the bacterial liquid according to the proportion of 1 percent to 600mL LB liquid test tube containing kanamycin (the final concentration is 50 mug/mL), culturing at 37 ℃ and 150rpm to OD₆₀₀The value is 1.0 to 1.2; adding inducer lactose (final concentration 1 g/L) into the bacterial liquid of the expression strain, and respectively inducing at 37 deg.C and 150rpm for 6, 8, 10, and 12 h;

③ centrifuging the induced bacterial solution at 4 ℃ and 9000rpm for 8min, then adding appropriate amount of ddH₂Blowing and uniformly mixing the mixture O, centrifuging the mixture at 9000rpm for 10min at 4 ℃, then keeping precipitate, blowing and uniformly mixing the mixture O with PBS buffer solution according to the proportion of 100:1, and then carrying out ultrasonic crushing;

fourthly, ultrasonic crushing: the power is 35%, the operation is carried out for 3s, the operation is stopped for 4s, each tube is used for 60-90 min, the crushing is carried out until the light transmission is achieved, and 80 mu L of whole protein is taken out from the sterilized EP tube. Centrifuging the rest whole protein at 13,000 rpm for 10min to obtain supernatant, and packaging the supernatant into sterilized EP tubes;

SDS-PAGE electrophoresis detection expression result (see figure 3), the target protein has the best expression effect under the conditions of 37 ℃, 150rpm and 1g/L lactose induction for 12 hours; therefore, the optimal induction conditions are 37 ℃, 150rpm and 1g/L lactose induction for 12 h;

sixthly, purifying by using an AKTA prime protein purification system after induction under the optimal condition, and detecting a purification result by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) after purification (see figure 4); the result shows that the elution band of the lyase Sly is single and the expression level is high, which indicates that the purification is successful.

Example 2: enzymology property and enzyme activity experiment of normal temperature lyase Sly

1. Determination of concentration and enzyme activity of lyase Sly protein

(1) Protein concentration determination Using the BCA (bicinchoninic acid) protein concentration assay

Under alkaline conditions, using Cu²⁺Can be reduced to Cu by protein⁺，Cu⁺The characteristic that the complex is combined with the BCA reagent to form purple complex is that the protein concentration of the sample to be detected can be calculated by measuring the OD value of the sample under 590nm wavelength and comparing the OD value with a protein standard curve.

The OD value at 590nm wavelength was measured by an enzyme-labeled meter according to the "microassay" in the BCA protein quantification kit. Protein concentration as abscissa, OD₅₉₀And drawing a standard curve with the value as a vertical coordinate, wherein the obtained standard curve equation is as follows: y = 0.0009x + 0.006 (R)²= 0.9914）。

(2) Enzyme activity determination by adopting Folin phenol method

Under alkaline conditions, the folin phenol reagent can be reduced by the phenolic compound to generate a blue compound. The protease hydrolyzes the casein substrate under the conditions of optimal action temperature and optimal action pH value, and amino acids (such as tyrosine, tryptophan and the like) containing phenolic groups are generated. Therefore, the enzyme activity can be measured by using the principle.

Definition of enzyme activity unit: under the conditions of certain temperature and pH value, the enzyme solution hydrolyzes casein substrate within 1min to generate 1 mu g of tyrosine as an enzyme activity unit, which is expressed by U.

(ii) reagent and solution

The reagent comprises a Folin phenol reagent, 0.4mol/L sodium carbonate solution, 0.4mol/L trichloroacetic acid solution, a phosphate buffer solution with the pH value of 7.2, 2% casein solution and 100 mu g/mL tyrosine solution.

② drawing of standard curve

Preparing various concentrations of the tyrosine solutions according to Table 5

TABLE 5 preparation of solutions of tyrosines with different concentrations

The determination step comprises: respectively sucking 1mL of tyrosine solution with different concentrations in the table above, and adding5mL of 0.4mol/L sodium carbonate solution, and then 1mL of forlin phenol reagent is added; shaking, placing in 40 deg.C water bath kettle, maintaining the temperature for 20min, and measuring OD with enzyme labeling instrument₆₈₀And (6) value and recording. Using tyrosine mass as abscissa, OD₆₈₀And (3) drawing a standard curve with the value as a vertical coordinate to obtain a standard curve equation: y = 0.003x + 0.0099 (R)²= 0.9959）。

③ enzyme activity determination step

2mL of the diluted solution (concentration of 46.67. mu.g/mL) of the enzyme solution was taken, preheated in a 40 ℃ water bath for 2min, then 1mL of the similarly preheated 2% casein solution was added, and the temperature was accurately maintained in the 40 ℃ water bath for 5 min. After the time is up, 1mL of 0.4mol/L trichloroacetic acid solution is added immediately to stop the reaction, and the reaction is kept in a water bath kettle at 40 ℃ for 20min to precipitate residual protein. Sucking the solution containing precipitate, filtering with 0.22 μm filter membrane, sucking filtrate 1mL, adding 0.4mol/L sodium carbonate solution 5mL, shaking Folin phenol reagent 1mL, maintaining temperature in water bath at 40 deg.C for 20min, and measuring OD₆₈₀The value is obtained.

Determination of the lyase OD₆₈₀The value was 0.317 and OD was added₆₈₀Values are substituted into the standard curve equation: y = 0.003x + 0.0099 (R)²= 0.9959), the tyrosine mass was calculated to be 102.37 μ g. Substituting tyrosine mass into the formula: y = (A x N)/(V x t) (Y: enzyme activity of sample, U; A: tyrosine mass, μ g; N: dilution multiple of sample; V: total volume of reaction reagent, mL; t: reaction time, min), and enzyme activity Y = (102.37 x 5)/(4 x 5) = 25.59U is calculated.

2. Influence of different action temperatures, pH values and metal ions on normal-temperature lyase Sly

The cleavage enzyme can hydrolyze the peptidoglycan, which, when hydrolyzed, results in a reaction substrate OD₆₀₀The value decreased significantly; the higher the enzyme activity, the higher the OD₆₀₀The larger the degree of decrease in the value, and thus the reaction substrate OD₆₀₀The change in value can be used to assess the activity of the lyase.

Enzyme activity (%) = [ (OD)_{Before reaction of 600}－OD_{After 600 reaction}) Initial OD₆₀₀］×100

The highest enzyme activity was determined to be 100%, and the relative enzyme activity (%) = (enzyme activity/highest enzyme activity at other temperatures) × 100.

(1) Preparation of reaction substrate

Dipping strain preservation solution of host bacteria DS26 frozen at-80 ℃ by using an aseptic inoculating loop, inoculating the strain preservation solution on an LB solid plate by a continuous line method, and culturing for 12-16 h at 37 ℃;

② picking the single colony of the plate by using sterile inoculating loop, placing it into 5mL LB liquid test tube, culturing at 37 deg.C and 150rpm to OD₆₀₀The value is 0.6-0.8, inoculating the bacterial liquid into a 600mL LB liquid test tube according to the proportion of 1%, culturing at 37 ℃ and 150rpm until the bacterial liquid is OD₆₀₀The value is 1.0 to 1.2;

thirdly, centrifuging the bacterial liquid at 9000rpm for 10min, and then discarding the supernatant. The precipitate was taken up in 10mL ddH₂And (3) blowing and uniformly mixing the mixture by using oxygen, carrying out ultrasonic crushing (the power is 35%, the work time is 3s, the stop time is 4s, and the total time is 120 min), and centrifuging at 13000rpm for 10min to obtain a precipitate. The pellet was reused with 10mL ddH₂Blowing, beating and uniformly mixing; in this case, the cell wall of the host strain DS26 was broken cells and used as a substrate for the following experiment.

(2) Optimum action temperature of normal temperature lyase Sly

Adding purified lyase Sly (with the concentration of 135.56 mu g/mL) into the prepared reaction substrate, and then placing the substrate at different temperatures for reaction for 30min, wherein the reaction temperatures are respectively 4 ℃, 20 ℃, 30 ℃, 35 ℃, 37 ℃, 40 ℃, 45 ℃, 50 ℃, 60 ℃ and 70 ℃. Wherein the initial OD of the reaction substrate₆₀₀=0.365 depending on OD before and after reaction₆₀₀Determines its optimum temperature (see fig. 5).

As can be seen from the analysis of FIG. 5, the optimum action temperature of the lyase is 37 ℃, and the enzyme activity is higher between 30 ℃ and 45 ℃.

(3) Optimum action pH value of normal temperature lyase Sly

Adding purified lyase Sly (the concentration is 135.56 mu g/mL) into the prepared reaction substrate, respectively reacting in phosphate buffer solutions with different pH values for 30min at the optimal action temperature, and measuring OD before and after the reaction₆₀₀Value, wherein the initial OD of the reaction substrate₆₀₀=0.295 in terms of OD before and after reaction₆₀₀The amount of change in (c) determines the optimum pH (see fig. 6).

As can be seen from the analysis of FIG. 6, the optimum pH value of the lyase is 7.5, and the activity of the lyase is higher between 6 and 10.

(4) Effect of different Metal ions on enzyme Activity

Adding the purified lyase Sly (the concentration is 145.56 mu g/mL) into the prepared reaction substrate, and adding different metal ions Mn into the mixed solution²⁺、Ca²⁺、Mg²⁺、Zn²⁺、Fe³⁺、Na⁺、K⁺Keeping the ion concentration at 1mmol/L, reacting for 30min under the conditions of optimum action temperature and optimum pH value, and determining OD before and after reaction₆₀₀Value, wherein the initial OD of the reaction substrate₆₀₀=0.411, control group ddH₂O replaces the metal ion. The metal ions capable of improving the enzyme activity are called activators of the enzyme, and conversely, the metal ions are called inhibitors of the enzyme (see figure 7).

As can be seen from FIG. 7, Mg²⁺、Na⁺、K⁺These three ions have an activating effect on the enzyme, Zn²⁺、Mn²⁺、Fe³⁺These three ions have an inhibitory effect on the enzyme, while Ca²⁺Has little influence on the enzyme.

3. Crystal violet staining result of normal temperature lyase Sly acting on escherichia coli CMCC (B)44102 and staphylococcus aureus ATCC6538

Escherichia coli CMCC (B)44102, Staphylococcus aureus ATCC6538 were cultured to OD₆₀₀The values are 0.531 and 0.519 respectively, the bacteria liquid is centrifuged for 10min at 8000rpm to obtain thallus, ddH is added₂O washing for 3 times, collecting the cells, diluting with PBS, and then 100 μ L of bacterial solution (2.37X 10)³CFU/mL) is added with 900 muL lyase Sly (the concentration is 141.11 mug/mL), the reaction is carried out for 30min at 37 ℃, the mixture is stained by ammonium oxalate crystal violet staining solution, microscopic examination is carried out under an optical microscope after flaking (see figure 8), and enzyme solution inactivated for 20min at 121 ℃ is adopted as contrast;

as can be seen from FIG. 8, the forms of some of the cells of E.coli CMCC (B)44102 and Staphylococcus aureus ATCC6538 on which lyase Sly acts cannot be stained with crystal violet staining solution, indicating that the cells are cleaved by lyase Sly; the shapes of the cells of the escherichia coli CMCC (B)44102 and the staphylococcus aureus ATCC6538 acted by the inactivated enzyme solution are not obviously changed.

4. Effect of Normal temperature lyase Sly on growth of Escherichia coli CMCC (B)44102, Staphylococcus aureus ATCC6538, Shigella DS26, Salmonella CMC (B)50094 strains

Inoculating the bacterial liquid of Escherichia coli CMCC (B)44102, staphylococcus aureus ATCC6538, shigella DS26 and salmonella CMC (B)50094 into LB liquid culture medium according to the inoculation amount of 1 percent, and culturing at 37 ℃ and 150rpm to OD₆₀₀The values are 0.506, 0.522, 0.537, 0.525, respectively. The enzyme solution (252.22. mu.g/mL) was filtered through a sterilized 0.22 μm filter and Mg was added²⁺、Na⁺、K⁺(final concentration is 1 mmol/L), 270. mu.L of enzyme solution and 30. mu.L of Escherichia coli CMCC (B)44102 bacterial solution (1.12X 10)³CFU/mL), Staphylococcus aureus ATCC6538 (1.43X 10 bacterial liquid³CFU/mL), Shigella DS26 bacterial liquid (1.08X 10)³CFU/mL), Salmonella CMC (B)50094 bacterial liquid (1.67X 10)³CFU/mL), reacting the mixed solution in a constant-temperature incubator at 37 ℃ for 0, 10, 20, 30, 40, 50 and 60min, coating 50 mu L of the reacted mixed solution on a flat plate, and inactivating the enzyme solution for 20min at 121 ℃ as a contrast; after coating, the mixture is placed in a constant-temperature incubator at 37 ℃ for overnight culture, the number of living cells of an experimental group and a control group is counted, and the antibacterial activity is calculated. (Note: 3 replicates each of the groups) antibacterial activity was defined as log relative inactivation unit log₁₀(N0/Ni), wherein N0 represents the number of living cells treated by the inactivated enzyme solution (control group), and Ni represents the number of living cells treated by the enzyme solution (experimental group);

as can be seen from fig. 9: under the condition of certain enzyme concentration and bacteria concentration, the killing effect of the lyase Sly on the strain changes along with the change of time. When the action time is 10min, the number of the living cells of the Escherichia coli CMCC (B)44102 is reduced most, the number of the living cells of the Shigella DS26 and the Salmonella CMC (B)50094 is reduced next time, and the number of the living cells of the Staphylococcus aureus ATCC6538 is reduced relatively less. In conclusion, the lyase has a killing effect on both gram-positive and gram-negative bacteria.

Sequence listing

<110> university of Kunming science

<120> a normal temperature lyase Sly and polynucleotide encoding the enzyme

<160>4

<170>SIPOSequenceListing 1.0

<210>1

<211>154

<212>PRT

<213> Phage SP26(Phage SP26)

<400>1

Met Ala Ile Ser Lys Asn Met Lys Ala Phe Leu Asp Met Leu Ala Tyr

1 5 10 15

Ser Glu Gly Thr Asp Asn Gly Arg Gln Lys Thr Asn Asn His Gly Tyr

20 25 30

Asp Val Ile Val Gly Gly Ser Leu Phe Thr Asp Tyr Ser Asp His Pro

35 40 45

Arg Lys Leu Ile Ser Leu Pro Lys Leu Gly Ile Lys Ser Thr Ala Ala

50 55 60

Gly Arg Tyr Gln Val Leu Ala Lys Phe Tyr Asp Ala Tyr Lys Lys Gln

65 70 75 80

Leu Arg Leu Pro Asp Phe Ser Pro Thr Ser Gln Asp Ala Ile Ala Met

85 90 95

Gln Leu Ile Arg Glu Cys Lys Ala Thr Ala Asp Ile Glu Ala Gly Arg

100 105 110

Ile Ala Asp Ala Ile His Lys Cys Arg Ser Arg Trp Ala Ser Leu Pro

115 120 125

Gly Ala Gly Tyr Gly Gln His Glu Gln Lys Leu Asp Lys Leu Ile Gln

130 135 140

Val Tyr Lys Glu Ala Gly Gly Ala Val Ala

145 150

<210>2

<211>465

<212>DNA

<213> Phage SP26(Phage SP26)

<400>2

atggctatta gcaaaaacat gaaggcgttt ctggatatgc tggcgtacag cgagggcacg 60

gataacgggc ggcagaaaac caataatcat ggctatgatg tgattgttgg tggctcactg 120

tttaccgact attccgacca cccgcgcaaa ctgattagcc tgcctaagct gggcatcaaa 180

tccaccgccg ccgggcgata tcaggtgctg gctaagtttt atgatgcgta caaaaagcag 240

ttgcgtttgc cggacttctc cccaacatcg caggacgcta ttgcaatgca gctaatccgt 300

gaatgcaagg ccaccgccga tattgaagct ggtcgcattg ctgatgctat tcataaatgc 360

cgctcccgct gggcttcatt gccgggtgct ggttatggtc agcacgaaca gaaactggat 420

aagctgattc aggtatataa agaggctggc ggagctgtgg catga 465

<210>3

<211>36

<212>DNA

<213> Artificial sequence (Artificial)

<400>3

catgccatgg caatggctat tagcaaaaac atgaag 36

<210>4

<211>33

<212>DNA

<213> Artificial sequence (Artificial)

<400>4

cggaattctg ccacagctcc gccagcctct tta 33

Claims

1. A normal temperature lyase Sly, which is characterized in that: the amino acid sequence is shown in SEQ ID NO. 1.

2. Polynucleotide encoding an orthothermolysin Sly according to claim 1, characterized in that: the nucleotide sequence is shown as SEQ ID NO. 2.