A mutant of N-acyl homoserine lactonase and its application

Abstract

The present invention discloses a mutant of N-acyl homoserine lactonase and its application. Through rational design and site-directed mutagenesis techniques, the corresponding mutant is obtained, which can greatly improve the thermal stability and AHL hydrolysis activity of AHL-lactonase AhlX. After transformation, the T m value of mutant M41 is increased by 12.13 °C, and the enzyme activity is 17.67 times that of the wild type. The mutant M41 is applied to the soft rot diseases of potatoes, zucchinis, green peppers, radishes, Chinese cabbages and eggplants caused by E. carotovora infection. The results show that the soft rot lesion areas of the co-treatment groups of mutant M41 and E. carotovora are significantly lower than those of the E. carotovora treatment group. The mutant M41 can effectively alleviate various plant soft rot diseases caused by E. carotovora.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein engineering, and particularly relates to a mutant of N-acyl homoserine lactonase and its application. Background Art

[0002] Bacterial infectious diseases of animals and plants pose a serious threat to the health of animals, plants, and even humans. Although the use of conventional antibiotic therapies is effective, drug resistance problems often arise because they directly interfere with the bacterial metabolic process. Quorum sensing (QS) is one of the bacterial mechanisms mediating animal and plant pathogenicity and has been proven to be able to regulate the expression of related genes including the production of virulence factors, antibiotic resistance, swarming motility, and biofilm formation. Targeting the quorum sensing system and interfering with quorum quenching (QQ) at stages such as the synthesis, accumulation, and response of signal molecules during quorum sensing has been proven to be an effective strategy for preventing and treating bacterial infectious diseases.

[0003] N N-acyl homoserine lactones ( N -acylhomoserine lactones, AHLs) are common signal molecules in the quorum sensing process of － animal and plant pathogenic bacteria. AHL-lactonase has high-efficiency and broad-spectrum AHLs degradation ability and can effectively prevent and treat bacterial infectious diseases of animals and plants. At the same time, because AHL-lactonase inhibits bacterial pathogenicity by degrading extracellular signal molecules through this special mechanism of action and does not directly act on bacterial cells, it does not directly exert pressure on the normal growth and metabolism of bacteria, so drug resistance problems are not likely to occur. Based on the above advantages, AHL-lactonase is expected to become an effective alternative to conventional antibiotics.

[0004] Although dozens of AHL-lactonases have been discovered at home and abroad, including AHL-lactonase AhlX (CN105543193A) from marine bacterium Salinicola salaries MCCC1A01339 which has high activity against a variety of AHLs, due to deficiencies in activity or stability, the practical application of such enzymes in the biological control of animal and plant pathogenic bacteria has not been realized. In order to overcome the limitations of AHL-lactonase in practical applications, in addition to using means such as chemical modification and immobilization to improve the stability of the enzyme, protein engineering modification of AHL-lactonase using techniques such as gene mutation, high-throughput screening, computer-aided protein structure prediction and design is also an effective means to improve its practical application value. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a group of mutants of AHL-lactonase with high enzyme activity and high thermal stability in view of the problems of insufficient thermal stability and activity of existing AHL-lactonase. The mutants are obtained by rational and semi-rational design and site-directed mutagenesis. The modified mutants have higher thermal stability and enzyme activity, and can achieve efficient degradation of N -acyl-homoserine lactone signal molecules in a complex environment.

[0006] In the first aspect of the present invention, a mutant of N-acyl-homoserine lactonase is provided, which has hydrolytic activity on molecules containing an ester bond. The N-acyl-homoserine lactonase is any one of the following proteins:

[0007] (a1) a protein with an amino acid sequence of SEQ ID NO.1;

[0008] (a2) a protein whose sequence contains the amino acid sequence defined in (a1);

[0009] (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);

[0010] The mutation sites of the mutant include one or more of the following mutations from the amino terminus to the carboxyl terminus: Glu at position 77 is mutated to Ile, Asp at position 157 is mutated to Gly, Thr at position 243 is mutated to Tyr, Thr at position 243 is mutated to Val, His at position 255 is mutated to Leu, and Ala at position 261 is mutated to Ile.

[0011] Preferably, the amino acid sequence of the N-acyl-homoserine lactonase is SEQ ID NO.1.

[0012] Preferably, the mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO.1 from the amino terminus to the carboxyl terminus through any one of the following situations:

[0013] 1) Glu at position 77 is mutated to Ile;

[0014] 2) Asp at position 157 is mutated to Gly;

[0015] 3) Thr at position 243 is mutated to Tyr;

[0016] 4) Thr at position 243 is mutated to Val;

[0017] 5) His at position 255 is mutated to Leu;

[0018] 6) Ala at position 261 is mutated to Ile;

[0019] 7) The Glu at the 77th position is mutated to Ile, and the Asp at the 157th position is mutated to Gly;

[0020] 8) The Glu at the 77th position is mutated to Ile, and the Thr at the 243rd position is mutated to Tyr;

[0021] 9) The Glu at the 77th position is mutated to Ile, and the His at the 255th position is mutated to Leu;

[0022] 10) The Glu at the 77th position is mutated to Ile, and the Ala at the 261st position is mutated to Ile;

[0023] 11) The Asp at the 157th position is mutated to Gly, and the Thr at the 243rd position is mutated to Tyr;

[0024] 12) The Asp at the 157th position is mutated to Gly, and the His at the 255th position is mutated to Leu;

[0025] 13) The Asp at the 157th position is mutated to Gly, and the Ala at the 261st position is mutated to Ile;

[0026] 14) The Thr at the 243rd position is mutated to Tyr, and the His at the 255th position is mutated to Leu;

[0027] 15) The Thr at the 243rd position is mutated to Tyr, and the Ala at the 261st position is mutated to Ile;

[0028] 16) The His at the 255th position is mutated to Leu, and the Ala at the 261st position is mutated to Ile;

[0029] 17) The Glu at the 77th position is mutated to Ile, the Asp at the 157th position is mutated to Gly, and the Thr at the 243rd position is mutated to Tyr;

[0030] 18) The Glu at the 77th position is mutated to Ile, the Asp at the 157th position is mutated to Gly, and the His at the 255th position is mutated to Leu;

[0031] 19) The Glu at the 77th position is mutated to Ile, the Asp at the 157th position is mutated to Gly, and the Ala at the 261st position is mutated to Ile;

[0032] 20) The Glu at the 77th position is mutated to Ile, the Thr at the 243rd position is mutated to Tyr, and the His at the 255th position is mutated to Leu;

[0033] 21) The Glu at the 77th position is mutated to Ile, the Thr at the 243rd position is mutated to Tyr, and the Ala at the 261st position is mutated to Ile;

[0034] 22) The Glu at the 77th position is mutated to Ile, the His at the 255th position is mutated to Leu, and the Ala at the 261st position is mutated to Ile;

[0035] 23) The 157th Asp is mutated to Gly, the 243rd Thr is mutated to Tyr, and the 255th His is mutated to Leu;

[0036] 24) The 157th Asp is mutated to Gly, the 243rd Thr is mutated to Tyr, and the 261st Ala is mutated to Ile;

[0037] 25) The 157th Asp is mutated to Gly, the 255th His is mutated to Leu, and the 261st Ala is mutated to Ile;

[0038] 26) The 243rd Thr is mutated to Tyr, the 255th His is mutated to Leu, and the 261st Ala is mutated to Ile;

[0039] 27) The 77th Glu is mutated to Ile, the 157th Asp is mutated to Gly, the 243rd Thr is mutated to Tyr, and the 255th His is mutated to Leu;

[0040] 28) The 77th Glu is mutated to Ile, the 157th Asp is mutated to Gly, the 243rd Thr is mutated to Tyr, and the 261st Ala is mutated to Ile;

[0041] 29) The 77th Glu is mutated to Ile, the 157th Asp is mutated to Gly, the 255th His is mutated to Leu, and the 261st Ala is mutated to Ile;

[0042] 30) The 77th Glu is mutated to Ile, the 243rd Thr is mutated to Tyr, the 255th His is mutated to Leu, and the 261st Ala is mutated to Ile;

[0043] 31) The 157th Asp is mutated to Gly, the 243rd Thr is mutated to Tyr, the 255th His is mutated to Leu, and the 261st Ala is mutated to Ile;

[0044] 32) The 77th Glu is mutated to Ile, the 157th Asp is mutated to Gly, the 243rd Thr is mutated to Tyr, the 255th His is mutated to Leu, and the 261st Ala is mutated to Ile.

[0045] The second aspect of the present invention provides a deoxyribonucleic acid molecule encoding the above-mentioned mutant.

[0046] According to the present invention, the sequence of the deoxyribonucleic acid molecule contains one or more mutants in which the G at the 229th position, the A at the 230th position, the A at the 470th position, the A at the 727th position, the A at the 727th position, the C at the 728th position, the C at the 728th position, the A at the 764th position, the G at the 781st position, the C at the 782nd position, and the T at the 783rd position are mutated to A, T, G, T, G, A, T, T, A, T, and C respectively in the deoxyribonucleotide sequence shown in SEQ ID NO.2 encoding the amino acid sequence of wild-type AHL-lactonase AhlX in the 5'-to-3' direction.

[0047] In some further specific embodiments of the present invention, the deoxyribonucleic acid molecule is the following DNA molecule:

[0048] (b1) A DNA molecule whose coding region encodes the protein described in the first aspect of the present invention;

[0049] (b2) A coding region that includes a DNA molecule encoding the protein described in the first aspect of the present invention;

[0050] (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;

[0051] (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.

[0052] The third aspect of the present invention provides an expression cassette, a recombinant vector, or a recombinant microorganism containing the deoxyribonucleic acid molecule described in the second aspect of the present invention.

[0053] The fourth aspect of the present invention provides the use of the mutant described in the first aspect of the present invention, or the mutant encoded by the deoxyribonucleic acid molecule described in the second aspect of the present invention, or the mutant prepared from the expression cassette, recombinant vector, or recombinant microorganism described in the third aspect of the present invention in the hydrolysis of ester bond-containing compounds and the biological control of bacterial infectious animal and plant diseases.

[0054] The beneficial effects of the present invention are as follows: The present invention provides a group of AHL-lactonase AhlX mutants. The AHL-lactonase AhlX described in the present invention is modified by rational design and site-directed mutagenesis techniques to obtain corresponding mutants, which can greatly improve the thermal stability and AHL hydrolysis activity of AHL-lactonase AhlX. After modification, the T m value of mutant M41 is increased by 12.13 °C, and the enzyme activity is 17.67 times that of the wild type. Applying mutant M41 to E. carotovoraSoft rot diseases of potatoes, zucchinis, green peppers, radishes, Chinese cabbages and eggplants caused by infection. The results show that the lesion areas of soft rot diseases of the mutant M41 and the E. carotovora co-treatment group are significantly lower than those of the E. carotovora treatment group. The mutant M41 can effectively alleviate the E. carotovora soft rot diseases of various plants caused by Description of the Drawings

[0055] Figure 1 is a schematic diagram of constructing an AhlX multi-site mutant expression plasmid by Gibson assembly method;

[0056] Figure 2 are the activity detection results of single-site mutants;

[0057] Figure 3 are the residual activity detection results of single-site mutants after heat treatment at 37 °C for 30 minutes;

[0058] Figure 4 are the activity detection results of multi-site mutants;

[0059] Figure 5 are the residual activity detection results of multi-site mutants after heat treatment at 37 °C for 30 minutes;

[0060] Figure 6 are the circular dichroism spectra and melting curves of AhlX wild type and mutant M41; among them, A is the 3D thermal denaturation circular dichroism spectra of AhlX wild type (left) and mutant M41 (right) between 29 °C and 95 °C, and B is the melting curves of AhlX wild type (left) and mutant M41 (right) between 29 °C and 95 °C;

[0061] Figure 7 is the biological control effect of AhlX mutant M41 on the E. carotovora soft rot diseases of various plants caused by Detailed Embodiments

[0062] To make the present invention easy to understand, the present invention will be described in detail below in conjunction with the drawings and embodiments. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terms used herein are only for describing specific embodiments and do not indicate restrictive.

[0063] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention, the preferred methods and materials are now described.

[0064] Ⅰ. Terms

[0065] As used herein, the term "HPLC" refers to high performance liquid chromatography, and " T m " refers to the melting temperature. An "amino acid mutant" refers to the smallest unit in the amino acid sequence of a protein that can undergo mutation.

[0066] II. Embodiments

[0067] As mentioned above, the current application of AHL-lactonase in infectious diseases of animals, plants and bacteria is limited by its insufficient activity and stability. To solve this problem, the inventors of the present invention have conducted extensive research on AHL-lactonase.

[0068] The inventors of the present invention have found through research and design that the thermal stability and ester hydrolysis activity of AHL-lactonase can be greatly improved through semi-rational design, rational design and site-directed mutagenesis. The mutation sites of the mutant include one or more of the following mutations of the amino acid sequence of wild-type AHL-lactonase from the amino terminus to the carboxyl terminus: Glu at position 77 is mutated to Ile, Asp at position 157 is mutated to Gly, Thr at position 243 is mutated to Tyr, Thr at position 243 is mutated to Val, His at position 255 is mutated to Leu, and Ala at position 261 is mutated to Ile. It has high hydrolysis activity towards molecules containing ester bonds and excellent thermal stability.

[0069] The wild-type AHL-lactonase to be modified is any one of the following (a1)-(a3):

[0070] (a1) A protein with the amino acid sequence of SEQ ID NO.1 (denoted as AHL-lactonase AhlX);

[0071] (a2) A protein whose sequence contains the amino acid sequence defined in (a1);

[0072] (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);

[0073] Preferably, the mutant is characterized in that it is obtained by mutating the amino acid sequence shown in SEQ ID NO.1 from the amino terminus to the carboxyl terminus through any one of the following situations:

[0074] 1) Glu at position 77 is mutated to Ile, denoted as AhlX mutant E77I;

[0075] 2) Asp at position 157 is mutated to Gly, denoted as AhlX mutant D157G;

[0076] 3) Thr at position 243 is mutated to Tyr, denoted as AhlX mutant T243Y;

[0077] 4) The 243rd Thr is mutated to Val, denoted as the AhlX mutant T243V;

[0078] 5) The 255th His is mutated to Leu, denoted as the AhlX mutant H255L;

[0079] 6) The 261st Ala is mutated to Ile, denoted as the AhlX mutant A261I;

[0080] 7) The 77th Glu is mutated to Ile and the 157th Asp is mutated to Gly, denoted as the AhlX mutant M20;

[0081] 8) The 77th Glu is mutated to Ile and the 243rd Thr is mutated to Tyr, denoted as the AhlX mutant M21;

[0082] 9) The 77th Glu is mutated to Ile and the 255th His is mutated to Leu, denoted as the AhlX mutant M22;

[0083] 10) The 77th Glu is mutated to Ile and the 261st Ala is mutated to Ile, denoted as the AhlX mutant M23;

[0084] 11) The 157th Asp is mutated to Gly and the 243rd Thr is mutated to Tyr, denoted as the AhlX mutant M24;

[0085] 12) The 157th Asp is mutated to Gly and the 255th His is mutated to Leu, denoted as the AhlX mutant M25;

[0086] 13) The 157th Asp is mutated to Gly and the 261st Ala is mutated to Ile, denoted as the AhlX mutant M26;

[0087] 14) The 243rd Thr is mutated to Tyr and the 255th His is mutated to Leu, denoted as the AhlX mutant M27;

[0088] 15) The 243rd Thr is mutated to Tyr and the 261st Ala is mutated to Ile, denoted as the AhlX mutant M28;

[0089] 16) The 255th His is mutated to Leu and the 261st Ala is mutated to Ile, denoted as the AhlX mutant M29;

[0090] 17) The 77th Glu is mutated to Ile, the 157th Asp is mutated to Gly, and the 243rd Thr is mutated to Tyr, denoted as the AhlX mutant M30;

[0091] 18) Glu at the 77th position is mutated to Ile, Asp at the 157th position is mutated to Gly, and His at the 255th position is mutated to Leu, denoted as AhlX mutant M31;

[0092] 19) Glu at the 77th position is mutated to Ile, Asp at the 157th position is mutated to Gly, and Ala at the 261st position is mutated to Ile, denoted as AhlX mutant M32;

[0093] 20) Glu at the 77th position is mutated to Ile, Thr at the 243rd position is mutated to Tyr, and His at the 255th position is mutated to Leu, denoted as AhlX mutant M33;

[0094] 21) Glu at the 77th position is mutated to Ile, Thr at the 243rd position is mutated to Tyr, and Ala at the 261st position is mutated to Ile, denoted as AhlX mutant M34;

[0095] 22) Glu at the 77th position is mutated to Ile, His at the 255th position is mutated to Leu, and Ala at the 261st position is mutated to Ile, denoted as AhlX mutant M35;

[0096] 23) Asp at the 157th position is mutated to Gly, Thr at the 243rd position is mutated to Tyr, and His at the 255th position is mutated to Leu, denoted as AhlX mutant M36;

[0097] 24) Asp at the 157th position is mutated to Gly, Thr at the 243rd position is mutated to Tyr, and Ala at the 261st position is mutated to Ile, denoted as AhlX mutant M37;

[0098] 25) Asp at the 157th position is mutated to Gly, His at the 255th position is mutated to Leu, and Ala at the 261st position is mutated to Ile, denoted as AhlX mutant M38;

[0099] 26) Thr at the 243rd position is mutated to Tyr, His at the 255th position is mutated to Leu, and Ala at the 261st position is mutated to Ile, denoted as AhlX mutant M39;

[0100] 27) Glu at the 77th position is mutated to Ile, Asp at the 157th position is mutated to Gly, Thr at the 243rd position is mutated to Tyr, and His at the 255th position is mutated to Leu, denoted as AhlX mutant M41;

[0101] 28) Glu at the 77th position is mutated to Ile, Asp at the 157th position is mutated to Gly, Thr at the 243rd position is mutated to Tyr, and Ala at the 261st position is mutated to Ile, denoted as AhlX mutant M42;

[0102] 29) The Glu at the 77th position is mutated to Ile, the Asp at the 157th position is mutated to Gly, the His at the 255th position is mutated to Leu, and the Ala at the 261st position is mutated to Ile, denoted as the AhlX mutant M43;

[0103] 30) The Glu at the 77th position is mutated to Ile, the Thr at the 243rd position is mutated to Tyr, the His at the 255th position is mutated to Leu, and the Ala at the 261st position is mutated to Ile, denoted as the AhlX mutant M44;

[0104] 31) The Asp at the 157th position is mutated to Gly, the Thr at the 243rd position is mutated to Tyr, the His at the 255th position is mutated to Leu, and the Ala at the 261st position is mutated to Ile, denoted as the AhlX mutant M45;

[0105] 32) The Glu at the 77th position is mutated to Ile, the Asp at the 157th position is mutated to Gly, the Thr at the 243rd position is mutated to Tyr, the His at the 255th position is mutated to Leu, and the Ala at the 261st position is mutated to Ile, denoted as the AhlX mutant M50.

[0106] The sequence of the deoxyribonucleic acid molecule contains one or more of the mutants in which the G at the 229th position, the A at the 230th position, the A at the 470th position, the A at the 727th position, the A at the 727th position, the C at the 728th position, the C at the 728th position, the A at the 764th position, the G at the 781st position, the C at the 782nd position, and the T at the 783rd position are mutated to A, T, G, T, G, A, T, T, A, C respectively, in the deoxyribonucleic acid sequence encoding the amino acid sequence of wild-type AHL-lactonase, in the 5'-to-3' direction;

[0107] The deoxyribonucleic acid molecule described above is the following DNA molecule:

[0108] (b1) A DNA molecule in which the coding region encodes the protein described in the first aspect of the present invention;

[0109] (b2) A coding region includes a DNA molecule encoding the protein described in the first aspect of the present invention;

[0110] (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;

[0111] (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.

[0112] Preferably, the deoxyribonucleic acid sequence encoding the amino acid sequence of the wild-type AHL-lactonase is the deoxyribonucleic acid sequence shown in SEQ ID NO.2 encoding the amino acid sequence of the wild-type AHL-lactonase shown in SEQ ID NO.1.

[0113] The deoxyribonucleic acid molecule with the deoxyribonucleic acid sequence shown in SEQ ID NO.2 encoding the wild-type AHL-lactonase with the amino acid sequence shown in SEQ ID NO.1 is denoted as the deoxyribonucleic acid molecule encoding AHL-lactonase AhlX in the present invention.

[0114] The present invention provides an expression cassette, a recombinant vector or a recombinant microorganism containing the above-mentioned deoxyribonucleic acid molecule, which can also be understood that the third aspect of the present invention provides an expression cassette, a recombinant vector or a recombinant microorganism containing the deoxyribonucleic acid molecule corresponding to the protein described in the first aspect of the present invention.

[0115] The present invention provides the application of the mutant described in the present invention, or the mutant encoded by the deoxyribonucleic acid molecule described in the present invention, or the mutant prepared from the expression cassette, recombinant vector or recombinant microorganism described in the present invention in the hydrolysis of ester bond-containing compounds and the biological control of bacterial infectious animal and plant diseases.

[0116] The present invention provides the application of the mutant described in the present invention, or the mutant encoded by the deoxyribonucleic acid molecule or the expression cassette, recombinant vector or recombinant microorganism described in the present invention in the hydrolysis of acyl-homoserine lactone and other esters.

[0117] Ⅲ. Specific Examples

[0118] The experimental methods described below are all conventional laboratory experimental methods unless otherwise specified. The experimental materials described below can all be obtained through commercial channels unless otherwise specified. The present invention will be further described below in combination with specific examples for better understanding of the present invention, but the protection scope of the present invention is not limited to the following description.

[0119] In some specific embodiments of the present invention, Escherichia coli BL21(DE3) is used as the expression host cell for the wild-type AHL-lactonase AhlX and its mutants. The medium formulations involved in the following examples are as follows:

[0120] LB liquid medium: Tryptone 10 g·L -1 、Yeast extract 5 g·L -1 、NaCl 10 g·L -1 ;

[0121] LB solid medium: Agar powder 18 g·L -1, Peptone 10 g·L -1 , Yeast extract 5 g·L -1 , NaCl 10 g·L -1 ;

[0122] 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 ;

[0123] Example 1: Construction of AHL-lactonase mutant expression strains

[0124] The AHL-lactonase used in this example is AhlX (GenBank: KY783591.1, CN105543193A), and the species source of its encoding gene is Salinicola salaries MCCC1A01339. The amino acid sequence of AhlX is shown in SEQ ID NO.1, and one of the deoxyribonucleic acid sequences of the encoding gene of AhlX is shown in SEQ ID NO.2.

[0125] Single-point mutations or multi-point mutations were performed on AhlX respectively:

[0126] Single-point mutations of AhlX were achieved by one-step site-directed mutagenesis. Using the expression plasmid pET-28a- of wild-type AhlX ahlX as a template, mutation primers were designed at the preset mutation sites, and mutations were introduced into the expression vector by PCR amplification. After digestion of the PCR amplification product with DpnI enzyme to remove the template plasmid, it was transformed into Escherichia coli BL21(DE3) host cells to construct the corresponding AhlX single-point mutant expression strains. Based on the single-point mutation plasmid, single-point mutations were performed again or Gibson assembly ( Figure 1 ) was used to construct the multi-point mutant expression plasmid of AhlX. The corresponding AhlX multi-point mutant expression plasmid and the molecular chaperone expression plasmid pGro7 were co-transformed into E. coli BL21(DE3) host cells to construct the corresponding AhlX multi-point mutant expression strains.

[0127] Example 2: Preparation of AHL-lactonase AhlX mutants

[0128] (1) Expression of wild-type and mutant AhlX

[0129] The AhlX single-point mutant expression strain and the AhlX wild-type expression strain constructed in Example 1 were inoculated into 5 mL of TB liquid medium containing 50 μg·mL -1 kanamycin, and the AhlX multi-point mutant expression strain was inoculated into 5 mL of TB liquid medium containing 50 μg·mL -1 kanamycin, 25 μg·mL -1 streptomycin, and 4 mg·mL -1 L-arabinose; after shaking culture at 37 °C and 220 rpm for 4 h, the temperature was adjusted to 30 °C and the rotation speed was adjusted to 150 rpm for continued culture for 16 h; after centrifuging to collect the bacterial cells, 5 mL of bacterial cell lysis buffer (50 mM K2HPO4, 300 mM NaCl, 5 mM imidazole, 10% glycerol, pH adjusted to 8.0 with NaOH) was added to resuspend the bacterial cells; ultrasonic disruption was performed on ice bath for 10 min, the supernatant of the cell lysate was collected by centrifugation, and the supernatant was filtered through a 0.45 μm filter membrane to collect the filtrate, and the obtained filtrate was the crude enzyme solution.

[0130] (2) Protein purification

[0131] Prepare a small-scale protein purification nickel column by filling Ni-NTA 6FF into a centrifugal microprotein purification empty column. The volume of Ni-NTA 6FF loaded into each column is approximately 350 μL; rinse and equilibrate the nickel column five times with 400 μL of 50 mM imidazole buffer (50 mM imidazole, 250 mM K2HPO4, 1.5 M NaCl, adjust the pH to 8.0) (the intermediate centrifugation conditions are 200 rpm and 15 s); aspirate 400 μL of the crude enzyme solution and add it to the nickel column, let it stand at room temperature for 1 min and then centrifuge at 200 rpm for 15 s, pour off the lower filtrate after centrifugation; repeat the above steps until all the crude enzyme solution is processed; aspirate 400 μL of 50 mM imidazole buffer and add it to the nickel column, centrifuge at 200 rpm for 15 s to elute the impurity proteins, and repeat this step 6 times; add 400 μL of 250 mM imidazole buffer (250 mM imidazole, 250 mM K2HPO4, 1.5 M NaCl, adjust the pH to 8.0), centrifuge at 200 rpm for 15 s to elute the target protein and collect the filtrate, repeat this step 6 times; rinse the PD-10 desalting column with 15 mL of ultrapure water filtered through a 0.45 μm filter membrane and then equilibrate the desalting column with 15 mL of protein desalting buffer (50 mM K2HPO4, 100 mM NaCl, 10% glycerol, 1 mM DTT, adjust the pH to 8.0); supplement the volume of the target protein eluate obtained in the previous step to 2.5 mL with the protein desalting buffer and transfer it to the equilibrated desalting column. After the 2.5 mL of the target protein eluate flows through the desalting column by gravity, add 3.5 mL of desalting buffer and collect the target protein solution that flows through by gravity.

[0132] (3)Concentration and Concentration Determination of Protein

[0133] Add the target protein solution purified by the nickel column and desalted by the desalting gel column to a 10 kDa ultrafiltration centrifugal tube, and centrifuge at 6000 rpm and 4 °C until the volume of the target protein solution in the concentrator tube remains about 2 mL. Use the improved Bradford protein concentration determination kit produced by Sangon Biotech (Shanghai) Co., Ltd. to determine the protein concentration.

[0134] Example 3: Enzyme Activity Assay of AHL-Lactonase AhlX Mutant

[0135] When using the HPLC method to determine the activity, it is not suitable for the determination of the activity of a large number of samples. According to the characteristic that AHL lactonase has lipase activity, a high-throughput screening method for the activity determination of wild-type AhlX and its mutants was constructed using 4-nitrophenyl butyrate (p-Nitrophenyl butyrate, pNPB), which is commonly used in the determination of lipase activity, as the substrate.

[0136] Drawing of the p-nitrophenol standard curve: Prepare a p-nitrophenol standard solution with a concentration of 10 - 100 μM using a 10 mM phosphate buffer solution at pH 8.0 as the solvent. Use the 10 mM phosphate buffer solution group at pH 8.0 as the blank control, measure the absorbance value at a wavelength of 405 nm, and draw the standard curve in the Origin Pro 2021 software with the p-nitrophenol concentration as the abscissa and OD 405 as the ordinate.

[0137] Using pNPB as the substrate, calculate the enzyme activity by measuring the concentration of p-nitrophenol generated after the enzyme hydrolyzes pNPB. The specific measurement method is as follows: Prepare a 100 μM pNPB solution using a 10 mM phosphate buffer solution at pH 8.0 as the solvent (this solution needs to be prepared and used immediately); add 5 μL of the purified 0.64 μM enzyme solution to a 96-well plate, then immediately add 200 μL of the 100 mM pNPB solution to the 96-well plate, and immediately place it in a microplate reader to measure the OD of the system after reacting at room temperature for 20 min 405 . Use the control group with 5 μL of ultrapure water added as the blank, and perform three replicates for each group. Calculate the concentration of p-nitrophenol generated within 20 min by measuring the OD 405 . Take the concentration of p-nitrophenol generated in the wild-type AhlX group as 100% activity, and calculate the relative activity of the mutants.

[0138] Before the thermal stability measurement, heat the enzyme at 70 °C for 30 min, and then immediately place it in normal-temperature water to cool to room temperature. Use the above method to measure the activity of the heat-treated enzyme. The relative thermal stability is calculated by dividing the concentration of p-nitrophenol generated during the enzyme activity measurement after heat treatment by the concentration of p-nitrophenol generated during the enzyme activity measurement before heat treatment, and then multiplying by 100%.

[0139] The enzyme activity detection results of wild-type AhlX and single-point mutants are as Figure 2 shown. Compared with wild-type AhlX, the enzyme activities of AhlX mutants E77I, D157G, T243Y, T243V, H255L, and A261I are all improved to different degrees, and their relative activities are 288%, 287%, 108%, 124%, 193%, and 614% of wild-type AhlX, respectively.

[0140] The thermal stability test results of wild-type AhlX and single-point mutants are as follows Figure 3 shown. After the wild-type AhlX was heat-treated at 70 °C for 30 min, the remaining activity was 70% of that before heat treatment. After the AhlX mutants E77I, D157G, T243Y, T243V, H255L, and A261I were heat-treated at 70 °C for 30 min, the remaining activities were 65%, 63%, 83%, 71%, 60%, and 64% of that before heat treatment, respectively.

[0141] The above results indicate that the enzyme activities of the six single-point mutants are all improved to varying degrees compared with the wild-type AhlX, and there is a trade-off effect between the stability and activity of the single-point mutants. The single-point mutants E77I, D157G, H255L, and A261I with relatively high enzyme activity all show varying degrees of decrease in stability. The enzyme activity of the single-point mutant T243Y with significantly improved thermal stability changes little compared with the wild-type AhlX.

[0142] The combined mutations of E77I, D157G, T243Y, H255L, and A261I were performed to construct 26 multi-point mutants. The enzyme activity test results are as follows Figure 4 shown, and the stability test results are as follows Figure 5 shown. From the stability results, the stabilities of most mutants are improved, while the stabilities of a few mutants are decreased. In terms of enzyme activity performance, except for the enzyme activity of the quadruple mutant M45 (D157G / T243Y / H255L / A261I) showing a drastic decrease, the enzyme activities of the other combined mutants are all higher than that of the wild-type. The mutant with the highest enzyme activity is the quadruple mutant M41 (E77I / D157G / T243Y / H255L), and its relative activity is 1767% of the wild-type, and its stability is also increased by 13% compared with the wild-type. The above results indicate that M41 is the AhlX multi-point mutant with the best comprehensive performance in terms of stability and activity.

[0143] Example 4: Analysis of the stability of AHL-lactonase AhlX mutants by circular dichroism

[0144] The circular dichroism spectrometer Chirascan™-plus was used to measure the changes in the secondary structure of wild-type AhlX and mutant M41 (E77I / D157G / T243Y / H255L) at different temperatures, and then analyze the effect of temperature on the stability of their secondary structure. Using 10 mM phosphate buffer solution at pH 8.0 as the solvent, the wild-type AhlX and mutant M41 were diluted to 0.25 mg·mL -1 ; The temperature was increased at a rate of 1 °C·min -1 in the temperature range of 29~95 °C, and 0.25 mg·mL -1Circular dichroism spectral data of wild-type AhlX and mutant M41 in the wavelength range of 185 - 260 nm; data processing and analysis were performed using CDtoolX, CDNN, and Origin Pro 2021 software.

[0145] To further explore the difference in thermodynamic stability between wild-type AhlX and its mutant M41, circular dichroism spectroscopy analysis was performed on both of them, and the results are as Figure 6 shown. The positive peak of the α-helix at 192 nm of wild-type AhlX and mutant M41 continuously decreased in peak intensity with the increase in temperature, and this positive peak showed a blue shift. This result indicates that the α-helix structure decreased in both wild-type AhlX and mutant M41 during the thermal denaturation process. However, different from wild-type AhlX, the rate of decrease in peak intensity and blue shift of the α-helix positive peak at 192 nm in mutant M41 was slower during the heating process, and the final decrease in peak intensity and blue shift value were both smaller than those of wild-type AhlX. This result shows that the rate and amount of decrease in the α-helix structure in mutant M41 were less than those of wild-type AhlX during the heating process. At the same time, the degree of change in the secondary structure of mutant M41 at the end point of thermal denaturation was lower than that of wild-type AhlX. It can be seen from the above results that the thermodynamic stability of mutant M41 is higher than that of wild-type AhlX. As Figure 6 shown in T m Figure B, the T m value of wild-type AhlX was 54.81 °C, and the T m value of mutant M41 was 66.94 °C. Compared with wild-type AhlX, the T m value of mutant M41 increased by 12.13 °C, further confirming that mutant M41 has better thermodynamic stability.

[0146] Example 5: Application of AHL-lactonase AhlX mutant in bacterial infectious diseases

[0147] (1) Bacterial concentration determination

[0148] The plant soft rot pathogen Erwinia carotovora was inoculated into an LB test tube and cultured overnight at 30 °C with shaking at 220 rpm. The colony-forming units (CFU) of the overnight cultured bacterial solution were measured. The E. carotovora culture solution was serially diluted with sterile water to a concentration of 10 -6 and 10 -7 times of the original solution. 100 μL of the above diluted bacterial solution was taken and spread on an LB culture dish, and then cultured overnight at 30 °C in an inverted position to calculate the colony-forming units, and the final bacterial concentration was calculated according to the dilution factor.

[0149] (2)Surface disinfection of vegetables

[0150] Rinse the zucchini, radish, green pepper, Chinese cabbage, eggplant and peeled potato thoroughly with running water. Inside the ultra-clean workbench, soak the above-mentioned vegetables in 75% ethanol for 10 s and then quickly rinse them with sterile water. Cut the above-mentioned vegetables into slices 0.3 - 0.4 cm thick with a sterile knife, place them in a sterilized glass petri dish lined with filter paper, and make a circular wound at the center of the slice with a sterilized awl.

[0151] (3)Biocontrol effect of mutant M41 on E. carotovora

[0152] E. carotovora Dilute the overnight culture broth with sterile water to a bacterial concentration of 3.0×10 7 , 1.5×10 8 and 1.5×10 9 CFU·mL -1 . At the circular wounds of the potato, zucchini, and green pepper slices, inoculate 11 μL of 10 mM phosphate buffer (pH 8.0), 1 μL of a mixture of 3.0×10 7 CFU·mL -1 E. carotovora bacterial broth and 10 μL of 10 mM phosphate buffer (pH 8.0), and 1 μL of a mixture of 3.0×10 7 CFU·mL -1 E. carotovora bacterial broth and 10 μL of 3.2 μM mutant M41; the potato and zucchini groups are placed at 30 °C for 5 days and then photographed to record the infection situation, and the green pepper group is placed at 30 °C for 3 days and then photographed to record the infection situation. At the circular wounds of the radish and Chinese cabbage slices, inoculate 11 μL of 10 mM phosphate buffer (pH 8.0), 1 μL of a mixture of 1.5×10 8 CFU·mL -1 E. carotovora bacterial broth and 10 μL of 10 mM phosphate buffer (pH 8.0), and 1 μL of a mixture of 1.5×10 8 CFU·mL -1 E. carotovora bacterial broth and 10 μL of 3.2 μM mutant M41; the radish group is placed at 30 °C for 3 days and then photographed to record the infection situation, and the Chinese cabbage group is placed at 30 °C for 2 days and then photographed to record the infection situation. At the circular wounds of the eggplant slices, inoculate 11 μL of 10 mM phosphate buffer (pH 8.0), 1 μL of 1.5×10 9 CFU·mL -1 E. carotovoraA mixture of the bacterial solution and 10 μL of 10 mM phosphate buffer (pH 8.0), 1 μL of 1.5×10 9 CFU·mL -1 E. carotovora A mixture of the bacterial solution and 10 μL of 3.2 μM mutant M41; Incubate at 30 °C for 2 days and then take pictures to record the infection situation. Each sample was set with three replicates.

[0153] The biocontrol results of mutant M41 against E. carotovora are as shown in Figure 7 The lesion areas of soft rot in the mutant M41 and E. carotovora the co-treatment group were significantly lower than those in the E. carotovora treatment group. Mutant M41 can effectively alleviate various plant soft rots caused by E. carotovora .

[0154] It should be noted that the above-described embodiments are only preferred embodiments of the present invention for the explanation of the present invention and do not constitute any limitation to the present invention. The present invention has been described by referring to typical embodiments, but it should be understood that the words used in the embodiments are descriptive and explanatory words rather than limiting words. The present invention can be modified within the scope of the claims of the present invention as specified, and can be revised without departing from the scope and spirit of the present invention. Although the description of the present invention involves specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific embodiments therein. On the contrary, the present invention can be extended to all other methods and applications with the same functions.

Claims

1. A mutant of N-acyl homoserine lactonase, characterized in that The N-acyl homoserine lactonase is any one of the following proteins: (a1): a protein with the amino acid sequence of SEQ ID NO.1; (a2): a fusion protein obtained by connecting a tag to the amino terminus and / or carboxyl terminus of the protein defined by (a1); The mutation sites of the mutant are that Glu at the 77th position from the amino terminus to the carboxyl terminus is mutated to Ile, Asp at the 157th position is mutated to Gly, Thr at the 243rd position is mutated to Tyr, and His at the 255th position is mutated to Leu.

2. A deoxyribonucleic acid molecule encoding the mutant according to claim 1.

3. Use of a mutant as described in claim 1 in hydrolysis of compounds containing an ester bond and biological control of bacterial infectious plant diseases, characterized in that, The ester bond-containing compound is 4-nitrophenyl butyrate, and the bacterial infectious plant disease is soft rot of zucchini, radish, green pepper, Chinese cabbage, eggplant and potato caused by the pathogen of plant soft rot.

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