Use of a hydantoinase PSDHP2 in the preparation of R-baclofen precursors and other analogs

Abstract

The application belongs to the technical field of pharmaceutical chemistry, and particularly relates to application of hydantoinase PSDHP2 in preparation of R-baclofen precursors and other analogues. The application provides a hydantoinase PSDHP2. The hydantoinase PSDHP2 is a kind of biological chiral enzyme for R-baclofen precursor synthesis, has the characteristics of high temperature resistance and strong alkali resistance, still has high chiral rate and high reaction rate under the environment of 70 DEG C and pH 10, when the hydantoinase PSDHP2 is used as a catalyst to produce R-CGM, the enantiomeric excess rate ee is greater than 90%, the reaction rate V max is 36.17 mu mol min ‑1 mg ‑1 . The hydantoinase PSDHP2 has the characteristics of low cost, environmental friendliness and no secondary pollution. The application provides a new effective way for producing R-baclofen precursor R-CGM.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, specifically relating to the application of a hydantoin PSDHP2 enzyme in the preparation of R-baclofen precursors and other analogues. Background Technology

[0002] Baclofen is a gamma-aminobutyric acid (GABA) B receptor agonist, primarily used clinically to relieve symptoms such as muscle spasms, clonus, rigidity, and accompanying pain associated with multiple sclerosis, spinal cord injury, cerebral palsy, and other central nervous system injuries. Its mechanism of action mainly involves activating GABA B receptors in the central nervous system, inhibiting the release of excitatory neurotransmitters, and reducing spinal reflex excitability, thereby exerting a central muscle relaxant effect. The baclofen molecule contains a chiral center, and the racemic mixture is commonly used clinically. R-baclofen (also known as arbaclofen) is considered the primary enantiomer. Related literature indicates that the pharmacological activity of baclofen mainly derives from the R-configuration enantiomer; therefore, obtaining high-optical-purity R-baclofen is crucial for clarifying the source of efficacy, reducing the burden of ineffective enantiomers, and improving drug quality control.

[0003] The intermediate 3-(4-chlorophenyl)glutarimide (CGI) for R-baclofen is readily prepared. CGI is hydrolyzed to its racemic form, R-3-(4-chlorophenyl)glutaric acid monoamide (R-CGM), which then undergoes a Hoffmann rearrangement to generate R-baclofen. Chemical hydrolysis typically requires high temperatures, strong acids, and strong bases, and involves the formation of large amounts of salts, making separation and purification difficult and causing environmental pollution. Compared to traditional chemical resolution methods, enzymatic hydrolysis offers advantages such as less environmental pollution, lower cost, and higher optical purity of the product. However, enzymatic hydrolysis also has drawbacks, including poor tolerance of proteases to complex environments, low enantiomeric excess of the hydrolysis product, and low catalytic rate. Therefore, how to provide a catalytic protease for the production of R-CGM that is highly tolerant to complex environments, has a high catalytic rate and high yield has become an urgent problem to be solved in this field. Summary of the Invention

[0004] To address the lack of a catalytic protease in the existing technology for the catalytic production of R-CGM, and to ensure that this protease possesses the characteristics of tolerance to complex environments, high catalytic rate, and high yield, this invention provides an application of hydantoin PSDHP2 in the preparation of R-baclofen precursors and other analogues, specifically including the following technical solutions: The present invention provides a hydantoin PSDHP2, the amino acid sequence of which is shown in SEQ ID NO:1.

[0005] Preferably, the nucleotide sequence encoding the hydantoin PSDHP2 is shown in SEQ ID NO:2.

[0006] The present invention also provides a hydantoin mutant PSDHP2M, the amino acid sequence of which is shown in SEQ ID NO:3.

[0007] Preferably, the nucleotide sequence encoding the hydantoin mutant PSDHP2M is shown in SEQ ID NO:4.

[0008] The present invention also provides an enzyme preparation, wherein the active ingredient of the enzyme preparation comprises the hydantoin PSDHP2 and / or the hydantoin mutant PSDHP2M as described above.

[0009] The present invention also provides the use of the hydantoin PSDHP2, the hydantoin mutant PSDHP2M, or the enzyme preparation described above in the synthesis of R-3-(4-chlorophenyl)glutaric acid monoamide.

[0010] The present invention also provides a method for synthesizing R-3-(4-chlorophenyl)glutaric acid monoamide, the steps of which include: Using 3-(4-chlorophenyl)glutaric acid imine as a substrate, a catalytic reaction was carried out to obtain R-3-(4-chlorophenyl)glutaric acid monoamide; The catalyst used in the catalytic reaction includes any one or more of the following: hydantoin PSDHP2, hydantoin mutant PSDHP2M, or enzyme preparations, as described above.

[0011] Preferably, the working concentration of the 3-(4-chlorophenyl)glutaric acid imine is 10~200mM; and the working concentration of the catalyst is 5~20μg / mL.

[0012] Preferably, the temperature of the catalytic reaction is 16~90℃; the pH of the catalytic reaction is 6~11; The reaction system for the catalytic reaction also includes Tris-HCl buffer and MnCl2; The concentration of the Tris-HCl buffer solution is 40~60 mM; The concentration of MnCl2 is 1.8~2.2mM.

[0013] The present invention also provides the application of the method described above in the preparation of R-3-(4-chlorophenyl)glutaric acid monoamide.

[0014] The present invention also provides the application of the method described above in the preparation of R-baclofen.

[0015] The beneficial effects of this invention are as follows: This invention provides a hydantoin PSDHP2, the amino acid sequence of which is shown in SEQ ID NO:1. Hydantoin PSDHP2 is a chiral bioenzyme for the synthesis of R-baclofen precursors, characterized by its high temperature and strong alkali resistance. It maintains high chirality and a high reaction rate even at 80℃ and pH 10. When using hydantoin PSDHP2 as a catalyst to produce R-CGM, the enantiomeric excess ee > 90% and the reaction rate Vc is high. max 36.17 μmol min -1 mg -1 The hydantoin PSDHP2 enzyme is characterized by low cost, environmental friendliness, and no secondary pollution. This invention provides a new and effective route for the production of the R-baclofen precursor R-CGM. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 The graph shows the catalytic rate detection results of heinz PSDHP2 and its mutant PSDHP2M. Figure 2 The graph shows the enantiomeric excess rate of hydantoin PSDHP2 and its mutant PSDHP2M. Figure 3 The graph shows the catalytic efficiency of hydantoin PSDHP2 and its mutant PSDHP2M at different pH values. Figure 4 The graph shows the catalytic rate detection results for comP1 and comP2. Detailed Implementation

[0018] The present invention provides a hydantoin PSDHP2, the amino acid sequence of which is shown in SEQ ID NO:1.

[0019] SEQ ID NO:1:MKKVIRGGTVVTAADTYRADVLIEGERITAIGSELQADGAEEIDAAGCYVLPGGIDPHTHLDMPFGGTVTADDFFTGTKAAAFGGTTSIIDFCLTKKGETLRSAIATWHEKAR GKAVIDYGFHLMIAEANDQVLDELESVVSTEGITSLKVFMAYKNVFQADDETLFKTLVRARELGALVQVHAENGDVLDYLTKKALAEGNTAPIYHALTRPPEAEGEATGRAIALTALA DAQLYVVHVSCAAAVQRIAEARERGLNVYGETCPQYLALDITALEKPDFEGAKYVWSPPLREKWNQDVLWNALKNGILQTVGSDHCAFNFFGQKELGKDDFTRIPNGGPLIEDRLAIL YSEGVRQGRISLNQFVDVCSTKAAKLFGMFPRKGTIAVGSDADVVIFDPRVKRTLSAQTHHMNVDYNPFEGMEVYGEVVSVLSRGAFVVRNKQFVGLAGAGQFIKRAAFKRPHHHHHHH.

[0020] As one embodiment, the nucleotide sequence encoding the hydantoin PSDHP2 is shown in SEQ ID NO:2.

[0021]

[0022] The hydantoin PSDHP2 described in this invention is a chiral bioenzyme for the synthesis of R-CGM, which is a precursor for the synthesis of R-baclofen. The hydantoin PSDHP2 can efficiently catalyze the hydrolysis of CGI into the racemic R-CGM, and it is resistant to high temperatures and strong alkaline environments, maintaining a high reaction rate even at 80°C and pH 10. Furthermore, its enzymatic hydrolysis products exhibit high chirality. In addition, traditional chemical processes using toxic reagents for mirror-image isomer separation pose a risk of secondary pollution due to the residue of these toxic reagents. This invention, however, uses a hydantoin biocatalysis method to produce only a single chiral product, eliminating the risk of pollution. Therefore, the hydantoin PSDHP2 described in this invention is also low-cost, environmentally friendly, and does not cause secondary pollution. This invention compares the efficiency of PSDHP2 in catalyzing CGI with that of existing chiral bioenzymes (comP1 and comP2) for R-CGM synthesis through verification experiments, all under the conditions of 80°C and pH 10. The results showed that the catalytic rate of hydantoin PSDHP2 was approximately 36.17 μmol of product per mg enzyme per min, while the catalytic rates of comP1 and comP2 were approximately 10.36 μmol of product per mg enzyme per min. g The enzyme produces 10.57 μmol of product per minute. The hydantoin PSDHP2 of this invention has a higher catalytic rate.

[0023] The present invention also provides a hydantoin mutant PSDHP2M, the amino acid sequence of which is shown in SEQ ID NO:3.

[0024] SEQ ID NO:3:MKKVIRGGTVVTAADTYRADVLIEGERITAIGSELQADGAEEIDAAGCYVLPGGIDPHTHLDAPHGGTVTADDFFTGTKAAAFGGTTSIIDFCLTKKGETLRSAIATWHEKAR GKAVIDYGFHLMIAEANDQVLDELESVVSTEGITSLKVFMAYKNVFQADDETLFKTLVRARELGALVQVHAENGDVLDYLTKKALAEGNTAPIYHALTRPPEAEGEATGRAIALTALA DAQLYVVHVSCAAAVQRIAEARERGLNVYGETCPQYLALDITALEKPDFEGAKYVWSPPLREKWNQDVLWNALKNGILQTVGSDHTAFNFFGQKELGKDDFTRIPNGGPLIEDRLAIL YSEGVRQGRISLNQFVDVCSTKAAKLFGMFPRKGTIAVGSDADVVIFDPRVKRTLSAQTHHMNVDYNPFEGMEVYGEVVSVLSRGAFVVRNKQFVGLAGAGQFIKRAAFKRPHHHHHHH.

[0025] As one embodiment, the nucleotide sequence encoding the hydantoin mutant PSDHP2M is shown in SEQ ID NO:4.

[0026]

[0027] The hydantoin mutant PSDHP2M is a mutant of hydantoin PSDHP2, containing substitutions at three amino acid residue sites. Specifically, the mutated sites are: amino acid 63 of hydantoin PSDHP2 is replaced by A instead of M; amino acid 65 is replaced by H instead of L; and amino acid 317 is replaced by T instead of C. PSDHP2M is also a chiral enzyme for R-CGM synthesis, capable of efficiently catalyzing the hydrolysis of CGI to racemic R-CGM, and is resistant to high temperatures and strong alkaline environments, maintaining high chirality and reaction rate even at 80℃ and pH 10. Compared to hydantoin PSDHP2, the mutant PSDHP2M exhibits higher chirality and reaction rate. This invention compared the efficiency of PSDHP2M in catalyzing CGI synthesis with existing chiral enzymes (comP1 and comP2) at 80℃ and pH 10. The results showed that the rate of R-CGM synthesis catalyzed by PSDHP2M is approximately 36.97 μmol / min. -1 mg -1 The enantiomeric excess ee > 90%. The hydantoin mutant PSDHP2M of the present invention has a higher catalytic rate.

[0028] The present invention also provides an enzyme preparation, wherein the active ingredient of the enzyme preparation comprises the hydantoin PSDHP2 and / or the hydantoin mutant PSDHP2M as described above.

[0029] In one embodiment, the enzyme preparation includes any one or more of the following: a recombinant vector expressing the hydantoin PSDHP2, a recombinant strain expressing the hydantoin PSDHP2, a recombinant vector expressing the hydantoin mutant PSDHP2M, and a recombinant strain expressing the hydantoin mutant PSDHP2M. In one embodiment, when the enzyme preparation includes a recombinant strain expressing the hydantoin PSDHP2 or a recombinant strain expressing the hydantoin mutant PSDHP2M, the enzyme preparation can be used directly or after purification. Direct use involves directly catalyzing a sample using whole cells of the recombinant strain. Purified use involves expressing the hydantoin PSDHP2 using the recombinant strain, and then purifying the hydantoin PSDHP2 before use. In one embodiment, the recombinant vector expressing the hydantoin PSDHP2 includes a backbone vector. In one embodiment, the backbone vector includes a plasmid vector. In one embodiment, the plasmid vector includes pET-28a. In one embodiment, the recombinant vector is obtained by cloning the coding gene of the hydantoin PSDHP2 into the backbone vector. In one embodiment, the cloning sites are EcoRI and HindIII. In another embodiment, the hydantoin PSDHP2 is obtained by recombinant expression followed by purification from a recombinant strain. In another embodiment, the composition and construction method of the recombinant vector expressing the hydantoin mutant PSDHP2M are the same as those for the recombinant vector expressing the hydantoin PSDHP2, and will not be repeated here. In yet another embodiment, the recombinant expression and purification are standard procedures in the art, and will not be described further.

[0030] In one embodiment, the recombinant strain expressing the hydantoin PSDHP2 includes a basic microorganism. In one embodiment, the basic microorganism includes *Escherichia coli*. In one embodiment, the recombinant strain expressing the hydantoin mutant PSDHP2M includes a basic microorganism. In one embodiment, the basic microorganism includes *Escherichia coli*.

[0031] The present invention also provides the use of the hydantoin PSDHP2, the hydantoin mutant PSDHP2M, or the enzyme preparation described above in the synthesis of R-3-(4-chlorophenyl)glutaric acid monoamide.

[0032] The hydantoin PSDHP2, the hydantoin mutant PSDHP2M, or the enzyme preparation of this invention are a type of chiral biological enzyme for the synthesis of R-baclofen precursors. They are characterized by high temperature and strong alkali resistance, low cost, environmental friendliness, and no secondary pollution. This invention provides a new and effective route for the production of the R-baclofen precursor R-CGM.

[0033] The present invention also provides a method for synthesizing R-3-(4-chlorophenyl)glutaric acid monoamide, the steps of which include: A catalytic reaction was carried out using 3-(4-chlorophenyl)glutaric acid imine as a substrate to obtain R-3-(4-chlorophenyl)glutaric acid monoamide; the catalyst used in the catalytic reaction included any one or more of the following: hydantoin PSDHP2, hydantoin mutant PSDHP2M, or enzyme preparations as described above.

[0034] In one embodiment, the working concentration of the 3-(4-chlorophenyl)glutaric acid imide is 10-200 mM. In a preferred embodiment, the working concentration of the 3-(4-chlorophenyl)glutaric acid imide is 10-100 mM. In a specific embodiment, the working concentration of the 3-(4-chlorophenyl)glutaric acid imide is 20 mM. The working concentration of the 3-(4-chlorophenyl)glutaric acid imide refers to the final concentration of the 3-(4-chlorophenyl)glutaric acid imide in the catalytic reaction system before the start of the catalytic reaction.

[0035] In one embodiment, the working concentration of the catalyst is 5-20 μg / mL. In an optional embodiment, the working concentration of the catalyst can be any one or an intermediate value between any two of the following: 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL, 11 μg / mL, 12 μg / mL, 13 μg / mL, 14 μg / mL, 15 μg / mL, 16 μg / mL, 17 μg / mL, 18 μg / mL, 19 μg / mL, and 20 μg / mL. In a preferred embodiment, the working concentration of the catalyst is 7-15 μg / mL. In a specific embodiment, the working concentration of the catalyst is 10 μg / mL. The working concentration refers to the final concentration of the catalyst in the catalytic reaction system before the catalytic reaction begins.

[0036] In one embodiment, the temperature of the catalytic reaction is 16~90°C. In a preferred embodiment, the temperature of the catalytic reaction is 75~85°C. In a further preferred embodiment, the temperature of the catalytic reaction is 78~82°C. In a specific embodiment, the temperature of the catalytic reaction is 80°C. In one embodiment, the pH of the catalytic reaction is 6~11. In a preferred embodiment, the pH of the catalytic reaction is 9.5~10.5. In a further preferred embodiment, the pH of the catalytic reaction is 9.8~10.2. In a specific embodiment, the pH of the catalytic reaction is 10.

[0037] In one embodiment, the reaction system of the catalytic reaction further includes Tris-HCl buffer and MnCl2. In one embodiment, the concentration of the Tris-HCl buffer is 40-60 mM. In an optional embodiment, the final concentration of the Tris-HCl buffer can be any one or an intermediate value between any two of the following: 40 mM, 45 mM, 50 mM, 55 mM, and 60 mM. In a specific embodiment, the final concentration of the Tris-HCl buffer is 50 mM. In one embodiment, the final concentration of the MnCl2 is 1.8-2.2 mM. In an optional embodiment, the concentration of the MnCl2 can be 1.8 mM, 1.9 mM, 2.0 mM, 2.1 mM, and 2.2 mM. It should be noted that the concentrations of the Tris-HCl buffer and MnCl2 are both 2 mM.

[0038] The present invention also provides the application of the method described above in the preparation of R-baclofen.

[0039] The method described above in this invention can be used to prepare R-3-(4-chlorophenyl)glutaric acid monoamide, which is an intermediate of R-baclofen. R-3-(4-chlorophenyl)glutaric acid monoamide can be converted into the drug R-baclofen through the Hoffmann rearrangement reaction.

[0040] The method described above can also be used to synthesize R-baclofen analogs. As one embodiment, the analog is a 3-substituted glutarimide analog. A 3-substituted glutarimide analog refers to a class of compounds with glutarimide as the parent nucleus and a substituent R attached to the carbon atom at position 3. The R can be any conventional substituent. As a preferred embodiment, the R is preferably derived from small molecule hydrocarbon groups, heterocyclic groups, halogenated groups, and derivatives of the aforementioned substituents.

[0041] To further illustrate the present invention, the application of the hydantoin PSDHP2 provided by the present invention in the preparation of R-baclofen precursor and other similar products is described in detail below with reference to the accompanying drawings and embodiments, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0042] In the embodiments of this invention, the pET-28a plasmid, Escherichia coli BL21 (D3), kanamycin, LB medium, Tris-HCl buffer, PBS buffer, MnCl2, and phosphate solution were all purchased from Sangon Biotech (Shanghai) Co., Ltd.; the IPTG was purchased from Diamond Biotech; the nickel column magnetic bead solution was purchased from Beijing TransGen Biotech Co., Ltd., with the trade name TransGen Ni-NTA Resin; and the acetonitrile, n-hexane, and ethanol were purchased from Sigma-Aldrich.

[0043] Centrifuges were purchased from Eppendorf (model 5804 R); protein disruptors were purchased from BRANSON (model S-450D); ultrafiltration tubes were purchased from Merck Group (Germany); vertical mixers were purchased from Hangzhou Ruicheng Instrument Co., Ltd.; water baths were purchased from Shanghai Jinghong Co., Ltd. (model DK-8D); aqueous microporous membranes were purchased from Shimadzu; HPLC systems were purchased from Agilent Technologies (model Agilent Technologies Infinity II 1260); and C18 columns were purchased from Agilent Technologies (model ZORBAX Eclipse XDB-C18 (4.6 × 250 mm, 5 μm)).

[0044] Example 1: Preparation method of enzyme solution of hydantoin PSDHP2 and its mutant PSDHP2M 1. Preparation of chiral enzyme expression bacteria The encoding genes of heynase PSDHP2 and the mutant PSDHP2M were respectively recombined into the EcoRI and HindIII restriction sites of plasmid pET-28a using the conventional double enzyme digestion method to obtain recombinant plasmids.

[0045] The recombinant plasmid was transformed into Escherichia coli BL21(DE3) using conventional methods to obtain the recombinant strain.

[0046] The recombinant strain was inoculated into an Erlenmeyer flask containing 1L of LB medium at a volume ratio of 1:100 and cultured with a final concentration of 50μg / mL kanamycin. The culture conditions for the recombinant strain were: 37℃, 200 rpm for 16h.

[0047] OD of the bacterial solution 600 When the cytokine concentration (nm) reached 0.8, 1 mL of 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) was added, and the culture was continued for 16 h. After the culture was completed, the bacterial culture was centrifuged at 6000 × g for 10 min at 4 °C to harvest the bacterial cells. The bacterial cells were washed twice with 50 mM Tris-HCl buffer at pH 10 to obtain recombinant strains expressing hydantoin PSDHP2 and recombinant strains expressing the mutant PSDHP2M.

[0048] 2. Purification of chiral enzymes Resuspend the recombinant strain expressing hydantoin PSDHP2 and the recombinant strain expressing the mutant PSDHP2M in 30 mL of pH 7.5 PBS buffer to obtain bacterial suspensions expressing hydantoin PSDHP2 and PSDHP2M respectively.

[0049] 300 μL of 10 mM benzyl sulfonyl fluoride (PMSF) was added to each of the two bacterial suspensions. The PMSF-containing suspensions were then sonicated in an ice bath using a protein disruptor to obtain cell lysate. The sonication conditions were: 12.5 W power, 4 s operation time, 5 s interval, and 20 min operation time.

[0050] Centrifuge the cell lysate at 10,000 rpm for 20 min at 4°C and collect the supernatant. Add 3 mL of nickel column magnetic bead buffer to the purification column. After the protective buffer has eluted naturally, pass the column through 5 column volumes of ultrapure water, followed by 5 column volumes of pH 7.5 PBS buffer. Add the nickel column magnetic beads to the supernatant, fix the column in a vertical mixer, and invert and mix for 2 h at 4°C. Pour the combined mixture into the purification column and allow it to stand, allowing the nickel column magnetic beads to settle naturally on the pad. Collect the eluent, and then elute the protein sequentially with 3 mL of 20 mM, 30 mM, 300 mM, and 500 mM imidazole solutions. Elute three times with each concentration of eluent and collect the eluent.

[0051] The effluent was poured into a 15 mL 3KD ultrafiltration tube, centrifuged at 4000g for 2 h at 4 °C, concentrated to 0.5 mL, and 5 mL of PBS buffer (pH=7.5) was added. The solution was then filtered through a 0.22 μm ultrafiltration membrane and centrifuged at 4000g for 2 h at 4 °C to concentrate to 0.5 mL. This process was repeated three times to obtain pure hydantoin PSDHP2 enzyme solution and mutant PSDHP2M enzyme solution.

[0052] Example 2: Method for synthesizing R-CGM from hydantoin PSDHP2 and mutant PSDHP2M enzymes Take 10 μg of the hydantoin PSDHP2 enzyme solution or the mutant PSDHP2M enzyme solution prepared in Example 1, and dissolve them in 1 mL of 50 mM Tris-HCl buffer at pH 10 to obtain the hydantoin PSDHP2 enzyme solution or the mutant PSDHP2M enzyme solution.

[0053] The above enzyme solution was mixed with 2 mM MnCl2 and different concentrations of CGI (10, 25, 50, 100, 150, 200 mM) and reacted for 20 min. The reaction was carried out under 80°C water bath heating conditions. After 20 min of water bath heating, 20 μL of 6M concentrated hydrochloric acid was added to stop the reaction, yielding a reaction solution. The reaction solution was centrifuged at 4°C and 10000g for 20 min, and the supernatant was collected. The supernatant was filtered through a 0.22 μm aqueous microporous membrane to obtain the sample to be tested. The amount of CGM generated and the proportion of R-CGM and S-CGM in the sample were analyzed by HPLC.

[0054] Wherein, CGI represents 3-(4-chlorophenyl)glutarimide; R-CGM represents (R)-3-(4-chlorophenyl)glutaric acid monoamide; and S-CGM represents (S)-3-(4-chlorophenyl)glutaric acid monoamide.

[0055] The conversion rate was calculated by detecting the peak area of ​​CGM using HPLC. The specific operation is as follows: The chromatographic column was a ZORBAX Eclipse XDB-C18 (4.6 mm × 250 mm, 5 μm); the flow rate was 1 mL / min; the column temperature was 35℃; and the detection wavelength was 210 nm. The HPLC program was as follows: aqueous phase (0.1% phosphoric acid solution), organic phase elution curve: 0 min 90% aqueous phase, 10% organic phase; 0-15 min 70% aqueous phase, 30% organic phase; 15-18 min 10% aqueous phase, 90% organic phase; 18-23 min 8% aqueous phase, 92% organic phase. After column equilibration, 5 μL of sample was injected, with an equilibration interval of 5 min between injections. The CGM production was measured using the Michaelis-Menten curve, and Vmax was calculated. Vmax was obtained through Michaelis-Menten nonlinear fitting. The obtained data were substituted into the Michaelis-Menten equation v = Vmax[S] / (Km + [S]) for nonlinear fitting, and the final Vmax was 36.17 μmol·min⁻¹. -1 ·mg -1 The Km value is 7.99 mM.

[0056] The content ratios of R-CGM and S-CGM were determined using an HPLC (Waters Alliance E2695) with a Chiralpak AD-H column. The flow rate was set to 0.5 mL / min, the column temperature to 35℃, and the absorbance to 223.1 nm. The elution program was an organic phase solution as the mobile phase for 50 min. After column equilibration, 10 μL of R-CGM was injected for detection. Finally, the enantiomeric excess was calculated using Formula I.

[0057] Enantiomer excess rate (ee) = [([R-CGM]-[S-CGM]) / ([R-CGM]+[S-CGM])]×100% (Formula I) Note: In Formula I, R-CGM is the molar concentration of R-CGM in the sample; S-CGM is the molar concentration of S-CGM in the sample.

[0058] The results of enzyme-catalyzed reaction kinetic parameters and enantiomeric excess are shown in the figure. Figures 1-2 And Tables 1-2.

[0059] Table 1. Enzymatic reaction kinetic parameters of hydantoin PSDHP2 and its mutant PSDHP2M enzyme.

[0060] Table 2 Enantiomeric excess rate (ee%) of hydantoin PSDHP2 and mutant PSDHP2M enzymes

[0061] The recombinant Escherichia coli strains carrying the hydantoin PSDHP2 or mutant PSDHP2M enzyme gene obtained in Example 1 were taken out of the -80°C ultra-low temperature freezer and subjected to the following operations: The recombinant strain was streaked onto LB agar containing kanamycin for activation and incubated upside down at 37°C for 8–10 h. A single colony was picked and inoculated into 5 mL of LB liquid agar containing kanamycin and cultured at 37°C with shaking at 200 rpm for 12–16 h. Subsequently, 1 mL of the seed culture was transferred to 200 mL of LB liquid agar containing kanamycin and cultured at 37°C with shaking at 200 rpm until OD500 reached. 600 The concentration was increased to 0.6-0.8. Isopropyl-β-D-1-thiogalactoside (IPTG) was added to a final concentration of 0.5 mM, and the mixture was induced at 200 rpm for 16 h at different induction temperatures.

[0062] After induction, bacterial cells were collected by centrifugation at 4°C and 6000 rpm, and the supernatant was discarded. The bacterial cells were resuspended in 30 mL of 50 mM Tris-HCl buffer (pH 7.0–10.0) and washed by centrifugation under the same conditions, repeating the washing once to thoroughly remove residual culture medium. The washed bacterial cells were then resuspended in 5 mL of 50 mM Tris-HCl buffer at different pH gradients, and the bacterial concentration was adjusted to OD using the appropriate buffer. 600 =80. The pH of the Tris-HCl buffer solution was 7, 7.5, 8, 8.5, 9, 9.5 or 10. Whole-cell reaction solutions were obtained in different pH environments.

[0063] The whole-cell catalytic reaction system consisted of 1 mL of the system, to which 20 mM CGI and 2 mM MnCl2 were added. Three biological replicates were set up, with BL21(DE3) carrying the empty vector as a negative control. The reaction was carried out at 50 °C and 200 rpm with shaking for 24 h. The CGM content was determined by reversed-phase high-performance liquid chromatography (HPLC). After centrifugation at 12000 rpm for 20 min, the supernatant was filtered through a hydrophilic mixed cellulose ester (MCE) microporous membrane, and the filtrate was transferred to a sample vial for HPLC analysis.

[0064] The content of CGM in the reaction solution was determined by high-performance liquid chromatography (HPLC) with external standard method. A series of CGM standard solutions of different concentrations were prepared and detected under the same chromatographic conditions. A standard curve was established with CGM concentration as the abscissa and the corresponding chromatographic peak area as the ordinate. The obtained standard curve showed good linearity within the linear range (R0). 2 =0.999). The reaction sample was analyzed by HPLC under the same conditions. The CGM concentration in the sample was calculated based on the CGM peak area and the standard curve. The CGI conversion rate was calculated by combining the initial amount of CGI in the reaction system.

[0065] The formula is CGM conversion rate (%) = C CGM / C CGI ×100%.

[0066] Among them, C CGM CGM concentration as measured by HPLC, C CGI This represents the initial concentration of CGI.

[0067] The results are as follows Figure 3 As shown in Table 3.

[0068] Table 3. CGM conversion rates (%) of hydantoin PSDHP2 and its mutant PSDHP2M at different pH values.

[0069] Figures 1-3 The results in Tables 1-3 indicate that the hydantoin PSDHP2 and the mutant PSDHP2M enzymes have high catalytic rates and enantiomeric excess rates.

[0070] Comparative Example 1 R-CGM was synthesized using the same method as in Example 2, except that the heinz PSDHP2 or the mutant PSDHP2M enzyme was replaced with comP1 or comP2.

[0071] The comP1 and comP2 enzymes are described in the literature “Nojiri M, Hibi M, Shizawa H, Horinouchi N, Yasohara Y, Takahashi S, Ogawa J. Imidase catalyzing desymmetric imidehydrolysis forming optically active 3-substituted glutaric acid monoamides for the synthesis of gamma-aminobutyric acid (GABA) analogs[J]. Applied Microbiology and Biotechnology, 2015, 99: 9961–9969.”.

[0072] The reaction rates of comP1 and comP2 were detected using the same method as in Example 2. The catalytic rate results for comP1 and comP2 are shown in [the table below]. Figure 4 And Table 4.

[0073] Table 4 Results of Enzyme-catalyzed reaction kinetic parameters determination

[0074] Depend on Figure 2 , 4 As shown in Tables 1 and 4, compared with existing R-CGM synthesis raw materials, hydantoin PSDHP2 and the mutant PSDHP2M have special tolerance to high temperature, and their catalytic rate at high temperature is significantly higher than that of comP1 and comP2.

[0075] In summary, this invention provides a hydantoin PSDHP2, the amino acid sequence of which is shown in SEQ ID NO:1. Hydantoin PSDHP2 is a chiral enzyme used for the synthesis of R-baclofen precursors, characterized by its high temperature and strong alkali resistance. It maintains high chirality and a high reaction rate even at 70℃ and pH 10. When using hydantoin PSDHP2 as a catalyst to produce R-CGM, the enantiomeric excess ee > 90%, and the reaction rate Vc max 36.17 μmol·min -1 ·mg -1 The hydantoin PSDHP2 enzyme is characterized by low cost, environmental friendliness, and no secondary pollution. This invention provides a new and effective route for the production of the R-baclofen precursor R-CGM.

[0076] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments without creative effort, as shown in these embodiments, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A hydantoin PSDHP2, characterized in that, The amino acid sequence of the hydantoin PSDHP2 is shown in SEQ ID NO:

1.

2. The hydantoin PSDHP2 as described in claim 1, characterized in that, The nucleotide sequence encoding the hydantoin PSDHP2 is shown in SEQ ID NO:

2.

3. A hydantoin mutant PSDHP2M, characterized in that, The amino acid sequence of the hydantoin mutant PSDHP2M is shown in SEQ ID NO:

3.

4. The hydantoin mutant PSDHP2M as described in claim 3, characterized in that, The nucleotide sequence encoding the hydantoin mutant PSDHP2M is shown in SEQ ID NO:

4.

5. An enzyme preparation, characterized in that, The active ingredient of the enzyme preparation includes the hydantoin PSDHP2 as described in claim 1 or 2 and / or the hydantoin mutant PSDHP2M as described in claim 3 or 4.

6. The use of the hydantoin PSDHP2 of claim 1 or 2, the hydantoin mutant PSDHP2M of claim 3 or 4, or the enzyme preparation of claim 5 in the synthesis of R-3-(4-chlorophenyl)glutaric acid monoamide.

7. A method for synthesizing R-3-(4-chlorophenyl)glutaric acid monoamide, characterized in that the step include: Using 3-(4-chlorophenyl)glutaric acid imine as a substrate, a catalytic reaction was carried out to obtain R-3-(4-chlorophenyl)glutaric acid monoamide; The catalyst used in the catalytic reaction includes any one or more of the following: the hydantoin PSDHP2 according to claim 1 or 2, the hydantoin mutant PSDHP2M according to claim 3 or 4, or the enzyme preparation according to claim 5.

8. The method as described in claim 7, characterized in that, The working concentration of the 3-(4-chlorophenyl)glutaric acid imine is 10~200mM; the working concentration of the catalyst is 5~20μg / mL; The temperature of the catalytic reaction is 16~90℃; the pH of the catalytic reaction is 6~11; The reaction system for the catalytic reaction also includes Tris-HCl buffer and MnCl2; The concentration of the Tris-HCl buffer solution is 40~60 mM; The concentration of MnCl2 is 1.8~2.2mM.

9. The use of the method of claim 7 or 8 in the preparation of R-3-(4-chlorophenyl)glutaric acid monoamide.

10. The use of the method of claim 7 or 8 in the preparation of R-baclofen.

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