A coding gene of bacterial protease, a recombinant expression vector, a recombinant engineering bacteria, a preparation method and application thereof

Abstract

The application discloses a coding gene of bacterial protease, a recombinant expression vector, a recombinant engineering bacterium and a preparation method and application thereof, and belongs to the technical field of genetic engineering. The application optimizes the structure of wild protease to obtain a recombinant mutant protease Cas800 1215R. In the preparation process, the expression time of the protein is shortened, the water solubility of the protein is improved, the yield of the recombinant protease is about 1.95 times that of the protease before recombination, the recombinant protease still has high activity, and the ability of the recombinant protease to enzymatically decompose a bacterial biofilm is maintained. The decomposition effect of the recombinant Cas800 1215R protease on extracellular polysaccharide is significantly improved, and the inhibition effect of the recombinant Cas800 1215R protease on the growth of a bacterial cell is higher than that of the original protease.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a bacterial protease encoding gene, a recombinant expression vector, a recombinant engineered bacterium, its preparation method, and its applications. Background Technology

[0002] Bacterial wilt is a soil-borne disease commonly affecting solanaceous plants, primarily caused by *Ralstonia solanacearum*, a highly resistant bacterium with a wide transmission route. It mainly invades the plant system through wounds on the roots or stems, affecting water transport and causing the crop to wilt and die. The pathogen first attaches to wounds on the plant surface and begins to grow and reproduce. Simultaneously, adjustments in gene expression cause it to secrete large amounts of extracellular polymeric substance (EPS), mainly composed of polysaccharides, proteins, and extracellular deoxyribonucleic acid (eDNA). EPS forms the three-dimensional framework of the biofilm, allowing individual bacteria to adhere and form flat or mushroom-like microcolonies. The accumulation of numerous colonies thickens the biofilm, enhancing the bacteria's adaptability to the environment. As the biofilm matures, the bacteria gradually detach and spread to fresh areas, initiating a new round of reproduction and expansion. Throughout the process, EPS and the biofilm it constructs are very important propagation materials for bacteria, and the biofilm is also a key structure for the bacterial pathogen to develop drug resistance.

[0003] Currently, the control of bacterial wilt is primarily preventative, employing methods such as chemical pesticides, crop rotation, and biological control to effectively prevent the disease and its retrograde growth. While chemical pesticides are fast-acting, they can negatively impact the rhizosphere soil environment, and long-term use can lead to pesticide resistance in the pathogen. Crop rotation, mainly involving different plant families, is costly and yields low economic returns. Biological control is currently the main control method, being safe, environmentally friendly, and significantly effective in inhibiting bacterial wilt. However, current research on bacterial wilt mainly focuses on its mechanisms, with limited research exploring and enhancing the protease-producing ability of the pathogen, particularly the pathogenic genes of the bacterium *Ralstonia solanacearum*. Summary of the Invention

[0004] To address the aforementioned limitations of existing technologies, the present invention aims to provide a bacterial protease encoding gene, a recombinant expression vector, a recombinant engineered bacterium, its preparation method, and its applications. This experiment screened for a protease that inhibits the growth of *Ralstonia solanacearum*. The protease was extracted, its phylogenetic tree was constructed to determine its position within the genus, and its phylogenetic relationships were analyzed. The antibacterial effect of the protease was verified by enzymatic hydrolysis of extracellular polysaccharides, determination of enzyme activity, and degradation of *Ralstonia solanacearum* biofilm. It was found that this protease can inhibit the growth of the pathogen. This protease not only possesses its own antibacterial effect but can also assist chemical pesticides in enhancing their ability to inhibit *Ralstonia solanacearum*. Because this protein is a membrane protein and is expressed in trace amounts during induction, a high-yield protease with unchanged function was subsequently designed to compensate for this deficiency.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a bacterial protease, the amino acid sequence of which is shown in SEQ ID NO:2.

[0006] In a second aspect, the present invention provides a gene encoding the bacterial protease, wherein the gene is a nucleic acid molecule as shown in (1) or (2) below: (1) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO:3; (2) Nucleic acid molecules other than (1) that encode the amino acid sequence shown in SEQ ID NO:2.

[0007] A third aspect of the present invention provides a recombinant expression vector comprising a nucleotide sequence encoding the bacterial protease.

[0008] In a fourth aspect, the present invention provides a recombinant engineered bacterium, the recombinant engineered bacterium comprising the encoding gene of the bacterial protease or the recombinant expression vector.

[0009] A fifth aspect of the present invention provides a method for preparing the bacterial protease, the method comprising: (1) Construct a recombinant expression vector for expressing the bacterial protease described above; (2) The obtained recombinant expression vector was transferred into the host bacteria, screened and verified, and the recombinant engineered bacteria were obtained. (3) The recombinant engineered bacteria were fermented and induced to express the bacterial protease.

[0010] Furthermore, the host bacterium is BL21, which is a competent cell.

[0011] A sixth aspect of the invention provides the use of the bacterial protease in any one of the following (1)-(3): (1) Enzymatic hydrolysis of Ralstonia solanacearum biofilm; (2) Enzymatic hydrolysis of Ralstonia solanacearum extracellular polysaccharide; (3) Inhibits the growth of Ralstonia solanacearum cells.

[0012] The beneficial effects of this invention are: This invention optimizes the structure of a wild-type protease to obtain a recombinant mutant protease, Cas800 1215R. The preparation process not only shortens the protein expression time but also improves its water solubility. The yield of the recombinant protease is approximately 1.95 times that of the original protease. It retains high activity and maintains its ability to enzymatically hydrolyze Ralstonia solanacearum biofilms. The recombinant Cas800 1215R protease significantly improves the degradation of extracellular polysaccharides and exhibits a higher inhibitory effect on Ralstonia solanacearum cell growth than the original protease. Attached Figure Description

[0013] Figure 1 The structure diagram of wild-type protease Cas800 1215.

[0014] Figure 2 To separate and purify the target protein sample.

[0015] Figure 3 Microscopic images of residual biofilm after enzymatic hydrolysis of Ralstonia solanacearum biofilm were obtained by laser confocal microscopy.

[0016] Figure 4 This is a laser confocal microscope image after co-culturing protease and Ralstonia solanacearum.

[0017] Figure 5 The images show the three-dimensional structures of the protein before and after reconstruction.

[0018] Figure 6 This is a comparison of the overlap of protein structures before and after recombination.

[0019] Figure 7 This is an SDS-PAGE image of the supernatant proteins before and after recombination.

[0020] Figure 8 The image shows the results of ultrasonic disruption of bacterial cells collected before and after recombination.

[0021] Figure 9 Crystal violet staining results of biomembranes treated with protease before and after recombination. Detailed Implementation

[0022] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0023] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0024] The test materials used in the embodiments of the present invention, unless otherwise specified, are all conventional test materials in the art and can be purchased through commercial channels.

[0025] Example 1: Obtaining wild-type protease Extracellular polymers are a major factor in the reproduction and pathogenicity of *Ralstonia solanacearum*. This bacterium colonizes the vascular bundles of plants, blocking and disrupting the transport of water within the plant, leading to dehydration and wilting. In the early stages of colonization, to expand its habitat, *Ralstonia solanacearum* first decomposes existing biofilms, then expands its conidial colonies and continuously forms new biofilms. Inspired by this, we screened and identified a biofilm-degrading *Ralstonia solanacearum* membrane protease from the Cas800 *Ralstonia solanacearum* genome.

[0026] After analyzing and comparing the protein, a phylogenetic tree was constructed. The results showed that the protein clustered with the Ralstonia solanacearum glycoside hydrolase family. The domain prediction by NCBI CD-Search identified it as Glycoside Hydrolase Family 114. This domain is usually associated with the hydrolysis of carbohydrates and may be involved in cell wall degradation or metabolic processes.

[0027] This protein has a TIM barrel fold structure, consisting of 8 β-strands surrounded by 8 α-helices. Figure 1 In classic TIM barrel enzymes, the key amino acid residues (such as nucleophilic groups) necessary for the catalytic reaction are usually located at the C-terminus of the β-sheet chain, and this region constitutes the enzyme's active pocket.

[0028] Using pET24a as the expression vector and BL21 as competent cells, an expression protein was constructed, with hydrophilic peptides and the molecular chaperone tf2 added to improve protein water solubility. The successfully constructed bacterial strain was activated in resistant LB liquid medium and cultured at 37°C and 200 rpm for 5 h. The activated bacterial solution was then inoculated at 1% into resistant LB liquid medium and shaken under the same conditions until OD600 = 0.4. A suitable amount of bacterial solution was used as the uninduced control group, and the remaining bacterial solution was inoculated with IPTG to a final concentration of 0.05 mM and cultured at 16°C and 120 rpm for 39 h. The bacterial solution was then centrifuged at 10,000 rpm and 4°C for 15 min. The supernatant was discarded, and the bacterial cells were resuspended in an appropriate amount of lysis buffer and sonicated at low temperature, followed by centrifugation at 10,000 rpm and 4°C for 15 min. The supernatant and precipitate samples were added to SDS loading buffer, boiled at 95-100°C for 10 min, and then centrifuged at 10,000 rpm for 2 min. 10 μL of the marker, control, and induced samples were loaded onto the culture medium. Protein electrophoresis was performed at 160 V for 50 min. The gel was then stained and destained until the background of the eluted gel was free of blue. The protein was purified using a nickel column, and the eluent containing the target protein was collected. After dialysis, the purified protein powder was collected by freeze-drying. The purified target protein was observed as a single band on a protein gel (RSC2241). Figure 2 ).

[0029] The obtained protein was named wild-type protease Cas800 1215. The amino acid sequence of wild-type protease Cas800 1215 is as follows (SEQ ID NO:1): MYQLQDLLTDAQINALDAQPYDMVVVEAGNTVKNQETFNTAGMVTRLHTKADGSPRKVIAYIDIGQAESFRTYWIDNQWKTPTATKPGVPDFILTADPDGWADDYPVAYWDQRWQDLWLGTNGAVAQLAREGFDGVYLDWVEGYTNAKV VAAAKKAGVNPAKAMVDFIAKIRAAGRAINPQFAVIQQNAPELIDAVGAAKLLPNLDAISQEDTWFGGDAGASWGDASGTDQATESADTQWTIEQLQKHCAGGLPVFTIDYALTSPNPRTVYTTSRSLGFRPLVSRVSLSKPTTTPPWNY Example 2: Performance determination of wild-type protease 1. Protease activity assay The reaction was performed in 1.5 mL polypropylene tubes, each containing 50 mM sodium phosphate buffer (pH 5.9), 100 mM sodium chloride, 5 mM 4-nitrophenyl-N-acetyl-β-D-glucosaminide, and 3.7 µg purified protein / mL, and incubated in a 37°C water bath. The reaction was terminated by adding 5 µl of 10 N sodium hydroxide. The absorbance change due to the release of p-nitrophenolate in each tube was measured using a UV-Vis spectrophotometer (wavelength set to 405 nm). One unit of enzyme activity was defined as an increase of 0.001 absorbance per minute, and the enzyme activity was calculated to be 67.5 U according to the formula.

[0030] 2. Enzymatic hydrolysis of Ralstonia solanacearum biofilm After overnight culture of Ralstonia solanacearum containing a green fluorescent probe, OD was modulated. 600 The concentration was 0.3, and the medium was added to 96-well and 24-well plates and incubated at 28°C for 72 h. After removal, the medium was aspirated, and obvious wrinkled biofilm of Ralstonia solanacearum was found at the bottom of the wells. After washing once with PBS buffer, PBS buffer, 0.2 mg / ml protease solution, 0.5 mg / ml protease solution, and protease-free solution were added respectively. The reaction was incubated at 37°C for 60 min. After removal, the fluorescence intensity was measured directly with a fluorescence microplate reader at 475 nm excitation wavelength and 509 nm emission wavelength, and detected by laser confocal microscopy. At the same time, after sampling, the supernatant liquid was aspirated, and the biofilm material at the bottom was retained. After washing once with PBS buffer, PBS buffer, 0.2 mg / ml protease solution, 0.5 mg / ml protease solution, and protease-free solution were added respectively. The reaction was incubated at 37°C for 60 min. After removal, 70% methanol was added for fixation for 15 min, followed by 1% crystal violet staining solution for 30 min. The residue was aspirated, washed three times with pure water, and the material in the wells was dissolved in anhydrous ethanol. The microplate was measured at 570 nm with a microplate reader.

[0031] Enzymatic hydrolysis of biofilms showed that higher protease concentrations resulted in lower overall fluorescence intensity after biofilm treatment. The fluorescence intensity measured by the microplate reader was comparable between enzyme-free and PBS-treated solutions (Table 1), indicating that the protease has a biofilm-degrading effect, while protein inactivation treatment did not. In a laser confocal microscope, cells treated with 0.5 mg / mL protease were observed at 40X magnification, revealing a significant decrease in fluorescence intensity, indicating that high-concentration protease effectively degrades cell membranes. Figure 3 ).

[0032] Table 1. Determination of residual fluorescence intensity before and after enzymatic hydrolysis of biomembranes 3. Enzymatic hydrolysis of Ralstonia solanacearum extracellular polysaccharides Extracellular polysaccharides of Ralstonia solanacearum Cas800 were collected. Dense streaks of Ralstonia solanacearum colonies were placed in NA medium and incubated at 30°C for 48 h. The colonies were rinsed with an appropriate amount of sterile water in a petri dish and transferred to a 50 ml sterile centrifuge tube. The tubes were centrifuged at 8000 rpm for 15 min. The bacterial cells were discarded and the supernatant was collected. Three volumes of anhydrous ethanol were added and the mixture was placed at 4°C overnight to precipitate the extracellular polysaccharides. The precipitate was then evaporated to remove the anhydrous ethanol. An appropriate amount of sterile water was added and the mixture was dialyzed with running water for 48 h. The precipitate was then freeze-dried into powder. The concentration of extracted extracellular polysaccharides was determined using the phenol-sulfuric acid method. Accurately pipette 0 mL, 0.4 mL, 0.8 mL, 1.2 mL, 1.6 mL, and 2.0 mL of 0.1 mg / mL glucose standard solution into EP tubes, and bring each tube to a final volume of 2.0 mL with distilled water. Add 1.0 mL of 5% phenol solution, mix well, and then quickly add 5 mL of 98% concentrated sulfuric acid. Mix well, incubate at 40℃ for 15 min, and then rapidly cool. Measure the absorbance (A) at 490 nm to obtain the glucose standard curve equation. Simultaneously, pipette 2 mL of the bacterial strain's extracellular polysaccharide test sample solution into a colorimetric tube and measure the absorbance. Calculate the mass concentration of extracellular polysaccharides based on the standard curve equation. Each group was measured three times, and the average value was taken. Alternatively, 0.1 mg / mL of extracellular polysaccharides was mixed with protease to a final protease concentration of 1.0 mg / mL. The reaction system was 1 mL, the reaction temperature was 37℃, and the reaction time was 60 min. The change in absorbance of the solution before and after the enzyme reaction was measured.

[0033] The concentration of the extracted polysaccharide was determined to be 0.2 mg / ml using the phenol-sulfuric acid method. It was then diluted to 0.1 and 0.01 mg / ml and measured at the full UV wavelength. The results showed a significant peak at 256 nm for the 0.1 mg / ml polysaccharide concentration; therefore, the wavelength for measuring the variation of this extracellular polysaccharide was 256 nm. Following the grouping in Table 2, different concentrations of enzyme were added to 0.2 mg / ml of extracellular polysaccharide and reacted for 60 min. The absorbance was measured at 256 nm using a UV spectrophotometer, and the results are shown in Table 2.

[0034] The absorbance results before and after the enzyme reaction showed that the final concentrations of 0.5 mg / ml and 1.0 mg / ml were higher than the initial concentrations after 60 min of enzyme reaction (Table 2). This indicates that the protease breaks down extracellular polysaccharides to obtain small substances or fragmented monosaccharides, which increases the refractive index at a specific wavelength.

[0035] Table 2. Absorbance determination of polysaccharides hydrolyzed by protease 4. Inhibits the growth of Ralstonia solanacearum cells After co-culturing protease and Ralstonia solanacearum containing GFP probe overnight in a 28°C incubator, the fluorescence intensity was measured. The results showed that the fluorescence intensity of the bacterial suspension with 1.0 mg / ml protease added was significantly lower than that without protease (Table 3), indicating that the growth and reproduction of Ralstonia solanacearum were indeed inhibited by the protease's degradation of the extracellular polysaccharide. Simultaneously, the protease and Ralstonia solanacearum were co-cultured and observed under a laser confocal microscope. Figure 4 The fluorescence intensity in the field of view was significantly reduced after treatment, indicating a decrease in fluorescent cells and that the protease inhibited cell growth. Simultaneously, samples from the laser confocal well were stained to observe the growth of the biofilm after cell inhibition. The results showed that the biofilm area was significantly reduced after protease treatment.

[0036] Table 3. Changes in residual fluorescence intensity after co-culturing of pathogens and proteases. Example 3: Optimization of wild-type protease structure During the preparation of the wild-type protease Cas800 1215, issues such as long expression time and poor water solubility were encountered. Therefore, further structural optimization was conducted. The ProteinMPNN algorithm was used for backfolding design to fix the functional domain backbone of the wild-type protein and synthesize the amino acid sequence de novo. The optimal parameter combination was calculated to optimize the hydrophobic structure on the surface of the wild-type protein into a hydrophilic group, enhancing its solubility. Finally, the recombinant mutant protease Cas800 1215R was obtained, with the following amino acid sequence (SEQ ID NO:2): MIVLKNLDTPEQIAALAATPDDLIIVTAMDLLKGKEDFDTAGMVATLKTRPDGTPRKVIAYLNIGYFDENWISWDPEKHKPPTATAPGEPPYILAADPYGRKGLYPVLFWDPRWQDIFLGPDGIVKRLAEKGFDGVLLDAVDIYRHPVV RAAAAAAGVDPAQAVVDFIGKVREAGRAVNPDFMVIAENAPELIDAVGADRLVPQLDAVIVRPLRYTGKEGAGWDDPAGYDIPVPADKTAATVAALAKWKAAGLPVYVLDFARTEPKPSIVYAEARALGFRPFVSHISLDRPTDVPAPDV The DNA nucleotide sequence corresponding to the Ralstonia solanacearum membrane protease Cas800 1215R is as follows (SEQ ID NO:3): ATGATCGTGCTGAAAAATCTGGATACCCCGGAACAGATTGCCGCACTGGCCGCAACCCCGGATGATCTGATTATTGTGACCGCCATGGATCTGCTGAAAGGTAAAGAAGATTTTGATACCGCAGGTATGGTGGCCACCCTGAAAACCCGCCCGGATGGCACCCCGCGTAAAGTGATTGCCTATCTGAATATTGGCTATTTTGATGAAAACTGGATCAGCTGGGATCCGGAAAAACATAAACCGCCGACCGCAACCGCCCCGGGTGAACCTCCTTATATTCTGGCAGCAGATCCGTATGGTCGTAAAGGCCTGTATCCGGTTCTGTTTTGGGATCCGCGTTGGCAGGATATTTTTCTGGGTCCGGATGGCATTGTTAAACGTCTGGCAGAAAAAGGCTTTGATGGCGTTCTGCTGGATGCCGTGGATATTTATCGCCATCCGGTTGTGCGCGCCGCAGCCGCAGCAGCAGGTGTTGATCCGGCACAGGCCGTGGTTGATTTTATTGGCAAAGTGCGTGAAGCCGGCCGCGCCGTGAATCCGGATTTTATGGTTATTGCCGAAAATGCCCCGGAACTGATTGATGCCGTGGGCGCCGATCGCCTGGTGCCTCAACTGGATGCCGTTATTGTTCGCCCGCTGCGTTATACCGGCAAAGAAGGTGCCGGCTGGGATGATCCGGCAGGCTATGATATTCCGGTTCCGGCAGATAAAACCGCAGCCACCGTTGCCGCACTGGCAAAATGGAAAGCCGCAGGCCTGCCGGTTTATGTGCTGGATTTTGCCCGCACCGAACCGAAACCGAGCATTGTTTATGCCGAAGCACGCGCCCTGGGCTTTCGCCCTTTTGTGAGCCATATTAGTCTGGATCGTCCGACCGATGTTCCGGCCCCGGATGTG The recombinant proteins before and after recombination are compared, wherein the recombinant protein retains the functional domain structural backbone of Cas800 1215, and the three-dimensional structure is shown in Figure 5The structure was predicted by comparing the sequences before and after recombination using PyMOL. The results showed an RMSD value of 0.333 Å. Generally, RMSD < 1.0 Å indicates that the two structures are highly similar, suggesting that the folded three-dimensional structure is almost unchanged from the wild type. Figure 6 (The gray structure represents the wild-type protein; the blue structure represents the recombinant protein). Therefore, they may have similar functions.

[0037] Example 4: Functional verification of protease before and after optimization 1. Protein expression Using pET24a as the expression vector, the pre- and post-mutation protein fragments (wild-type protease Cas800 1215 and recombinant mutant protease Cas800 1215R) were ligated into the pET24a vector, and BL21 was used as competent cells to construct the expressed protein. The successfully constructed bacterial strain was activated in Kan-resistant LB liquid medium and cultured at 37°C and 200 rpm for 5 h. The activated bacterial solution was then inoculated at 1% into Kan-resistant LB liquid medium and shaken to OD under the same conditions. 600 =0.4. A suitable amount of bacterial culture was taken as the uninduced control group. The remaining bacterial culture was added to IPTG at a final concentration of 0.05 mM and incubated at 16℃, 120 rpm for 16-39 h. At different time points, the bacterial culture was centrifuged at 10000 rpm, 4℃ for 15 min. The supernatant was discarded, and the bacterial cells were resuspended in an appropriate amount of lysis buffer and sonicated at low temperature. The mixture was then centrifuged at 10000 rpm, 4℃ for 15 min. The supernatant and precipitate samples were added to SDS loading buffer, boiled at 95-100℃ for 10 min, and then centrifuged at 10000 rpm for 2 min. 10 μL of the marker, control, and post-induction samples were loaded. Protein electrophoresis was performed at 160 V for 50 min. The gel was stained and destained until the background of the eluted gel was free of blue. The protein was purified using a nickel column, and the eluent containing the target protein was collected. After dialysis, the purified protein powder was collected by freeze-drying under vacuum.

[0038] SDS protein electrophoresis image is shown below. Figure 7 ,according to Figure 7 As a result, under the same expression conditions, the band width of the wild-type protein Cas800 1215 was smaller than that of the recombinant protein Cas800 1215R, indicating that the expression level of the wild-type protein was lower than that of the recombinant protein. Screening of the induction conditions for the recombinant protein revealed that a stable expression of the protein could be obtained by shortening the induction time from 39 h to 16 h. Furthermore, during ultrasonic disruption, the recombinant bacterial cells showed clearer expression after disruption. Figure 8The study revealed a reduction in the number of inclusion bodies produced. For every 1 L of culture medium obtained in the experiment, 591.8 mg of purified powder was obtained from wild-type protein Cas800 1215 and 1152 mg of purified powder was obtained from recombinant protein Cas800 1215R, meaning that the yield of the recombinant protease was approximately 1.95 times that of the unrecombinant protease.

[0039] 2. Protease activity assay Following the protease activity assay method in Example 2, 4-nitrophenyl-N-acetyl-β-D-glucosaminide was used as a substrate to verify whether there was a change in protease activity before and after recombinant synthesis. The activity values ​​of the wild-type protease Cas800 1215 collected in this induction were 81.08 U and the activity value of the recombinant protease Cas800 1215R were 76.58 U. The results showed that the protease still had high activity after recombinant synthesis.

[0040] 3. Enzymatic hydrolysis of Ralstonia solanacearum biofilm experiment Based on the enzymatic hydrolysis method for Ralstonia solanacearum biofilm in Example 2, the function of recombinant protease on Ralstonia solanacearum was further verified: Ralstonia solanacearum containing green fluorescent probe was picked and cultured overnight, and OD was modulated. 600 The medium concentration was 0.3, and the medium was added to 96-well and 24-well plates and incubated at 28°C for 72 h. After removal, the medium was aspirated, and obvious wrinkled biofilm of Ralstonia solanacearum was found at the bottom of the wells. The control group was washed once with PBS buffer and then PBS buffer was added. The Cas800 1215 group was washed once with PBS buffer and then 0.5 mg / ml wild-type protease Cas800 1215 was added. The Cas800 1215R group was washed once with PBS buffer and then 0.5 mg / ml recombinant mutant protease Cas800 1215R was added. The reaction was incubated at 37°C for 60 min. After removal, the fluorescence intensity was measured directly using a fluorescence microplate reader at an excitation wavelength of 475 nm and an emission wavelength of 509 nm. The results are shown in Table 5. Simultaneously, after sampling, the supernatant liquid was aspirated, while the biofilm material at the bottom was retained. The control group was washed once with PBS buffer followed by the addition of PBS buffer. The Cas800 1215 group was washed once with PBS buffer followed by the addition of 0.5 mg / ml wild-type protease Cas800 1215. The Cas800 1215R group was washed once with PBS buffer followed by the addition of 0.5 mg / ml recombinant mutant protease Cas800 1215R. The reaction was incubated at 37℃ for 60 min. After incubation, 70% methanol was added for fixation for 15 min, followed by 1% crystal violet staining for 30 min. The residue was then aspirated, washed three times with pure water, and dried. The morphology of the residual biofilm at the bottom was observed. The results are shown in [Figure number missing]. Figure 8 .

[0041] Table 4. Fluorescence intensity determination of Ralstonia solanacearum biofilm after enzymatic hydrolysis before and after recombinant treatment. according to Figure 9 As a result, no biofilm residue was observed in the Cas800 1215 and Cas800 1215R groups, while significant biofilm residue was observed in the control group. According to Table 4, the fluorescence intensity of the Cas800 1215 and Cas800 1215R groups was lower than that of the control group. The recombinant protease Cas800 1215R still possessed enzymatic activity and exhibited a decomposition effect on Ralstonia solanacearum biofilm.

[0042] 4. Recombinant protease hydrolysis of extracellular polysaccharides experiment The concentration of the extracted polysaccharide was determined to be 0.2 mg / ml using the phenol-sulfuric acid method. It was then diluted to 0.1 and 0.01 mg / ml and measured at the full UV wavelength. The results showed a significant peak at 256 nm for the 0.1 mg / ml polysaccharide concentration. Following the groupings in Tables 5-6, different concentrations of enzyme were added to 0.1 mg / ml of extracellular polysaccharide and reacted for 60 min. The absorbance was then measured at 256 nm using a UV spectrophotometer. The results are shown in Tables 5-6.

[0043] The absorbance results before and after the enzyme reaction showed that the final concentrations of 0.5 mg / ml and 1.0 mg / ml after 60 min of enzyme reaction were higher than the initial concentrations, indicating that the protease decomposes extracellular polysaccharides into smaller substances or fragmented monosaccharides, resulting in an increase in refractive index at a specific wavelength. Furthermore, the change in absorbance indicates that the recombinant Cas800 1215R protease significantly improves the decomposition effect on extracellular polysaccharides.

[0044] Table 5. Absorbance determination of polysaccharides hydrolyzed by Cas800 1215 protease Table 6. Absorbance determination of polysaccharides hydrolyzed by Cas800 1215R protease 5. Recombinant proteases inhibit the growth of Ralstonia solanacearum cells. The protease was mixed with a bacterial suspension of Ralstonia solanacearum containing the GFP probe and cultured overnight at 28°C. The changes in fluorescence intensity were then measured. The groups were as follows: experimental group, with Cas800 1215R enzyme added; control group, with Cas800 1215 enzyme added or NB medium added; the bacterial suspension used in each group was the same. The specific dosage and results are shown in Table 7.

[0045] According to the results in Table 7, the fluorescence intensity of the bacterial suspension cultured with 1.0 mg / ml protease was significantly lower than that without protease, indicating that the growth and reproduction of Ralstonia solanacearum were inhibited by the decomposition of extracellular polysaccharides by protease. Furthermore, the recombinant Cas800 1215R protease showed a higher inhibitory effect on the growth of Ralstonia solanacearum cells than the original protease.

[0046] Table 7. Changes in residual fluorescence intensity after co-culturing of pathogens and proteases. In summary, optimizing the structure of the wild-type protease yielded the recombinant mutant protease Cas800 1215R. The preparation process not only shortened the protein expression time but also improved its water solubility. The yield of the recombinant protease was approximately 1.95 times that of the original protease. It retained high activity, maintaining its ability to enzymatically hydrolyze Ralstonia solanacearum biofilms. The recombinant Cas800 1215R protease showed significantly improved decomposition of extracellular polysaccharides and a higher inhibitory effect on Ralstonia solanacearum cell growth compared to the original protease.

[0047] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

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

2.

2. The gene encoding the bacterial protease according to claim 1, characterized in that, The coding gene is a nucleic acid molecule as shown in (1) or (2) below: (1) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO:3; (2) Nucleic acid molecules other than (1) that encode the amino acid sequence shown in SEQ ID NO:

2.

3. A recombinant expression vector, characterized in that, The recombinant expression vector contains a nucleotide sequence encoding the bacterial protease of claim 1.

4. A recombinant engineered bacterium, characterized in that, The recombinant engineered bacteria contains the encoding gene of the bacterial protease as described in claim 1 or the recombinant expression vector as described in claim 3.

5. The method for preparing the bacterial protease according to claim 1, characterized in that, The preparation method includes: (1) Construct a recombinant expression vector expressing the bacterial protease of claim 1; (2) The obtained recombinant expression vector was transferred into the host bacteria, screened and verified, and the recombinant engineered bacteria were obtained. (3) The recombinant engineered bacteria were fermented and induced to express the bacterial protease.

6. The preparation method according to claim 5, characterized in that, The host bacteria is BL21 cells.

7. The use of the bacterial protease of claim 1 in any one of the following (1)-(3): (1) Enzymatic hydrolysis of Ralstonia solanacearum biofilm; (2) Enzymatic hydrolysis of Ralstonia solanacearum extracellular polysaccharide; (3) Inhibits the growth of Ralstonia solanacearum cells.

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