A thermostable mutant of AHL-lactonase and its application in biological control of fire blight in fruit trees

Abstract

The present invention discloses a highly thermostable mutant of AHL-lactonase and its application in biological control of fire blight of fruit trees. The mutation site of the mutant is to mutate the amino acid sequence from position 176 to position 181 of the AHL-lactonase shown in SEQ ID NO.3 from the amino terminus to the carboxyl terminus. The mutated amino acid sequence is shown in SEQ ID NO.1, denoted as mutant A19-9, or to mutate the amino acid sequence from position 176 to position 184 from the amino terminus to the carboxyl terminus. The mutated amino acid sequence is shown in SEQ ID NO.2, denoted as mutant C13-1. The thermostability of both mutants has been greatly improved compared to the wild type. When the two mutants are applied to prepare a biological pathogenic bacteria inhibitor, the inhibitory Erwinia amylovora effect on fire blight of apple caused by infection is better than that of the wild type, and it provides a potential biological control agent for E.amylovora fire blight of fruit trees caused by 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 provides a high-thermal-stability 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:

[0007] The present invention provides a high-thermal-stability mutant of AHL-lactonase, and the AHL-lactonase is any one of the following proteins:

[0008] (a1) a protein with an amino acid sequence as shown in SEQ ID NO.3;

[0009] (a2) a protein whose sequence contains the amino acid sequence in (a1);

[0010] (a3) a fusion protein obtained by connecting a tag to the amino terminus and / or carboxy terminus of any one of the proteins in (a1) and (a2).

[0011] The mutation site of the mutant is that the amino acid sequence LysArgHisGluAspAsn from the 176th to the 181st position of the amino acid sequence shown in SEQ ID NO.3 is mutated to ThrGluHisAspAsnAsp from the amino terminus to the carboxy terminus, and the mutated amino acid sequence is as 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 carboxy terminus is mutated to ArgAspArgHisGlyGlySerPheGluAsnIleProGly, and the mutated amino acid sequence is as shown in SEQ ID NO.2, denoted as mutant C13-1.

[0012] Also provided is a deoxyribonucleic acid molecule encoding the high-thermal-stability mutant of AHL-lactonase. The DNA sequence of mutant A19-9 is as shown in SEQ ID NO.4, the DNA sequence of mutant C13-1 is as shown in SEQ ID NO.5, a recombinant vector containing the above deoxyribonucleic acid sequence, and a recombinant bacterium containing the recombinant vector.

[0013] Also provided is an application of the high-thermal-stability 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 .

[0014] 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 infection, and provide potential biological control agents for E. amylovora the fruit tree fire blight caused by infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 shows the purification result of the GcL mutant protein;

[0016] Figure 2 shows the non-denaturing polyacrylamide gel electrophoresis pattern of the GcL mutant;

[0017] Figure 3 shows the activity detection result of the GcL mutant;

[0018] Figure 4 shows the melting curves of the GcL wild type and mutants A19-9 and C13-1;

[0019] Figure 5 shows the thermal inactivation curves of GcL and mutant A19-9 fitted with the Boltzmann function at 70 °C;

[0020] Figure 6 shows the thermal inactivation curves of mutant C13-1 at 70 and 90 °C;

[0021] Figure 7 shows the control effects of the GcL wild type and mutants A19-9 and C13-1 on E. amylovora the apple fire blight caused. DETAILED DESCRIPTION OF THE INVENTION

[0022] In order to clearly illustrate the content of the present invention, the present invention will be explained in detail below with reference to the accompanying drawings of the specification and specific examples. However, before introducing in depth, it should be clearly pointed out that the scope of the present invention is not limited to these specifically described examples. 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.

[0023] 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 commonly 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.

[0024] Ⅰ. TERMS

[0025] In the present invention, the term "HPLC" specifically refers to high performance liquid chromatography; "T m”is explicitly expressed as the melting temperature.

[0026] II. Embodiments

[0027] As mentioned above, considering that the current application of AHL-lactonase in the prevention and treatment of animal and plant bacterial infectious diseases is limited due to its insufficient thermal stability, to solve this key problem, the researchers of the present invention adopted a computer-aided de novo design strategy of protein motifs to improve AHL-lactonase.

[0028] The inventors of the present invention 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 thermostability-enhanced mutant M41 of AHL-lactonase AhlX. Experimental verification by 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 design tool RFdiffusion, this special three-histidine interaction structure was transplanted into the AHL-lactonase GcL Parageobacillus caldoxylosilyticus derived from a thermophilic bacterium 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 highly thermostable AHL-lactonase mutant. The obtained mutant has N high hydrolytic activity towards 3-oxo-octanoyl-L-homoserine lactone and excellent thermal stability at the same time.

[0029] The wild-type AHL-lactonase to be modified is any one of the following (a1)-(a3):

[0030] (a1) A protein with the amino acid sequence of SEQ ID NO.3 (denoted as AHL-lactonase GcL);

[0031] (a2) A protein whose sequence contains the amino acid sequence defined in (a1);

[0032] (a3) A fusion protein obtained by connecting a tag to the amino terminus and / or carboxyl terminus of the protein defined in any one of (a1) and (a2);

[0033] The mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO.3 from the amino terminus to the carboxyl terminus through any of the following situations:

[0034] The mutant in which the amino acid sequence LysArgHisGluAspAsn at positions 176 to 181 is mutated to ThrGluHisAspAsnAsp, denoted as GcL mutant A19-9, and its amino acid sequence is as shown in SEQ ID NO.1;

[0035] The mutant in which the amino acid sequence LysArgHisGluAspAsnIleLeuLeu at positions 176 to 184 is mutated to ArgAspArgHisGlyGlySerPheGluAsnIleProGly, denoted as GcL mutant C13-1, and its amino acid sequence is as shown in SEQ ID NO.2.

[0036] The DNA molecule described above is the following DNA molecule:

[0037] (b1) A DNA molecule in which the coding region encodes the protein described in the first aspect of the present invention;

[0038] (b2) A coding region including a DNA molecule encoding the protein described in the first aspect of the present invention;

[0039] (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;

[0040] (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.

[0041] 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 that encodes the amino acid sequence of wild-type AHL-lactonase shown in SEQ ID NO.3.

[0042] In the present invention, the DNA sequence as shown in SEQ ID NO.6, which encodes the wild-type AHL-lactonase with the amino acid sequence as shown in SEQ ID NO.3, is hereby defined as the DNA molecule encoding the AHL-lactonase GcL; the DNA sequence as shown in SEQ ID NO.4, which encodes the wild-type AHL-lactonase with the amino acid sequence as shown in SEQ ID NO.1, is hereby defined as the DNA molecule encoding the GcL mutant A19-9; the DNA sequence as shown in SEQ ID NO.5, which encodes the wild-type AHL-lactonase with the amino acid sequence as shown in SEQ ID NO.2, is hereby defined as the DNA molecule encoding the GcL mutant C13-1;

[0043] 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.

[0044] The present invention discloses the application scope, specifically relating to the practical application of the following mutants: the mutants described in the present invention, the mutants encoded by the DNA molecules 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 ester bond-containing compounds and the biological control of bacterial infectious plant diseases.

[0045] The present invention provides the application of the following mutants, which include: the mutants described in the present invention, the mutants encoded by the DNA molecules 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 in the hydrolysis process of acyl-homoserine lactones and other ester compounds.

[0046] Ⅲ. Specific Examples

[0047] Unless otherwise specified, all experimental methods mentioned below follow the conventional laboratory operation specifications. Similarly, the experimental materials described below can be obtained through commercial channels without special indication. To further elaborate the present invention, the following will be described in detail in combination with specific examples, but please note that the protection scope of the present invention is not limited to the description of these specific examples.

[0048] In some specific examples of the present invention, Escherichia coli BL21(DE3) is selected as the host cell for expressing the wild-type AHL-lactonase GcL and its mutants. The medium formulations used in the following examples are listed in detail as follows:

[0049] LB liquid medium: Peptone 10 g·L -1 、Yeast extract 5 g·L -1 、NaCl 10 g·L -1 ;

[0050] LB solid medium: Agar powder 18 g·L -1 、Peptone 10 g·L -1 、Yeast extract 5 g·L -1 、NaCl 10g·L -1 ;

[0051] TB solid medium: KH2PO4 2.31 g·L -1 、K2HPO4 12.54 g·L -1 、Glycerol 4 g·L -1 、Tryptone 12 g·L -1 、Yeast extract 24 g·L -1 。

[0052] Example 1: Construction of a strain expressing the AHL-lactonase mutant

[0053] 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.

[0054] The random coil structure of GcL was replaced using the one-step site-directed mutagenesis technique. 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 are to mutate the amino acid sequence LysArgHisGluAspAsn from positions 176 to 181 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 and is denoted as mutant A19-9, or to mutate the amino acid sequence LysArgHisGluAspAsnIleLeuLeu from positions 176 to 184 from the amino terminus to the carboxyl terminus to ArgAspArgHisGlyGlySerPheGluAsnIleProGly. The mutated amino acid sequence is shown in SEQ ID NO.2 and is denoted as mutant C13-1. Subsequently, the mutation was introduced into the expression vector by PCR amplification technology. The amplification product was subjected to DpnTreat with I enzyme to remove the original template plasmid. The treated product was transformed into E. coli BL21(DE3) host cells, thus successfully constructing the corresponding GcL mutant expression strain.

[0055] Example 2: Preparation of AHL-lactonase GcL mutant

[0056] (1) Heterologous expression and purification of wild-type GcL and its mutants

[0057] The GcL mutant expression strain and the wild-type GcL expression strain constructed in Example 1 were respectively inoculated into 5 mL of TB liquid medium containing 50 μg / mL kanamycin. After all strains were cultured with shaking at 37 °C and 220 rpm for 4 hours, then 0.2 mM IPTG 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 composition was 50 mM K2HPO4, 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.

[0058] Pack Ni-NTA 6FF filler into a centrifugal micro protein purification empty column to prepare a small-scale protein purification nickel column. The volume of Ni-NTA 6FF filled in each column is about 350 μL. Next, use 400 μL of 50 mM imidazole buffer (containing 50 mM imidazole, 250 mM K2HPO4, 1.5 M NaCl, pH adjusted to 8.0) to wash and balance the nickel column five times. After each wash, centrifuge at 200 rpm for 15 seconds. Then, take 400 μL of crude enzyme solution and add it to the nickel column. After standing at room temperature for 1 minute, centrifuge at 200 rpm for 15 seconds, and discard the lower filtrate after centrifugation. This step needs to be repeated until all the crude enzyme solution is processed. Subsequently, add 400 μL of 50 mM imidazole buffer to the nickel column again, centrifuge at 200 rpm for 15 seconds to elute the impurity proteins, and this step needs to be repeated 6 times. Then, add 400 μL of 250 mM imidazole buffer (containing 250 mM imidazole, 250 mM K2HPO4, 1.5 M NaCl, pH adjusted to 8.0), centrifuge at 200 rpm for 15 seconds to elute the target protein, and collect the filtrate. This step also needs to be repeated 6 times. After that, pre-treat the PD-10 desalting column. First, wash it with 15 mL of ultrapure water filtered through a 0.45 μm filter membrane, and then balance it with 15 mL of protein desalting buffer (containing 50 mM K2HPO4, 100 mM NaCl, 10% glycerol, 1 mM DTT, pH adjusted to 8.0). Finally, make up the target protein eluate obtained in the previous step to 2.5 mL with protein desalting buffer and transfer it to the balanced desalting column. After 2.5 mL of the target protein eluate completely passes through the desalting column, add 3.5 mL of desalting buffer and collect the flowing target protein solution. The purification results of GcL wild type and its mutants are as Figure 1 shown, and the corresponding target proteins are successfully purified.

[0059] (2)Concentration and concentration determination of proteins

[0060] Transfer the target protein solution after nickel column purification and desalting gel column treatment to a 10 kDa molecular weight cut-off ultrafiltration centrifugal tube, and then centrifuge at 6000 rpm at 4°C until the volume of the target protein solution in the concentration tube is reduced to about 2 mL. Next, use the improved Bradford protein concentration determination kit provided by Sangon Biotech (Shanghai) Co., Ltd. to determine the concentration of the obtained protein solution.

[0061] Example 3: Native polyacrylamide gel electrophoresis

[0062] According to the instructions of the One-Step PAGE Gel Fast Preparation Kit (12%)-BOX2 (Nanjing Novoprotein Science & Technology Co., Ltd.), prepare a separating gel with a concentration of 12%. Install two clean glass electrophoresis plates on the protein gel preparation rack and fix them with a clamping device. Subsequently, slowly add 5 mL of the prepared separating gel between the assembled glass electrophoresis plates to ensure uniform distribution. Then, prepare the stacking gel according to the kit instructions. Evenly cover the separating gel with the stacking gel and slowly insert a comb at an appropriate position, taking care to avoid generating bubbles. After the stacking gel has completely solidified, carefully remove it and install it into the protein electrophoresis tank. Subsequently, add an appropriate amount of protein electrophoresis buffer to the electrophoresis tank.

[0063] 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. Place it in a boiling water bath and heat for 5 minutes. After cooling to room temperature, pipette 10 μL of the sample from the mixture into the loading wells of the Native-PAGE gel, and add 5 μL of the protein prestained marker (Marker) to the empty loading wells. Subsequently, at a stable voltage, perform electrophoresis at an initial voltage of 90 V. When the sample bands in the stacking gel show a slender linear distribution, increase the voltage to 190 V and continue electrophoresis until the indicator reaches the bottom of the separating gel. After electrophoresis is completed, remove the gel and prepare for subsequent processing.

[0064] 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 destaining solution and continue to gently shake it on the shaker for destaining. Replace the destaining solution every 15 minutes until the protein bands are clear and the background is transparent. Finally, remove the gel and take an image for photography.

[0065] 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 BroadSubstrate Specificity of the Quorum Quenching Lactonase GcL. Chembiochem : a European journal of chemical biology , 20(14), 1848–1855.). The protein monomer 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 to be slightly less than 250 kDa, which is consistent with the molecular weight of the hexameric structure ( Figure 2 ). 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.

[0066] Example 4: Enzyme Activity Assay of Mutants

[0067] 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. Immediately after the water bath, 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. The relative activity of the mutants was calculated based on the amount of N -3-oxo-octanoyl-L-homoserine lactone degraded by the GcL group as 100% activity.

[0068] N The high performance liquid chromatography (HPLC) detection conditions for -3-oxo-octanoyl-L-homoserine lactone are as follows: Use a 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.

[0069] N Drawing of the standard curve for -3-oxo-octanoyl-L-homoserine lactone:

[0070] Dilute a 200 mM N -3-oxo-octanoyl-L-homoserine lactone solution 100-fold with ultrapure water to prepare a 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, and 2 mM according to the gradient dilution method. According to N-3-oxo-octanoyl-L-homoserine lactone was detected by HPLC. Standard solutions of various concentrations were obtained, and with the concentration of N -3-oxo-octanoyl-L-homoserine lactone as the abscissa and the detected peak area as the ordinate, a standard curve of N -3-oxo-octanoyl-L-homoserine lactone was plotted on OriginPro 2021.

[0071] 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 mutants A19-9 and C13-1 were both improved, and their relative activities were both 2.5 times that of the GcL wild type.

[0072] Example 5: Circular dichroism analysis of the stability of AHL-lactonase GcL mutants

[0073] The circular dichroism spectrometer Chirascan™-plus was used to quantitatively determine the secondary structure changes of the GcL wild-type protein and its mutants under different temperature conditions. The specific experimental steps are as follows: First, using a 10 mM, pH 8.0 phosphate buffer solution 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 of 29 °C to 95 °C, the samples were heated at a constant rate of 1 °C·min⁻¹, and at the same time, 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 in the wavelength range of 185-260 nm were continuously collected and recorded. In order to comprehensively analyze the collected spectral data, the present invention uses a software combination of CDtoolX, CDNN, and Origin Pro 2021 for data processing and analysis.

[0074] The results of circular dichroism analysis are as Figure 4 shown. The T m value of mutant A19-9 was increased by 10.49 °C compared with the wild type, and the T m value of mutant C13-1 was increased by 3.75 °C compared with the wild type.

[0075] Example 6: Determination of the half-life of AHL-lactonase GcL mutants at 70 °C and 90 °C

[0076] 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 p-Nitrophenyl butyrate (pNPB) to p-nitrophenol. The specific operation steps are as follows: First, a 100 μM pNPB solution was prepared with 10 mM PB at pH 8.0 as the solvent (this solution needs to be prepared immediately before use). 5 μL of 0.64 μM enzyme sample was added to a 96-well plate. Subsequently, 200 μL of 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 taking the activity of the wild-type GcL group as 100%, the relative activity of the mutant was calculated. With 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.

[0077] The results are as Figure 5 shown. The half-life of mutant A19-9 at 70 °C was extended by one hour compared to 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 temperature on the enzyme activity of C13-1. The results showed that after 10 days of incubation at 90 °C, 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.

[0078] Example 6: Application of AHL-lactonase GcL mutants in the treatment of bacterial infectious diseases

[0079] (1) Disinfection of apple surface

[0080] 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 a wound with a diameter of about 1 cm on the apple surface for subsequent experiments.

[0081] (2) Mutants A19-9 and C13-1 onE. amylovora Biocontrol effect

[0082] Dilute the overnight culture E. amylovora bacterial solution with sterile water to prepare a bacterial solution with a concentration of 1.2×10 12 CFU·mL -1 . Mix 1.2×10 12 CFU·mL -1 E. amylovora bacterial solution with 2.5 μΜ GcL, 2.5 μΜ A19-9 or 2.5 μΜ C13-1 in equal volume, and pipette 2 μL of the above mixture and inoculate it at the round wound of the apple. Pipette 2 μL of 10 mM PB (pH 8.0) and 2 μL of 6.0×10 11 CFU·mL -1 E. amylovora bacterial solution and inoculate them at the round wound of the apple as controls respectively. Place the inoculated petri dishes in a constant temperature incubator at 30 °C and incubate them statically for 10 days.

[0083] The biocontrol results of mutants A19-9 and C13-1 against E. amylovora are as Figure 7 shown. The fire blight lesion area of the co-treatment group of wild-type GcL and E. amylovora is lower than that of the E. amylovora single-treatment group. No obvious fire blight lesions appeared in the co-treatment groups of mutants A19-9 and C13-1 and E. amylovora . This result indicates that both mutants A19-9 and C13-1 can effectively inhibit E. amylovora the apple fire blight caused by infection.

[0084] 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 form of limitation to it. 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 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. An AHL-lactonase high thermal stability mutant, characterized in that, The amino acid sequence of the AHL-lactonase is shown in SEQ ID NO. 3; the mutation site of the mutant is that the amino acid sequence LysArgHisGluAspAsn from the 176th to the 181st positions of the AHL-lactonase shown in SEQ ID NO. 3 from the amino terminus to the carboxyl terminus 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 positions 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.

2. A deoxyribonucleic acid molecule encoding the thermostable mutant of the AHL-lactonase described in claim 1, 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 described in claim 2.

4. A recombinant bacterium containing the recombinant vector described in claim 3.

5. Use of an AHL-lactonase thermostable mutant as described in claim 1 in the preparation of a biological pathogenic bacteria inhibitor, characterized in that, The biological pathogen is Erwinia amylovora.

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