A penicillin acylase mutant and its use in preparing tranexamic acid

Abstract

The application provides a penicillin acylase mutant and application thereof in preparation of trans-aminomethylcyclohexane acid, the amino acid sequence of the penicillin acylase mutant is an amino acid sequence obtained by mutation of the amino acid sequence shown in SEQ ID NO: 1, and the aspartic acid at the 357th position of the penicillin acylase mutant is mutated into lysine. The penicillin acylase mutant in the application takes a gene sequence encoding a penicillin acylase of Providencia rettgeri as a template to construct a genetically engineered bacterial strain Escherichia coli BL21 (DE3)-pET28a (+)- Pr PGA, and through reaction condition optimization, the final conversion rate can reach 87-96%.

Description

Technical Field

[0001] This invention belongs to the field of biochemical engineering, and in particular relates to a penicillin acylase mutant and its application in the preparation of trans-tranexamic acid. Background Technology

[0002] Tranexamic acid, also known as tranexamic acid, is chemically named trans-4-aminomethylcyclohexanecarboxylic acid. Studies have confirmed that its active ingredient is trans-tranexamic acid, while the effect of cis-tranexamic acid is only 1 / 50th that of the trans isomer. Tranexamic acid is a derivative of tranexamic acid and is a classic antifibrinolytic hemostatic drug. Its hemostatic mechanism is the same as that of aminocaproic acid and tranexamic acid, but its effect is stronger, 7-10 times more potent than aminocaproic acid and twice as potent as tranexamic acid, while its toxicity is similar. It is widely used in clinical trials for patients with bleeding tendencies, such as those with hemophilia, oral surgery, orthopedics, postpartum hemorrhage, menorrhagia, and traumatic brain injury.

[0003] Another important use of tranexamic acid is in cosmetics, where it has two main effects: improving rough skin and acne, and resisting pigmentation (whitening), especially effective for melasma.

[0004] Currently, the main methods for preparing trans-tranexamic acid are as follows:

[0005] The preparation method in Chinese invention patent application number CN2022100677295 (application date January 20, 2022) involves mixing cis isomer or a mixture of cis / trans isomers of tranexamic acid with barium hydroxide octahydrate, heating at 220℃ for 6 hours, cooling to 30-60℃, adjusting the pH to 7-8 with concentrated hydrochloric acid, and then slurrying at 20-30℃ to obtain trans tranexamic acid, with a cis / trans ratio of 7.9 / 92.1.

[0006] The preparation method in Chinese invention patent application CN2025106636733 (application date May 22, 2025) involves adding sodium hydroxide to a mixture of cis / trans isomers of tranexamic acid, passing it through a cation exchange resin at 205°C for 10 hours, concentrating and crystallizing to obtain trans-tranexamic acid.

[0007] In the above methods, cis / trans mixtures of tranexamic acid are converted to trans-tranexamic acid through high-temperature and high-pressure conversion. This method has many problems, such as numerous reaction steps, complex operation, long reaction time, and a large amount of post-treatment waste. It also places high demands on equipment and poses significant safety risks. Summary of the Invention

[0008] In view of this, the present invention aims to overcome the defects in the prior art and proposes a penicillin acylase mutant and its application in the preparation of trans-tranexamic acid.

[0009] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0010] The present invention provides a penicillin acylase mutant, wherein the amino acid sequence of the penicillin acylase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO: 1, wherein the aspartic acid at position 357 of the penicillin acylase mutant is mutated to lysine, and the amino acid sequence is shown in SEQ ID NO: 2.

[0011] The present invention also provides a nucleic acid molecule that encodes the penicillin acylase mutant.

[0012] The present invention also provides a recombinant vector comprising the aforementioned nucleic acid molecule.

[0013] The present invention also provides a recombinant bacterium, wherein the recombinant bacterium comprises the recombinant vector.

[0014] Furthermore, the expression vector of the recombinant bacteria is pET28a(+), and the starting strain of the recombinant bacteria is Escherichia coli BL21(DE3).

[0015] The present invention also provides the application of the described penicillin acylase mutant in the preparation of trans-tranexamic acid.

[0016] The present invention also provides a method for preparing trans-tranexamic acid using the aforementioned penicillin acylase mutant, comprising the following steps:

[0017] Step 1 involves mixing the cis-trans isomers of tranexamic acid with a solvent and a base, then adding phenylacetyl chloride dropwise to initiate an N-phenylacetylation reaction. The pH of the reaction system is adjusted, and the mixture is then filtered to obtain N-phenylacetyl-tranexamic acid.

[0018] Step 2 involves dissolving the N-phenylacetyl-tranexamic acid in deionized water, adjusting the pH of the solution, adding the penicillin acylase mutant to it, and performing an enzymatic resolution reaction. After the reaction is completed, the solution is centrifuged, concentrated, crystallized, and filtered to obtain cis-tranexamic acid and N-phenylacetyl-trans-tranexamic acid solutions.

[0019] Step 3 involves adjusting the pH of the N-phenylacetyl-trans-tranexamic acid solution, then concentrating, crystallizing, and filtering to obtain N-phenylacetyl-trans-tranexamic acid. An acid is then added to the N-phenylacetyl-trans-tranexamic acid to initiate a hydrolysis reaction, followed by concentration, crystallization, filtration, drying, and recrystallization to obtain the trans-tranexamic acid.

[0020] The preparation route of the trans-tranexamic acid is shown in Formula I:

[0021]

[0022] Formula I

[0023] Further, the purity of the cis-trans isomer of tranexamic acid in step 1 is 95.0-99.8%, wherein the mass ratio of cis-tranexamic acid to trans-tranexamic acid is (40.0-60.0):(40.0-60.0); the solvent in step 1 is deionized water, and the base is sodium hydroxide; the molar ratio of the cis-trans isomer of tranexamic acid to phenylacetyl chloride in step 1 is 1:(1.05-1.3); the molar ratio of the cis-trans isomer of tranexamic acid to sodium hydroxide in step 1 is 1:(1.1-1.4); the mass ratio of the cis-trans isomer of tranexamic acid to deionized water in step 1 is 1:(10-20); the reaction conditions for the N-acetylation reaction in step 1 are: stirring at 0-5℃ for 2-5 h, and stirring at room temperature for 1-3 h; the pH value of the reaction system in step 1 is 3.0-4.5.

[0024] Furthermore, in step 2, the mass ratio of N-phenylacetyl-tranexamic acid to deionized water is 1:(30-60); the pH of the solution in step 2 is 7.0-8.0; the amount of penicillin acylase mutant added in step 2 is 0.15-1.2 times the mass of N-phenylacetyl-tranexamic acid; the temperature of the enzymatic resolution reaction in step 2 is 25-35℃, the time is 24-48h, and the pH is 7.0-8.0.

[0025] Furthermore, the pH value of the N-phenylacetyl-trans-tranexamic acid solution in step 3 is 3-3.5; the acid in step 3 is 3-6M hydrochloric acid; the solid-liquid ratio of N-phenylacetyl-trans-tranexamic acid to acid in step 3 is 1g:(8-12)mL; the temperature of the hydrolysis reaction in step 3 is 50-70℃, and the time is 1.5-4.5h.

[0026] Compared with the prior art, the present invention has the following advantages:

[0027] The penicillin acylase mutant described in this invention encodes Providencia retrieval (… Providencia rettgeri The gene sequence of penicillin acylase ( Pr Using PGA (GenBank: M86533.1) as a template, a genetically engineered strain of *Escherichia coli* BL21(DE3)-pET28a(+)- expressing penicillin acylase was constructed. Pr PGA, and through optimization of reaction conditions, the final conversion rate can reach 87-96%.

[0028] The method for preparing trans-tranexamic acid described in this invention uses the cells of genetically engineered bacteria that efficiently express penicillin acylase mutants as the enzyme source to carry out an enzymatic reaction. Compared with the chemical method, the reaction conditions are milder, the operation is simpler, the conversion rate is higher, and the product purity is higher. Attached Figure Description

[0029] Figure 1 The transformation result of the original enzyme (PGA) described in Example 1 of this invention;

[0030] Figure 2 The transformation result of the mutant enzyme (PGA-1) described in Example 1 of this invention;

[0031] Figure 3 The detection results of the cis-trans isomers of tranexamic acid described in Example 2 of this invention;

[0032] Figure 4 The detection results are those of trans-tranexamic acid as described in Example 2 of this invention. Detailed Implementation

[0033] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0034] The present invention will be described in detail below with reference to the embodiments.

[0035] Example 1: Construction of genetically engineered Escherichia coli and mutation and expression of penicillin acylase

[0036] according to Pr Primers were designed based on the PGA gene sequence: upstream primer F: 5'-TCGCGGATCCATGAAAAAACACC-3' (SEQ ID NO.3), downstream primer R: 5'-GGCCGCAAGCTTTTATCTCTC-3' (SEQ ID NO.4), to amplify the target gene and construct the recombinant expression plasmid pET-28a(+)- Pr PGA, restriction site is BamH I and Hind III;

[0037] Penicillin acylase was simulated using the Swiss Model online software to obtain its spatial structure. Molecular docking analysis was performed using Autodock 4.2 to construct a substrate binding model and screen key amino acid residues within the substrate's 4 Å range. Finally, the acidic amino acid aspartic acid (ASP) at position 357, closest to the substrate, was mutated to the basic amino acid lysine (LYS). The upstream primer D357KF is 5'-CTACCGCTGGGTTTGGTAAAGGTGTTGATATATTTG-3' (SEQ ID NO.5), and the downstream primer D357KR is 5'-CAAATATATCAACACCTTTACCAAACCCAGCGGTAG-3' (SEQ ID NO.6).

[0038] The PCR system is shown in Table 1;

[0039] Table 1 PCR system

[0040]

[0041] PCR amplification conditions: 98 ℃ pre-denaturation for 5 min; 95 ℃ denaturation for 30 s, 64 ℃ annealing for 1 min, 68 ℃ extension for 10 min, 15 cycles, incubation at 8 ℃.

[0042] The successfully sequenced recombinant plasmid was transformed into E. coli BL21(DE3), and after culturing in LB medium at 37 °C for 4 h, expression was induced by adding IPTG at low temperature. Pr PGA, 18 ℃ for 16 h. Collect the cultured cells by centrifugation, wash with PBS and store at -20℃ for later use.

[0043] The bacterial sludge was resuspended in 50 mM phosphate buffer to a final cell suspension concentration of 10 g / L. Then, 50 mM substrate was added, and the pH was controlled at 7.5. The reaction was carried out at 30°C for 24 h. Samples were taken for analysis, and the HPLC results are as follows: Figures 1-2 As shown.

[0044] The substrate is N-phenylacetyl-cis-tranexamic acid (ratio 1:1). A decrease in the content of N-phenylacetyl-cis-tranexamic acid indicates... Pr PGA is active. The conversion effect of penicillin acylase can be reflected by the peak area change of N-phenylacetyl-cis-tranexamic acid. Figures 1-2As shown, the penicillin acylase mutant exhibits a significant improvement in transformation efficiency after optimization. By comparing peak areas, the original enzyme (PGA) retained 28.2% of N-phenylacetyl-cis-tranexamic acid after transformation (cis-trans ratio 1:2.5), while the mutant enzyme (PGA-1) retained only 8.39% of N-phenylacetyl-cis-tranexamic acid after transformation (cis-trans ratio 1:10.9), indicating that… Pr The PGA mutant exhibits enzymatic activity, and the conversion efficiency of the mutated PGA is significantly improved, indicating its value for industrial application.

[0045] Example 2: Application of Trans-tranexamic Acid Preparation

[0046] A method for preparing trans-tranexamic acid includes the following steps:

[0047] (1) In a 1L single-necked bottle, add the cis-trans isomers of tranexamic acid sequentially (purity 99.8%, mass ratio of cis to trans tranexamic acid isomers 50.4:49.6, such as...). Figure 3 The following solutions were prepared: (45.0 g, 0.29 mol) of sodium hydroxide (675 mL) and sodium hydroxide (14.5 g, 0.36 mol). The solution was stirred at 0°C until completely dissolved. Phenylacetyl chloride (53.8 g, 0.38 mol) was slowly added dropwise over 40 min. The mixture was stirred at 0°C for 3 h, allowed to rise naturally to room temperature, and stirred for 2 h. 3.0% hydrochloric acid was slowly added dropwise until the pH value was 3.5-4.0. A large amount of solid precipitated out. The solid was filtered to obtain a white powdery solid, N-phenylacetyl-tranexamic acid (71.6 g), with an HPLC purity of 99.2%, a content of 98.6%, and a yield of 89.7%.

[0048] (2) In a 5L single-necked flask, add N-phenylacetyl-tranexamic acid (63.5g) and deionized water (2.54L), adjust the pH to 7.5 with 20% NaOH solution, add penicillin acylase (25.4g) from Example 1 at 28℃, maintain the pH of the system at 7.3-7.5 with 20% NaOH solution, keep warm at 28-30℃, centrifuge after 36h, concentrate the supernatant to 500mL, add 500mL isopropanol, crystallize in an ice-water bath for 3h, filter to obtain cis-tranexamic acid (16.7g), HPLC purity 98.8%, ee value 98.2%;

[0049] (3) The filtrate was an N-phenylacetyl-trans-tranexamic acid solution. The pH was adjusted to 3-3.5 with 3.0% hydrochloric acid. Concentration was stopped when a solid appeared. Crystallization was carried out in an ice-water bath for 4 hours. After filtration, N-phenylacetyl-trans-tranexamic acid (29.7 g) was obtained. In a 500 mL single-necked flask, N-phenylacetyl-trans-tranexamic acid (27.5 g) and 4M hydrochloric acid (275 mL) were added. The mixture was heated at 60 °C for 3 hours. After concentration, crystallization, filtration, drying, and recrystallization, trans-tranexamic acid (14.7 g) was obtained. The HPLC purity was 99.7%, the ee value was 99.6%, and the yield was 93.6%. Figure 4 As shown, trans-tranexamic acid prepared using commercially available penicillin acylase had an HPLC purity of 87.3%, an ee value of 91.6%, and a yield of 89.7%.

Claims

1. A penicillin acylase mutant, characterized in that: The amino acid sequence of the penicillin acylase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO:

1. The penicillin acylase mutant is obtained by mutating the aspartic acid at position 357 of the amino acid shown in SEQ ID NO: 1 to lysine. The amino acid sequence of the penicillin acylase mutant is shown in SEQ ID NO:

2.

2. A nucleic acid molecule, characterized in that: The nucleic acid molecule encodes the penicillin acylase mutant of claim 1.

3. A recombinant vector, characterized in that: The recombinant vector comprises the nucleic acid molecule of claim 2.

4. A recombinant bacterium, characterized in that: The recombinant bacteria comprise the recombinant vector as described in claim 3.

5. The recombinant bacteria according to claim 4, characterized in that: The expression vector of the recombinant bacteria is pET28a(+), and the starting strain of the recombinant bacteria is Escherichia coli BL21(DE3).

6. The use of the penicillin acylase mutant of claim 1 in the preparation of trans-tranexamic acid.

7. A method for preparing trans-tranexamic acid using the penicillin acylase mutant of claim 1, characterized in that: Includes the following steps: Step 1 involves mixing the cis-trans isomers of tranexamic acid with a solvent and a base, then adding phenylacetyl chloride dropwise to initiate an N-phenylacetylation reaction. The pH of the reaction system is adjusted, and the mixture is then filtered to obtain N-phenylacetyl-tranexamic acid. Step 2 involves dissolving the N-phenylacetyl-tranexamic acid in deionized water, adjusting the pH of the solution, adding the penicillin acylase mutant described in claim 1, and performing an enzymatic resolution reaction. After the reaction is completed, the solution is centrifuged, concentrated, crystallized, and filtered to obtain cis-tranexamic acid and N-phenylacetyl-trans-tranexamic acid solutions. Step 3 involves adjusting the pH of the N-phenylacetyl-trans-tranexamic acid solution, then concentrating, crystallizing, and filtering to obtain N-phenylacetyl-trans-tranexamic acid. An acid is then added to the N-phenylacetyl-trans-tranexamic acid to initiate a hydrolysis reaction, followed by concentration, crystallization, filtration, drying, and recrystallization to obtain the trans-tranexamic acid.

8. The method for preparing trans-tranexamic acid according to claim 7, characterized in that: The purity of the cis-trans isomers of tranexamic acid in step 1 is 95.0-99.8%, wherein the mass ratio of cis-tranexamic acid to trans-tranexamic acid is (40.0-60.0):(40.0-60.0); the solvent in step 1 is deionized water, and the base is sodium hydroxide; the molar ratio of the cis-trans isomers of tranexamic acid to phenylacetyl chloride in step 1 is 1:(1.05-1.3); the molar ratio of the cis-trans isomers of tranexamic acid to sodium hydroxide in step 1 is 1:(1.1-1.4); the mass ratio of the cis-trans isomers of tranexamic acid to deionized water in step 1 is 1:(10-20); the reaction conditions for the N-phenylacetylation reaction in step 1 are: stirring at 0-5℃ for 2-5 h, and stirring at room temperature for 1-3 h; the pH value of the reaction system in step 1 is 3.0-4.

5.

9. The method for preparing trans-tranexamic acid according to claim 7, characterized in that: In step 2, the mass ratio of N-phenylacetyl-tranexamic acid to deionized water is 1:(30-60); the pH of the solution in step 2 is 7.0-8.0; the amount of penicillin acylase mutant added in step 2 is 0.15-1.2 times the mass of N-phenylacetyl-tranexamic acid; the temperature of the enzymatic resolution reaction in step 2 is 25-35℃, the time is 24-48h, and the pH is 7.0-8.

0.

10. The method for preparing trans-tranexamic acid according to claim 7, characterized in that: The pH value of the N-phenylacetyl-trans-tranexamic acid solution in step 3 is 3-3.5; the acid in step 3 is 3-6M hydrochloric acid; the solid-liquid ratio of N-phenylacetyl-trans-tranexamic acid to acid in step 3 is 1g:(8-12)mL; the temperature of the hydrolysis reaction in step 3 is 50-70℃, and the time is 1.5-4.5h.

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