AHL-lactonase high-thermal-stability mutant and application thereof in biological control of fruit tree fire blight

Abstract

The invention discloses an AHL-lactonase high thermal stability mutant and application thereof in biological control of fruit tree fire blight, the mutation site of the mutant is obtained by mutating the amino acid sequence of AHL-lactonase as shown in SEQ ID NO.3 from the 176th amino acid sequence to the 181th amino acid sequence from the amino terminal to the carboxyl terminal, the mutated amino acid sequence is as shown in SEQ ID NO.1, the mutant is marked as a mutant A19-9, and the mutant A19-9 has a nucleotide sequence shown in SEQ ID NO.1. The amino acid sequence from the amino terminal to the carboxyl terminal is mutated, or the amino acid sequence from the 176th site to the 184th site from the amino terminal to the carboxyl terminal is mutated, the mutated amino acid sequence is shown as SEQ ID NO.2 and is marked as the mutant C13-1, and the thermal stability of the two mutants is greatly improved compared with that of the wild type. The two mutants are applied to preparation of a biological pathogenic bacterium inhibitor, the effect of inhibiting apple fire blight caused by Erwinia amylovora infection is better than that of a wild type, and a potential biological control agent is provided for fruit tree fire blight caused by Erwinia amylovora infection.

Description

Technical Field

[0001] The invention belongs to the technical field of protein engineering, and particularly relates to an AHL-lactonase high-thermostability mutant and application thereof in biological control of fruit tree fire blight. Background Art

[0002] Animal and plant diseases caused by bacterial infections are posing a serious challenge to global health and food security. Although current treatment options that rely on conventional antibiotics have achieved remarkable results in controlling such diseases, the increasing problem of drug resistance has become an issue that cannot be ignored. Quorum sensing (QS), a key mechanism for regulating bacterial pathogenicity, has been widely studied and proven to play an important role in regulating the release of virulence factors, the development of antibiotic resistance, group movement, and biofilm synthesis. In response to this mechanism, the quorum quenching (QQ) strategy that interferes with quorum sensing signal molecules has emerged and has been proven to be an effective method for preventing and treating bacterial infections.

[0003] In particular, through N -Acyl homoserine lactonase degrades N -Acylhomoserine lactone ( N -acylhomoserine lactones (AHLs) signal molecules can effectively block the quorum sensing process of pathogenic bacteria, thereby achieving disease prevention and control. AHL-lactonases have great potential in preventing and controlling bacterial animal and plant diseases with their high efficiency and broad-spectrum AHLs degradation ability. Their unique mechanism of action - that is, only degrading extracellular signal molecules instead of directly interfering with bacterial growth and metabolism - greatly reduces the risk of inducing bacterial resistance. Therefore, AHL-lactonases are regarded as potential alternatives to traditional antibiotics and have broad application prospects.

[0004] Although new AHL-lactonases have been discovered in the past two decades, including those from thermophilic bacteria Parageobacillus caldoxylosilyticus GcL shows high activity against a variety of AHLs. Unfortunately, however, these AHL-lactonases that perform well under laboratory conditions are often difficult to fully realize their potential when actually applied to the biological control of animal and plant pathogens due to insufficient thermal stability. In order to overcome this bottleneck, modern biotechnology has become the key to improving the practical application efficiency of AHL-lactonases. Deep protein engineering of AHL-lactonases through computer-aided design, gene mutation, high-throughput screening and other technical means can effectively improve their thermal stability, promote the practical application of AHL-lactonases in the field of animal and plant disease prevention and control, and provide new and more effective solutions to health and food security problems caused by bacterial infections. Summary of the Invention

[0005] The present invention is dedicated to solving the problem of insufficient thermal stability of current AHL-lactonases, and proposes a thermostable mutant of AHL-lactonase and its application in the biological control of fire blight of fruit trees. These mutants are obtained by modifying based on computer-aided de novo design of protein motifs and site-directed mutagenesis techniques, aiming to improve N -the thermal stability of acyl-homoserine lactonase. Importantly, while improving the thermal stability, the activity of the enzyme molecule is ensured not to be damaged.

[0006] The specific technical solutions are as follows: The present invention provides a thermostable mutant of AHL-lactonase, and the AHL-lactonase is any one of the following proteins: (a1) A protein with the amino acid sequence shown in SEQ ID NO.3; (a2) A protein whose sequence contains the amino acid sequence in (a1); (a3) A fusion protein obtained by connecting a tag to the amino terminus and / or carboxyl terminus of any one of the proteins in (a1) and (a2).

[0007] The mutation site of the mutant is that the amino acid sequence LysArgHisGluAspAsn from the 176th to the 181st position from the amino terminus to the carboxyl terminus of the amino acid sequence shown in SEQ ID NO.3 is mutated to ThrGluHisAspAsnAsp, and the mutated amino acid sequence is shown in SEQ ID NO.1, denoted as mutant A19-9, or the amino acid sequence LysArgHisGluAspAsnIleLeuLeu from the 176th to the 184th position from the amino terminus to the carboxyl terminus is mutated to ArgAspArgHisGlyGlySerPheGluAsnIleProGly, and the mutated amino acid sequence is shown in SEQ ID NO.2, denoted as mutant C13-1.

[0008] It also provides a deoxyribonucleic acid molecule encoding the thermostable mutant of AHL-lactonase. The DNA sequence of mutant A19-9 is shown in SEQ ID NO.4, the DNA sequence of mutant C13-1 is shown in SEQ ID NO.5, a recombinant vector containing the above deoxyribonucleic acid sequence, and a recombinant bacterium containing the recombinant vector.

[0009] It also provides an application of a thermostable mutant of AHL-lactonase in the preparation of a biological pathogenic bacteria inhibitor, and the biological pathogenic bacteria is the pathogenic bacteria of fire blight of fruit trees Erwinia amylovora .

[0010] The beneficial effects of the present invention are as follows: Mutants A19-9 and C13-1 can effectively inhibit E. amylovora the apple fire blight caused by E. amylovora infection, and provide potential biological control agents for Description of the Drawings

[0011] Figure 1 is the purification result of the GcL mutant protein; Figure 2 is the non-denaturing polyacrylamide gel electrophoresis pattern of the GcL mutant; Figure 3 is the activity detection result of the GcL mutant; Figure 4 is the melting curve of the GcL wild type and mutants A19-9 and C13-1; Figure 5 is the thermal inactivation curve of GcL and mutant A19-9 at 70 °C fitted by the Boltzmann function; Figure 6 is the thermal inactivation curve of mutant C13-1 at 70 and 90 °C; Figure 7 is the control effect of the GcL wild type and mutants A19-9 and C13-1 on E. amylovora the apple fire blight caused by Detailed Embodiments

[0012] In order to clearly elaborate the content of the present invention, the present invention will be explained in detail with the help of the accompanying drawings of the specification and specific embodiments. However, before the in-depth introduction, it should be clearly pointed out that the scope of the present invention is not limited to these specifically described embodiments. At the same time, the terms used in this article are only intended to accurately describe specific embodiments, rather than constituting any restrictive interpretation or definition.

[0013] Unless otherwise clearly defined, the terms used in this article follow the generally accepted technical meanings in the field to which the present invention belongs, and are consistent with the meanings generally understood by those of ordinary skill in the art. Although other methods and materials similar or equivalent to those described in this article can be used when implementing or testing the present invention, the preferred methods and materials are specifically described in this article.

[0014] Ⅰ. Terms In the present invention, the term "HPLC" specifically refers to high performance liquid chromatography; "T m " is clearly defined as the melting temperature.

[0015] Ⅱ. Embodiment Modes As described above, in view of the current application of AHL-lactonase in the prevention and treatment of plant and animal bacterial infectious diseases being limited due to its insufficient thermal stability, to solve this key problem, the researchers of the present invention adopted a computer-aided de novo protein motif design strategy to improve AHL-lactonase.

[0016] The inventor found through structural analysis that a special three-histidine interaction structure can be formed at the subunit interaction interface in the protein structure of the heat-stable mutant M41 of AHL-lactonase AhlX. Experiments of heat treatment and non-denaturing polyacrylamide gel electrophoresis confirmed that this three-histidine interaction structure plays an important role in maintaining the thermal stability of its hexameric structure. Using the computer-aided de novo protein motif design tool RFdiffusion, this special three-histidine interaction structure was transplanted into the AHL-lactonase GcL Parageobacillus caldoxylosilyticus derived from thermophilic bacteria Chembiochem : a European journal of chemical biology , 20 (14), 1848–1855.), and a random coil structure adapted to this three-histidine interaction structure was generated de novo to construct a high-thermal-stability AHL-lactonase mutant. The obtained mutant has high hydrolytic activity against N 3-oxo-octanoyl-L-homoserine lactone and excellent thermal stability at the same time.

[0017] The wild-type AHL-lactonase to be modified is any one of the following (a1)-(a3): (a1) a protein with the amino acid sequence of SEQ ID NO.3 (denoted as AHL-lactonase GcL); (a2) a protein whose sequence contains the amino acid sequence defined in (a1); (a3) a fusion protein obtained by connecting a tag to the amino terminus and / or carboxy terminus of the protein defined in either (a1) or (a2); The mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO.3 from the amino terminus to the carboxy terminus through any one of the following cases: A mutant with the amino acid sequence LysArgHisGluAspAsn at positions 176 to 181 mutated to ThrGluHisAspAsnAsp, denoted as GcL mutant A19-9, and its amino acid sequence is as shown in SEQ ID NO.1; A mutant with the amino acid sequence LysArgHisGluAspAsnIleLeuLeu at positions 176 to 184 mutated to ArgAspArgHisGlyGlySerPheGluAsnIleProGly, denoted as GcL mutant C13-1, and its amino acid sequence is as shown in SEQ ID NO.2.

[0018] The DNA molecule described above is the following DNA molecule: (b1) A DNA molecule in the coding region encoding the protein described in the first aspect of the present invention; (b2) A coding region including a DNA molecule encoding the protein described in the first aspect of the present invention; (b3) A DNA molecule having 75% or more similarity to the deoxyribonucleotide sequence described in (b1) or (b2) and encoding the protein described in the first aspect of the present invention; (b4) A DNA molecule that hybridizes with the deoxyribonucleotide sequence described in (b1) or (b2) under stringent conditions and encodes the protein described in the first aspect of the present invention.

[0019] Preferably, the DNA sequence encoding the amino acid sequence of wild-type AHL-lactonase specifically refers to the DNA sequence shown in SEQ ID NO.6 encoding the wild-type AHL-lactonase amino acid sequence shown in SEQ ID NO.3.

[0020] In the present invention, the DNA sequence shown in SEQ ID NO.6 encoding the wild-type AHL-lactonase with the amino acid sequence shown in SEQ ID NO.3 is hereby defined as the DNA molecule encoding AHL-lactonase GcL; the DNA sequence shown in SEQ ID NO.4 encoding the wild-type AHL-lactonase with the amino acid sequence shown in SEQ ID NO.1 is hereby defined as the DNA molecule encoding GcL mutant A19-9; the DNA sequence shown in SEQ ID NO.5 encoding the wild-type AHL-lactonase with the amino acid sequence shown in SEQ ID NO.2 is hereby defined as the DNA molecule encoding GcL mutant C13-1; The present invention provides an expression cassette, a recombinant vector or a recombinant microorganism, all of which contain the aforementioned DNA molecule. It can also be understood that the present invention specifically provides an expression cassette, a recombinant vector or a recombinant microorganism in the third aspect, and the DNA molecule corresponding to the protein described in the first aspect of the present invention is integrated into these constructs.

[0021] The present invention discloses the scope of application, specifically relating to the use of the following mutants: the mutants described in the present invention, the mutants encoded by the DNA molecule described in the present invention, or the mutants prepared by the expression cassette, recombinant vector or recombinant microorganism described in the present invention, in the hydrolysis reaction of compounds containing ester bonds and the biological control of bacterial infectious plant diseases.

[0022] The present invention provides the use of the following mutants, which include: the mutants described in the present invention, the mutants encoded by the DNA molecule described in the present invention, and the mutants prepared by the expression cassette, recombinant vector or recombinant microorganism described in the present invention. These mutants are applied to the hydrolysis process of acyl-homoserine lactones and other ester compounds.

[0023] Ⅲ. Specific Examples Unless otherwise specified, all experimental methods mentioned below follow the conventional operating specifications of the laboratory. Similarly, the experimental materials described below can be obtained through commercial channels without special indication. To further illustrate the present invention, the following will be described in detail with specific examples, but please note that the protection scope of the present invention is not limited to the description of these specific examples.

[0024] In some specific examples of the present invention, Escherichia coli BL21(DE3) was selected as the host cell for expressing wild-type AHL-lactonase GcL and its mutants. The medium formulations used in the following examples are listed in detail as follows: LB liquid medium: Tryptone 10 g·L -1 、Yeast extract 5 g·L -1 、NaCl 10 g·L -1 ; LB solid medium: Agar powder 18 g·L -1 、Tryptone 10 g·L -1 、Yeast extract 5 g·L -1 、NaCl 10g·L -1 ; TB solid medium: KH 2 PO 4 2.31 g·L -1 、K 2 HPO 412.54 g·L -1 、 glycerol 4 g·L -1 、 tryptone 12 g·L -1 、 yeast extract 24 g·L -1 。

[0025] Example 1: Construction of a Strain Expressing AHL-Lactonase Mutant The AHL-lactonase used in this example is GcL, and its encoding gene is derived from Parageobacillus caldoxy losilyticus 。 The amino acid sequence of GcL is shown in SEQ ID NO.3, and one of the DNA sequences of its encoding gene is shown in SEQ ID NO.6.

[0026] The random coil structure of GcL was replaced by one-step site-directed mutagenesis. The specific process is as follows: Using the expression plasmid pET-28a- gcL of wild-type GcL as a template, specific mutagenic primers were designed for the preset mutation sites. Specifically, the mutation sites of the mutant were to mutate the amino acid sequence LysArgHisGluAspAsn from the 176th to the 181st amino acid sequence of the AHL-lactonase shown in SEQ ID NO.3 from the amino terminus to the carboxyl terminus to ThrGluHisAspAsnAsp. The mutated amino acid sequence is shown in SEQ ID NO.1, denoted as mutant A19-9, or to mutate the amino acid sequence LysArgHisGluAspAsnIleLeuLeu from the 176th to the 184th amino acid sequence from the amino terminus to the carboxyl terminus to ArgAspArgHisGlyGlySerPheGluAsnIleProGly. The mutated amino acid sequence is shown in SEQ ID NO.2, denoted as mutant C13-1. Subsequently, the mutation was introduced into the expression vector by PCR amplification technology. The amplified product was treated with Dpn I enzyme to remove the original template plasmid. The treated product was transformed into E. coli BL21(DE3) host cells, thereby successfully constructing the corresponding GcL mutant expression strain.

[0027] Example 2: Preparation of AHL-Lactonase GcL Mutant (1) Heterologous Expression and Purification of Wild-Type GcL and Its Mutants The GcL mutant expression strain constructed in Example 1 and the GcL wild-type expression strain were respectively inoculated into 5 mL of TB liquid medium containing 50 μg / mL kanamycin. After all the strains were cultured with shaking at 37 °C and 220 rpm for 4 hours, subsequently, IPTG with a final concentration of 0.2 mM was added in a laminar flow hood, the temperature was adjusted to 30 °C, and the rotation speed was reduced to 150 rpm, and the culture was continued for 16 hours. Subsequently, the cells were collected by centrifugation, and 5 mL of cell lysis buffer (the components were 50 mM K 2 HPO 4 、300 mM NaCl, 5 mM imidazole, 10% glycerol, and the pH was adjusted to 8.0 with NaOH) was added to resuspend the cells. Then, ice bath ultrasonic disruption treatment was carried out for 10 minutes, and centrifugation was carried out again to collect the supernatant of the cell lysate. After the supernatant was filtered through a 0.45 μm filter membrane, the obtained filtrate was the crude enzyme solution.

[0028] The Ni-NTA 6FF packing material was filled into a centrifugal microprotein purification empty column to prepare a small-scale protein purification nickel column, and the volume of Ni-NTA 6FF filled in each column was about 350 μL. Next, 400 μL of 50 mM imidazole buffer (containing 50 mM imidazole, 250 mM K 2 HPO 4 、1.5 M NaCl, pH adjusted to 8.0) was used to wash and equilibrate the nickel column five times, and after each wash, centrifugation was carried out at 200 rpm for 15 seconds. Then, 400 μL of the crude enzyme solution was taken and added to the nickel column. After standing at room temperature for 1 minute, centrifugation was carried out at 200 rpm for 15 seconds, and the lower-layer filtrate after centrifugation was discarded. This step was repeated until all the crude enzyme solution was processed. Subsequently, 400 μL of 50 mM imidazole buffer was added to the nickel column again, and centrifugation was carried out at 200 rpm for 15 seconds to elute the impurity proteins, and this step was repeated 6 times. Then, 400 μL of 250 mM imidazole buffer (containing 250 mM imidazole, 250 mM K 2 HPO 4 、1.5 M NaCl, pH adjusted to 8.0) was added, and centrifugation was carried out at 200 rpm for 15 seconds to elute the target protein, and the filtrate was collected. This step was also repeated 6 times. After that, the PD-10 desalting column was pretreated, first rinsed with 15 mL of ultrapure water filtered through a 0.45 μm filter membrane, and then with 15 mL of protein desalting buffer (containing 50 mM K 2 HPO 4, 100 mM NaCl, 10% glycerol, 1 mM DTT, pH adjusted to 8.0) for equilibration. Finally, the eluate of the target protein obtained in the previous step was made up to 2.5 mL with protein desalting buffer and transferred to the equilibrated desalting column. After 2.5 mL of the target protein eluate completely passed through the desalting column, 3.5 mL of desalting buffer was added, and the eluted target protein solution was collected. The purification results of GcL wild type and its mutants are as Figure 1 shown, and the corresponding target proteins were successfully purified.

[0029] (2) Protein concentration and determination of concentration The target protein solution after nickel column purification and desalting gel column treatment was transferred to an ultrafiltration centrifugal tube with a molecular weight cut-off of 10 kDa, and then centrifuged at 6000 rpm at 4 °C until the volume of the target protein solution in the concentration tube was reduced to approximately 2 mL. Next, the concentration of the obtained protein solution was determined using the improved Bradford protein concentration determination kit provided by Sangon Biotech (Shanghai) Co., Ltd.

[0030] Example 3: Native polyacrylamide gel electrophoresis According to the instructions of the One-Step PAGE Gel Fast Preparation Kit (12%)-BOX2 (Nanjing Novoprotein Scientific Co., Ltd.), a separating gel with a concentration of 12% was prepared. Two clean glass electrophoresis plates were installed on the protein gel preparation rack and fixed with a clamping device. Subsequently, 5 mL of the prepared separating gel was slowly added between the assembled glass electrophoresis plates to ensure uniform distribution. Then, the stacking gel was prepared according to the kit instructions. The stacking gel was evenly covered on the surface of the separating gel, and a comb was slowly inserted at an appropriate position, taking care to avoid generating bubbles. After the stacking gel was completely solidified, it was carefully removed and installed into the protein electrophoresis tank. Subsequently, an appropriate amount of protein electrophoresis buffer was added to the electrophoresis tank.

[0031] Take 20 μL of the protein sample solution to be tested and mix it thoroughly with 5 μL of the pre-prepared 5× protein loading buffer, and place it in a boiling water bath for 5 minutes. After cooling to room temperature, 10 μL of the sample was aspirated from the mixture and added to the loading well of the Native-PAGE gel, and 5 μL of protein prestained marker (Marker) was added to the empty loading well. Subsequently, under the condition of stable voltage, electrophoresis was carried out at an initial voltage of 90 V. When the sample bands in the stacking gel showed a slender linear distribution, the voltage was increased to 190 V and electrophoresis was continued until the indicator reached the bottom of the separating gel. After electrophoresis, the gel was taken out and prepared for subsequent treatment.

[0032] Place the Native-PAGE gel in a Ø150 mm glass Petri dish containing Coomassie Brilliant Blue staining solution, and gently shake it at a low speed on a shaker for about 20 minutes, then discard the staining solution. Next, wash the gel with ultrapure water to remove the residual staining solution. Subsequently, transfer the gel to the decolorizing solution and continue to gently shake it on the shaker for decolorization. Replace the decolorizing solution every 15 minutes until the protein bands are clear and the background is transparent. Finally, take out the gel and perform imaging and photography.

[0033] It has been reported that the GcL protein has a homodimeric structure (Bergonzi, C., Schwab, M., Naik, T., & Elias, M. (2019). The Structural Determinants Accounting for the Broad Substrate Specificity of the Quorum Quenching Lactonase GcL. Chembiochem : a European journal of chemical biology , 20 (14), 1848–1855.). The protein monomer size of the GcL mutant is approximately 35 kDa. The molecular weights of mutants A19-9 and C13-1 were verified by non-denaturing protein gel electrophoresis experiments to be slightly less than 250 kDa, which is consistent with the molecular weight of the hexameric structure ( Figure 2 ). Thus, it can be seen that the transplantation of the three-histidine interaction structure and the generation of the thermostable protein motif enable GcL to form a hexameric structure.

[0034] Example 4: Enzyme Activity Assay of Mutants Add 5 μL of 0.05 μM free GcL to 200 μL of 10 mM PB (pH 8.0) containing 1.25 mM N -3-oxo-octanoyl-L-homoserine lactone, and react at 30 °C in a water bath for 30 min. After the water bath, immediately add 50 μL of 10% (m / V) SDS solution to the reaction system to terminate the reaction. The remaining N -3-oxo-octanoyl-L-homoserine lactone in the reaction system was detected by HPLC. All reactions were set up in triplicate. Calculate the relative activity of the mutant with the amount of N -3-oxo-octanoyl-L-homoserine lactone degraded by the GcL group as 100% activity.

[0035] N-3-oxo-octanoyl-L-homoserine lactone high performance liquid chromatography (HPLC) detection conditions are as follows: Use Welch Ultimate AQ-C18 Yuexu aqueous chromatographic column (C18, 5 μm, 4.6×250 mm) as the stationary phase; Use methanol: water (6:4, V / V) as the mobile phase with a flow rate of 1 mL·min -1 ; The column temperature is controlled at 30 °C; The sample injection volume is 20 μL.

[0036] N -3-oxo-octanoyl-L-homoserine lactone standard curve drawing: Dilute the 200 mM N -3-oxo-octanoyl-L-homoserine lactone solution 100 times with ultrapure water to prepare 2 mM N -3-oxo-octanoyl-L-homoserine lactone standard solution. Then prepare 3OC8-HSL standard solutions of 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM in a gradient dilution manner. According to N -3-oxo-octanoyl-L-homoserine lactone high performance liquid chromatography detection conditions, detect each concentration of the standard solution obtained, and use N -3-oxo-octanoyl-L-homoserine lactone concentration as the abscissa and the detected peak area as the ordinate to draw N -3-oxo-octanoyl-L-homoserine lactone standard curve on OriginPro 2021.

[0037] The enzyme activity detection results of GcL wild type and mutants are as Figure 3 shown. Compared with the GcL wild type, the enzyme activities of the constructed A19-9 and C13-1 mutants have both increased, and their relative activities are both 2.5 times that of the GcL wild type.

[0038] Example 5: Circular dichroism analysis of the stability of AHL-lactonase GcL mutants The secondary structure changes of the GcL wild-type protein and its mutants under different temperature conditions were quantitatively determined using a circular dichroism spectrometer, Chirascan™-plus. The specific experimental steps are as follows: First, using a phosphate buffer solution with a concentration of 10 mM and a pH of 8.0 as the solvent, the GcL wild-type protein and mutants A19-9 and C13-1 were diluted to a solution with a concentration of 0.25 mg·mL⁻¹. Subsequently, in the temperature range from 29 °C to 95 °C, the samples were heated at a constant rate of 1 °C·min⁻¹, and simultaneously, the circular dichroism spectral data of the wild-type GcL and mutants A19-9 and C13-1 with a concentration of 0.25 mg·mL⁻¹ were continuously collected and recorded in the wavelength range of 185 - 260 nm. To comprehensively analyze the collected spectral data, the present invention used a software combination of CDtoolX, CDNN, and Origin Pro 2021 for data processing and analysis.

[0039] The results of the circular dichroism spectrum analysis are as Figure 4 shown. The T m value of mutant A19-9 was increased by 10.49 °C compared to the wild-type, and the T m value of mutant C13-1 was increased by 3.75 °C compared to the wild-type.

[0040] Example 6: Determination of the half-life of the AHL-lactonase GcL mutant at 70 °C and 90 °C The enzyme samples were placed in a water bath at 70 °C and 90 °C for heat treatment for different times. Subsequently, they were quickly transferred to normal-temperature water and cooled to room temperature. The enzyme activity was calculated by measuring the ability of the enzyme to catalyze the conversion of 4-nitrophenyl butyrate (p-Nitrophenyl butyrate, pNPB) to p-nitrophenol. The specific operation steps are as follows: First, a 100 μM pNPB solution was prepared using 10 mM pH 8.0 PB as the solvent (this solution needs to be prepared freshly before use). 5 μL of a 0.64 μM enzyme sample was added to a 96-well plate. Subsequently, 200 μL of the 100 μM pNPB solution was immediately added to the well, and it was quickly transferred to a microplate reader. The absorbance value of the system at 405 nm was measured after 20 min of reaction at room temperature. Ultra-pure water without added enzyme was used as the blank control group. Three parallel samples were set for each group. By detecting the concentration of the generated p-nitrophenol, the amount of product generated within 20 min was calculated, and the relative activity of the mutant was calculated with the activity of the wild-type GcL group as 100%. Using the heat treatment time as the abscissa and the relative activity as the ordinate, the obtained data were curve-fitted using the Boltzmann equation in Origin Pro 2021 software.

[0041] The results are as Figure 5As shown, the half-life of mutant A19-9 at 70 °C was extended by one hour compared to the wild-type GcL. When analyzing the half-life of C13-1, it was found that its activity remained between 50% and 60% after incubation in a 70 °C water bath for 0.5 to 9 hours ( Figure 6 ). Based on this result, the experimental conditions were further increased to 90 °C to explore the effect of higher temperatures on the enzyme activity of C13-1. The results showed that after incubation at 90 °C for 10 days, the enzyme activity performance of C13-1 was similar to that at 70 °C, and its relative enzyme activity still remained between 50% and 60%. This result indicates that compared with GcL and A19-9, C13-1 has better heat resistance under extreme high temperature conditions and shows higher enzyme activity stability.

[0042] Example 6: Application of AHL-lactonase GcL mutants in the treatment of bacterial infectious diseases (1) Apple surface disinfection The apples were thoroughly rinsed under running tap water. Subsequently, the processed apples were immersed in a 75% (v / v) ethanol solution in a laminar flow hood for 10 seconds for surface disinfection, and then immediately rinsed thoroughly with sterile water to remove the residual ethanol. Then, a sterile knife was used to cut wounds with a diameter of about 1 cm on the apple surface for subsequent experiments.

[0043] (2) Biological control effects of mutants A19-9 and C13-1 on E. amylovora The overnight culture E. amylovora bacterial solution was diluted with sterile water to prepare a bacterial solution with a concentration of 1.2×10 12 CFU·mL -1 . 1.2×10 12 CFU·mL -1 E. amylovora bacterial solution was mixed with an equal volume of 2.5 μΜ GcL, 2.5 μΜ A19-9, or 2.5 μΜ C13-1, and 2 μL of the above mixture was inoculated at the circular wounds on the apples. 2 μL of 10 mM PB (pH 8.0) and 2 μL of 6.0×10 11 CFU·mL -1 E. amylovora bacterial solution were respectively inoculated at the circular wounds on the apples as controls. The inoculated petri dishes were placed in a 30 °C constant temperature incubator and incubated statically for 10 days.

[0044] The biological control results of mutants A19-9 and C13-1 on E. amylovora are as shown in Figure 7 . The fire blight lesion area of the co-treatment group of wild-type GcL and E. amylovora was lower than that of E. amylovoraThe single treatment group. Mutants A19-9 and C13-1 and E. amylovora There were no obvious fire blight lesions in the co-treatment group. This result indicates that both mutants A19-9 and C13-1 can effectively inhibit E. amylovora Apple fire blight caused by infection.

[0045] It should be clear that the above embodiments are only a part of many preferred embodiments of the present invention, aiming to elaborate and explain the core idea of the present invention, rather than constituting any formal limitation thereto. The terms used in the description are all descriptive and explanatory terms, aiming to help understanding, rather than setting strict boundaries. Within the scope of the rights covered by the present invention, appropriate modifications and adjustments are allowed and encouraged according to actual needs, as long as these changes do not violate the core principles and spirit of the present invention. Although the present invention is described in combination with specific methods, materials and implementation details, this does not mean that the application scope of the present invention is limited to these specific examples. On the contrary, the present invention has wide applicability and can be extended to all methods and application fields with similar functional requirements.

Claims

1. A highly thermostable mutant of AHL-lactonase, characterized in that: The amino acid sequence of AHL-lactonase is shown in SEQ ID NO.3; the mutation site of the mutant is to mutate the amino acid sequence LysArgHisGluAspAsn from the 176th to the 181st amino acid sequence of AHL-lactonase from the amino terminus to the carboxyl terminus as shown in SEQ ID NO.3 to ThrGluHisAspAsnAsp, and the amino acid sequence after mutation is shown in SEQ ID NO.1, which is recorded as mutant A19-9, or to mutate the amino acid sequence LysArgHisGluAspAsnIleLeuLeu from the 176th to the 184th amino acid sequence from the amino terminus to the carboxyl terminus to ArgAspArgHisGlyGlySerPheGluAsnIleProGly, and the amino acid sequence after mutation is shown in SEQ ID NO.2, which is recorded as mutant C13-1.

2. A deoxyribonucleic acid molecule encoding the AHL-lactonase high thermostable mutant according to claim 1, wherein the DNA sequence of mutant A19-9 is shown in SEQ ID NO.4, and the DNA sequence of mutant C13-1 is shown in SEQ ID NO.

5.

3. A recombinant vector containing the deoxyribonucleic acid sequence according to claim 2.

4. A recombinant bacterium containing the recombinant vector according to claim 3.

5. Use of the AHL-lactonase high thermostable mutant according to claim 1 in the preparation of a biological pathogenic bacteria inhibitor, characterized in that: The biological pathogenic bacteria are fire blight bacteria of fruit trees.

Images