Erythromycin esterase mutant d245p with improved thermal stability and application thereof

Abstract

The application discloses an erythromycin esterase mutant D245P with improved thermal stability and application thereof, and belongs to the technical field of enzyme engineering. The erythromycin esterase mutant is obtained by mutating the aspartic acid at the 245th position to proline through molecular modification of the erythromycin esterase from Enterobacter hormaechei. Compared with the wild enzyme, the specific enzyme activity of the mutant is increased to 3.03 times of that of the wild enzyme, the half-life at 40 DEG C is increased to 10.75 times of that of the wild enzyme, the degradation efficiency of the mutant on erythromycin and clarithromycin within 30 min is 100% and 22.4% respectively, and the degradation efficiency is increased by 13.41% and 4.21% respectively compared with the wild enzyme. The mutant shows a good industrial application prospect.

Description

Technical Field

[0001] This invention relates to an erythromycin esterase mutant D245P with improved thermal stability and its applications, belonging to the field of enzyme engineering technology. Background Technology

[0002] Macrolide antibiotics, as a class of broad-spectrum antibiotics, can inhibit the growth of Gram-positive bacteria and some Gram-negative bacteria, and are widely used in clinical practice and animal husbandry. Among them, the most commonly used macrolide antibiotics are erythromycin (Ery), clarithromycin (Clr), and roxithromycin (Rox). The structure of macrolide antibiotics consists of a 12-18 membered lactone ring and different hexose groups at the 3- and 5-positions. Furthermore, the sugar group at the 3-position of macrolide compounds is replaced by a ketone group. Erythromycin (Ery), clarithromycin (Clr), and roxithromycin (Rox) are all macrolides with a 14-membered or less lactone ring.

[0003] Despite the widespread use of macrolide antibiotics, their overuse leads to environmental pollution and antibiotic resistance. Therefore, strict control of antibiotic concentrations in the environment is necessary to protect human and animal health and maintain ecological balance. Esterases (EC 3.1.1.X) are a class of enzymes that hydrolyze ester compounds, including broad-spectrum and specific esterases. Esterases hydrolyze the ester bonds in macrolide antibiotic molecules, thereby rendering them inactive and breaking them down into non-toxic metabolites. Some bacteria also utilize macrolide antibiotics as carbon sources, and their metabolic pathways involve the action of esterases. Bacteria have developed many methods to combat macrolide drugs, among which erythromycin esterase (Ere) catalyzing the cleavage of the lactone ring is one of the important bacterial resistance mechanisms.

[0004] Currently, various Ere families have been discovered and characterized, including members of the EreA, EreB, EreC, and EreD families. Among them, members of the EreC family exhibit the highest enzymatic activity, but relatively speaking, EreC also has poor thermal stability. There are currently no reports on mutating EreC to improve its thermal stability and achieve efficient degradation of macrolide antibiotics. Summary of the Invention

[0005] To address the aforementioned deficiencies in the prior art, this invention molecularly modifies erythromycin esterase from Enterobacter hormaechei to improve its thermal stability and obtain a macrolide antibiotic degrading enzyme that is better adapted to environmental requirements.

[0006] This invention provides an erythromycin esterase mutant, wherein the mutant is based on erythromycin esterase with the amino acid sequence shown in SEQ ID NO.2 as the parent, and the aspartic acid at position 245 of the parent is mutated to proline.

[0007] In one embodiment, the amino acid sequence of the erythromycin esterase mutant is shown in SEQ ID NO.4.

[0008] In one embodiment, the erythromycin esterase is derived from Enterobacter hormaechei.

[0009] This invention provides a gene encoding the erythromycin esterase mutant.

[0010] In one embodiment, the nucleotide sequence of the gene for the erythromycin esterase mutant is shown in SEQ ID NO.3.

[0011] The present invention provides a recombinant plasmid carrying the gene encoding the erythromycin esterase mutant.

[0012] In one embodiment, the recombinant plasmid includes, but is not limited to, pET series, Duet series, pGEX series, pHY300, pHY300PLK, pPIC3K or pPIC9K as vectors.

[0013] In one embodiment, the pET series carrier is preferred.

[0014] In one embodiment, the recombinant plasmid is pET-22b(+) as a vector, and the gene encoding the erythromycin esterase mutant is inserted into the multiple cloning site of the pET-22b(+) vector.

[0015] The present invention provides microbial cells that express the erythromycin esterase mutant, or carry the gene, or carry the recombinant plasmid.

[0016] In one embodiment, the host cell of the microbial cell includes, but is not limited to, Escherichia coli, Bacillus subtilis, Pichia pastoris, or Saccharomyces cerevisiae.

[0017] In one embodiment, the host cell is Escherichia coli BL21(DE3), or the host cell is E. coli JM109(DE3).

[0018] In one embodiment, the microbial cells use Escherichia coli BL21(DE3) as the host and pET-22b(+) as the vector to express any of the erythromycin esterase mutants described above.

[0019] The present invention provides a method for improving the thermostability of erythromycin esterase, wherein the aspartic acid at position 245 of the erythromycin esterase, as shown in SEQ ID NO.2, is mutated to proline.

[0020] The present invention provides a method for degrading macrolide antibiotics, wherein the method involves adding the erythromycin esterase mutant to a system containing macrolide antibiotics and reacting therein.

[0021] In one embodiment, the macrolide antibiotic includes erythromycin and / or erythromycin derivatives.

[0022] In one embodiment, the erythromycin derivative includes clarithromycin.

[0023] In one embodiment, the reaction is carried out at 30°C to 70°C.

[0024] In one embodiment, the amount of the macrolide antibiotic added is 5-15 mg / mL.

[0025] In one embodiment, the weight ratio of the amount of the erythromycin esterase mutant added to the amount of the macrolide antibiotic added is 1:(40-100).

[0026] The present invention provides a product containing the erythromycin esterase mutant or the microbial cells.

[0027] In one embodiment, the product includes, but is not limited to, enzyme preparations or compositions containing the erythromycin esterase mutant.

[0028] This invention provides the erythromycin esterase mutant, or the microbial cell, or the method for degrading macrolide antibiotics, or the application of the product in the degradation of substances containing macrolide antibiotics.

[0029] In one embodiment, the macrolide antibiotic includes erythromycin and / or erythromycin derivatives.

[0030] In one embodiment, the erythromycin derivative includes clarithromycin.

[0031] Beneficial effects:

[0032] (1) The thermostability of the erythromycin esterase mutant of the present invention is significantly improved compared with that of the wild-type enzyme before mutation. Compared with the wild-type enzyme before mutation, the optimal catalytic temperature of the mutant is increased from 50°C to 60°C. The specific enzyme activity at 60°C is increased from 1.89 U / mg to 5.72 U / mg, which is 3.03 times that of the wild-type enzyme. The half-life of the mutant at 40°C is 77.1 min, which is 10.75 times that of the wild-type enzyme.

[0033] (2) The erythromycin esterase mutant of the present invention significantly improved the catalytic efficiency of erythromycin and its derivatives. Under the conditions of a reaction system of 100 μL of DMSO solution of erythromycin or clarithromycin (concentration of 8 mg / mL), 100 μL of enzyme (0.1 mg / mL), and 500 μL of Tris-HCl buffer (50 mM, pH 7.5), and a reaction temperature of 50 °C, the degradation efficiency of erythromycin reached 100% within 30 min, and the degradation efficiency of clarithromycin reached 22.4%, which were 13.41% and 4.21% higher than those of the wild-type enzyme, respectively. Attached Figure Description

[0034] Figure 1 Comparison of the optimal temperatures of erythromycin esterase mutant and wild-type enzyme;

[0035] Figure 2 Comparison of the half-inactivation temperatures of erythromycin esterase mutant and wild-type enzyme;

[0036] Figure 3 Comparison of the half-life of erythromycin esterase mutant and wild-type enzyme at 40°C;

[0037] Figure 4 Electrophoresis diagrams of the original enzyme (lane 1) and the mutant enzyme D245P (lane 2). Detailed Implementation

[0038] All experimental reagents and materials used in the following examples were purchased from Sinopharm Group.

[0039] The culture media involved in the following examples:

[0040] LB medium: yeast extract 5 g / L; tryptone 10 g / L; sodium chloride 10 g / L, pH 7.0. Sterilize at 121°C for 20 min. Add ampicillin to a final concentration of 100 μg / mL before use. If preparing a solid medium, add 1.5% (w / v) agar powder before sterilization.

[0041] Plasmid transformation methods:

[0042] The main steps of transferring recombinant plasmids into competent cells of genetically engineered bacteria via heat shock include:

[0043] (1) Take out the competent cells stored in the -80℃ freezer and quickly insert them into the ice.

[0044] (2) Take 2 μL of the company's synthetic plasmid or 6 μL of PCR-related product and slowly inject it into the lysed competent cells. Gently stir to mix and let stand in an ice bath for 20 min.

[0045] (3) Place the tube containing competent cells in a water bath at 42°C and heat shock for 90 seconds. After the heat shock, quickly place it in an ice box to cool and let it stand for 5 minutes.

[0046] (4) Take 1 mL of sterilized LB liquid culture medium that has been preheated in an oven at 50°C for 30 min, add it to the tube containing competent cells, mix well by blowing and aspiration, and incubate at 37°C and 200 r / min on a shaker for about 1 h.

[0047] (5) If a non-circular PCR construct plasmid is used, centrifuge at 3500 r / min after the culture is completed, discard 960 μL of supernatant, and resuspend the bacterial cells.

[0048] (6) Take 60 μL of bacterial culture from the tube and spread it on an LB solid plate with the corresponding resistance. Invert the plate and incubate it overnight at 37°C for 8–12 h.

[0049] Methods for determining erythromycin esterase activity and definition of enzyme activity:

[0050] Reaction system: 100 μL DMSO solution of erythromycin (8 mg / mL erythromycin), 100 μL enzyme (0.1 mg / mL) and 500 μL Tris-HCl buffer (50 mM, pH 7.5). The reaction was carried out at 50 °C for 15 min, and the reaction was terminated by adding 500 μL acetonitrile.

[0051] Enzyme activity (U): The amount of enzyme required to consume 1 μmol of substrate per minute.

[0052] HPLC detection conditions for erythromycin:

[0053] Equipped with Hypersi1 at 40°C TM An ODS C18 column (250 × 4.6 mm, 5 μm) was used to detect substrate residue via gradient elution. Gradient elution was performed over 20 min using 0.02 M phosphate buffer (solution A, pH 8.0) and acetonitrile (solution B) as the mobile phase. The gradient was as follows: 20% solution B was used from 0 to 2 min; from 2 to 10 min, the proportion of solution B was linearly increased to 95%; from 10 to 12 min, the proportion of solution B was maintained at 95%; and from 12 to 16 min, the proportion of solution B was linearly decreased to 20%. The mobile phase flow rate was maintained at 1 mL / min throughout.

[0054] Protein concentration determination method:

[0055] Protein content was determined using a BCA kit with bovine serum albumin (BSA) as the standard protein. The maximum detectable concentration was 0.5 mg / mL, and the standard curve was prepared fresh for each use. The BSA standard solution (5 mg / mL) was aliquoted into 50 μL tubes and stored frozen at -20°C to minimize freeze-thaw cycles. Before use, the solution was thoroughly mixed by pipetting. Solution A and solution B in the kit were mixed thoroughly according to the instructions to prepare the working solution for the protein reaction. The freshly prepared working solution was mixed with protein at a 10:1 volume ratio and reacted in a 37°C water bath for 30 min. The absorbance of the reaction solution of the standard protein and the sample was measured at 560 nm. A standard curve fitting formula was established to calculate the corresponding target protein concentration.

[0056] Example 1: Preparation and purification of erythromycin esterase mutant enzyme from Enterobacter hormaechei

[0057] (1) Construction of plasmids:

[0058] Construction of recombinant plasmid pET22b-Enho containing wild-type Enho: Gene synthesis of the erythromycin esterase gene fragment Enho shown in SEQ ID No. 1, and ligation between the restriction sites Nde I and Xho I of the vector pET-22b(+) to obtain recombinant plasmid pET22b(+)-Enho.

[0059] Using pET-22b(+)-Enho plasmid as a template, D245P site-directed mutation was introduced by PCR and digestion. Sequencing verification results showed that no random mutations appeared except for the required mutation site, indicating that the mutant plasmid pET-22b(+)-D245P was successfully constructed.

[0060] The mutant primers are shown below: (Underlined primers indicate mutants)

[0061] Upstream primer F: 5'-GCACTTCACTC CCT GGTGACACTTCCGTT-3',

[0062] Downstream primer R: 5'-GTGTCACC AGG GAGTGAAGTGCCATCA-3'.

[0063] Table 1 Composition of PCR 1 reaction system

[0064]

[0065] PCR reaction conditions: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 2.5 min, 28 cycles, 72℃ extension for 5 min. The reaction solution was purified and recovered for PCR 2 reaction.

[0066] Table 2 Composition of the digestion reaction system:

[0067]

[0068] Digestion reaction conditions: Digestion at 37℃ for 2 hours.

[0069] The digestion product was transformed into E. coli DH5α competent cells and cultured overnight at 37°C in LB solid medium containing 100 μg / mL ampicillin. Single clones were then picked and cultured overnight at 37°C in LB liquid medium containing 100 μg / mL ampicillin. Subsequently, the mutant plasmid pET-22b(+)-D245P was extracted using a plasmid miniprep kit (Tiangen, DP103-03). The mutant plasmid was identified as the correct mutation by sequencing.

[0070] (2) Expression and purification of erythromycin esterase mutant from Enterobacter hormaechei

[0071] The mutant plasmid and wild-type plasmid pET22b-Enho, verified by sequencing in step (1), were transformed into E. coli BL21(DE3) cells, respectively. Positive transformants were picked and cultured in 4 mL of LB liquid medium at 37°C and 200 rpm for 12 h, followed by inoculation into 200 mL of LB medium and cultured at 37°C until OD. 600 The value was 0.6 to 0.8. After the temperature of the bacterial culture was lowered to 26°C, IPTG was added to a final concentration of 1 mM and induced for 18 h to obtain the fermentation broth.

[0072] The fermentation broth was centrifuged at 4°C and 6000 rpm for 5 min to collect the bacterial cells. 15 mL of buffer (50 mM Tris-HCl, 100 mM NaCl, adjusted to pH approximately 7.2) was added to fully resuspend the cells. The centrifuge tubes were then placed in an ice bath and placed in an ultrasonic cell disruptor. The ultrasonic disruption conditions were: 1 second on, 2 seconds off, repeated for a total of 20 min. The resulting disrupted solution was centrifuged at low temperature and high speed at 4°C and 8000 rpm for 15 min to obtain the crude enzyme solution. This solution was filtered through a 0.45 μm microporous membrane for later use, yielding crude enzyme solutions of the erythromycin esterase mutant and the wild-type erythromycin esterase.

[0073] Prepare a nickel-ion affinity chromatography column, with a UV detector connected downstream. First, flush the column with deionized water (approximately 6-12 column volumes) using a constant flow pump at room temperature. Then, equilibrate the column with buffer A (500 mM NaCl, 50 mM Tris-HCl, pH 7.2). Once the effluent from the bottom of the column has stabilized at the UV detector reading (approximately 5 column volumes of buffer are needed), add the obtained crude enzyme solution to the column. Next, wash with buffer B (500 mM NaCl, 50 mM imidazole, 50 mM Tris-HCl, pH 7.2) until the UV detector reading stabilizes at baseline. Elute with a high concentration of imidazole (500 mM NaCl, 500 mM imidazole, 50 mM Tris-HCl, pH 7.2). Collect the eluent with the UV absorption peak to obtain the purified target protein. The purified protein band is shown below. Figure 4 As shown, the purified erythromycin esterase mutant enzyme and wild-type enzyme reached electrophoretic purity.

[0074] Example 2: Comparison of thermostability between mutant enzyme D245P and wild-type enzyme

[0075] The changes in the thermostability of the enzyme before and after mutation were determined. The wild-type enzyme refers to the erythromycin esterase derived from *Enterobacter holmieae* prepared in Example 1 (amino acid sequence shown in SEQ ID NO. 2). The mutant enzyme refers to the mutant enzyme obtained by mutating aspartic acid at position 245 of the erythromycin esterase prepared in Example 1 to proline (amino acid sequence shown in SEQ ID NO. 4), which is abbreviated as mutant enzyme D245P for convenience.

[0076] (1) Determination of the optimal reaction temperature between mutant enzyme D245P and wild-type enzyme

[0077] The mutant enzyme D245P and the wild-type enzyme were subjected to enzymatic reactions within a temperature range of 30–70 °C (with temperature points set at 5 °C intervals). The reaction system consisted of 100 μL of erythromycin in DMSO solution (8 mg / mL erythromycin), 100 μL of enzyme (0.1 mg / mL), and 500 μL of Tris-HCl buffer (50 mM, pH 7.5). The reaction was carried out for 15 min, and 500 μL of acetonitrile was added to terminate the reaction. The residual amount of erythromycin was detected by HPLC, and the relative enzyme activities at different reaction temperatures were calculated. The enzyme activities at the optimal reaction temperature for both the wild-type and mutant enzymes D245P were set as 100% relative enzyme activities to calculate the relative enzyme activities at other temperatures. The optimal reaction temperature is the temperature corresponding to the highest enzyme activity.

[0078] The results are as follows Figure 1As shown in Table 3, the optimal reaction temperature for the wild-type enzyme is 50℃, while the optimal temperature for the mutant enzyme D245P is increased to 60℃, a 10℃ increase compared to the wild-type enzyme. At 50℃, the specific enzyme activity of the wild-type enzyme is calculated to be 3.22 U / mg, while that of the mutant enzyme D245P is 4.83 U / mg, which is 1.5 times that of the wild-type enzyme. At 60℃, the specific enzyme activity of the wild-type enzyme is 1.89 U / mg, while that of the mutant enzyme D245P is 5.72 U / mg, which is 3.03 times that of the wild-type enzyme.

[0079] Table 3 Comparison of specific enzyme activities of mutant and wild-type enzymes at different temperatures.

[0080]

[0081]

[0082] (2) The half-inactivation temperature (T50) of the enzyme before and after mutation 5 )Compare

[0083] The wild-type enzyme and mutant enzyme D245P obtained according to the method in Example 1 were incubated for 5 min at a temperature gradient range of 30-60℃ (each temperature point was set at 5℃) before enzyme reaction. The reaction system consisted of 100 μL of erythromycin in DMSO solution (8 mg / mL erythromycin), 100 μL of enzyme (0.1 mg / mL), and 500 μL of Tris-HCl buffer (50 mM, pH 7.5). The reaction was carried out at 50℃ for 15 min, and then 500 μL of acetonitrile was added to terminate the reaction. The residual amount of erythromycin in the reaction system was detected by HPLC, and the specific enzyme activity data after incubation at different temperatures were calculated. The initial enzyme activity without heat treatment was set as 100% relative enzyme activity to calculate the residual enzyme activity of the wild-type enzyme and mutant enzyme after 5 min of heat treatment at each temperature.

[0084] By performing nonlinear fitting using the Boltzmann function, the temperature at which the residual enzyme activity decreases to half of the initial enzyme activity is obtained, which is T50. 5 Value. Result as follows Figure 2 As shown, the half-inactivation temperature (T50) of the mutant enzyme D245P is... 5 The temperature value increased from 40.04℃ for the wild-type enzyme to 46.93℃, which is 6.89℃ higher than that for the wild-type enzyme.

[0085] (3) Comparison of enzyme half-life before and after mutation

[0086] The wild-type enzyme and mutant enzyme D245P obtained according to the method in Example 1 were incubated at 40°C, and samples were taken at 5-minute intervals. Enzyme activity was then measured. The reaction system for enzyme activity measurement consisted of 100 μL of erythromycin in DMSO solution (8 mg / mL erythromycin), 100 μL of enzyme (0.1 mg / mL), and 500 μL of Tris-HCl buffer (50 mM, pH 7.5). The reaction was carried out at 50°C, and 500 μL of acetonitrile was added to terminate the reaction. The residual amount of erythromycin in the reaction system was detected by HPLC, and the specific enzyme activity data were calculated. To observe the change in enzyme activity over time when the wild-type enzyme and mutant enzyme D245P of the present invention were incubated at 40°C for different times, the enzyme activity of the pure enzyme solution without 40°C incubation was used as a control (i.e., 100%).

[0087] The half-life was determined based on the Arrhenius equation, where the relative enzyme activity at time 0 (before incubation) was denoted as E0, and the residual enzyme activity after incubation time t was denoted as E2. t Plotting the heat preservation time t on the x-axis, ln(E) t Using / E0 as the ordinate, a straight line can be fitted. The slope of this line can then be used to calculate the half-life (t). 1 / 2 ).

[0088] The specific calculation formula is as follows;

[0089]

[0090]

[0091] Where A0 is the initial enzyme activity, A t It is the residual enzyme activity after incubation time t. 1 / 2 This is the half-life at which the enzyme becomes inactive at this temperature.

[0092]

[0093] Where k is the reaction rate constant, R is 8.31 J / (mol·k), and Ea is the activation energy that causes the enzyme to be thermally inactivated.

[0094] The results are as follows Figure 3 As shown, the residual enzyme activity of mutant enzyme D245P after 10 min at 40℃ increased from 31.8% of the wild-type enzyme to 90.7%. The half-life of mutant enzyme D245P at 40℃ increased from 7.17 min of the wild-type enzyme to 77.1 min, an increase of 10.75 times, indicating that the thermostability of mutant enzyme D245P is significantly improved compared with that of wild-type enzyme.

[0095] (4) Comparison of the reaction processes of mutant enzyme degradation of erythromycin and clarithromycin

[0096] The degradation of erythromycin and clarithromycin at different reaction times, specifically the degradation rate of the two macrolide antibiotics by the mutant enzyme at 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 45 min and 60 min.

[0097] The mutant enzyme D245P prepared in Example 1 and the wild-type enzyme were reacted with erythromycin or clarithromycin, respectively. The reaction system consisted of 100 μL of DMSO solution of erythromycin or clarithromycin (concentration of 8 mg / mL), 100 μL of enzyme (0.1 mg / mL), and 500 μL of Tris-HCl buffer (50 mM, pH 7.5), and the reaction was carried out at 50 °C. Samples were taken at 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 45 min, and 60 min after the start of the reaction. The residual amount of erythromycin or clarithromycin at different reaction times was detected by HPLC, and the degradation rate of erythromycin or clarithromycin by the mutant enzyme D245P and the wild-type enzyme was calculated.

[0098] The results are shown in Table 4. Within 30 min, the mutant D245P achieved degradation efficiencies of 100% and 22.4% for erythromycin and clarithromycin, respectively, which were 13.41% and 4.21% higher than those of the wild-type enzyme.

[0099] Table 4 Comparison of degradation rates of erythromycin and clarithromycin by mutant and wild-type enzymes

[0100]

[0101]

[0102] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. An erythromycin esterase mutant, characterized in that, Using erythromycin esterase with the amino acid sequence shown in SEQ ID NO.2 as the parent, the aspartic acid at position 245 of the parent was mutated to proline.

2. The gene encoding the erythromycin esterase mutant of claim 1.

3. A recombinant plasmid carrying the gene described in claim 2.

4. The recombinant plasmid according to claim 3, characterized in that, The recombinant plasmids are based on pET series, Duet series, pGEX series, pHY300, pHY300PLK, pPIC3K, or pPIC9K vectors.

5. Microbial cells expressing the erythromycin esterase mutant of claim 1, or carrying the gene of claim 2, or carrying the recombinant plasmid of claim 3 or 4.

6. The microbial cell according to claim 5, characterized in that, The host cells of the microbial cells are Escherichia coli, Bacillus subtilis, Pichia pastoris, or Saccharomyces cerevisiae.

7. A method for improving the thermostability of erythromycin esterase, wherein the method involves mutating aspartic acid at position 245 of the erythromycin esterase, as shown in SEQ ID NO.2, to proline.

8. A method for degrading macrolide antibiotics, the method comprising adding the erythromycin esterase mutant of claim 1 to a system containing macrolide antibiotics and reacting therewith, wherein the macrolide antibiotics are erythromycin and / or clarithromycin.

9. A product containing the erythromycin esterase mutant of claim 1, or the microbial cells of claim 5 or 6.

10. The use of the erythromycin esterase mutant of claim 1, or the microbial cell of claim 5 or 6, or the method of claim 8, or the product of claim 9 in the degradation of a substance containing a macrolide antibiotic, wherein the macrolide antibiotic is erythromycin and / or clarithromycin.

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