Alpha / beta-hydrolase superfamily macrolide esterase mutants and uses thereof

Abstract

The application discloses an alpha / beta-hydrolase superfamily macrolide esterase mutant and application thereof, wherein the alpha / beta-hydrolase superfamily macrolide esterase mutant is mutated from Met at the 30th position of the amino acid sequence shown in SEQ ID NO:1 to Ala, mutated from Gly at the 31st position to Ala, mutated from Ala at the 32nd position to Asp, mutated from Gln at the 33rd position to Ala, and mutated from Trp at the 38th position to Ala, and the rest positions remain unchanged, and the amino acid sequence is shown in SEQ ID NO:6; mutated from Phe at the 77th position to Ala, mutated from Met at the 101st position to Ala, and mutated from Phe at the 130th position to Ala. The mutant of the alpha / beta-hydrolase superfamily macrolide esterase is significantly superior to the wild-type enzyme in terms of protein expression level and catalytic activity, and the recombinant expression amount is increased, and the specific enzyme activity is obviously enhanced. The successful construction of the mutant provides an excellent candidate for the application of the alpha / beta-hydrolase superfamily in the biological catalysis of macrolide compounds, and expands the enzyme resource library that can be used for the degradation of the compounds.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a mutant of an α / β-hydrolase superfamily of macrocyclic lactone esterases and its applications. Background Technology

[0002] Since their introduction in the 1950s, macrolide antibiotics have been widely used in agriculture and hospitals. Macrolide antibiotics are a class of antibiotics widely used clinically and can effectively treat a variety of bacterial infections.

[0003] However, the extensive use of macrolide antibiotics has had a significant impact on the environment. Macrolide antibiotics that are not adequately treated are mainly 16-membered ring antibiotics. They are discharged into the environment through livestock and poultry waste and sewage, leading to their long-term accumulation in the natural environment. This promotes the development of antibiotic resistance in bacteria and the transfer of antibiotic resistance genes, thus arousing widespread concern about the environment and public health.

[0004] Currently, biodegradation, especially enzymatic degradation, is considered a powerful means of treating this type of pollution due to its advantages of low cost, high efficiency, and no secondary pollution. Therefore, finding macrolide esterases with high efficiency, high enzyme activity, and broad substrate spectrum is particularly important for subsequent enzymatic degradation.

[0005] The currently used α / β-hydrolase superfamily of macrolide esterases is derived from the Gram-positive bacterium *Brevibacillus brevis*. These esterases can significantly degrade 16-membered ring human and veterinary antibiotics, including tylosin, tilmicosin, tebuconazole, josamycin, midecamycin, spiramycin, and acetylspiramycin. However, the low heterologous expression levels and insufficient enzyme activity of these esterases severely limit their potential as biocatalytic tools, making it difficult to utilize them to expand the library of macrolide compounds available for synthesis. Summary of the Invention

[0006] The purpose of this invention is to provide a mutant of the α / β-hydrolase superfamily of macrolide esterases and its applications. The mutant exhibits significantly increased expression levels and effectively enhances enzyme activity.

[0007] The objective of this invention is achieved as follows:

[0008] A mutant of the α / β-hydrolase superfamily of macrolide esterases, obtained by performing any of the following steps on the amino acid sequence shown in SEQ ID NO: 1:

[0009] Mutant 1, wherein mutant 1 is based on the amino acid sequence shown in SEQ ID NO: 1, wherein the 30th position Met is mutated to Ala, and the other positions remain unchanged, and its amino acid sequence is shown in SEQ ID NO: 2;

[0010] Mutant 2, wherein mutant 2 is based on the amino acid sequence shown in SEQ ID NO: 1, wherein the 31st position Gly is mutated to Ala, and the other positions remain unchanged, and its amino acid sequence is shown in SEQ ID NO: 3;

[0011] Mutant 3, wherein mutant 3 is based on the amino acid sequence shown in SEQ ID NO: 1, wherein the 32nd position Ala is mutated to Asp, while the other positions remain unchanged, and its amino acid sequence is shown in SEQ ID NO: 4;

[0012] Mutant 4, wherein mutant 4 is based on the amino acid sequence shown in SEQ ID NO: 1, wherein the 33rd position Gln is mutated to Ala, and the other positions remain unchanged, and its amino acid sequence is shown in SEQ ID NO: 5;

[0013] Mutant 5, wherein mutant 5 is based on the amino acid sequence shown in SEQ ID NO: 1, wherein the Trp at position 38 is mutated to Ala, and the other positions remain unchanged, and its amino acid sequence is shown in SEQ ID NO: 6;

[0014] Mutant 6, wherein mutant 6 is based on the amino acid sequence shown in SEQ ID NO: 1, wherein Phe at position 77 is mutated to Ala, while the other positions remain unchanged, and its amino acid sequence is shown in SEQ ID NO: 7;

[0015] Mutant 7, wherein mutant 7 is based on the amino acid sequence shown in SEQ ID NO: 1, wherein the 101st Met position is mutated to Ala, and the other positions remain unchanged, and its amino acid sequence is shown in SEQ ID NO: 8;

[0016] Mutant 8 is based on the amino acid sequence shown in SEQ ID NO: 1, in which Phe at position 130 is mutated to Ala, while the other positions remain unchanged, and its amino acid sequence is shown in SEQ ID NO: 9.

[0017] The application of α / β-hydrolase superfamily macrolide esterase mutants as biocatalysts for the degradation of macrolide antibiotics.

[0018] The mutants are mutant 1 (Met at position 30 mutated to Ala), mutant 5 (Trp at position 38 mutated to Ala), mutant 6 (Phe at position 77 mutated to Ala), mutant 7 (Met at position 101 mutated to Ala), and mutant 8 (Phe at position 130 mutated to Ala). Mutants 1, 5, 6, 7, and 8 all increase the activity of macrolide esterase.

[0019] The beneficial effects of this invention are as follows: The mutant of the α / β-hydrolase superfamily macrolide esterase is significantly superior to the wild-type enzyme in terms of both protein expression level and catalytic activity. Its recombinant expression level is increased, and its specific enzyme activity is significantly enhanced. The successful construction of this mutant provides an excellent candidate for the application of the α / β-hydrolase superfamily in the biocatalysis of macrolide compounds, and expands the enzyme resource library that can be used for the degradation of these compounds. Attached Figure Description

[0020] Figure 1 This is a standard curve for enzyme activity determination according to the present invention;

[0021] Figure 2 This is a bar chart showing the relative enzyme activity of BRB-1 and different mutants of the present invention;

[0022] Figure 3 This is a diagram of the inhibition zone of the mutant W38A of this invention before reaction with tylosin;

[0023] Figure 4 This is a diagram of the inhibition zone after the mutant W38A of this invention reacts with tylosin.

[0024] Figure 5 This is a mass spectrometry analysis of the mutant W38A of this invention before and after reaction with tylosin. Detailed Implementation

[0025] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0026] A mutant of the α / β-hydrolase superfamily of macrolide esterases, obtained by performing any of the following steps on the amino acid sequence shown in SEQ ID NO: 1:

[0027] Mutant 1, wherein mutant 1 is based on the amino acid sequence shown in SEQ ID NO: 1, wherein the 30th position Met is mutated to Ala, and the other positions remain unchanged, and its amino acid sequence is shown in SEQ ID NO: 2;

[0028] Mutant 2, wherein mutant 2 is based on the amino acid sequence shown in SEQ ID NO: 1, wherein the 31st position Gly is mutated to Ala, and the other positions remain unchanged, and its amino acid sequence is shown in SEQ ID NO: 3;

[0029] Mutant 3, wherein mutant 3 is based on the amino acid sequence shown in SEQ ID NO: 1, wherein the 32nd position Ala is mutated to Asp, while the other positions remain unchanged, and its amino acid sequence is shown in SEQ ID NO: 4;

[0030] Mutant 4, wherein mutant 4 is based on the amino acid sequence shown in SEQ ID NO: 1, wherein the 33rd position Gln is mutated to Ala, and the other positions remain unchanged, and its amino acid sequence is shown in SEQ ID NO: 5;

[0031] Mutant 5, wherein mutant 5 is based on the amino acid sequence shown in SEQ ID NO: 1, wherein the Trp at position 38 is mutated to Ala, and the other positions remain unchanged, and its amino acid sequence is shown in SEQ ID NO: 6;

[0032] Mutant 6, wherein mutant 6 is based on the amino acid sequence shown in SEQ ID NO: 1, wherein Phe at position 77 is mutated to Ala, while the other positions remain unchanged, and its amino acid sequence is shown in SEQ ID NO: 7;

[0033] Mutant 7, wherein mutant 7 is based on the amino acid sequence shown in SEQ ID NO: 1, wherein the 101st Met position is mutated to Ala, and the other positions remain unchanged, and its amino acid sequence is shown in SEQ ID NO: 8;

[0034] Mutant 8 is based on the amino acid sequence shown in SEQ ID NO: 1, in which Phe at position 130 is mutated to Ala, while the other positions remain unchanged, and its amino acid sequence is shown in SEQ ID NO: 9.

[0035] The application of α / β-hydrolase superfamily macrolide esterase mutants as biocatalysts for the degradation of macrolide antibiotics. The mutants are mutant 1 (Met at position 30 mutated to Ala), mutant 5 (Trp at position 38 mutated to Ala), mutant 6 (Phe at position 77 mutated to Ala), mutant 7 (Met at position 101 mutated to Ala), and mutant 8 (Phe at position 130 mutated to Ala). Mutants 1, 5, 6, 7, and 8 all increase the activity of macrolide esterases.

[0036] The materials used in the experiment are as follows:

[0037] (1) Deionized water; (2) Water bath; (3) Timer; (4) Beaker; (5) Graduated cylinder; (6) Snowflake ice maker; (7) Ice box; (8) Pipette; (9) Pipette tip; (10) Ultraviolet spectrophotometer; (11) Quartz cuvette; (12) 2 mL EP tube; (13) 15 mL centrifuge tube; (14) 50 mL centrifuge tube; (15) EP tube rack; (16) Centrifuge tube rack; (17) pH meter; (18) Ni-smart beads, dialysis bag; (19) LB medium: Weigh 10.0 g of peptone, 5.0 g of yeast powder, and 10.0 g of sodium chloride, add 1 L of deionized water, mix thoroughly, and sterilize at 121℃ for 15 min.

[0038] (20) LB agar medium: Weigh 10.0 g of peptone, 5.0 g of yeast powder, 10.0 g of sodium chloride and 15.0 g of agar powder, add 1 L of deionized water, mix thoroughly and sterilize at 121℃ for 15 min.

[0039] (21) LB medium with kanamycin resistance: Weigh 10.0 g of peptone, 5.0 g of yeast powder and 10.0 g of sodium chloride and add 1 L of deionized water. After mixing thoroughly, sterilize at 121℃ for 15 min and then cool naturally to 50-60℃. Add kanamycin to a final concentration of 100 μg / mL.

[0040] (22) 1x SDS-PAGE buffer: Weigh 3.00 g Tris, 14.40 g g glycine and 1.00 g SDS, add deionized water to dissolve and bring the volume to 1 L.

[0041] (23) 5x Loading Buffer: Component concentrations: 1.00 M Tris-HCl (pH 6.8), 10% SDS, 0.5% (W / V) BPB, 50% (V / V) glycerol, 5% (W / V) 2-ME. Measure 1.25 mL of 1 M Tris-HCl (pH 6.8), 5 mL of SDS, 25 mg of BPB, 2.5 mL of glycerol, and 250 μL of 2-ME into a 15 mL plastic centrifuge tube. Dissolve the contents in deionized water, bring the volume to 5 mL, aliquot (0.5 mL / part), and store at 4°C for later use.

[0042] (24) 0.1% (w / v) Coomassie Brilliant Blue R-250: Measure 0.25 L of methanol, 0.65 L of deionized water, 0.1 L of glacial acetic acid, and 1.0 g of Coomassie Brilliant Blue R250 and mix them to dissolve for later use.

[0043] (25) Coomassie staining decolorizing solution: Measure 0.24 L of ethanol and 0.08 L of glacial acetic acid, and add deionized water to make up to 1 L.

[0044] (26) 10% ammonium persulfate solution: Weigh 0.1g of ammonium persulfate into a 2 mL centrifuge tube, add 1mL of deionized water and blow to dissolve, and store at 4℃.

[0045] (27) Buffer A (20 mmol / L Tris, 500 mmol / L NaCl, pH 7.0): Weigh 2.42 g Tris and 29.25 g NaCl into a 1 L reagent bottle, add 0.8 L of deionized water to dissolve, adjust the pH to 7.0 with 30% HCl solution and a pH meter, bring the volume to 1 L with deionized water, and filter with a 0.2 μm aqueous filter membrane.

[0046] (28) Buffer B (20 mmol / L Tris, 500 mmol / L NaCl, 500 mmol / L imidazole, pH=7.0): Weigh 2.42 g Tris, 29.25 g NaCl, and 34.04 g imidazole into a 1 L reagent bottle, add 0.8 L of deionized water to dissolve, adjust the pH to 7.0 with 30% HCl solution and a pH meter, and bring the volume to 1 L with deionized water. Filter the solution through a 0.2 μm aqueous filter membrane.

[0047] (29) 20 mmol / L tris-HCl: Weigh 2.42 g of Tris and dissolve it in 0.8 L of deionized water. Adjust the pH to 7.0 with 30% HCl and then bring the volume to 1 L with deionized water. Filter the solution using a 0.2 μm aqueous filter membrane.

[0048] (30) 10 mmol / L imidazole: Weigh 0.14 g imidazole, 0.48 g Tris and 5.85 g NaCl and dissolve them in 0.15 L of deionized water. Adjust the pH to 7.0 with 30% HCl and then make up to 0.2 L with deionized water. Filter the solution using a 0.2 μm aqueous filter membrane.

[0049] (31) 20 mmol / L imidazole: Weigh 0.27 g imidazole, 0.48 g Tris and 5.85 g NaCl and dissolve them in 0.15 L of deionized water. Adjust the pH to 7.0 with 30% HCl and then make up to 0.2 L with deionized water. Filter the solution using a 0.2 μm aqueous filter membrane.

[0050] (32) 50 mmol / L imidazole: Weigh 0.68 g imidazole, 0.48 g Tris and 5.85 g NaCl and dissolve them in 0.15 L of deionized water. Adjust the pH to 7.0 with 30% HCl and then make up to 0.2 L with deionized water. Filter the solution using a 0.2 μm aqueous filter membrane.

[0051] (33) 100 mmol / L imidazole: Weigh 1.36 g imidazole, 0.48 g Tris and 5.85 g NaCl and dissolve them in 0.15 L of deionized water. Adjust the pH to 7.0 with 30% HCl and then make up to 0.2 L with deionized water. Filter the solution using a 0.2 μm aqueous filter membrane.

[0052] (34) 200 mmol / L imidazole: Weigh 2.72 g imidazole, 0.48 g Tris and 5.85 g NaCl and dissolve them in 0.15 L of deionized water. Adjust the pH to 7.0 with 30% HCl and then make up to 0.2 L with deionized water. Filter the solution using a 0.2 μm aqueous filter membrane.

[0053] (35) Plasmid extraction kit

[0054] (36) 2 mM p-NPB (4-Nitrophenyl butyrate) was used as the substrate. The specific dissolution method of the substrate was as follows: 2 mM p-NPB, 20 mM Tris-HCl (pH=7.0), 0.1% Triton X-100, placed on ice for later use.

[0055] Plasmid extraction: Inoculate the bacterial strain containing the plasmid into LB agar medium with kanamycin resistance and incubate at 37°C for 8-12 h. Pick a single colony and inoculate it into 5 mL of LB liquid medium and incubate at 37°C on a shaker for 8-12 h. Collect the bacterial cells by centrifugation and extract the plasmid according to the instructions in the plasmid extraction kit.

[0056] Transformation: The extracted plasmid was introduced into BL 21(DE3) competent cells.

[0057] (1) To make ice, take out a tube (100 uL) of competent bacteria from the -80℃ ultra-low temperature freezer and insert it into the ice, and wait for the competent bacterial cells to dissolve.

[0058] (2) Add 5 μL of the ligated plasmid to 100 μL of competent cells, gently shake, and place on ice for 30 min.

[0059] (4) After gently shaking, insert it into a 42℃ water bath for 90 seconds for heat shock, then quickly put it back on ice and let it stand for 10 minutes.

[0060] (5) Add 800 uL of LB culture medium to each of the above tubes in a clean bench and mix gently. Then fix the tubes at 37 ℃ and shake for 1 h.

[0061] (6) After centrifugation, take 50 μL of the above conversion mixture in a clean bench and drop it onto solid LB medium containing kanamycin. Spread it evenly with a spreader.

[0062] (7) Mark the coated petri dish, place it in a 37°C constant temperature incubator for 30-60 minutes until the liquid on the surface has penetrated into the culture medium, then invert it and place it in a 37°C constant temperature incubator overnight.

[0063] Induction of expression: Positive single clones were picked and cultured overnight at 37°C in 5 mL LB medium as seed culture. 100 μL of seed culture was inoculated into 100 mL of medium (500 mL Erlenmeyer flask). 100 μg / mL kanamycin was always present in the medium. The culture was incubated at 37°C in a shaker until the OD value reached 0.6-0.8. Then, IPTG (1 mM) was added for induction at 25°C for 16 h to obtain macrolide esterase BRB-1 and its mutant.

[0064] Protein purification

[0065] (1) Collect the bacterial cells by centrifugation after induction. Centrifugation conditions: 6500 rpm, 5 min.

[0066] (2) Resuspension and cell disruption: The centrifuged bacterial solution was resuspended with Buffer A and placed in a cell disruptor to disrupt the cell until the resuspension was transparent.

[0067] (3) After the resuspension is broken, it is dispensed and centrifuged to collect the supernatant.

[0068] (4) Column Packing: Vertically fix the empty chromatography column on the iron stand, clamping the lower outlet with a clip or capping it. Add approximately 1 / 3 column volume of Buffer A to the empty column. Slowly pour the prepared, uniformly suspended Ni-smartbeads into the column one at a time, continuously along the inner wall. The packing will settle naturally within the column bed. Once all the packing has settled, a clear interface will be visible. Attach the extension tube or cap to the top of the column (but do not tighten it, leaving it open to the atmosphere). Compact the column bed. Compact until the column height no longer changes and the interface is smooth. A high-quality column bed should be uniform, crack-free, and bubble-free.

[0069] (5) Buffer A Equilibration: After the column bed is compacted and leveled, switch to normal operating flow rate and flow equilibration buffer A through the column. Ensure that the environment inside the column is completely consistent with the sample environment to create optimal conditions for subsequent protein binding. After equilibration (after the UV absorbance of the effluent stabilizes), turn off the peristaltic pump and prepare for sample loading.

[0070] (6) Sample loading: After the cell wall is broken, the supernatant is fed into the nickel column through a peristaltic pump for sample loading.

[0071] (7) Buffer A rinsing: After all the sample has entered the column, continue to rinse the column with Buffer A.

[0072] (8) Rinse off contaminating proteins with 10-20mM imidazole.

[0073] (9) Elute the target protein with 100-200mM imidazole and collect the target protein eluent.

[0074] (10) Buffer B rinse: Finally, rinse the column with a high concentration of imidazole.

[0075] (11) The protein elution buffer was dialyzed three times in a 20 mmol / L tris-HCl liquid environment to remove imidazole.

[0076] The mutant with higher enzyme activity was obtained through the following experiments:

[0077] A crucial prerequisite for accurate enzyme activity assays is a highly reliable p-NP standard curve. First, it's necessary to explore the relationship between OD405nm value and p-NP concentration. Precisely measured p-NP standards were prepared into solutions of 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, and 0.1 mg / mL. These solutions were then measured at OD405nm. A linear regression analysis was performed using OD405nm absorbance versus p-NP concentration to obtain the standard curve. (See [link to curve]). Figure 1 .

[0078] The results are as above. Figure 1 As shown, the equation of the curve is Y = 1.019*X + 0.02817, R... 2= A value of 0.9979 shows good fitting and can be used for subsequent detection of macrolide esterase activity.

[0079] Detection:

[0080] p-Nitrophenylbutyrate (p-NPB) was used as the substrate to determine enzyme activity, with a final concentration fixed at 2 mM. The experiment was conducted in 1.5 mL centrifuge tubes, with three parallel experimental tubes and one blank control tube per group, for a total of four reaction tubes. Each group of sample tubes was numbered and clearly labeled.

[0081] The specific procedures are as follows: Accurately pipette 990 μL of p-NPB substrate solution into each reaction tube using a 1 mL pipette, then add 10 μL of wild-type and mutant enzyme solutions to bring the total reaction volume to 1 mL. Incubate the reaction system in a 37°C water bath for 10 minutes. Immediately after the reaction, remove the tube and quickly transfer the reaction solution to a quartz cuvette. Measure the absorbance (OD value) at 405 nm using a UV-Vis spectrophotometer. Before each measurement, zero the sample by using an equal volume of Tris-HCl buffer (pH 7.0) as a blank to subtract background absorption. In the blank control group, an equal volume of Tris-HCl buffer (pH 7.0) is used instead of the enzyme solution; the remaining steps are identical to the sample group. Each experiment is independently repeated at least three times to ensure reliable results. An enzyme activity unit is defined as the amount of enzyme required to catalyze the production of 1 μmol of p-nitrophenol (p-NP) per minute under the assay conditions. Based on the measured OD value and the standard molar extinction coefficient of p-NP, its generation rate and sample enzyme activity can be calculated.

[0082] Table 1 OD of BRB-1 and different mutants after reacting with substrates 405nm

[0083] protein name control group Experimental group 1 Experimental group 2 Experimental group 3 protein concentration Wild type (BRB-1) 0.333 0.594 0.628 0.598 2.4 Mutant 1 (M30A) 0.403 0.677 0.687 0.694 6.86 Mutant 2 (G31A) 0.325 0.486 0.537 0.544 5.09 Mutant 3 (A32D) 0.328 0.331 0.325 0.321 7.14 Mutant 4 (Q33A) 0.523 0.628 0.713 0.747 5.56 Mutant 5 (W38A) 0.311 0.695 0.677 0.684 6.31 Mutant 6 (F77A) 0.354 0.695 0.688 0.692 5.77 Mutant 7 (M101A) 0.33 0.637 0.640 0.655 3.76 Mutant 8 (F130A) 0.412 0.744 0.744 0.743 8.32

[0084] As shown in Table 1, this invention measured the absorbance values ​​of wild-type esterase and its mutants in reaction with the substrate p-NPB at a wavelength of 405 nm, and detected the total protein concentration after cell lysis. Based on the obtained data, the specific enzyme activity of each enzyme sample was further determined by calculating the amount of p-nitrophenol (p-NP) generated per unit time, and the relative enzyme activity was obtained by converting it to wild-type enzyme activity as a benchmark.

[0085] like Figure 2As shown, the results indicate that site-directed mutations at key conserved sites in the α / β-hydrolase superfamily esterases resulted in significant differences in enzyme activity among different mutants. Mutant 5 (W38A) exhibited the highest catalytic efficiency, with a relative activity reaching 141% of the wild type (BRB-1), confirming that this mutation significantly enhances enzyme activity. This improvement in catalytic efficiency may be achieved by reducing side chain volume, enhancing substrate binding channel accessibility, or optimizing the microenvironment of the active site. Mutants 6 (F77A), 8 (F130A), and 7 (M101A) also showed some degree of activity enhancement (relative activities of 126%, 123%, and 116%, respectively). In contrast, mutants 2 (G31A) and 4 (Q33A) showed significantly reduced enzyme activity, with relative activities of only 69% and 59%, respectively, suggesting that these sites may play a crucial role in the catalytic process of this esterase. Of particular note is that mutant 3 (A32D) showed no detectable enzyme activity (ND) under the assay conditions, indicating that mutations at this site may lead to severe disruption of the enzyme structure or failure of the active site. The enzyme activity of mutant 1 (M30A) was close to that of the wild type (104%), suggesting that amino acid substitutions at this site had a relatively small impact on enzyme function. Mutations at sites in mutants 2 (G31A), 4 (Q33A), and 3 (A32D) resulted in a sharp decrease in enzyme activity, or even complete inactivation, indicating that these residues play an irreplaceable role in maintaining enzyme structural stability, transition state binding, or the formation of the catalytic ternary structure. These experimental results clearly demonstrate the effectiveness of the site-directed mutagenesis strategy. Not only was a higher-efficiency esterase mutant 5 (W38A) successfully obtained, providing an excellent candidate enzyme for subsequent industrial biocatalysis applications, but loss-of-function mutations also revealed several conserved sites crucial to the activity of this superfamily of enzymes, providing experimental evidence for a deeper understanding of its catalytic mechanism.

[0086] This invention utilizes p-nitrophenylbutyrate (p-NPB) as a substrate and conducts an enzymatic reaction in a Tris-HCl buffer system at pH 7.0 and 37°C. By employing site-directed mutagenesis to modify the α / β-hydrolase superfamily esterases, mutants with significantly enhanced enzyme activity were successfully obtained. The optimal mutant, 5 (W38A), exhibits a tryptophan-to-alanine mutation at position 38, achieving a soluble expression level 2.63 times that of the wild type, a specific enzyme activity 1.41 times higher than the wild type, and a relative enzyme activity of 141%. This mutant demonstrates excellent catalytic efficiency and application potential, suitable for the biocatalytic processes of macrolides.

[0087] The preparation method for tylosin stock solution is as follows: Accurately weigh 0.020 g of tylosin standard, dissolve it in 1 mL of ethanol to prepare a stock solution with a concentration of 20 mg / mL, and store it at -20 °C for later use. Working concentration tylosin solutions can be obtained by serially diluting the above stock solution using Buffer A.

[0088] To verify the degradation ability of the esterase mutant W38A on tylosin, its antibacterial activity was assessed by assay and electrospray mass spectrometry.

[0089] The specific steps are as follows:

[0090] To assess the antibacterial activity of antibiotics before and after degradation, *Staphylococcus aureus* strain ATCC 25923, sensitive to macrolides, was selected. The specific procedure was as follows: 15 μL of overnight cultured *Staphylococcus aureus* (OD600≈1.0) was mixed with 15 mL of LB agar containing 1.5% agar, preheated to approximately 40°C. After thorough mixing, the agar was poured into 9 cm × 9 cm petri dishes. After the agar solidified, three 4.5 mm diameter wells were punched on the surface using a sterile punch. Then, 30 μL of enzyme-treated 10 μg / mL tylosin solution (and a corresponding control solution without enzyme) was added to each well. The petri dishes were incubated at 37°C for 24 hours, and the antibacterial activity was assessed by measuring the diameter of the inhibition zone.

[0091] 900 μL of a 50 μg / mL tylosin solution was mixed with the esterase mutant W38A. The reaction mixture was incubated overnight at 37°C, with an inactivated enzyme (inactivated by adding methanol to a final concentration of 50% and reacting for 10 minutes) as a control. Antibiotic degradation products were analyzed by electrospray ionization mass spectrometry (ESI-MS). The mass spectrometry scan range was 100 m / z to 1200 m / z. The ion source parameters were as follows: curtain gas pressure 40 psi, ion source gas 1 pressure 30 psi, ion source gas 2 pressure 30 psi, ESI+ voltage 5500 V, ESI- voltage 4500 V. The drying gas temperature and flow rate were maintained at 450°C and 5 L / min, respectively.

[0092] It was observed that the inhibition zone of the tylosin solution completely disappeared after degradation treatment. Figure 3 This indicates that its antibacterial activity has been largely lost. Furthermore, mass spectrometry analysis was used to identify the degradation products, and the results showed that ( Figure 5 Compared to the undegraded substrate (tylosin: 916.30→934.40 m / z), the degradation substrate showed a +18 Da mass shift, consistent with the lactone ring hydrolysis reaction. Figure 4This indicates that the esterase mutant W38A can effectively degrade tylosin through ester bond ring opening.

[0093] This invention utilizes p-nitrophenylbutyrate (p-NPB) as a substrate and conducts an enzymatic reaction in a Tris-HCl buffer system at pH 7.0 and 37°C. Site-directed mutagenesis was used to modify the α / β-hydrolase superfamily esterases, successfully obtaining mutants with significantly enhanced enzyme activity. The optimal mutant, W38A, exhibits a tryptophan-to-alanine mutation at position 38, achieving a 2.63-fold increase in soluble expression and a 1.41-fold increase in specific activity compared to the wild type, with a relative enzyme activity of 141%. This mutant demonstrates good catalytic efficiency and application potential, suitable for the biocatalytic processes of macrolides. Subsequent inhibition zone and mass spectrometry analysis confirmed the effective degradation of tylosin by the esterase mutant W38A. This mutant provides a novel, highly efficient, and specific biocatalyst for the green removal of tylosin residues from the environment, and has potential value in controlling the spread of antibiotic resistance.

[0094] SEQ ID NO: 1

[0095] MAEQILKVNGVEICAESFGKPTDPAILLIMGAQMSMLWWEEEFCQRIADAGRFVIRFDNRDVGRSTTYEVGQPGYTFEDMADDAVHVLDAFGVQQAHFVGMSMGGMLTQMIALRHPERVRTITLHATSNFAPGLPPIDEKL MEFFSKMGEINWEDEKEALEAAVASWKVLSGSKHPFDESRVRELAKIDIARSNHYASRNNHAFVTASEPYLLRTAEIAVPALVIHGTEDLLIPFAHALHLANTIPGAVLLTLEGTGHELPYGDWDVVIEAILKHTSGRRVRL

[0096] SEQ ID NO: 2

[0097] MAEQILKVNGVEICAESFGKPTDPAILLIAGAQMSMLWWEEEFCQRIADAGRFVIRFDNRDVGRSTTYEVGQPGYTFEDMADDAVHVLDAFGVQQAHFVGMSMGGMLTQMIALRHPERVRTITLHATSNFAPGLPPIDEKLMEFFSKMGEINWEDEKEALEAAVASWKVLSGSKHPFDESRVRELAKIDIARSNHYASRNNHAFVTASEPYLLRTAEIAVPALVIHGTEDLLIPFAHALHLANTIPGAVLLTLEGTGHELPYGDWDVVIEAILKHTSGRRVRL

[0098] SEQ ID NO:3

[0099] MAEQILKVNGVEICAESFGKPTDPAILLIMAAQMSMLWWEEEFCQRIADAGRFVIRFDNRDVGRSTTYEVGQPGYTFEDMADDAVHVLDAFGVQQAHFVGMSMGGMLTQMIALRHPERVRTITLHATSNFAPGLPPIDEKLMEFFSKMGEINWEDEKEALEAAVASWKVLSGSKHPFDESRVRELAKIDIARSNHYASRNNHAFVTASEPYLLRTAEIAVPALVIHGTEDLLIPFAHALHLANTIPGAVLLTLEGTGHELPYGDWDVVIEAILKHTSGRRVRL

[0100] SEQ ID NO:4

[0101] MAEQILKVNGVEICAESFGKPTDPAILLIMGDQMSMLWWEEEFCQRIADAGRFVIRFDNRDVGRSTTYEVGQPGYTFEDMADDAVHVLDAFGVQQAHFVGMSMGGMLTQMIALRHPERVRTITLHATSNFAPGLPPIDEKLMEFFSKMGEINWEDEKEALEAAVASWKVLSGSKHPFDESRVRELAKIDIARSNHYASRNNHAFVTASEPYLLRTAEIAVPALVIHGTEDLLIPFAHALHLANTIPGAVLLTLEGTGHELPYGDWDVVIEAILKHTSGRRVRL

[0102] SEQ ID NO:5

[0103] MAEQILKVNGVEICAESFGKPTDPAILLIMGAAMSMLWWEEEFCQRIADAGRFVIRFDNRDVGRSTTYEVGQPGYTFEDMADDAVHVLDAFGVQQAHFVGMSMGGMLTQMIALRHPERVRTITLHATSNFAPGLPPIDEKLMEFFSKMGEINWEDEKEALEAAVASWKVLSGSKHPFDESRVRELAKIDIARSNHYASRNNHAFVTASEPYLLRTAEIAVPALVIHGTEDLLIPFAHALHLANTIPGAVLLTLEGTGHELPYGDWDVVIEAILKHTSGRRVRL

[0104] SEQ ID NO:6

[0105] MAEQILKVNGVEICAESFGKPTDPAILLIMGAQMSMLAWEEEFCQRIADAGRFVIRFDNRDVGRSTTYEVGQPGYTFEDMADDAVHVLDAFGVQQAHFVGMSMGGMLTQMIALRHPERVRTITLHATSNFAPGLPPIDEKLMEFFSKMGEINWEDEKEALEAAVASWKVLSGSKHPFDESRVRELAKIDIARSNHYASRNNHAFVTASEPYLLRTAEIAVPALVIHGTEDLLIPFAHALHLANTIPGAVLLTLEGTGHELPYGDWDVVIEAILKHTSGRRVRL

[0106] SEQ ID NO:7

[0107] MAEQILKVNGVEICAESFGKPTDPAILLIMGAQMSMLWWEEEFCQRIADAGRFVIRFDNRDVGRSTTYEVGQPGYTAEDMADDAVHVLDAFGVQQAHFVGMSMGGMLTQMIALRHPERVRTITLHATSNFAPGLPPIDEKLMEFFSKMGEINWEDEKEALEAAVASWKVLSGSKHPFDESRVRELAKIDIARSNHYASRNNHAFVTASEPYLLRTAEIAVPALVIHGTEDLLIPFAHALHLANTIPGAVLLTLEGTGHELPYGDWDVVIEAILKHTSGRRVRL

[0108] SEQ ID NO:8

[0109] MAEQILKVNGVEICAESFGKPTDPAILLIMGAQMSMLWWEEEFCQRIADAGRFVIRFDNRDVGRSTTYEVGQPGYTFEDMADDAVHVLDAFGVQQAHFVGASMGGMLTQMIALRHPERVRTITLHATSNFAPGLPPIDEKLMEFFSKMGEINWEDEKEALEAAVASWKVLSGSKHPFDESRVRELAKIDIARSNHYASRNNHAFVTASEPYLLRTAEIAVPALVIHGTEDLLIPFAHALHLANTIPGAVLLTLEGTGHELPYGDWDVVIEAILKHTSGRRVRL

[0110] SEQ ID NO:9

[0111] MAEQILKVNGVEICAESFGKPTDPAILLIMGAQMSMLWWEEEFCQRIADAGRFVIRFDNRDVGRSTTYEVGQPGYTFEDMADDAVHVLDAFGVQQAHFVGMSMGGMLTQMIALRHPERVRTITLHATSNAAPGLPPIDEKLMEFFSKMGEINWEDEKEALEAAVASWKVLSGSKHPFDESRVRELAKIDIARSNHYASRNNHAFVTASEPYLLRTAEIAVPALVIHGTEDLLIPFAHALHLANTIPGAVLLTLEGTGHELPYGDWDVVIEAILKHTSGRRVRL

Claims

1. A macrocyclic lactone esterase mutant of the α / β-hydrolase superfamily, characterized in that, α / β-hydrolase superfamily macrocyclic lactone esterase mutants are obtained by performing any of the following steps on the amino acid sequence shown in SEQ ID NO: 1: Mutant 1, wherein mutant 1 is based on the amino acid sequence shown in SEQ ID NO: 1, wherein the 30th position Met is mutated to Ala, and the other positions remain unchanged, and its amino acid sequence is shown in SEQ ID NO: 2; Mutant 2, wherein mutant 2 is based on the amino acid sequence shown in SEQ ID NO: 1, wherein the 31st position Gly is mutated to Ala, and the other positions remain unchanged, and its amino acid sequence is shown in SEQ ID NO: 3; Mutant 3, wherein mutant 3 is based on the amino acid sequence shown in SEQ ID NO: 1, wherein the 32nd position Ala is mutated to Asp, while the other positions remain unchanged, and its amino acid sequence is shown in SEQ ID NO: 4; Mutant 4, wherein mutant 4 is based on the amino acid sequence shown in SEQ ID NO: 1, wherein the 33rd position Gln is mutated to Ala, and the other positions remain unchanged, and its amino acid sequence is shown in SEQ ID NO: 5; Mutant 5, wherein mutant 5 is based on the amino acid sequence shown in SEQ ID NO: 1, wherein the Trp at position 38 is mutated to Ala, while the other positions remain unchanged, and its amino acid sequence is shown in SEQ ID NO: 6; Mutant 6, wherein mutant 6 is based on the amino acid sequence shown in SEQ ID NO: 1, wherein Phe at position 77 is mutated to Ala, while the other positions remain unchanged, and its amino acid sequence is shown in SEQ ID NO: 7; Mutant 7, wherein mutant 7 is based on the amino acid sequence shown in SEQ ID NO: 1, wherein the 101st Met position is mutated to Ala, and the other positions remain unchanged, and its amino acid sequence is shown in SEQ ID NO: 8; Mutant 8 is based on the amino acid sequence shown in SEQ ID NO: 1, in which Phe at position 130 is mutated to Ala, while the other positions remain unchanged, and its amino acid sequence is shown in SEQ ID NO:

9.

2. The application of the α / β-hydrolase superfamily macrocyclic lactone esterase mutant as described in claim 1, characterized in that, It can be used as a biocatalyst to degrade macrolide antibiotics.

3. The application of the α / β-hydrolase superfamily macrocyclic lactone esterase mutant according to claim 2, characterized in that, The mutants are mutant 1 (Met at position 30 mutated to Ala), mutant 5 (Trp at position 38 mutated to Ala), mutant 6 (Phe at position 77 mutated to Ala), mutant 7 (Met at position 101 mutated to Ala), and mutant 8 (Phe at position 130 mutated to Ala). Mutants 1, 5, 6, 7, and 8 all increase the activity of macrolide esterase.

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