Serine protease and preparation method and application thereof
By screening and optimizing the preparation method of serine proteases, the problem of poor thermostability of existing enzymes has been solved, and the effect of effectively inhibiting and removing biofilms in the food industry has been achieved, ensuring food safety.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHENZHEN UNIV
- Filing Date
- 2025-02-17
- Publication Date
- 2026-07-21
AI Technical Summary
There are few existing enzymes for biofilms, and their thermal stability is poor, making it difficult to effectively inhibit or eliminate methicillin-resistant Staphylococcus aureus biofilms in environments with fluctuating temperatures.
A serine protease was screened and optimized. By synthesizing the encoding gene, inserting it into an expression vector, expressing it in host cells, and purifying it, a stable serine protease was obtained and applied to the removal and inhibition of biofilms in the food industry.
This serine protease is stable at different temperatures, can effectively inhibit and remove biofilms, and is not likely to cause pathogens to develop drug resistance. It is harmless to food safety and is suitable for food industry environments with fluctuating temperatures.
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Figure CN119979510B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of bioenzyme technology, and in particular to a serine protease, its preparation method, and its application. Background Technology
[0002] Food safety has long been a major factor affecting public health, and the incidence of foodborne illnesses caused by microbial pathogens is on the rise worldwide. Methicillin-resistant Staphylococcus aureus (MRSA) is a common foodborne pathogen, and foodborne illnesses caused by it have become a global public health problem.
[0003] Currently, the main strategies for preventing and controlling foodborne diseases are the use of microbial disinfectants or exposure to low doses of ultraviolet A (UVA). However, direct killing methods may lead to 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 allow them to continue to survive in the environment.
[0004] While strategies for controlling biofilms in the food industry, such as cleaning and disinfection, plasma treatment, and ultrasonic treatment, are effective, they are 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 varies with temperature, making them unsuitable for use in temperature-fluctuating environments. Therefore, screening and developing more stable enzymes is crucial for combating biofilms from foodborne pathogens under temperature-variable conditions. Summary of the Invention
[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a serine protease, its preparation method and application, in order to solve the problems of the limited availability and poor thermal stability of existing enzymes for biofilm clearance.
[0006] The technical solution of the present invention is as follows:
[0007] In a first aspect, a serine protease is provided, the amino acid sequence of which is shown in SEQ ID NO.1 or SEQ ID NO.2.
[0008] In a second aspect, a nucleic acid is provided that encodes a serine protease as described in the first aspect.
[0009] In a preferred embodiment, the nucleotide sequence of the nucleic acid is shown in SEQ ID NO.3.
[0010] Thirdly, a recombinant vector is provided that expresses the serine protease as described in the first aspect.
[0011] Fourthly, a host cell is provided, said host cell containing the recombinant vector as described in the third aspect.
[0012] Fifthly, a composition is provided that contains the serine protease as described in the first aspect.
[0013] Sixthly, a method for preparing a serine protease as described in the first aspect is provided, comprising the steps of:
[0014] A gene encoding a serine protease as described in the first aspect is synthesized, and the gene is inserted into an expression vector to obtain a recombinant vector;
[0015] The recombinant vector was transferred into host cells for protein expression, and the product of the protein expression was purified to obtain the serine protease.
[0016] In a preferred embodiment, the expression vector is pET-28a(+).
[0017] In a preferred embodiment, the host cell is Escherichia coli.
[0018] In a seventh aspect, the use of a serine protease as described in the first aspect, a nucleic acid as described in the second aspect, a recombinant vector as described in the third aspect, a host cell as described in the fourth aspect, a composition as described in the fifth aspect, or a serine protease prepared by the preparation method as described in the sixth aspect, in the preparation of a product for clearing biofilms or inhibiting biofilm formation.
[0019] Beneficial Effects: This invention provides a novel serine protease that exhibits stable activity at different temperatures. In practical applications, this protease better maintains its activity and efficacy, ensuring effective inhibition of biofilm formation or removal of mature biofilms even under fluctuating temperature conditions. Experimental results show that this serine protease significantly inhibits and removes methicillin-resistant Staphylococcus aureus (MRSA) biofilms; it also shows no antibacterial activity and is unlikely to induce drug resistance in pathogens; furthermore, it is non-toxic and has no effect on the germination rate or sprout length of alfalfa seeds. Therefore, the application of this serine protease in the food industry is safe and reliable, offering significant advantages for food applications. Attached Figure Description
[0020] Figure 1 This is a phylogenetic tree of strain XY-144 from Example 1 and the reference strain.
[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 graph showing the results of verifying the scavenging and inhibitory activity of the fermentation supernatant of XY-144 on MRSA biofilm in Example 3.
[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 scavenging 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 XY-144 salting-out component in Example 6.
[0026] Figure 7 This is a graph showing the results of the activity verification of each component in removing MRSA biofilm after the fermentation supernatant of XY-144 and the ultrafiltration component of XY-144 were treated by the Sevage method in Example 7.
[0027] Figure 8 This is a graph showing the SDS-PAGE analysis results of each component of the fermentation supernatant of XY-144 in Example 8.
[0028] Figure 9 This is a phylogenetic tree of protein 500784 in Example 8 and reported serine proteases.
[0029] Figure 10 This is a graph showing the enzyme digestion detection results of the recombinant vector in Example 8.
[0030] Figure 11 This is a diagram showing the purification results of the recombinant serine protease in Example 9.
[0031] Figure 12 This is a graph showing the results of verifying the activity of recombinant serine protease in clearing MRSA biofilm in Example 10.
[0032] Figure 13 This is a graph showing the results of the activity verification of recombinant serine protease in clearing MRSA biofilm in Example 11. Detailed Implementation
[0033] This invention provides a serine protease, its preparation method, and its application. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below.
[0034] Biofilm removal is a current research hotspot. In clinical settings, strategies such as enzyme treatment, small molecule drugs, and surgical resection have been successfully applied, enabling bacteria to transition from a biofilm state to a more vulnerable planktonic state. Compared to other biofilm removal strategies, enzyme treatment does not present the drug resistance problem encountered with other small molecule drugs. Drug resistance often leads to disease progression and treatment failure, thus enzyme treatment has a greater advantage in practical applications. Furthermore, enzyme treatment can achieve high specificity and effectiveness against target biofilms at relatively low concentrations. However, few enzymes have been reported for biofilm control, and the thermal instability of some enzymes limits their application.
[0035] Based on this, embodiments of the present invention provide a serine protease, the amino acid sequence of which is shown in SEQ ID NO.1 or SEQ ID NO.2.
[0036] Specifically, the inventors initially collected a sample of a manatee from the shallow waters of the Dapeng Nature Reserve in Shenzhen and isolated approximately 200 strains. Activity screening of these strains revealed that strain XY-144 exhibits biofilm scavenging and biofilm inhibition activities. Further, through the isolation, purification, and analysis of the active ingredient in strain XY-144, a serine protease with the amino acid sequence shown in SEQ ID NO.1 was screened. The amino acid sequence of the serine protease in strain XY-144 was optimized by removing the 19-amino acid signal peptide at the N-terminus and adding a His6 tag at the C-terminus, resulting in a recombinant serine protease with the amino acid sequence shown in SEQ ID NO.2. Both the serine protease in strain XY-144 and the recombinant serine protease possess biofilm scavenging and biofilm inhibition activities.
[0037] This invention provides a nucleic acid that encodes a serine protease as described above.
[0038] In one embodiment, the nucleotide sequence of the nucleic acid is shown in SEQ ID NO.3.
[0039] This invention provides a recombinant vector that expresses the serine protease as described above.
[0040] This invention provides a host cell containing the recombinant vector described above.
[0041] This invention provides a composition containing the serine protease described above.
[0042] In one embodiment, the composition further contains an antibiotic.
[0043] This invention provides a method for preparing the serine protease as described above, comprising the following steps:
[0044] A gene encoding the serine protease as described above was synthesized, and the gene was inserted into an expression vector to obtain a recombinant vector;
[0045] The recombinant vector was transferred into host cells for protein expression, and the product of the protein expression was 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] This invention provides the application of the serine protease prepared 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 preparation method as described above in the preparation of products that clear biofilms or inhibit biofilm formation.
[0050] In one embodiment, the product is a product that removes MRSA biofilms or inhibits MRSA biofilm formation.
[0051] The present invention will be further described below through specific embodiments.
[0052] In this embodiment of the invention, the culture medium is prepared with deionized water, and the formula is as follows:
[0053] LB liquid medium: 10 g / L tryptone, 1 g / L yeast extract, 5 g / L sodium chloride.
[0054] LB solid medium: tryptone 10g / L, yeast extract 1g / L, sodium chloride 5g / L, agar 15g / L.
[0055] MA solid medium: peptone 5 g / L, yeast extract 1 g / L, ferric citrate 0.1 g / L, sodium chloride 19.45 g / L, magnesium chloride 5.98 g / L, sodium sulfate 3.24 g / L, calcium chloride 1.8 g / L, potassium chloride 0.55 g / L, sodium carbonate 0.16 g / L, potassium bromide 0.08 g / L, strontium chloride 0.034 g / L, boric acid 0.022 g / L, sodium silicate 0.004 g / L, sodium fluoride 0.0024 g / L, sodium nitrate 0.0016 g / L, disodium hydrogen phosphate 0.008 g / L, agar 15 g / L.
[0056] SGTYP liquid medium: glucose 5g / L, soluble starch 5g / L, tryptone 1g / L, yeast extract 1g / L, peptone 1g / L.
[0057] SGTYP solid medium: glucose 5g / L, soluble starch 5g / L, tryptone 1g / L, yeast extract 1g / L, peptone 1g / L, agar 15g / L.
[0058] In this embodiment of the invention, the preparation process of the fermentation supernatant of XY-144 and the overnight seed culture of MRSA is as follows:
[0059] Fermentation supernatant of XY-144: A small amount of frozen XY-144 cells were scraped onto MA solid medium and streaked, and cultured at 28°C for 3 days. A single colony was then placed in 3 mL of SGTYP liquid medium and incubated at 28°C and 200 rpm for 3 days. After centrifugation at 10,000 rpm for 20 minutes at 4°C, the fermentation supernatant of XY-144 was obtained.
[0060] Overnight seed culture of MRSA: Scrape a small amount of frozen MRSA cells onto LB solid medium and streak. Incubate at 37°C for 1 day. Take a single colony and place it in 3 mL of LB liquid medium. Incubate overnight at 37°C and 200 rpm to obtain the overnight seed culture of MRSA.
[0061] Example 1: Isolation and Identification of Strain XY-144
[0062] Strain XY-144 was collected in March 2017 from Dapeng Nature Reserve in Shenzhen. It is a symbiotic bacterium of the marine invertebrate manatee.
[0063] DNA was extracted from XY-144 using the TIANamp Bacteria DNA Kit (TIANGEN). The 16S rDNA region of XY-144 was amplified by PCR using standard forward primer 27F and reverse primer 1492R. Sequencing was then performed to obtain the 16S rRNA sequence data of XY-144. Comparative analysis showed that the 16S rRNA sequence of XY-144 is similar to that of Fangiahongkongensis FSC776. T The 16S rRNA sequence of XY-144 showed a similarity of 99.37%, thus identifying XY-144 as belonging to Fangiahongkongensis. The 16S rRNA sequence of XY-144 has been submitted to the GenBank database, accession number OR262794.1.
[0064] DNA was extracted from XY-144 using the TIANamp Bacteria DNA Kit (TIANGEN), and sequenced to obtain the whole genome data of XY-144. A phylogenetic tree was then constructed based on the sequencing results, as detailed below. Figure 1 As shown. By Figure 1 It can be seen that XY-144 belongs to the genus Fangia, and the most closely related strain is Fangia hongkongensis FSC776. T The core gene homology is 78%, therefore it is speculated that XY-144 is a new strain.
[0065] XY-144 was deposited on June 26, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo: 31090 and classified as Fangiahongkongensis.
[0066] Example 2: Antibacterial activity of strain XY-144
[0067] The overnight seed culture of MRSA was diluted 1000 times with LB liquid medium, and 40 μL was spread onto LB solid medium, with circular paper discs placed on top. 5 μL 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 onto circular paper discs, and then incubated at 37°C for 1 day. The experimental results are as follows: Figure 2 As shown. Figure 2 In this context, 1 refers to the addition of XY-144 fermentation supernatant; 2 refers to the addition of SGTYP liquid medium; and 3 refers to the addition of SGTYP liquid 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 scavenging and inhibitory activities of strain XY-144
[0069] Overnight MRSA seed culture was diluted 100-fold by volume in LB liquid medium containing 0.5% (w / v) glucose, and 1 mL / well was added to each well of a 24-well plate. After incubation at 37°C for 1 day, the bacterial culture was slowly aspirated using a pipette to obtain 24-well plates containing MRSA biofilm. XY-144 fermentation supernatant was diluted with LB liquid medium containing 0.5% (w / v) glucose to obtain different concentrations (75%, 50%, 25%, 12.5%, and 6.25% of the XY-144 fermentation supernatant volume). 1 mL / well of these dilutions was slowly added to each well of the 24-well plate containing MRSA biofilm. SGTYP liquid medium was also diluted in the same proportion and added as a negative control. After incubation at 37°C for 1 day, the bacterial culture was slowly aspirated using a pipette, washed once with physiological saline, dried at 37°C for 30 minutes, fixed at 65°C for 30 minutes, and then cooled to room temperature. Add 1 mL of 0.1% (w / v) crystal violet solution to each well for staining. Wash off the stain after 5 minutes, dry, and observe the biofilm removal effect. (Specific details are as follows...) Figure 3 As shown in (a). Figure 3 In (a), 144 75% refers to a dilution containing 75% (v / v) XY-144 in the fermentation supernatant; 144 50% refers to a dilution containing 50% (v / v) XY-144 in the fermentation supernatant; 144 12.5% refers to a dilution containing 12.5% (v / v) XY-144 in the fermentation supernatant; 144 6.25% refers to a dilution containing 6.25% (v / v) XY-144 fermentation supernatant; SG75% refers to a dilution containing 75% (v / v) SGTYP liquid medium; SG50% refers to a dilution containing 50% (v / v) SGTYP liquid medium; SG12.5% refers to a dilution containing 12.5% (v / v) SGTYP liquid medium; SG6.25% refers to a dilution containing 6.25% (v / v) SGTYP liquid medium. Figure 3 As shown in (a), the fermentation supernatant of XY-144 has a scavenging effect on MRSA biofilm, and the scavenging effect increases with the increase of the proportion of fermentation supernatant.
[0070] Overnight MRSA seed culture was diluted 100-fold by volume with LB liquid medium containing 0.5% (w / v) glucose, and 1 mL / well was added to each well of a 24-well plate to obtain MRSA-containing 24-well plates. XY-144 fermentation supernatant was diluted with LB liquid medium containing 0.5% (w / v) glucose to obtain different concentrations (75%, 50%, 25%, 12.5%, and 6.25% of the XY-144 fermentation supernatant volume). 1 mL / well of each dilution was slowly added to each well of the MRSA-containing 24-well plates. SGTYP liquid medium was also diluted in the same proportion and added as a negative control. After incubation at 37°C for 1 day, the bacterial culture was slowly aspirated with a pipette, washed once with physiological saline, dried at 37°C for 30 minutes, fixed at 65°C for 30 minutes, and then cooled to room temperature. Add 1 mL of 0.1% (w / v) crystal violet solution to each well for staining. Wash off the stain after 5 minutes, dry, and observe the inhibitory effect on the biofilm. (Specific details are as follows...) Figure 3 As shown in (b). Figure 3 In (b), 144 75% refers to a dilution containing 75% (v / v) XY-144 fermentation supernatant; 144 50% refers to a dilution containing 50% (v / v) XY-144 fermentation supernatant; 144 12.5% refers to a dilution containing 12.5% (v / v) XY-144 fermentation supernatant; 144 6.25% refers to a dilution containing 6.25% (v / v) XY-144 fermentation supernatant; SG 75% refers to a dilution containing 75% (v / v) SGTYP liquid medium; SG 50% refers to a dilution containing 50% (v / v) SGTYP liquid medium; SG 12.5% refers to a dilution containing 12.5% (v / v) SGTYP liquid medium; SG 6.25% refers to the addition of a dilution containing 6.25% (v / v) SGTYP liquid medium. Figure 3 As shown in Figure (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 of the proportion of 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 percentage of Tris-HCl buffer was 10%. Ammonium sulfate powder was then slowly added until the mass concentration was 50%. The mixture was then incubated overnight at 4°C to precipitate the protein, resulting in a precipitated protein solution. The precipitated protein solution was centrifuged at 10,000 rpm for 20 minutes at 4°C, the supernatant was discarded, and the precipitate was resuspended in ddH2O to obtain the precipitated sample. Salting out the precipitated sample was then performed. Before salting out, the dialysis bag (Beyotime, 44mm, 14kDa) was sequentially boiled for 10 minutes with dialysis bag treatment solution (2% (w / v) NaHCO3, 1M EDTA, pH 8), ddH2O, 1M EDTA, and ddH2O, followed by cooling. After leak testing, the above-mentioned precipitated sample was added to a dialysis bag and dialyzed for 20 hours at 4°C using ddH2O as the dialysate on a horizontal shaker at 80 rpm. The dialysate was replaced 3 times during the process. After the last replacement, the sample was dialyzed for another 2 hours to obtain the XY-144 salting-out fraction.
[0073] The XY-144 salting-out fraction was concentrated using an ultrafiltration tube (Millipore, 3 kDa). The resulting filtrate and concentrate were designated as XY-144 filtrate and XY-144 ultrafiltration fraction, respectively. The XY-144 ultrafiltration fraction was then further purified using an anion exchange column. A 3 mL column of DEAE homogenate resin was packed into the column, washed sequentially with 3 column volumes of 2M NaCl solution and ddH2O, and then equilibrated with 3 column volumes of 20 mM Tris-HCl buffer (pH 8). Finally, 6 mL of the XY-144 ultrafiltration fraction was loaded onto the column. The sample was washed with three column volumes of 20 mM Tris-HCl (pH 8) solution containing 0.1 M NaCl to obtain the XY-144 sample pass-through, denoted as FT. Then, it was eluted sequentially with three column volumes of 20 mM Tris-HCl (pH 8) solutions containing 0.1 M, 0.25 M, 0.5 M, and 1 M NaCl to obtain the XY-144 eluent, denoted as E. 0.1 E 0.25 E 0.5 And E1; 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 medium (negative control) were diluted with LB liquid medium containing 0.5% (w / v) glucose to verify their activity in scavenging MRSA biofilms. For specific procedures, please refer to Example 3. The experimental results are as follows: Figure 4 As shown. Figure 4In (a), 20% salting out refers to the addition of a diluent containing 20% (v / v) XY-144 salting out component; 10% salting out refers to the addition of a diluent containing 10% (v / v) XY-144 salting out component; 20% ultrafiltration refers to the addition of a diluent containing 20% (v / v) XY-144 ultrafiltration component; 10% ultrafiltration refers to the addition of a diluent containing 10% (v / v) XY-144 ultrafiltration component; 20% negative control refers to the addition of a diluent containing 20% (v / v) SGTYP liquid medium; 10% negative control refers to the addition of a diluent containing 10% (v / v) SGTYP liquid medium; 10% filtrate refers to the addition of a diluent containing 10% (v / v) XY-144 filtrate; and the blank control did not contain any component. Figure 4 In (b), FT 10% refers to the addition of a diluent containing 10% (v / v) of the XY-144 sample pass solution; E 0.1 5% refers to the addition of 5% (v / v) XY-144 elution component E. 0.1 diluent; E 0.25 5% refers to the addition of 5% (v / v) XY-144 elution component E. 0.25 diluent; E 0.5 5% refers to the addition of 5% (v / v) XY-144 elution component E. 0.5 The diluent; E15% refers to the diluent containing 5% (v / v) XY-144 elution fraction E1; the blank control did not contain any components. Figure 4 It can be seen that the XY-144 salt-precipitated fraction after salting out and the XY-144 ultrafiltration fraction after salting out and ultrafiltration concentration still have a scavenging effect on MRSA biofilm. However, the XY-144 filtrate after ultrafiltration did not show a scavenging effect on MRSA biofilm. The results of anion exchange purification of the ultrafiltration fraction showed that among the XY-144 eluents after DEAE column elution, only the XY-144 eluent E eluted with 20mM Tris-HCl (pH 8) solution containing 0.25M NaCl was effective. 0.25 It has the activity of scavenging MRSA biofilm.
[0075] Example 5: Thermal stability test of active ingredients in strain XY-144
[0076] The fermentation supernatant of XY-144 was incubated at 28℃, 40℃, 60℃, 80℃, and 100℃ for 30 minutes, then cooled to room temperature for activity verification in scavenging MRSA biofilm. Overnight MRSA seed culture was diluted 100-fold by volume with LB liquid medium containing 0.5% (w / v) glucose, and 1 mL / well was added to a 24-well plate. After incubation at 37℃ for 1 day, the bacterial culture was slowly aspirated using a pipette to obtain 24-well plates containing MRSA biofilm. Fermentation supernatants of XY-144 treated at different temperatures were diluted with LB liquid medium containing 0.5% (w / v) glucose to a volume ratio of 50% of the treated XY-144 fermentation supernatant. 1 mL / well was then slowly added to each well of a 24-well plate containing MRSA biofilm. SGTYP liquid medium was also diluted and added as a negative control. After incubation at 37°C for 1 day, the bacterial culture was slowly aspirated with a pipette, washed once with physiological saline, dried at 37°C for 30 minutes, fixed at 65°C for 30 minutes, and then 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. The plate was then dried. The biofilm removal effect was as follows: Figure 5 As shown in (a). Then, the pigment was dissolved in 5 mL of 30% (v / v) acetic acid solution, mixed well, and 100 μL was taken for OD measurement using a microplate reader. 550 The test results are as follows Figure 5 As shown in (b). From Figure 5 It can be seen that the fermentation supernatant of XY-144 has good thermal stability, and its activity in scavenging MRSA biofilm remains very good after incubation at 60℃ for 30 minutes.
[0077] Example 6: Toxicity test of the active ingredient in strain XY-144
[0078] Tris-HCl buffer (1M, pH 8) was added to the fermentation supernatant of XY-144 until the volume percentage of Tris-HCl buffer was 10%. Ammonium sulfate powder was then slowly added until the mass concentration was 50%. The mixture was then incubated overnight at 4°C to precipitate the protein, resulting in a precipitated protein solution. The precipitated protein solution was centrifuged at 10,000 rpm for 20 minutes at 4°C, the supernatant was discarded, and the precipitate was resuspended in ddH2O to obtain the precipitated sample. Salting out the precipitated sample was then performed. Before salting out, the dialysis bag (Beyotime, 44mm, 14kDa) was sequentially boiled for 10 minutes with dialysis bag treatment solution (2% (w / v) NaHCO3, 1M EDTA, pH 8), ddH2O, 1M EDTA, and ddH2O, followed by cooling. After leak testing, the precipitated sample was added to a dialysis bag and dialyzed for 20 hours at 4°C using ddH2O as the dialysate on a horizontal shaker at 80 rpm, with the dialysate replaced three times. After the last dialysate replacement, dialyzing was performed for another 2 hours to obtain the XY-144 salting-out fraction. Alfalfa seeds were soaked in water, rinsed thoroughly with water on a filter screen, then soaked in sodium hypochlorite (10-fold diluted 84 disinfectant) for 10 minutes, washed 4-5 times with sterile water, air-dried, and stored in 50 mL EP tubes with desiccant. Fifty seeds were weighed into a petri dish, and 20 mL of sterile water containing 10% (v / v) XY-144 salting-out fraction was added. The mixture was incubated at 25°C. The negative control group consisted of 20 mL of sterile water. Observations and records were made at 24 and 48 hours. The results are as follows: Figure 6 As shown. By Figure 6 It is known that the XY-144 salting-out component has no effect on the germination of alfalfa, neither on the germination rate nor the length of the shoots, and is not toxic.
[0079] Example 7: Property Analysis of Active Components in Strain XY-144
[0080] The Sevage method was used to verify whether the active ingredient of strain XY-144 was a polysaccharide. 30 mL of the fermentation supernatant of XY-144 and 3 mL of the XY-144 ultrafiltration fraction from Example 4 were taken, and 10 mL and 1 mL of the prepared Sevage reagent (chloroform: n-butanol = 4:1) were added respectively. After thorough mixing for 30 minutes, the mixture was centrifuged at 5000 rpm for 20 minutes. The upper aqueous phase was transferred to a 15 mL centrifuge tube and treated at -80℃ for 20 minutes, followed by lyophilization to remove residual organic reagents. The lyophilized powder was resuspended in ddH2O and its activity in scavenging MRSA biofilm was verified. The experimental results are as follows: Figure 7 As shown. Figure 7In this study, 1. XY-144 ultrafiltration fraction treated with the Sevage method was added; 2. XY-144 ultrafiltration fraction was added; 3. SGTYP liquid medium ultrafiltration fraction was added; 4. Fermentation supernatant of XY-144 treated with the Sevage method was added; 5. Fermentation supernatant of XY-144 was added; 6. Fermentation supernatant of SGTYP liquid medium was added; 7. A blank control was prepared without any added components. Figure 7 It is known that neither the fermentation supernatant of XY-144 nor the ultrafiltration fraction of XY-144 has the activity of removing MRSA biofilm after protein removal treatment. Therefore, the possibility that the active ingredient of XY-144 is polysaccharide can be ruled out, and it is more likely to be protein.
[0081] Example 8: Gene analysis and identification of the active ingredient in strain XY-144
[0082] To identify the gene encoding the active ingredient of XY-144, its genomic sequence information was analyzed. XY-144 strain was activated on MA solid medium and incubated upside down at 28°C for 3 days. Bacterial genome was extracted using a bacterial genomic DNA extraction kit (TIANGEN). Electrophoresis was performed on a 1% (w / v) agarose gel at 120V for 15 minutes. The quality of the extracted genome was confirmed by observing the bands using a gel imaging system and then sent to Sangon Biotech (Shanghai) Co., Ltd. for genome sequencing analysis. Long, scattered repeat sequences and tandem repeat sequences were annotated and identified using RepeatMasker 4.0.5 and Tandem repeats finder 4.07b, respectively. The coding gene was annotated using GeneMarkS 4.17 with default parameters. Genome component prediction involved using the following databases to predict coding genes, repetitive sequences, non-coding RNAs, genomic islands, transposons, prophages, and regularly spaced clustered short palindromic repeats (CRISPR): GO (Gene Ontology), KEGG (Kyoto Encyclopedia of Genes and Genomes), COG (Clusters of Orthologous Groups), NR (Non-Redundant Protein Database), TCDB (Transporter Classification Database), and Swiss-Prot database. A genome-wide BLAST search was performed using these databases (E value less than 1 × 10⁻⁶). -5 (Minimum alignment length percentage greater than 40%). Proteins were searched in the aforementioned database using local BLASTP to identify potential active protein-coding genes from whole-genome sequencing data. Simultaneously, the elution fraction E from XY-144 in Example 4 was...0.25 The sample was sent to Shenzhen MicroNanobio Biotechnology Co., Ltd. for mass spectrometry analysis. The analysis results were compared with the genome sequencing results of XY-144 to obtain the sequence that may encode the active ingredient of XY-144, denoted as gene 500784. The protein encoded by gene 500784 is denoted as protein 500784, and its amino acid sequence is shown in SEQ ID NO.1.
[0083] In addition, the components in Example 4 were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the results are as follows. Figure 8 As shown. Figure 8 The specific substances added to each channel are as follows: 1 is the XY-144 salting-out component; 2 is the XY-144 ultrafiltration component; 3 is the XY-144 sample loading and passing solution; 4 is the XY-144 elution component E. 0.1 ; 5 is the elution fraction E of XY-144 0.25 ; 6 is the elution fraction E of XY-144 0.5 7 is the elution fraction E1 from XY-144. (From...) Figure 8 It can be seen that the XY-144 salting-out fraction, ultrafiltration fraction, and elution fraction E 0.25 All samples showed a distinct band at a size of 39 kDa, while this band was not found in other components. Therefore, it is speculated that the size of the active ingredient XY-144 is approximately 39 kDa. The size of the active ingredient XY-144 differs somewhat from that of the 500784 protein, possibly because the active ingredient XY-144 contains other smaller extraneous proteins.
[0084] A maximum likelihood phylogenetic tree (Bootstrap 1000) was constructed using RaxML software to analyze the protein encoded by the 500784 gene. Specifically, the analysis was performed as follows: Figure 9 As shown. By Figure 9 It can be seen that protein 500784 and protein WP_018298764.1 belong to the same evolutionary branch, but are relatively distantly related. Their amino acid identity is 44% and their similarity is 61%, indicating that protein 500784 is unique in 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 by removing the 19-amino acid signal peptide at the N-terminus and adding a His6 tag at the C-terminus to make it more suitable for recombinant expression. The optimized amino acid sequence of the 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. Sufficient PCR products were amplified by PCR. The PCR products and the pET-28a(+) plasmid vector were digested with NcoI and XhoI restriction enzymes, respectively. The digested PCR products and the pET-28a(+) plasmid vector were ligated using a ligase to obtain the recombinant vector. The ligated solution was transformed into BL21(DE3) competent cells, and positive clones were screened for verification. The recombinant vector was obtained by gel electrophoresis, and the results are shown below. Figure 10 As shown. Figure 10 The specific substances added to each channel are as follows: M is DNA Marker; 1 is the gene after double digestion with ApaI-XhoI (size: 4197bp / 2472bp); 2 is the recombinant vector.
[0088] The recombinant vector was transformed into BL21(DE3) competent cells, plated, and cultured upside down at 37°C overnight. Single clones were selected and cultured at 37°C in 1L of LB medium containing 30 μg / mL kanamycin until OD600 reached 0.6–0.8. Isopropyl thiogalactoside (IPTG) was added to a final concentration of 1 mM, and induction was performed at 37°C for 4 h. The cells were collected by centrifugation, resuspended, and sonicated. After centrifugation, the precipitate was dissolved in lysis buffer (50 mM Tris, 300 mM NaCl, 8 M urea, pH 8) and centrifuged again to obtain the lysed precipitate and supernatant. The supernatant was purified by nickel affinity chromatography. The nickel column affinity chromatography purification process includes: equilibration, sample loading, washing, and elution. The equilibration buffer is a "Tris-NaCl-Urea" buffer (50 mM Tris, 300 mM NaCl, 2 M urea), pH 8; the washing buffer is a "Tris-NaCl-Urea" buffer containing 50 mM imidazole, pH 8; and the elution buffer is a "Tris-NaCl-Urea" buffer containing 500 mM imidazole, pH 8. The purified fractions from the nickel column affinity chromatography include: the eluent collected after sample loading, the wash sample collected after washing, and the elution sample collected after elution. The SDS-PAGE results for each fraction are shown below. Figure 11 As shown. Figure 11 The specific substances added to each channel are as follows: M is protein marker; 1 is precipitate after disruption; 2 is supernatant after disruption; 3 is eluent; 4 is washing sample; 5 is elution sample. Figure 11 It can be seen that the target protein, namely recombinant serine protease, can be obtained after purification by nickel column affinity chromatography.
[0089] Example 10: Validation of biomembrane scavenging activity of recombinant serine protease
[0090] The overnight seed culture of MRSA was diluted 100-fold by volume in LB liquid medium containing 0.5% (w / v) glucose, and 1 mL / well was added to a 24-well plate. After incubation at 37°C for 1 day, the bacterial culture was slowly aspirated with a pipette 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 concentrations of 84 μg / mL, 42 μg / mL, 21 μg / mL, and 10.5 μg / mL, respectively, to obtain protein dilutions of different concentrations. The protein dilutions were slowly added 1 mL / well to a 24-well plate containing MRSA biofilm. After incubation at 37°C for 1 day, the solution was slowly aspirated with a pipette, washed once with physiological saline, dried at 37°C for 30 minutes, fixed at 65°C for 30 minutes, and then cooled to room temperature. Add 1 mL of 0.1% (w / v) crystal violet solution to each well for staining. Wash off the stain after 5 minutes, dry, and observe the biofilm removal effect as follows: Figure 12 As shown. Figure 12 In the above, 1 is a blank control, with no components added; 2 is a protein dilution containing 84 μg / mL recombinant serine protease; 3 is a protein dilution containing 42 μg / mL recombinant serine protease; 4 is a protein dilution containing 100 μg / mL proteinase K as a positive control; 5 is a protein dilution containing 21 μg / mL recombinant serine protease; and 6 is a protein dilution containing 10.5 μg / mL recombinant serine protease. Figure 12 It can be seen that the recombinant serine protease prepared in Example 9 has a scavenging effect on MRSA biofilm, and it also has a scavenging effect at low concentrations.
[0091] Example 11: Thermal stability test of recombinant serine protease
[0092] The recombinant serine protease solution was incubated at 25℃, 40℃, 55℃, and 70℃ for 30 minutes, then cooled to room temperature, and its activity in scavenging MRSA biofilms was verified. The overnight seed culture of MRSA was diluted 100-fold by volume with LB liquid medium containing 0.5% (w / v) glucose, and 1 mL / well was added to each well of a 24-well plate. After incubation at 37℃ for 1 day, the bacterial culture was slowly aspirated using a pipette to obtain 24-well plates containing MRSA biofilms. Recombinant serine protease solutions treated at different temperatures were diluted to a concentration of 42 μg / mL using LB liquid medium containing 0.5% (w / v) glucose. The solutions were then slowly added at 1 mL / well to 24-well plates containing MRSA biofilms. Dimethyl sulfoxide (DMSO) treated at 25°C served as a negative control, and proteinase K treated at 25°C served as a positive control. After incubation at 37°C for 1 day, the solutions were slowly aspirated with a pipette, washed once with physiological saline, dried at 37°C for 30 minutes, fixed at 65°C for 30 minutes, and then cooled to room temperature. 1 mL of 0.1% (w / v) crystal violet solution was added to each well for staining. After 5 minutes, the stain was washed off, and the plates were dried. The pigment was then dissolved in 5 mL of 30% (v / v) acetic acid solution, mixed well, and 100 μL was taken for OD measurement using a microplate reader. 550 The test results are as follows Figure 13 As shown. By Figure 13 It can be seen that the recombinant serine protease prepared in Example 9 has good thermostability, and its activity in clearing MRSA biofilm remains very good 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 examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.
[0094] The amino acid and nucleotide sequences involved in this 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 as described in 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 as described in claim 1.
5. A host cell, characterized in that, The host cell contains the recombinant vector as described in claim 4.
6. A composition, characterized in that, The composition contains the serine protease as described in claim 1.
7. A method for preparing a serine protease as described in claim 1, characterized in that, Including the following steps: A gene encoding the serine protease as described in claim 1 is synthesized, and the gene is inserted into an expression vector to obtain a recombinant vector; The recombinant vector was transferred into host cells for protein expression, and the product of the protein expression was 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. The use of a serine protease as described in claim 1, a nucleic acid as described in claim 2 or 3, a recombinant vector as described in claim 4, a host cell as described in claim 5, a composition as described in claim 6, or a serine protease prepared by any one of the preparation methods described in claims 7 to 9 in the preparation of products that eliminate methicillin-resistant Staphylococcus aureus (MRSA) biofilms or inhibit the formation of MRSA biofilms.