CN111876400B - Normal temperature lyase Sly and polynucleotide for coding same - Google Patents

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

A room temperature lyase Sly and a polynucleotide encoding the enzyme

technical field

The invention belongs to the field of biotechnology, in particular to a room temperature lyase Sly and a nucleotide sequence encoding the enzyme.

Background technique

In recent years, due to the irrational use of antibiotics, problems such as multi-drug resistance of bacteria, drug residues, food safety, and environmental flora imbalance have been caused, which seriously threaten the health of humans and animals. There is an urgent need to study new antibacterial reagents, and phages, the natural enemies of bacteria, provide new ideas for people to resist bacterial invasion. More and more people are trying to use phage lyases to treat bacterial infections.

Phage lyase (hereinafter referred to as lyase) is a type of peptidoglycan hydrolase expressed at the end of phage infection of the host, which can rapidly degrade the peptidoglycan on the cell wall of the host bacteria, lyse the infected bacteria and release progeny phage particles. Phage lyases can be classified into glucosidases, amidases, endopeptidases, and transglycosylases according to their role in the covalent bond sites of peptidoglycan. In addition, lyases have a typical modular structure, including N-terminal catalytic domain (CatalyticDomain, CD) and C-terminal cell-wall binding domain (Cell-wall binding domain, CBD). The catalytic domain structure has catalytic activity and can specifically cut the chemical bonds of peptidoglycan on the bacterial cell wall; the binding domain structure can bind to specific substrates on the host bacterial cell wall, mediating the effective binding and cleavage of the catalytic domain structure and the target. . In addition, according to Rehman's calculations, there are about 10 ³² bacteriophages on the earth, which is about 10 times the number of bacteria. It can be said that wherever there is a distribution of bacteria, there will be bacteriophages. Therefore, lyases have attracted the attention of many researchers as a wide-ranging alternative to antibiotics.

Related studies have shown that lyases have many advantages. First, the specificity of lyases is better than that of antibiotics, and the spectrum of lysis is expanded compared to phages. The study found that penicillin-resistant Streptococcus pneumoniae strains can be killed by lyase without affecting the normal flora of the human body, while antibiotics kill pathogenic bacteria and damage part of the normal flora. Secondly, the action of lyase is efficient and rapid, and the lyase rapidly ruptures bacterial cells at the moment of contact with bacteria in vitro, which can rapidly decrease bacterial turbidity. Schuch et al. added 2 units (2 μg) of Bacillus anthracis γ phage lyase PlyG to 1.0×10 ⁴ streptomycin-resistant Bacillus cereus RSVF1, and the bacteria could be lysed within 10s. Finally, there is a synergistic effect either between lyases or between lyases and antibiotics. Jado et al. found that when low concentrations of Dp-1 and low concentrations of Cpl-1 were used together, all mice could survive, while when Dp-1 or Cpl-1 was used alone, all mice died due to severe bacteremia. Djurkovic et al found that the combination of lyase Cpl-1 and gentamicin can reduce the minimum inhibitory concentration of penicillin against Streptococcus pneumoniae. When Cpl-1 is used in combination with penicillin, it can also synergistically kill penicillin-resistant strains.

Extensive studies have shown that lyase has high efficiency, high stability, potential broad bactericidal and safety, can kill bacteria specifically and is not easy to make bacteria resistant to bacteria; lyase has great development in antibacterial. It is expected to become a substitute for antibiotics and has broad application prospects.

SUMMARY OF THE INVENTION

The present invention aims to provide a room temperature lyase Sly, which can inhibit the growth of some Gram-positive and negative bacteria, has catalytic activity in the range of 20°C to 50°C, and is derived from bacteriophage SP26. The amino acid sequence of the room temperature lyase Sly is shown in SEQ ID NO: 1, or a polypeptide, analog or derivative having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1.

Another object of the present invention is to provide a polynucleotide encoding a room temperature lyase Sly, the nucleotide sequence of which is shown in SEQ ID NO: 2, or its complementary sequence, or a nucleotide with the nucleotide shown in SEQ ID NO: 2 Polynucleotides with at least 80% sequence identity and their complements.

Beneficial effects of the present invention:

The lyase of the present invention has catalytic activity in the range of 20°C to 50°C, the highest catalytic activity at 37°C, ^and higher ^enzymatic activity when the pH value is between 6 ^and 10. The ion has an activating effect on the enzyme, and the room temperature lyase of the present invention can efficiently lyse bacteria, so that it is expected to replace the traditional antibiotic treatment, and can also be used as a bacteriostatic agent for the sterilization of the environment; its nucleotide sequence can be used to construct and produce the lyase genetically engineered strains.

Description of drawings

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

Fig. 2 is a schematic diagram of the double-enzyme digestion map of the recombinant plasmid pET28a-Sly in the present invention, wherein lane 1 represents Marker, and lane 2 is the recombinant plasmid pET28a-Sly band without double-enzyme digestion; swimming lane 3 is the recombinant plasmid pET28a after double-enzyme digestion - two bands produced by Sly;

Figure 3 is a schematic diagram of the protein expression detection of the room temperature lyase Sly in the present invention, wherein the lane 1 is the GenStarM221-01 protein marker; the lane 2 is the uninduced whole protein of pET28a-Sly/Rosetta; the lane 3 is pET28a-Sly/Rosetta at 37°C , 150rpm, lactose (1g/L) induced 6h whole protein; Lane 4 is pET28a-Sly/Rosetta at 37℃, 150rpm, lactose (1g/L) induced 6h supernatant; Lane 5 is pET28a-Sly/Rosetta at 37℃ , 150rpm, lactose (1g/L) for 8h induction of whole protein; lane 6 is the supernatant of pET28a-Sly/Rosetta induced at 37℃, 150rpm, lactose (1g/L) for 8h; lane 7 is pET28a-Sly/Rosetta at 37℃ , 150 rpm, lactose (1 g/L) induced 10h whole protein; lane 8 is the supernatant of pET28a-Sly/Rosetta induced by lactose (1 g/L) at 37℃, 150rpm for 10h; lane 9 is pET28a-Sly/Rosetta 37°C, 150rpm, lactose (1g/L) for 12h induction of whole protein; lane 10 is the supernatant of pET28a-Sly/Rosetta induced at 37°C, 150rpm, lactose (1g/L) for 12h;

Fig. 4 is a schematic diagram of the detection of the purification result of the room temperature lyase Sly protein of the present invention, wherein 1 is the GenStar M221-01 protein Marker, and 2 is the second tube after the imidazole buffer of 500 mM after the column is passed through the column;

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

6 is a schematic diagram of the effect of different pH values on the activity of room temperature lyase Sly in the present invention;

7 is a schematic diagram of the effect of different metal ions on the activity of room temperature lyase Sly in the present invention;

FIG. 8 is a schematic diagram of the crystal violet staining results of the action of the room temperature lyase Sly of the present invention on Escherichia coli CMCC(B)44102 and Staphylococcus aureus ATCC6538. A is the cell shape of Escherichia coli CMCC(B)44102 under the action of lyase after inactivation; B is the cell shape of Escherichia coli CMCC(B)44102 under the action of lyase; C is the shape of gold under the action of lyase after inactivation The bacterial morphology of Staphylococcus aureus ATCC6538; D is the bacterial morphology of Staphylococcus aureus ATCC6538 under the action of lyase;

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

Detailed ways

The present invention will be described in further detail below through the examples, but the content of the present invention is not limited to this. The methods in this embodiment are operated by conventional methods unless otherwise specified, and the reagents used are conventional reagents or Reagents prepared according to conventional methods.

Example 1: Cloning and expression of room temperature lyase Sly

1. Amplification of the lyase Sly gene (using the genomic DNA of phage SP26 as a template)

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

Forward primer: 5'-CATG CCATGG CAATGGCTATTAGCAAAAAACATGAAG-3'

Reverse primer: 5'-CG GAATTC TGCCACAGCTCCGCCAGCCTCTTTA-3'

Restriction site: Nco I and EcoR I

(2) The amplification system is as follows:

Table 1 Components of the amplification system

；

;

(3) The amplification conditions are as follows:

The reaction system was mixed and pre-denatured at 94 °C for 10 min, then denatured at 94 °C for 45 s, annealed at 58 °C for 45 s, extended at 72 °C for 90 s, and then extended at 72 °C for 10 min after 30 cycles. After amplification, 5 μL of the product was taken and analyzed by electrophoresis in a 1.2% agarose gel (see Figure 1). The size of the PCR product was 465 bp.

2. Gel recovery of lyase Sly gene PCR product

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

(2) Spot electrophoresis of the PCR product to be separated and purified, and stop the electrophoresis at an appropriate position;

(3) Cut the gel containing the target gene fragment under UV light and transfer it to a 1.5mL EP tube;

(4) Use the StarPrep rapid DNA gel recovery kit to recover the target gene fragments. The recovery method is carried out according to the instructions.

3. Construction of recombinant plasmids

In order to connect the target gene fragment to the plasmid pET28a, it is necessary to make the target gene fragment and the plasmid pET28a have sticky ends, that is, with an enzyme cutting site.

(1) Extract plasmid pET28a

① Strain activation: dip the sterile inoculation loop into the strain preservation solution of plasmid pET28a frozen at -80℃, and inoculate it on LB solid plate containing kanamycin (final concentration 50µg/mL) by continuous line method at 37℃ Culture for 12~16h;

② Enriched bacterial solution: Pick a single colony with an inoculation loop and put it in a 5 mL LB liquid test tube containing kanamycin (final concentration 50 µg/mL), and cultivate at 37 °C and 150 rpm for 12-16 h;

③ Extraction of plasmid pET28a: Extract plasmid pET28a with StarPrep Rapid Plasmid Extraction Kit, and the extraction method is carried out according to the instructions.

(2) Double enzyme digestion of the target gene fragment and plasmid pET28a

① The enzyme digestion system is as follows:

Table 2 Enzyme digestion system components

② Reaction conditions: digested in a metal bath at 37°C for 3 hours, and recovered the target gene fragment and plasmid pET28a after digested.

(3) Construction of recombinant plasmids

The target gene fragment with sticky ends and the plasmid pET28a were transformed to construct a recombinant plasmid by ligation;

① The connection system is as follows:

Table 3 Components of the linking system

② Reaction conditions: connect in a metal bath at 16°C for 12h, and recover the recombinant plasmid.

4. Expression of recombinant plasmids

(1) Transform the recombinant plasmid into E. coli DH5α competent cells

①Transfer 10μL of recombinant plasmid into competent cells of 100μL system;

②After ice bath for 30min, heat shock at 42℃ for 90s, and then ice bath for 30min;

③ Add 890 μL of LB liquid medium, incubate at 37°C, 100 rpm shaker for 1 h;

④Put the cultured liquid medium into a centrifuge at 10,000 rpm for 10 min;

⑤ Discard the supernatant, keep about 100 μL of the precipitate, gently pipette and mix, spread it on the LB solid plate containing kanamycin (final concentration 50 μg/mL), and culture at 37°C for 12~16h;

⑥ Observe the single colony on the above plate, randomly pick a single colony with an inoculation loop, and place it in a 5 mL LB liquid test tube containing kanamycin (final concentration 50 µg/mL), and incubate at 37°C and 150 rpm for 12 hours.

(2) Double enzyme digestion verification

① Extract the recombinant plasmid with the StarPrep Rapid Plasmid Extraction Kit, and the extraction method is carried out according to the instructions;

②Verify the extracted recombinant plasmid by double-enzyme digestion. The double-enzyme digestion system is as follows:

Table 4 Components of double enzyme digestion system

Reaction conditions: Enzymatic digestion was performed in a metal bath at 37°C for 3 hours, and the recombinant plasmids after digestion were recovered and verified by electrophoresis (see Figure 2).

(3) Transform the recombinant plasmid into E. coli Rosetta strain

Transfer the verified correct recombinant plasmid into E. coli Rosetta strain, the method is the same as above; the expression strain pET28a-Sly/Rosetta can be obtained.

5. Induction, expression and purification of recombinant protein

①Inoculate the expression strain pET28a-Sly/Rosetta into 5mL LB liquid test tube containing kanamycin (final concentration 50µg/mL), and cultivate it at 37℃ and 150rpm until the OD ₆₀₀ value is 0.6~0.8;

②Inoculate 1% of the above bacterial solution into 600mL LB liquid test tube containing kanamycin (final concentration 50µg/mL), cultivate at 37℃, 150rpm until the OD ₆₀₀ value is 1.0~1.2; then add the inducer lactose (final concentration 50µg/mL). Concentration of 1g/L) to the bacterial solution of the expression strain, placed at 37 °C and 150 rpm to induce 6, 8, 10, and 12 h respectively;

③ After the induced bacterial solution was centrifuged at 4°C and 9000rpm for 8min, the precipitate was left. Add an appropriate amount of ddH ₂ O to mix by blowing and beating. After centrifugation at 4°C and 9000rpm for 10min, the precipitate was left. The PBS buffer was mixed at a ratio of 100:1. After ultrasonic crushing;

④Ultrasonic crushing: the power is 35%, work for 3s, stop for 4s, 60~90min per tube, crush until light is transparent, take 80µL of whole protein into a sterilized EP tube. The remaining whole protein was centrifuged at 13,000 rpm for 10 min to get the supernatant, and the supernatant was divided into sterilized EP tubes;

⑤ The expression results were detected by SDS-PAGE electrophoresis (see Figure 3). The expression of the target protein was the best at 37°C, 150rpm, and 1g/L lactose for 12 h. Therefore, the best induction conditions were 37°C, 150rpm, 1g/L. Lactose induction for 12h;

⑥ After induction under optimal conditions, AKTA prime protein purification system was used for purification, and SDS-PAGE electrophoresis was used to detect the purification results (see Figure 4); the results showed that the elution band of lyase Sly was single and the expression level was high, indicating purification success.

Example 2: Enzymatic properties and enzymatic activity experiments of room temperature lyase Sly

1. Determination of lyase Sly protein concentration and enzyme activity

(1) Determination of protein concentration by BCA (Bicinchoninic Acid) protein concentration detection method

Under alkaline conditions, Cu ²⁺ can be reduced to Cu ⁺ by protein, and Cu ⁺ combines with BCA reagent to form a purple complex. By measuring the OD value of the sample at a wavelength of 590 nm, and comparing it with the protein standard curve By comparison, the protein concentration of the sample to be tested can be calculated.

According to the "micro method" in the instructions of the BCA protein quantification kit, the OD value at the wavelength of 590nm was measured with a microplate reader. Taking the protein concentration as the abscissa and the OD ₅₉₀ value as the ordinate, draw the standard curve, and the obtained standard curve equation is: y= 0.0009x+0.006 (R ² = 0.9914).

(2) Determination of enzyme activity by Folin phenol method

Under alkaline conditions, the Folin phenol reagent can be reduced by phenolic compounds to form blue compounds. The protease hydrolyzes the casein substrate under the conditions of the optimum temperature and pH, and produces amino acids containing phenolic groups (such as: tyrosine, tryptophan, etc.). Therefore, this principle can be used to measure enzyme activity.

The definition of enzyme activity unit: Under certain temperature and pH value conditions, the enzyme solution hydrolyzes the casein substrate within 1min to produce 1 μg of tyrosine as an enzyme activity unit, which is represented by U.

①Reagents and solutions

Folin phenol reagent, 0.4mol/L sodium carbonate solution, 0.4mol/L trichloroacetic acid solution, pH 7.2 phosphate buffer, 2% casein solution, 100 μg/mL tyrosine solution.

②Drawing of standard curve

Prepare tyrosine solutions of different concentrations according to Table 5

Table 5 Preparation methods of tyrosine solutions with different concentrations

Determination steps: draw 1 mL of tyrosine solutions of different concentrations in the table above, add 5 mL of 0.4 mol/L sodium carbonate solution to each, and then add 1 mL of Folin phenol reagent; shake well and place in a 40°C water bath, keep warm for color development 20min, the microplate reader measures the OD ₆₈₀ value and records. Taking the mass of tyrosine as the abscissa and the OD ₆₈₀ value as the ordinate, draw a standard curve, and the obtained standard curve equation: y=0.003x+0.0099 (R ² =0.9959).

③ Enzyme activity determination steps

Draw 2 mL of the diluted enzyme solution (concentration of 46.67 μg/mL), place it in a 40°C water bath to preheat for 2 minutes, then add 1 mL of the same preheated 2% casein solution, and keep it in a 40°C water bath for 5 minutes. . After the time was up, 1 mL of 0.4 mol/L trichloroacetic acid solution was added immediately to terminate the reaction, and the mixture was kept in a 40°C water bath for 20 min to precipitate residual protein. Aspirate the solution containing the precipitate, filter the solution with a 0.22 μm filter membrane, then absorb 1 mL of the filtrate, add 5 mL of 0.4 mol/L sodium carbonate solution, and 1 mL of Folin phenol reagent, shake well, and measure the OD in a water bath at 40°C for 20 minutes. ₆₈₀ value.

The OD ₆₈₀ value of the lyase was determined to be 0.317, and the OD ₆₈₀ value was substituted into the standard curve equation: y = 0.003x + 0.0099 (R ² = 0.9959), and the calculated mass of tyrosine was 102.37 μg. Substitute the mass of tyrosine into the formula: Y=(A×N)/(V×t) (Y: enzyme activity of the sample, U; A: mass of tyrosine, μg; N: dilution ratio of the sample; V: reaction The total volume of the reagents, mL; t: reaction time, min), the calculated enzyme activity Y=(102.37×5)/(4×5)=25.59 U.

2. The effect of different temperature, pH value and metal ions on the room temperature lyase Sly

Lyases can hydrolyze peptidoglycan. After peptidoglycan is hydrolyzed, the OD ₆₀₀ value of the reaction substrate will decrease significantly; the higher the enzyme activity, the greater the decrease in the OD ₆₀₀ value, so the change in the OD ₆₀₀ value of the reaction substrate Can be used to evaluate lyase activity.

Enzyme activity (%)=[( _before OD _{600 reaction - after} OD 600 reaction)/initial OD ₆₀₀ ]×100

Set the highest enzyme activity measured as 100%, relative enzyme activity (%) = (enzyme activity at other temperatures/maximum enzyme activity) × 100.

(1) Preparation of reaction substrates

① Dip the strain preservation solution of host bacteria DS26 frozen at -80°C with a sterile inoculation loop, inoculate it on LB solid plate by continuous line method, and cultivate at 37°C for 12~16h;

②Pick a single colony on the above plate with a sterile inoculating loop and put it in a 5mL LB liquid test tube, incubate at 37°C and 150rpm until the OD ₆₀₀ value is 0.6~0.8, and inoculate the bacterial liquid at a ratio of 1% into a 600mL LB liquid test tube, at 37°C , 150rpm culture to OD ₆₀₀ value of 1.0~1.2;

③ After centrifuging the above bacterial solution at 9000rpm for 10min, discard the supernatant. After the precipitate was mixed with 10 mL of ddH ₂ O by blowing and beating, ultrasonically disrupted (power was 35%, working for 3 s, stopping for 4 s, total time 120 min), and centrifuging at 13,000 rpm for 10 min to obtain the precipitate. The pellet was mixed with 10 mL of ddH ₂ O again by pipetting; at this time, the cell walls of the host bacteria DS26 were all cell debris, which was used as the reaction substrate for the following experiments.

(2) The optimum temperature for the action of the lyase Sly at room temperature

The purified lyase Sly (concentration of 135.56 μg/mL) was added to the prepared reaction substrate, and then placed at different temperatures for 30 min. The reaction temperatures were 4 °C, 20 °C, 30 °C, 35 °C, and 37 °C. , 40℃, 45℃, 50℃, 60℃, 70℃. Among them, the initial OD ₆₀₀ of the reaction substrate is 0.365, and the optimum temperature is determined according to the change of OD ₆₀₀ before and after the reaction (see Figure 5).

It can be seen from the analysis in Figure 5 that the optimal temperature of the lyase is 37 °C, and the enzyme activity is higher between 30 °C and 45 °C.

(3) The optimum pH value of room temperature lyase Sly

The purified lyase Sly (concentration of 135.56 μg/mL) was added to the prepared reaction substrate, and at the optimum temperature, the reaction was carried out in phosphate buffers with different pH values for 30 min, and the OD before and after the reaction was measured. ₆₀₀ value, where the initial OD ₆₀₀ of the reaction substrate = 0.295, and its optimum pH value was determined according to the change in OD ₆₀₀ before and after the reaction (see Figure 6).

It can be seen from the analysis in Figure 6 that the optimal pH value of the lyase is 7.5, and the enzyme activity is higher between pH 6 and 10.

(4) Effects of different metal ions on enzyme activity

The purified lyase Sly (concentration of 145.56μg/mL) was added to the prepared reaction substrate, and different metal ions Mn ²⁺ , Ca ²⁺ , Mg ²⁺ , Zn ²⁺ , Fe ³⁺ were added to the mixture. , Na ⁺ , K ⁺ , and keep the ion concentration at 1 mmol/L, under the conditions of the optimum temperature and pH value, react for 30 min, and measure the OD ₆₀₀ value before and after the reaction, wherein the initial OD ₆₀₀ of the reaction substrate =0.411, The control group used ddH ₂ O instead of metal ions. We call the metal ions that can increase the enzyme activity as enzyme activators, and vice versa, as enzyme inhibitors (see Figure 7).

It can be seen from Figure 7 that the three ions Mg ²⁺ , Na ⁺ , and K ⁺ have an activating effect on the enzyme, and the three ions Zn ²⁺ , Mn ²⁺ , and Fe ³⁺ have an inhibitory effect on the enzyme, while Ca ^{2 +} Little effect on enzymes.

3. Crystal violet staining results of room temperature lyase Sly acting on Escherichia coli CMCC(B)44102 and Staphylococcus aureus ATCC6538

Escherichia coli CMCC (B) 44102 and Staphylococcus aureus ATCC6538 were cultured to OD ₆₀₀ values of 0.531 and 0.519, respectively, and the bacterial solution was centrifuged at 8000 rpm for 10 min to obtain bacterial cells, washed three times with ddH ₂ O, collected and diluted with PBS. , and then 900µL of lyase Sly (concentration of 141.11µg/mL) was added to 100µL bacterial solution (2.37×10 ³ CFU/mL), reacted at 37°C for 30min, stained with ammonium oxalate crystal violet staining solution, sliced and placed under a light microscope Perform microscopic examination (see Figure 8), and use the enzyme solution at 121°C for 20 minutes of inactivation as the control;

It can be seen from Fig. 8 that the cell morphology of Escherichia coli CMCC (B) 44102 and Staphylococcus aureus ATCC6538 acted by lyase Sly cannot be stained by crystal violet staining solution, indicating that this part has been lysed by lyase Sly; The bacterial morphology of Escherichia coli CMCC(B)44102 and Staphylococcus aureus ATCC6538 treated with the live enzyme solution did not change significantly.

4. The effect of normal temperature lyase Sly on the growth of Escherichia coli CMCC(B)44102, Staphylococcus aureus ATCC6538, Shigella DS26 and Salmonella CMC(B)50094 strains with time

According to 1% inoculation amount, inoculate the bacterial liquid of Escherichia coli CMCC(B)44102, Staphylococcus aureus ATCC6538, Shigella DS26, Salmonella CMC(B)50094 into LB liquid medium, and cultivate to OD at 37°C and 150rpm. The ₆₀₀ values are 0.506, 0.522, 0.537, 0.525, respectively. The enzyme solution (concentration of 252.22 μg/mL) was filtered through a sterilized 0.22 μm filter, and Mg ²⁺ , Na ⁺ , K ⁺ (the final concentration was all 1 mmol/L) were added, and 270 μL of the enzyme solution and 30 μL of the large intestine were drawn. Bacillus CMCC(B)44102 (1.12×10 ³ CFU/mL), Staphylococcus aureus ATCC6538 (1.43×10 ³ CFU/mL), Shigella DS26 (1.08×10 ³ CFU/mL) ) and Salmonella CMC(B) 50094 bacterial solution (1.67×10 ³ CFU/mL), the mixture was reacted in a constant temperature incubator at 37°C for 0, 10, 20, 30, 40, 50, and 60 min, and 50 μL of the reacted The mixture was coated on the plate, and the enzyme solution was inactivated at 121 °C for 20 min as the control; after coating, it was placed in a constant temperature incubator at 37 °C for overnight incubation, and the number of viable cells in the experimental group and the control group was counted to calculate the antibacterial activity. (Note: 3 parallels in each group) Antibacterial activity is defined as logarithmic relative inactivation unit log ₁₀ (N0/Ni), N0 is the number of live cells treated with inactivated enzyme solution (control group), and Ni is after enzyme solution treatment The number of viable cells (experimental group);

It can be seen from Fig. 9 that the killing effect of the lyase Sly on the strain changes with the change of time when the enzyme concentration and the bacterial concentration are constant. When the action time was 10 min, the number of viable cells of Escherichia coli CMCC(B)44102 decreased the most, followed by that of Shigella DS26 and Salmonella CMC(B)50094, and the number of viable cells of Staphylococcus aureus ATCC6538 decreased the second. The decrease is relatively small. In conclusion, this lyase has a certain killing effect on both Gram-positive and negative bacteria.

sequence listing

<110> Kunming University of Science and Technology

<120> A room temperature lyase Sly and a 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 (2)

1. A room temperature lyase Sly, characterized in that: its amino acid sequence is as shown in SEQ ID NO:1. 2. The polynucleotide encoding the room temperature lyase Sly of claim 1, wherein the nucleotide sequence is as shown in SEQ ID NO: 2.

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