A method for synthesizing dexketoprofen using amidase

Abstract

The application provides a method for synthesizing dextroketoprofen by using an amidase. The method comprises selectively hydrolyzing ketoprofen amide by using an amidase to obtain dextroketoprofen, wherein the amidase is from Rhodococcus erythropolis CCM2595. The amino acid sequence of the amidase is shown as SEQ ID NO. 1. The amidase in the application has significant catalytic activity, substrate affinity and regioselectivity on ketoprofen amide, and has high tolerance to substrates and products. The enzyme bottom of the amidase reaction is 1 / 20, the substrate concentration is 100 g / L, the conversion rate is more than 43%, and the optical purity of the product is greater than 99.5%, which shows good potential for industrial application.

Description

Technical Field

[0001] This invention relates to the field of enzyme hydrolysis technology, and in particular to a method for synthesizing dextro-ketoprofen using amidase. Background Technology

[0002] Dextro-ketoprofen, also known as (S)-(+)-ketoprofen, (S)-ketoibuprofen, or (S)-(+)-2-(3-benzoylphenyl)propionic acid, is a 2-arylpropionic acid class of nonsteroidal anti-inflammatory drugs (NSAIDs). Its commercial form is dextro-ketoprofen tromethamine. Ketoprofen has one chiral center and two enantiomers, only of which dextro-ketoprofen possesses anti-inflammatory, antirheumatic, and analgesic effects. Levo-ketoprofen has almost no pharmacological activity and toxic side effects. Dextro-ketoprofen was developed by the Italian company Menarini in 1996 and first marketed in Spain. Its anti-inflammatory and analgesic effects are twice that of the racemic mixture, indicating that levo-ketoprofen is ineffective. Therefore, the synthesis and production of the single isomer dextro-ketoprofen has significant application value.

[0003] Currently, the main synthetic methods for dextro-ketoprofen include chemical asymmetric synthesis, chemical resolution, and enzymatic resolution (including esterases, lipases, nitrile hydrolases, nitrile hydratases, and amidases).

[0004] Chemical asymmetric synthesis methods require heavy metal chiral catalysts and high temperature and pressure conditions. Most catalysts are highly toxic and cause environmental pollution, and chiral catalysts are also expensive. Chemical resolution involves recrystallization, which is time-consuming, increases costs, and reduces yield. In addition, the resolving agent is used in large quantities, resulting in high costs and a lot of wastewater and waste solvent. Lipase or esterase catalytic resolution has disadvantages such as low raw material concentration, large enzyme dosage, environmentally unfriendly reaction medium, low ee% value of product, and difficulty in separating product mixtures. Nitrile hydrolase hydrolysis resolution yields products with very low ee% values. The two-step enzymatic hydrolysis resolution of ketoprofen nitrile to synthesize dextro-ketoprofen using nitrile hydratase and amidase mostly results in low concentration and low yield. The two-step enzymatic method increases costs, and the post-processing steps are cumbersome.

[0005] There are numerous reports on the hydrolysis and resolution of ketoprofen amide by amidases to synthesize dextro-ketoprofen. For example, Appl. Microbiol. Biotechnol., 1994, 42:1-7 reported that the reaction using Agrobacterium tumefaciens strain d3 amidase resulted in a conversion rate of 5% and an ee% value of 97%. BIOTECHNOLOGY LETTERS, 1995, 17(2):187-192 introduced microbial amidases, showing that Rhodococcus sp. C3II cells converted 2mM feedstock achieved a conversion rate of 53% after 20.5 hours, with a dextro-ketoprofen ee% value of 97%; Rhodococcus erythropolis MP 50 cells converted 0.2mM feedstock achieved a conversion rate of 49%, with a dextro-ketoprofen ee% value of 99%. Protein & Peptide Letters, 2008, 15, 617-623, reported the Sulfolobus solfataricus amidase reaction: 1.5 g ketoprofen amide was dissolved in 100 mL of methanol, 500 mL of Tris-HCl buffer was added, 12 mg of purified enzyme was added, and the reaction was carried out at 70 °C for 24 h, with an optical purity of 99.6% ee. CN111363736 reported the preparation of dextro-ketoprofen using Klebsiella amidase, as in Example 6: 0.8 g of enzyme powder was resuspended in 100 mL of sodium phosphate buffer, 4 g of ketoprofen amide was dissolved in 10 mL of toluene and added to the reaction flask, and the reaction was incubated for 10 h, with a product ee% of 100% and a conversion rate of 50.85%. CN111378637 describes the preparation of dextro-ketoprofen using Agrobacterium amidase, as in Example 6. 1g of enzyme powder was resuspended in 100mL of sodium phosphate buffer, and 4g of amido-ketoprofen dissolved in 10mL of toluene was added dropwise to the reaction flask. The reaction was incubated for 10 hours, yielding a product ee% of 99.8% and a conversion rate of 50.75%. CN114134132 describes the mutation of Rhodococcus erythropolis MP50 amidase, yielding the highest activity mutant, as in Example 9. 0.37g of AMD03-38 enzyme powder was added dropwise to 100mL of sodium phosphate buffer, and 6g of amido-ketoprofen dissolved in 10mL of ethyl acetate. The reaction was incubated for 24 hours, yielding a conversion rate of 49% and a product ee% of 99.5%.

[0006] Of the various amidases from different sources used in the synthesis of dextrokeprofen, only those with low enzyme activity, low reaction concentration, and low product optical purity were reported. Only the optimal mutant amidases, namely Klebsiella (CN111363736), Agrobacterium (CN111378637), and Rhodococcus erythropolis MP50 (CN114134132), achieved a product ee% > 99.5% and a feed concentration of 40-60 g / L. However, these results are still not ideal. Industrial-scale enzymatic processes require higher feed concentrations and lower enzyme-to-feed ratios (mass ratio of enzyme to feedstock) to reduce wastewater discharge, increase space-time yield, and lower costs. Therefore, it is necessary to develop new amidases for the enzymatic production of dextrokeprofen.

[0007] Rhodococcus erythropolis CCM2595 is a Rhodococcus erythropolis strain that has been shown to degrade aromatic compounds such as phenol, hydroxybenzoic acid, p-chlorophenol, and aniline (Cejkova et al. 2005). Studies have found that this strain can catalyze the degradation of nitrile compounds, particularly adiponitrile, exhibiting high regioselectivity and substrate affinity (Liu Shengxian, 2019). However, the hydrolytic resolution of ketoprofen amide by the amidase in this strain has not yet been reported. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a method for synthesizing dextro-ketoprofen using amidase, which solves the problems of high enzyme-to-feed ratio, low feed concentration, high cost, and unsuitability for industrial production in existing technologies.

[0009] In one aspect, the present invention provides a method for synthesizing dextro-ketoprofen using an amidase, wherein the amidase catalyzes the selective hydrolysis of ketoprofen amide to obtain dextro-ketoprofen, wherein the amidase is derived from Rhodococcus erythropolis CCM2595.

[0010] Furthermore, the amino acid sequence of the amidase is shown in SEQ ID NO.1.

[0011] Furthermore, the amidase is prepared using the following method:

[0012] (1) Synthesize the nucleotide sequence encoding the amidase and construct engineered bacteria;

[0013] (2) Inducing culture of engineered bacteria, collecting bacterial cells by centrifugation, breaking the bacterial cells, and preparing enzyme preparations;

[0014] The nucleotide sequence is shown in SEQ ID NO.2.

[0015] Furthermore, the enzyme preparation includes one of enzyme solution, enzyme powder, bacterial cells, and immobilized enzyme.

[0016] Furthermore, the reaction involves adding a solubilizer and ketoprofen amide to the reaction medium, followed by selective hydrolysis with amidase to obtain dextrorotatory ketoprofen. The reaction process is shown below:

[0017]

[0018] Furthermore, the co-solvent is dimethyl sulfoxide, and the reaction medium is one of water and Tris-HCl buffer.

[0019] Furthermore, the selective hydrolysis temperature is 20-60℃, preferably 40℃.

[0020] Furthermore, the pH value of the selective hydrolysis is 6-10, preferably 8.0.

[0021] Furthermore, the concentration of the ketoprofenamide is 20-150 g / L, preferably 100 g / L.

[0022] Furthermore, the mass ratio of the amidase to ketoprofenamide is 1 / 1 to 1 / 50, preferably 1 / 20.

[0023] The technical principle of this invention is as follows: In the prior art, amidases derived from Rhodococcus erythropolis MP50, Agrobacterium, and Klebsiella can achieve asymmetric hydrolysis of ketoprofen amide to obtain dextro-ketoprofen. However, due to the different molecular structures of amidases from different sources, there are certain differences in their amino acid sequences. These differences are likely the reason for their different substrate specificities, leading to different catalytic performances. This invention investigated the hydrolytic resolution of ketoprofen amide by an amidase derived from *Rhodococcus erythropolis* CCM2595 to synthesize dextro-ketoprofen. It was found that the highest feed concentration of ketoprofen amide catalyzed by this amidase was 150 g / L, higher than that of other amidases from different sources (maximum feed concentration 60 g / L, reference CN114134132). Furthermore, the enzyme-to-substrate ratio was 1 / 20, lower than that of other amidases from different sources (lowest enzyme-to-substrate ratio 1 / 16.3, reference CN114134132), indicating that this enzyme exhibits stronger catalytic activity towards the ketoprofen amide substrate and can withstand the inhibitory effect of high substrate concentrations. This may be related to the enzyme's stronger affinity for the substrate, thus providing a possibility for the large-scale industrial production of dextro-ketoprofen via enzyme catalysis.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) This invention first constructs a genetically engineered *Escherichia coli* containing the *Rhodococcus erythropolis* CCM2595 amidase gene, then cultivates and induces amidase expression, prepares various forms of enzyme preparations, and finally applies them to catalyze the hydrolysis of ketoprofen amide to synthesize dextro-ketoprofen. The amidase-catalyzed hydrolysis reaction has a feed concentration of 100 g / L, an enzyme-to-substrate ratio of 1 / 20, a feed conversion rate >43%, and a product optical purity >99.5%.

[0026] (2) The amidase in this invention has significant catalytic activity, substrate affinity and regioselectivity for ketoprofen amide, and has high tolerance to substrate and product. It has high feed concentration for catalytic reaction, low enzyme dosage, high space-time yield, low waste and low cost, showing good industrialization potential. Detailed Implementation

[0027] The technical solutions of the present invention will be further described below with reference to the embodiments.

[0028] Example 1: Preparation of amidase from Rhodococcus erythropolis CCM2595

[0029] 1. Constructing engineered bacteria that produce amidase

[0030] Based on the amino acid sequence of the amidase from *Rhodococcus erythropolis* CCM2595 published on the NCBI website (as shown in SEQ ID NO.1), the nucleic acid sequence was optimized using codons. An amidase gene (nucleotide sequence shown in SEQ ID NO.2) was then artificially designed and synthesized. NdeI and EcoRI sites were introduced at both ends of the gene, and the gene was cloned into the pET21a vector. The constructed recombinant plasmid was chemically transformed into *E. coli* BL21(DE3) competent cells, plated on LB agar plates containing 50 μg / ml ampicillin resistance, and cultured at 37°C for 8–12 h. Single clones were then picked to obtain a genetically engineered strain capable of inducibly expressing amidase.

[0031] 2. Fermentation tank cultivation

[0032] BL21(DE3) cells containing the amidase gene were inoculated into LB liquid medium containing 50 μg / mL ampicillin resistance and cultured overnight at 37°C to obtain a seed culture. The seed culture was then transferred at a 3% inoculation rate to fermentation medium containing 50 μg / mL ampicillin resistance. Fermentation was carried out at 37°C with an initial stirring speed of 300 rpm, maintaining dissolved oxygen at over 30% by stirring speed. Feed was continuously added at a flow rate of 20 mL / h (10L fermenter) for approximately 3 hours. When the OD reached 0.8-1.0, the temperature was lowered to 25°C, and IPTG was added at a concentration of 0.2 mM for induced expression for 20 hours. After the culture was completed, the fermentation broth was centrifuged at 4800 rpm for 10 min, and the precipitate was collected to obtain amidase cells, which were then stored at -70°C for long-term preservation.

[0033] Fermentation medium formula: glucose 10g / L, yeast extract 30g / L, disodium hydrogen phosphate 5.08g / L, potassium dihydrogen phosphate 3g / L, magnesium sulfate 2g / L, calcium chloride 1g / L, sodium citrate 0.23g / L, 0.7g / L antifoaming agent, pH 7.0.

[0034] Feeding solution formula: yeast extract 50g / L, glucose 300g / L, magnesium sulfate 10g / L;

[0035] 3. Preparation of amidase

[0036] 3.1 Preparation of enzyme solution

[0037] The amidase cells obtained after centrifuging the fermentation broth were resuspended evenly in 0.1M pH7.0 Tris-HCl (the amount used was 5 times the weight of the cells, V / W), and then the cells were disrupted in an ultrasonic homogenizer or a high-pressure homogenizer to obtain the cell disruption solution, which is the enzyme solution, and stored at -70℃ for a long time.

[0038] 3.2 Preparation of enzyme powder

[0039] Take the enzyme solution prepared in 3.1, place it in a vacuum freeze dryer, and freeze dry it under vacuum to finally obtain enzyme powder. Store the enzyme powder at 4℃.

[0040] 3.3 Preparation of immobilized enzymes

[0041] After centrifuging or filtering the enzyme solution prepared in 3.1, obtain the enzyme clear solution. Add resin to the enzyme clear solution for immobilization, filter, freeze dry under vacuum to obtain the immobilized enzyme, and store at 4℃.

[0042] Example 2 Synthesis of dextro-ketoprofen catalyzed by different forms of amidase

[0043]

[0044] In a 500ml reaction flask, 100mL of 0.1M Tris-HCl buffer (pH 7.5) was added. 5g of ketoprofen amide was dissolved in 20mL of DMSO and added to the reaction flask. Different forms of amidase were then added, and the pH was adjusted to 7.5 with sodium hydroxide aqueous solution. The reaction was carried out at 35℃ with stirring for 24h. Samples were taken for analysis to determine the conversion rate and the ee% value of the product dextro-ketoprofen. The test results for different forms of amidase are shown in Table 1.

[0045] Table 1. Conversion rate and ee value of different forms of amidase reaction

[0046] Bacterial cells 0.90g 33.82％ 99.75％ enzyme solution 5.0g 43.61％ 99.78％ enzyme powder 250mg 42.35％ 99.71％ Immobilized enzymes 1.0g 35.41％ 99.73％

[0047] As shown in Table 1, different forms of amidase preparations exhibit varying activities, while their selectivity remains unchanged.

[0048] Example 3: Synthesis of dextro-ketoprofen catalyzed by amidase at different temperatures

[0049] 100 mL of 0.1 M Tris-HCl buffer (pH 7.5) was added to a 500 mL reaction flask. 5 g of ketoprofen amide was dissolved in 20 mL of DMSO and added to the reaction flask. Then, 0.25 g of amidase powder was added, and the pH was adjusted to 7.5 with sodium hydroxide aqueous solution. The reaction was stirred for 24 h at different temperatures. Samples were taken for analysis to determine the conversion rate and the ee% value of the product dextro-ketoprofen. The results of the amidase reaction at different temperatures are shown in Table 2.

[0050] Table 2. Conversion rate and ee value of amidase reaction at different temperatures.

[0051] 20℃ 25.34％ 99.77％ 30℃ 37.49％ 99.71％ 40℃ 43.74％ 99.73％ 50℃ 41.43％ 99.77％ 60℃ 33.47％ 99.63％

[0052] As shown in Table 2, the conversion rate is highest at a reaction temperature of 40℃.

[0053] Example 4: Synthesis of dextro-ketoprofen catalyzed by amidase at different initial pH levels

[0054] 100 mL of 0.1 M Tris-HCl buffer solution with different pH values ​​was added to a 500 mL reaction flask. 5 g of ketoprofen amide was dissolved in 20 mL of DMSO and added to the reaction flask. Then, 0.25 g of amidase powder was added, and the pH was adjusted to the set value with sodium hydroxide aqueous solution. The reaction was stirred at 40 °C for 24 h. Samples were taken for analysis to determine the conversion rate and the ee% value of the product dextro-ketoprofen. The amidase reaction results at different initial pH values ​​are shown in Table 3.

[0055] Table 3. Conversion rate and ee value of amidase at different initial pH values

[0056] 6.0 27.37％ 99.75％ 7.0 39.37％ 99.72％ 8.0 44.64％ 99.76％ 9.0 42.71％ 99.76％ 10.0 39.81％ 99.74％

[0057] As shown in Table 3, the conversion rate is highest when the reaction pH is 8.0.

[0058] Example 5: Synthesis of dextro-ketoprofen catalyzed by amidase at different feed concentrations

[0059] 100 mL of 0.1 M Tris-HCl buffer solution at different pH 8.0 was added to a 500 mL reaction flask. Ketoprofen amide was dissolved in 20 mL of DMSO and added to the reaction flask. Then, amidase powder was added, and the pH was adjusted to the set value with sodium hydroxide aqueous solution. The reaction was stirred at 40 °C for 24 h. Samples were taken for analysis to determine the conversion rate and the ee% value of the product dextro-ketoprofen. The amidase reaction results at different feed concentrations are shown in Table 4.

[0060] Table 4. Conversion rate and ee value of amidase reaction at different feed concentrations

[0061] 20g / L 0.1g 2g 49.77％ 99.73％ 50g / L 0.25g 5g 45.37％ 99.77％ 80g / L 0.4g 8g 43.72％ 99.76％ 100g / L 0.5g 10g 42.81％ 99.76％ 150g / L 0.75g 15g 35.59％ 99.71％

[0062] As shown in Table 4, the conversion rate exceeds 40% when the feed concentration is 100 g / L.

[0063] Example 6: Synthesis of dextro-ketoprofen catalyzed by amidase at different enzyme-to-base ratios

[0064] 100 mL of 0.1 M Tris-HCl buffer (pH 8.0) was added to a 500 mL reaction flask. 10 g of ketoprofen amide was dissolved in 20 mL of DMSO and added to the reaction flask. Then, amidase powder was added according to different enzyme-to-raw material ratios (mass ratio of enzyme to raw material). The pH was adjusted to the set value with sodium hydroxide aqueous solution. The reaction was stirred at 40 °C for 24 h. Samples were taken for analysis to determine the conversion rate and the ee% value of the product dextro-ketoprofen. The reaction results of amidase at different feed concentrations are shown in Table 5.

[0065] Table 5. Conversion rate and product ee value of amidase reaction at different enzyme-to-base ratios.

[0066] 1 / 50 0.2g 10g 18.33％ 99.75％ 1 / 20 0.5g 10g 43.15％ 99.77％ 1 / 10 1g 10g 49.67％ 99.76％ 1 / 5 2g 10g 50.07％ 99.73％ 1 / 1 10g 10g 50.17％ 99.67％

[0067] As shown in Table 5, considering both enzyme usage cost and reaction efficiency, the enzyme-to-bottom ratio is determined to be 1 / 20.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for synthesizing dextro-ketoprofen using amidase, characterized in that: Ketoprofen amide was selectively hydrolyzed using an amidase derived from Rhodococcus erythropolis CCM2595 to obtain dextro-ketoprofen; the amino acid sequence of the amidase is shown in SEQ ID NO.1; the selective hydrolysis reaction was carried out in Tris-HCl buffer with dimethyl sulfoxide as a co-solvent at a reaction temperature of 40°C and a pH of 8.0, the concentration of ketoprofen amide was 100-150 g / L, and the mass ratio of the amidase to ketoprofen amide was 1 / 20 to 1 / 50.

2. The method for synthesizing dextro-ketoprofen using amidase as described in claim 1, characterized in that: The concentration of ketoprofenamide is 100 g / L, and the mass ratio of the amidase to ketoprofenamide is 1 / 20.

3. The method for synthesizing dextro-ketoprofen using amidase as described in claim 1, characterized in that: The amidase is an enzyme powder.

4. The method for synthesizing dextro-ketoprofen using amidase as described in claim 1, characterized in that, The amidase was prepared using the following method: (1) Synthesize the nucleotide sequence encoding the amidase, the nucleotide sequence being shown in SEQ ID NO.2; introduce NdeI and EcoRI sites at both ends of the gene respectively, clone it into the pET21a vector, transform it into Escherichia coli BL21(DE3) competent cells, plate it on LB plates containing 50 μg / ml ampicillin resistance, pick single clones after culture, culture at 37℃ for 8-12 h, pick single clones to obtain engineered bacteria; (2) The engineered bacteria were inoculated into LB liquid medium containing 50 μg / mL ampicillin resistance and cultured overnight at 37°C to obtain seed culture. The seed culture was transferred to fermentation medium containing 50 μg / mL ampicillin resistance at an inoculation rate of 3%. The culture was cultured at 37°C with a stirring speed of 300 rpm and dissolved oxygen controlled at 30% or higher. Feed was added continuously throughout the process. When the OD reached 0.8-1.0, the temperature was lowered to 25°C, and IPTG was added to a final concentration of 0.2 mM to induce expression for 20 h. The amidase cells were collected by centrifugation. The fermentation medium consists of: 10 g / L glucose, 30 g / L yeast extract, 5.08 g / L disodium hydrogen phosphate, 3 g / L potassium dihydrogen phosphate, 2 g / L magnesium sulfate, 1 g / L calcium chloride, and 0.23 g / L sodium citrate. The composition of the feed solution is: yeast extract 50g / L, glucose 300g / L, magnesium sulfate 10g / L; (3) The amidase cells obtained in step (2) are suspended in 0.1M pH7.0 Tris-HCl buffer and the cells are broken in an ultrasonic homogenizer or a high-pressure homogenizer to obtain cell lysate, which is enzyme solution; the enzyme solution is placed in a vacuum freeze dryer and freeze-dried to obtain amidase powder.

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