Serine protease as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological enzymes, and discloses serine protease as well as a preparation method and application thereof. Wherein the amino acid sequence of the serine protease is as shown in SEQ ID NO. 1 or SEQ ID NO. 2. The serine protease provided by the invention has stable activity at different temperatures, can better maintain the activity and efficacy in practical application, and can still effectively inhibit the formation of a biological membrane or remove a mature biological membrane under the condition of variable temperatures. Experimental results show that the serine protease can obviously inhibit the formation of the methicillin-resistant staphylococcus aureus biological membrane and remove the methicillin-resistant staphylococcus aureus biological membrane; no antibacterial activity is found, so that pathogenic bacteria are not easy to generate drug resistance; meanwhile, toxicity is avoided, and the germination rate and bud length of alfalfa seeds are not affected. Therefore, the serine protease is safe and reliable in application in the food industry, and has great advantages in food application.

Description

Technical Field

[0001] The present invention relates to the technical field of bioenzymes, and in particular to a serine protease and a preparation method and application thereof. Background Art

[0002] Food safety has long been one of the main factors affecting public health, and the incidence of foodborne diseases caused by microbial pathogens has been on the rise worldwide. Methicillin-resistant Staphylococcus aureus (MRSA) is a common foodborne pathogen, and the foodborne diseases caused by it have become a global public health issue.

[0003] Currently, the prevention and control strategies for foodborne diseases mainly use microbial disinfectants or exposure to low doses of ultraviolet A (UVA). However, direct killing methods may cause problems such as the development of antibiotic resistance. In addition, these foodborne pathogens often have the ability to form biofilms, which can protect the pathogens from the cleaning process and continue to survive in the environment.

[0004] Some strategies used in the food industry to control biofilms, such as cleaning and disinfection, plasma treatment, and ultrasonic treatment, are effective but still difficult to completely control biofilms. In contrast, enzyme treatment has greater advantages in practical applications due to its high specificity and effectiveness. However, few enzymes have been reported for controlling biofilms, and the activity of most enzymes changes with temperature, which is not conducive to their application in temperature-variable environments. Therefore, screening and developing more stable enzymes is essential for combating biofilms of foodborne pathogens under temperature-variable conditions. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a serine protease and a preparation method and application thereof, aiming to solve the problem that the existing enzymes used for biofilm removal are relatively few and have poor thermal stability.

[0006] The technical solution of the present invention is as follows:

[0007] In a first aspect, a serine protease is provided, wherein the amino acid sequence of the serine protease is shown as SEQ ID NO.1 or SEQ ID NO.2.

[0008] In a second aspect, a nucleic acid is provided, wherein the nucleic acid encodes the serine protease as described in the first aspect.

[0009] In a preferred technical solution, the nucleotide sequence of the nucleic acid is shown as SEQ ID NO.3.

[0010] In a third aspect, a recombinant vector is provided, wherein the recombinant vector expresses the serine protease as described in the first aspect.

[0011] In a fourth aspect, a host cell is provided, wherein the host cell contains the recombinant vector as described in the third aspect.

[0012] In a fifth aspect, a composition is provided, wherein the composition contains the serine protease as described in the first aspect.

[0013] In a sixth aspect, a method for preparing the serine protease according to the first aspect is provided, comprising the steps of:

[0014] Synthesizing a gene encoding the serine protease as described in the first aspect, and inserting the gene into an expression vector to obtain a recombinant vector;

[0015] The recombinant vector is transferred into a host cell for protein expression, and the product of the protein expression is purified to obtain the serine protease.

[0016] In a preferred technical solution, the expression vector is pET-28a(+).

[0017] In a preferred technical solution, the host cell is Escherichia coli.

[0018] In the seventh aspect, there is provided a use of the serine protease as described in the first aspect, the nucleic acid as described in the second aspect, the recombinant vector as described in the third aspect, the host cell as described in the fourth aspect, the composition as described in the fifth aspect, or the serine protease prepared by the preparation method as described in the sixth aspect in the preparation of products for removing biofilms or inhibiting biofilm formation.

[0019] Beneficial effects: The present invention provides a new serine protease, which has stable activity at different temperatures and can better maintain activity and efficacy in practical applications, ensuring that the formation of biofilms can still be effectively inhibited or mature biofilms can be removed under temperature-changing conditions. Experimental results show that the serine protease can significantly inhibit the formation of methicillin-resistant Staphylococcus aureus biofilms and remove methicillin-resistant Staphylococcus aureus biofilms; and no antibacterial activity was found, and it is not easy for pathogens to develop drug resistance; at the same time, it is not toxic and has no effect on the germination rate and sprout length of alfalfa seeds. Therefore, the application of the serine protease in the food industry is safe and reliable, which has great advantages for food applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the evolutionary tree of strain XY-144 and the reference strain in Example 1.

[0021] Figure 2This is a graph showing the antibacterial activity test results of the fermentation supernatant of XY-144 against MRSA in Example 2.

[0022] Figure 3 This is a diagram showing the verification results of the fermentation supernatant of XY-144 in Example 3 for the removal and inhibition activity of MRSA biofilm.

[0023] Figure 4 This is a graph showing the results of the activity verification of each component of the fermentation supernatant of XY-144 in Example 4 in removing MRSA biofilm.

[0024] Figure 5 This is a graph showing the thermal stability test results of the fermentation supernatant of XY-144 in Example 5.

[0025] Figure 6 This is a graph showing the toxicity test results of the salting-out component of XY-144 in Example 6.

[0026] Figure 7 This is a graph showing the results of the verification of the activity of the fermentation supernatant of XY-144 and the ultrafiltration fraction of XY-144 in Example 7 in removing MRSA biofilm after being treated by the Sevage method.

[0027] Figure 8 This is a diagram showing the results of SDS-PAGE analysis of the components of the fermentation supernatant of XY-144 in Example 8.

[0028] Fig. 9 This is the evolutionary tree of protein 500784 in Example 8 and reported serine proteases.

[0029] Fig.10 This is a diagram showing the result of enzyme digestion detection of the recombinant vector in Example 8.

[0030] Fig.11 This is a diagram showing the purification results of the recombinant serine protease in Example 9.

[0031] Fig.12 This is a graph showing the results of the activity verification of the recombinant serine protease in Example 10 in clearing MRSA biofilm.

[0032] Fig.13 This is a graph showing the results of the activity verification of the recombinant serine protease in Example 11 in clearing MRSA biofilm. DETAILED DESCRIPTION

[0033] The present invention provides a serine protease and a preparation method and application thereof. To make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below.

[0034] Removing biofilms is one of the current research hotspots. In clinical settings, strategies such as enzyme treatment, small molecule drugs, and surgical resection have been successfully applied to transform bacteria from a biofilm state to a more fragile planktonic state. Compared with other strategies for removing biofilms, enzyme treatment does not have the drug resistance problem faced by other small molecule drugs, and the development of drug resistance often leads to worsening of the disease and treatment failure. Therefore, enzyme treatment has greater advantages in practical applications. In addition, enzyme treatment can achieve high specificity and effectiveness against target biofilms at relatively low concentrations. However, few enzymes have been reported for controlling biofilms, and the thermal instability of some enzymes limits their application.

[0035] Based on this, an embodiment of the present invention provides a serine protease, the amino acid sequence of the serine protease is shown as SEQ ID NO.1 or SEQ ID NO.2.

[0036] Specifically, the inventor collected a sample of a sea cow in the shallow waters of Shenzhen Dapeng Nature Reserve and isolated the strains to obtain about 200 strains. The isolated strains were screened for activity and found that strain XY-144 has the activity of removing biofilms and inhibiting biofilm formation. Further, by separating, purifying and analyzing the active components of strain XY-144, a serine protease with an amino acid sequence as shown in SEQ ID NO.1 was screened. The amino acid sequence of the serine protease in strain XY-144 was optimized, the signal peptide of 19 amino acids at the N-terminus was removed, and a His6 tag was added to the C-terminus to obtain a recombinant serine protease with an amino acid sequence as shown in SEQ ID NO.2. The serine protease or recombinant serine protease in strain XY-144 has the activity of removing biofilms and inhibiting biofilm formation.

[0037] An embodiment of the present invention provides a nucleic acid encoding the serine protease as described above.

[0038] In one embodiment, the nucleotide sequence of the nucleic acid is shown as SEQ ID NO.3.

[0039] An embodiment of the present invention provides a recombinant vector, wherein the recombinant vector expresses the serine protease described above.

[0040] An embodiment of the present invention provides a host cell, wherein the host cell contains the recombinant vector as described above.

[0041] An embodiment of the present invention provides a composition, wherein the composition contains the serine protease as described above.

[0042] In one embodiment, the composition further comprises an antibiotic.

[0043] The present invention provides a method for preparing the serine protease as described above, comprising the steps of:

[0044] Synthesizing a gene encoding the serine protease described above, and inserting the gene into an expression vector to obtain a recombinant vector;

[0045] The recombinant vector is transferred into a host cell for protein expression, and the product of the protein expression is purified to obtain the serine protease.

[0046] In one embodiment, the expression vector is pET-28a(+).

[0047] In one embodiment, the host cell is Escherichia coli.

[0048] In a more specific embodiment, the host cell is a BL21 (DE3) competent cell.

[0049] An embodiment of the present invention provides a use of the serine protease as described above, the nucleic acid as described above, the recombinant vector as described above, the host cell as described above, the composition as described above, or the serine protease prepared by the preparation method as described above in the preparation of a product for removing biofilm or a product for inhibiting biofilm formation.

[0050] In one embodiment, the product is a product for eliminating MRSA biofilm or a product for inhibiting the formation of MRSA biofilm.

[0051] The present invention will be further described below by means of specific examples.

[0052] In the embodiment of the present invention, the culture medium involved is prepared with deionized water, and the formula is as follows:

[0053] LB liquid culture medium: tryptone 10 g / L, yeast powder 1 g / L, sodium chloride 5 g / L.

[0054] LB solid medium: tryptone 10 g / L, yeast powder 1 g / L, sodium chloride 5 g / L, agar 15 g / L.

[0055] MA solid medium: peptone 5g / L, yeast extract 1g / L, ferric citrate 0.1g / L, sodium chloride 19.45g / L, magnesium chloride 5.98g / L, sodium sulfate 3.24g / L, calcium chloride 1.8g / L, potassium chloride 0.55g / L, sodium carbonate 0.16g / L, potassium bromide 0.08g / L, strontium chloride 0.034g / L, boric acid 0.022g / L, sodium silicate 0.004g / L, sodium fluoride 0.0024g / L, sodium nitrate 0.0016g / L, disodium hydrogen phosphate 0.008g / L, agar 15g / L.

[0056] SGTYP liquid culture medium: glucose 5 g / L, soluble starch 5 g / L, tryptone 1 g / L, yeast extract 1 g / L, peptone 1 g / L.

[0057] SGTYP solid culture medium: glucose 5 g / L, soluble starch 5 g / L, tryptone 1 g / L, yeast extract 1 g / L, peptone 1 g / L, agar 15 g / L.

[0058] In the embodiment of the present invention, the preparation process of the fermentation supernatant of XY-144 and the overnight seed solution of MRSA is as follows:

[0059] Fermentation supernatant of XY-144: Scrape a small amount of frozen bacterial cells of strain XY-144 onto MA solid medium for streaking and culture at 28°C for 3 days. Take a single colony and place it in 3 mL of SGTYP liquid medium, shake culture at 28°C and 200 rpm for 3 days, and centrifuge at 10,000 rpm for 20 minutes at 4°C to obtain the fermentation supernatant of XY-144.

[0060] Overnight seed solution of MRSA: Scrape a small amount of frozen cells of strain MRSA onto LB solid medium for streaking and culture at 37°C for 1 day. Take a single colony and place it in 3 mL of LB liquid medium, shake and culture overnight at 37°C and 200 rpm to obtain the overnight seed solution of MRSA.

[0061] Example 1 Isolation and identification of strain XY-144

[0062] Strain XY-144 was collected from Shenzhen Dapeng Nature Reserve in March 2017 and is a symbiotic bacterium of the marine invertebrate Dugong.

[0063] The DNA of XY-144 was extracted using TIANamp Bacteria DNA Kit (TIANGEN), and the 16S rDNA region of XY-144 was amplified by PCR using conventional forward primer 27F and reverse primer 1492R. The samples were sent for sequencing to obtain the 16S rRNA sequence data of XY-144. After comparison and analysis, the 16S rRNA sequence of XY-144 was similar to that of Fangia hongkongensis FSC776. T The 16S rRNA sequence of XY-144 was 99.37% similar, so XY-144 was identified as Fangia hongkongensis. The 16S rRNA sequence of XY-144 has been submitted to the GenBank database with the accession number OR262794.1.

[0064] The DNA of XY-144 was extracted using TIANamp Bacteria DNA Kit (TIANGEN) and sent for sequencing to obtain the whole genome data of XY-144. The genome evolution tree was established based on the sequencing results. Figure 1 As shown. Figure 1 It can be seen that XY-144 belongs to the genus Fangia, and the closest evolutionary relative is Fangiahongkongensis FSC776 T The core gene homology number is 78%, so it is speculated that XY-144 is a new strain.

[0065] XY-144 was deposited in the China General Microbiological Culture Collection Center (CGMCC) on June 26, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No: 31090, and the classification name is Fangia hongkongensis.

[0066] Example 2 Antibacterial activity of strain XY-144

[0067] The overnight seed solution of MRSA was diluted 1000 times with LB liquid medium, and then 40 μL was spread on the LB solid medium, and then a circular paper was placed on it. 5 μL of each of the fermentation supernatant of XY-144, SGTYP liquid medium (negative control), and SGTYP liquid medium containing 5 μg / mL vancomycin (positive control) were spotted on the circular paper, and then cultured at 37°C for 1 day. The experimental results are as follows: Figure 2 shown. Figure 2 In the figure, 1 refers to the addition of the fermentation supernatant of XY-144; 2 refers to the addition of SGTYP liquid culture medium; 3 refers to the addition of SGTYP liquid culture medium containing 5 μg / mL vancomycin. Figure 2 It can be seen that the fermentation supernatant of XY-144 has no antibacterial activity against MRSA.

[0068] Example 3 Verification of biofilm removal and inhibitory activity of strain XY-144

[0069] The overnight seed solution of MRSA was diluted 100 times by volume with LB liquid medium containing 0.5% (w / v) glucose, and 1 mL / well was added to a 24-well plate. After culturing at 37°C for 1 day, the bacterial solution was slowly aspirated with a spray gun to obtain a 24-well plate containing MRSA biofilm. The fermentation supernatant of XY-144 was diluted with LB liquid medium containing 0.5% (w / v) glucose to 75%, 50%, 25%, 12.5%, and 6.25% of the fermentation supernatant of XY-144, and different concentrations of dilutions were obtained. The dilution was slowly added to a 24-well plate containing MRSA biofilm at 1 mL / well. The SGTYP liquid medium was also diluted in the same proportion and added as a negative control. After culturing at 37°C for 1 day, the bacterial solution was slowly aspirated with a spray gun, washed once with saline, dried at 37°C for 30 minutes, and then fixed at 65°C for 30 minutes before being taken out and cooled to room temperature. Each well was stained with 1 mL of 0.1% (w / v) crystal violet solution. After 5 minutes, the stain was washed off and dried. The removal effect of the biofilm was observed. Figure 3 As shown in (a). Figure 3 In (a), 144 75% refers to the addition of a dilution containing 75% (v / v) XY-144 in the fermentation supernatant; 144 50% refers to the addition of a dilution containing 50% (v / v) XY-144 in the fermentation supernatant; 144 12.5% ​​refers to the addition of a dilution containing 12.5% ​​(v / v) XY-144 in the fermentation supernatant; 144 6.25% refers to the addition of a dilution containing 6.25% (v / v) XY-144 in the fermentation supernatant; SG75% refers to the addition of a dilution containing 75% (v / v) SGTYP liquid culture medium; SG50% refers to the addition of a dilution containing 50% (v / v) SGTYP liquid culture medium; SG12.5% ​​refers to the addition of a dilution containing 12.5% ​​(v / v) SGTYP liquid culture medium; SG6.25% refers to the addition of a dilution containing 6.25% (v / v) SGTYP liquid culture medium. Figure 3 As shown in (a), the fermentation supernatant of XY-144 has a clearing effect on MRSA biofilm, and the clearing effect increases with the increase in the proportion of fermentation supernatant.

[0070] The overnight seed solution of MRSA was diluted 100 times by volume with LB liquid medium containing 0.5% (w / v) glucose, and 1 mL / well was added to a 24-well plate to obtain a 24-well plate containing MRSA. The fermentation supernatant of XY-144 was diluted with LB liquid medium containing 0.5% (w / v) glucose to 75%, 50%, 25%, 12.5%, and 6.25% of the fermentation supernatant of XY-144, and dilutions of different concentrations were obtained. The dilution was slowly added to the 24-well plate containing MRSA at 1 mL / well, and the SGTYP liquid medium was also diluted in the same proportion and added as a negative control. After culturing at 37°C for 1 day, the bacterial solution was slowly aspirated with a spray gun, washed once with saline, dried at 37°C for 30 minutes, and then fixed at 65°C for 30 minutes and then taken out and cooled to room temperature. Each well was stained with 1 mL of 0.1% (w / v) crystal violet solution. After 5 minutes, the stain was washed off and dried. The biofilm inhibition effect was observed. Figure 3 As shown in (b). Figure 3 In (b), 144 75% refers to the addition of a dilution containing 75% (v / v) XY-144 in the fermentation supernatant; 144 50% refers to the addition of a dilution containing 50% (v / v) XY-144 in the fermentation supernatant; 144 12.5% ​​refers to the addition of a dilution containing 12.5% ​​(v / v) XY-144 in the fermentation supernatant; 144 6.25% refers to the addition of a dilution containing 6.25% (v / v) XY-144 in the fermentation supernatant; SG 75% refers to the addition of a dilution containing 75% (v / v) SGTYP liquid culture medium; SG 50% refers to the addition of a dilution containing 50% (v / v) SGTYP liquid culture medium; SG12.5% ​​refers to the addition of a dilution containing 12.5% ​​(v / v) SGTYP liquid culture medium; SG 6.25% refers to the addition of a diluent containing 6.25% (v / v) SGTYP liquid culture medium. Figure 3 As shown in (b), the fermentation supernatant of XY-144 has an inhibitory effect on the formation of MRSA biofilm, and the inhibitory effect increases with the increase in the proportion of the fermentation supernatant.

[0071] Example 4 Isolation and purification of active ingredients from strain XY-144

[0072] Tris-HCl buffer (1M, pH 8) was added to the fermentation supernatant of XY-144 until the volume of Tris-HCl buffer was 10%, and then ammonium sulfate powder was slowly added until its mass concentration was 50%, and then placed at 4°C overnight to precipitate protein to obtain a precipitated protein solution. The precipitated protein solution was centrifuged at 10000 rpm for 20 minutes at 4°C to remove the supernatant, and then heated with ddH 2O to resuspend the precipitate to obtain a precipitate sample. The precipitate sample was subjected to salting out. Before salting out, the dialysis bag (Biyuntian, 44 mm, 14 kDa) was sequentially treated with a dialysis bag treatment solution (2% (w / v) NaHCO 3 , 1M EDTA, pH 8), ddH 2 O, 1M EDTA and ddH 2 O for 10 minutes and then cool. After leak testing, add the above precipitate sample into the dialysis bag and dialysis with ddH 2 O was used as the dialysate and dialyzed on a horizontal shaker at 80 rpm for 20 hours, during which the dialysate was replaced 3 times. After the last replacement, the dialysate was dialyzed for another 2 hours to obtain the XY-144 salting-out fraction.

[0073] The XY-144 salting-out fraction was ultrafiltered and concentrated using an ultrafiltration tube (Millipore, 3 kDa). The filtrate and concentrate obtained were recorded as XY-144 filtrate and XY-144 ultrafiltration fraction, respectively. The XY-144 ultrafiltration fraction was then further purified by anion exchange column. The DEAE homogenate resin was loaded into the column (column volume was 3 mL), and 3 times the column volume of 2M NaCl solution, ddH 2 O, and then equilibrated with 3 column volumes of 20mM Tris-HCl buffer (pH8), and then 6mL of XY-144 ultrafiltration fraction was loaded. The impurities were washed with 3 column volumes of 20mM Tris-HCl (pH8) solution containing 0.1M NaCl to obtain the XY-144 loading flowthrough, which was recorded as FT; then eluted with 3 column volumes of 20mM Tris-HCl (pH8) solution containing 0.1M, 0.25M, 0.5M, and 1M NaCl to obtain XY-144 elution fractions, which were recorded as E and E respectively. 0.1 、E 0.25 、E 0.5 and E 1 ; Finally, the column was regenerated with 4 column volumes of 20 mM Tris-HCl (pH 8) solution containing 2 M NaCl.

[0074] The collected components and SGTYP liquid culture medium (negative control) were diluted with LB liquid culture medium containing 0.5% (w / v) glucose to verify the activity of removing MRSA biofilm. The specific operation is shown in Example 3. The experimental results are shown in Figure 4 shown. Figure 4In (a), salting out 20% refers to the addition of a diluent containing 20% ​​(v / v) XY-144 salting out component; salting out 10% refers to the addition of a diluent containing 10% (v / v) XY-144 salting out component; ultrafiltration 20% refers to the addition of a diluent containing 20% ​​(v / v) XY-144 ultrafiltration component; ultrafiltration 10% refers to the addition of a diluent containing 10% (v / v) XY-144 ultrafiltration component; negative control 20% refers to the addition of a diluent containing 20% ​​(v / v) SGTYP liquid culture medium; negative control 10% refers to the addition of a diluent containing 10% (v / v) SGTYP liquid culture medium; filtrate 10% refers to the addition of a diluent containing 10% (v / v) XY-144 filtrate; and the blank control did not add any component. Figure 4 In (b), FT 10% refers to the addition of a diluent containing 10% (v / v) XY-144 load flowthrough; E 0.1 5% means adding elution fraction E containing 5% (v / v) XY-144 0.1 dilution; E 0.25 5% means adding elution fraction E containing 5% (v / v) XY-144 0.25 dilution; E 0.5 5% means adding elution fraction E containing 5% (v / v) XY-144 0.5 dilution; E 1 5% means adding elution fraction E containing 5% (v / v) XY-144 1 The blank control had no components added. Figure 4 It can be seen that the XY-144 salting-out fraction after salting-out purification and the XY-144 ultrafiltration fraction after salting-out ultrafiltration concentration still have a clearing effect on MRSA biofilm. However, the XY-144 filtrate after ultrafiltration did not show a clearing effect on MRSA biofilm. The results of anion exchange purification of the ultrafiltration fractions showed that among the XY-144 elution fractions after the DEAE column, only the XY-144 elution fraction E eluted with 20mM Tris-HCl (pH8) solution containing 0.25M NaCl 0.25 It has activity in clearing MRSA biofilm.

[0075] Example 5 Thermal stability test of active ingredients of strain XY-144

[0076] The fermentation supernatant of XY-144 was incubated at 28°C, 40°C, 60°C, 80°C and 100°C for 30 minutes, cooled to room temperature, and then the activity of removing MRSA biofilm was verified. The overnight seed solution of MRSA was diluted 100 times by volume with LB liquid culture medium containing 0.5% (w / v) glucose, and 1 mL / well was added to a 24-well plate. After culturing at 37°C for 1 day, the bacterial solution was slowly sucked away with a spray gun to obtain a 24-well plate containing MRSA biofilm. The fermentation supernatant of XY-144 treated at different temperatures was diluted with LB liquid medium containing 0.5% (w / v) glucose to a volume ratio of 50% of the fermentation supernatant of XY-144 treated, and then 1 mL / well was slowly added to the 24-well plate containing MRSA biofilm. The SGTYP liquid medium was also diluted in the same proportion and added as a negative control. After culturing at 37°C for 1 day, the bacterial solution was slowly aspirated with a spray gun, washed once with saline, dried at 37°C for 30 minutes, and then fixed at 65°C for 30 minutes before being taken out and cooled to room temperature. 1 mL of 0.1% (w / v) crystal violet solution was added to each well for staining, and the stain was washed off after 5 minutes and dried. The removal effect of the biofilm was as follows: Figure 5 Then, 5 mL of 30% (v / v) acetic acid solution was used to dissolve the pigment, and 100 μL was taken after mixing to measure OD using an enzyme marker. 550 , the test results are as follows Figure 5 As shown in (b). Figure 5 It can be seen that the fermentation supernatant of XY-144 has good thermal stability and its activity in removing MRSA biofilm is still very good after incubation at 60°C for 30 minutes.

[0077] Example 6 Toxicity test of active ingredients of strain XY-144

[0078] Tris-HCl buffer (1M, pH 8) was added to the fermentation supernatant of XY-144 until the volume of Tris-HCl buffer was 10%, and then ammonium sulfate powder was slowly added until its mass concentration was 50%, and then placed at 4°C overnight to precipitate protein to obtain a precipitated protein solution. The precipitated protein solution was centrifuged at 10000 rpm for 20 minutes at 4°C to remove the supernatant, and then heated with ddH 2 O to resuspend the precipitate to obtain a precipitate sample. The precipitate sample was subjected to salting out. Before salting out, the dialysis bag (Biyuntian, 44 mm, 14 kDa) was sequentially treated with a dialysis bag treatment solution (2% (w / v) NaHCO 3 , 1M EDTA, pH 8), ddHO 2 O, 1M EDTA and ddH 2 O for 10 minutes and then cool. After leak testing, add the above precipitate sample into the dialysis bag and dialysis with ddH 2O was used as the dialysate and dialyzed on a horizontal shaker at 80 rpm for 20 hours. During this period, the dialysate was replaced 3 times. After the last change of the liquid, the dialysate was dialyzed for another 2 hours to obtain the XY-144 salting-out component. After soaking alfalfa seeds in water, rinse them continuously with water on the filter screen, then soak them in sodium hypochlorite (84 disinfectant diluted 10 times) for 10 minutes, wash them with sterile water 4 to 5 times, then air-dry them and put them into a 50 mL EP tube, add a desiccant and store them for later use. Weigh 50 seeds in a culture dish, add 20 mL of sterile water containing 10% (v / v) XY-144 salting-out component, and culture them at 25°C. The negative control group was 20 mL of sterile water. Observation and recording were performed at the 24th and 48th hours. The results are as follows Figure 6 As shown. Figure 6 It can be seen that the salting-out component of XY-144 has no effect on the germination of alfalfa, whether it is the germination rate or the length of the sprout, and is not toxic.

[0079] Example 7 Analysis of the properties of the active ingredients of strain XY-144

[0080] The Sevage method was used to verify whether the active ingredient of strain XY-144 was polysaccharide. Take 30 mL of the fermentation supernatant of XY-144 and 3 mL of the ultrafiltration fraction of XY-144 in Example 4, add 10 mL and 1 mL of the prepared Sevage reagent (chloroform: n-butanol = 4:1) respectively, mix thoroughly for 30 minutes, centrifuge at 5000 rpm for 20 minutes, take the upper aqueous phase and transfer it to a 15 mL centrifuge tube, place it at -80°C for 20 minutes, and then freeze-dry it to remove the residual organic reagent. The freeze-dried powder was lyophilized with ddH 2 After resuspending, the activity of removing MRSA biofilm was verified. The experimental results are as follows Figure 7 shown. Figure 7 In the above, 1 is added with the ultrafiltration fraction of XY-144 treated by Sevage method; 2 is added with the ultrafiltration fraction of XY-144; 3 is added with the ultrafiltration fraction of SGTYP liquid culture medium; 4 is added with the fermentation supernatant of XY-144 treated by Sevage method; 5 is added with the fermentation supernatant of XY-144; 6 is added with the fermentation supernatant of SGTYP liquid culture medium; 7 is the blank control, without adding any component. Figure 7 It can be seen that the fermentation supernatant of XY-144 and the ultrafiltration fraction of XY-144 have no activity in removing MRSA biofilm after protein removal, so the possibility that the active ingredient of XY-144 is polysaccharide can be ruled out, and it is speculated that protein is more likely.

[0081] Example 8 Genetic analysis and identification of active components of strain XY-144

[0082] In order to identify the gene encoding the active ingredient of XY-144, its genomic sequence information was analyzed. The strain XY-144 was activated with MA solid medium and inverted at 28°C for 3 days. The bacterial genome was extracted using a bacterial genomic DNA extraction kit (TIANGEN). 1% (w / v) agarose gel was electrophoresed at 120V for 15 minutes, and then the bands were observed with a gel imager to confirm the quality of the extracted genome, and then sent to Sangon Biotech (Shanghai) Co., Ltd. for genome sequencing analysis. RepeatMasker 4.0.5 and Tandem repeats finder 4.07b were used to annotate and identify long interspersed repeat sequences and tandem repeat sequences, respectively. GeneMarkS 4.17 and default parameters were used to annotate the coding genes. The prediction of genome components includes the prediction of coding genes, repetitive sequences, noncoding RNA, genomic islands, transposons, prophages, and clustered regularly interspaced short palindromic repeats (CRISPR) using the following databases: GO database (Gene Ontology), KEGG database (Kyoto Encyclopedia of Genes and Genomes), COG database (Clusters of Orthologous Groups), NR database (Non-Redundant Protein Database), TCDB database (Transporter Classification Database), and Swiss-Prot database. The whole genome BLAST search (E value less than 1×10 -5 , the minimum alignment length percentage is greater than 40%). The protein was searched in the above database by local BLASTP to find potential active protein coding genes from the whole genome sequencing data. 0.25 The sample was sent to Shenzhen Micron Biotechnology Co., Ltd. for mass spectrometry analysis. The analysis results were compared with the genome sequencing results of XY-144 to obtain a sequence that may encode the active ingredient of XY-144, which was recorded as gene 500784. The protein encoded by gene 500784 was recorded as protein 500784, and its amino acid sequence is shown in SEQ ID NO.1.

[0083] In addition, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on each component in Example 4. The analysis results are as follows: Figure 8 shown. Figure 8 The substances added in each channel are as follows: 1 is the salting-out component of XY-144; 2 is the ultrafiltration component of XY-144; 3 is the loading flow-through of XY-144; 4 is the elution component E of XY-144 0.1; 5 is XY-144 elution component E 0.25 ; 6 is XY-144 elution component E 0.5 ; 7 is XY-144 elution component E 1 .Depend on Figure 8 It can be seen that the salting-out component, ultrafiltration component and elution component E of XY-144 0.25 Both have obvious bands at the position of 39 kDa, but this band is not found in other components, so it is estimated that the size of the active component of XY-144 is about 39 kDa. There is a certain difference between the size of the active component of XY-144 and the size of the 500784 protein, which may be due to the fact that the active component of XY-144 also contains other smaller impurities.

[0084] The maximum likelihood evolutionary tree (Bootstrap 1000) was constructed using RaxML software to analyze the 500,784 proteins encoded by the 500,784 genes. Fig. 9 As shown. Fig. 9 It can be seen that protein 500784 and protein WP_018298764.1 are in the same evolutionary branch, but their evolutionary relationship is distant. Their amino acid identity is 44% and their similarity is 61%, indicating that protein 500784 is unique among Fangia strains and belongs to a novel S8 family serine protease.

[0085] Example 9 Expanded Expression and Purification of Serine Protease in Strain XY-144

[0086] The amino acid and nucleotide sequences of the serine protease in strain XY-144 were optimized, the signal peptide of 19 amino acids at the N-terminus was removed, and a His6 tag was added at the C-terminus to make it more suitable for recombinant expression. The amino acid sequence of the optimized serine protease is shown in SEQ ID NO.2, and the optimized serine protease gene is shown in SEQ ID NO.3.

[0087] Primers were designed based on the optimized serine protease gene. The nucleotide sequence of the upstream primer (FP) is shown in SEQ ID NO.4, and the nucleotide sequence of the downstream primer (RP) is shown in SEQ ID NO.5. A sufficient amount of PCR product was amplified by PCR, and the PCR product and the pET-28a(+) plasmid vector were digested with NcoI and XhoI restriction enzymes, respectively. The PCR product after digestion and the pET-28a(+) plasmid vector were connected by ligase to obtain a recombinant vector. The connected solution was transferred into BL21(DE3) competent cells, and positive clones were detected and screened for verification. The recombinant vector was obtained by gel detection, and the detection results are shown in FIG. Fig.10 shown. Fig.10The added substances in each channel are as follows: M is DNA Marker; 1 is the gene after ApaI-XhoI double digestion (size: 4197bp / 2472bp); 2 is the recombinant vector.

[0088] The recombinant vector was transformed into BL21 (DE3) competent cells, and the plates were inverted and cultured overnight at 37°C. A monoclonal strain was selected and expanded in 1L LB medium containing 30μg / mL kanamycin at 37°C to OD600 = 0.6-0.8, and isopropylthiogalactoside (IPTG) was added to a final concentration of 1mM, and induced at 37°C for 4h; the bacteria were collected by centrifugation, resuspended, and ultrasonically disrupted; centrifuged, the precipitate was dissolved in lysis buffer (50mMTris, 300mM NaCl, 8M urea, pH 8) and centrifuged again to obtain the precipitate and supernatant after disruption; the supernatant after disruption was purified by nickel column affinity chromatography. The process of nickel column affinity chromatography purification includes: equilibration, loading, washing and elution; wherein, the equilibration buffer is "Tris-NaCl-Urea" buffer (50mM Tris, 300mM NaCl, 2M urea), pH8; the washing buffer is "Tris-NaCl-Urea" buffer containing 50mM imidazole, pH8; the elution buffer is "Tris-NaCl-Urea" buffer containing 500mM imidazole, pH8. The components after nickel column affinity chromatography purification include: the effluent collected after loading, the washing sample collected after washing and the elution sample collected after elution. The SDS-PAGE test results of each component are shown as follows: Fig.11 shown. Fig.11 In the figure, the substances added in each channel are as follows: M is protein marker; 1 is the precipitate after crushing; 2 is the supernatant after crushing; 3 is the effluent; 4 is the washing sample; 5 is the elution sample. Fig.11 It can be seen that the target protein, namely the recombinant serine protease, can be obtained after purification by nickel column affinity chromatography.

[0089] Example 10 Verification of biofilm removal activity of recombinant serine protease

[0090] The overnight seed solution of MRSA was diluted 100 times by volume with LB liquid medium containing 0.5% (w / v) glucose, and 1 mL / well was added to a 24-well plate. After culturing at 37°C for 1 day, the bacterial solution was slowly sucked away with a discharge gun to obtain a 24-well plate containing MRSA biofilm. The recombinant serine protease prepared in Example 9 was diluted with LB liquid medium containing 0.5% (w / v) glucose to a concentration of 84 μg / mL, 42 μg / mL, 21 μg / mL, and 10.5 μg / mL of serine protease to obtain protein dilutions of different concentrations. The protein dilutions were slowly added to a 24-well plate containing MRSA biofilm at 1 mL / well. After culturing at 37°C for 1 day, the solution was slowly sucked away with a discharge gun, washed once with saline, dried at 37°C for 30 minutes, and then fixed at 65°C for 30 minutes and then taken out and cooled to room temperature. Each well was stained by adding 1 mL of 0.1% (w / v) crystal violet solution. After 5 minutes, the stain was washed off and dried. The removal effect of biofilm was as follows: Fig.12 shown. Fig.12 In the figure, 1 is a blank control without any component added; 2 is a protein diluent containing 84μg / mL recombinant serine protease; 3 is a diluent containing 42μg / mL recombinant serine protease; 4 is a protein diluent containing 100μg / mL proteinase K as a positive control; 5 is a protein diluent containing 21μg / mL recombinant serine protease; 6 is a protein diluent containing 10.5μg / mL recombinant serine protease. Fig.12 It can be seen that the recombinant serine protease prepared in Example 9 has a clearing effect on MRSA biofilm, and has a clearing effect even at a lower concentration.

[0091] Example 11 Thermal stability test of recombinant serine protease

[0092] The recombinant serine protease solution was incubated at 25°C, 40°C, 55°C and 70°C for 30 minutes and then cooled to room temperature, and then the activity of removing MRSA biofilm was verified. The overnight seed solution of MRSA was diluted 100 times by volume with LB liquid medium containing 0.5% (w / v) glucose, and 1 mL / well was added to a 24-well plate. After culturing at 37°C for 1 day, the bacterial solution was slowly aspirated with a spray gun to obtain a 24-well plate containing MRSA biofilm. The recombinant serine protease solutions treated at different temperatures were diluted with LB liquid medium containing 0.5% (w / v) glucose to a recombinant serine protease concentration of 42 μg / mL, and then slowly added to the 24-well plate containing MRSA biofilm at 1 ml / well. Dimethyl sulfoxide (DMSO) treated at 25°C was used as a negative control, and proteinase K treated at 25°C was used as a positive control. After culturing at 37°C for 1 day, the solution was slowly aspirated with a spray gun, washed once with physiological saline, dried at 37°C for 30 minutes, and then fixed at 65°C for 30 minutes before being taken out and cooled to room temperature. 1 mL of 0.1% (w / v) crystal violet solution was added to each well for staining, the stain was washed off after 5 minutes, dried, and then 5 mL of 30% (v / v) acetic acid solution was used to dissolve the pigment. After mixing, 100 μL was taken out and measured with an enzyme marker for OD 550 , the test results are as follows Fig.13 As shown. Fig.13 It can be seen that the recombinant serine protease prepared in Example 9 has good thermal stability and is still very active in removing MRSA biofilm after incubation at 25-70° C. for 30 minutes.

[0093] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

[0094] The amino acid and nucleotide sequences involved in the present invention are as follows:

[0095] SEQ ID NO.1:

[0096] MKKVILSTIAASLALGAYANNSSNQVELIVKYKKSANTMLMASANNAEDKNYSIVKQINPTTAIVSVQQTAPQNVSLSTSALAESGSSNSAQDQAYATAKAFMDENPNVLYAIPKDSKMSAYQLPTSSAQSTSGQNVNSWDKQWDMKNTKAGLGAEGAWNYVNSGNDVTVAVVDSGLAPNAPEDITKKLNLNNTYYFTLSGNDIVVTRDITDNGSYHGTHVAGTIAANGPNVSGVAGPVNGVTVMPVRALGDDGSGSTYAILDAVKWAAGGVPDGVTTENGGSIDANTANVKVINLSLGMSRINPYTNLPQISKKKWESDYMGTLCPAWKDAIDTAHQNGVTVVIAAGNDNHSVYNDIPAGCQDIDAVVVEAAGPTGKLSSYSTYLDSDWSINSLVVRAPGGDSRATYTDESGQTVSYGESGEIYSTMNGGYGYMQGTSMATPHVAGLVSLIYSLDANATPSFVQTVLKNAANPQNPDIVNAQAAVEYTLNSVSSAAA.

[0097] SEQ ID NO.2:

[0098] MNNSSNQVELIVKYKKSANTMLMASANNAEDKNYSIVKQINPTTAIVSVQQTAPQNVSLSTSALAESGSSNSAQDQAYATAKAFMDENPNVLYAIPKDSKMSAYQLPTSSAQSTSGQNVNSWDKQWDMKNTKAGLGAEGAWNYVNSGNDVTVAVVDSGLAPNAPEDITKKLNLNNTYYFTLSGNDIVVTRDITDNGSYHGTHVAGTIAANGPNVSGVAGPVNGVTVMPVRALGDDGSGSTYAILDAVKWAAGGVPDGVTTENGGSIDANTANVKVINLSLGMSRINPYTNLPQISKKKWESDYMGTLCPAWKDAIDTAHQNGVTVVIAAGNDNHSVYNDIPAGCQDIDAVVVEAAGPTGKLSSYSTYLDSDWSINSLVVRAPGGDSRATYTDESGQTVSYGESGEIYSTMNGGYGYMQGTSMATPHVAGLVSLIYSLDANATPSFVQTVLKNAANPQNPDIVNAQAAVEYTLNSVSSAAALEHHHHHH。

[0099] SEQ ID NO.3:

[0100]

[0101] SEQ ID NO.4:

[0102] taagaaggagatataccatgaataatagcagcaatcaggt.

[0103] SEQ ID NO.5:

[0104] tggtggtggtggtgctcgagggctgctgcgctactaacac。

Claims

1. A serine protease, characterized in that The amino acid sequence of the serine protease is shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. A nucleic acid, characterized in that The nucleic acid encodes the serine protease according to claim 1.

3. The nucleic acid according to claim 2, characterized in that The nucleotide sequence of the nucleic acid is shown in SEQ ID NO.

3.

4. A recombinant vector, characterized in that: The recombinant vector expresses the serine protease according to claim 1.

5. A host cell, characterized in that The host cell contains the recombinant vector according to claim 4.

6. A composition, characterized in that The composition contains the serine protease according to claim 1.

7. A method for preparing the serine protease according to claim 1, characterized in that: Includes steps: Synthesizing a gene encoding the serine protease according to claim 1, and inserting the gene into an expression vector to obtain a recombinant vector; The recombinant vector is transferred into a host cell for protein expression, and the product of the protein expression is purified to obtain the serine protease.

8. The preparation method according to claim 7, characterized in that: The expression vector is pET-28a(+).

9. The preparation method according to claim 7, characterized in that: The host cell is Escherichia coli.

10. Use of the serine protease according to claim 1, the nucleic acid according to claim 2 or 3, the recombinant vector according to claim 4, the host cell according to claim 5, the composition according to claim 6 or the serine protease prepared by the preparation method according to any one of claims 7 to 9 in the preparation of a product for removing biofilm or a product for inhibiting biofilm formation.

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