Dihydropyrimidine enzyme mutants and their application in the production of (R)-3-(carbamoylmethyl)-5-methylhexanoic acid

Abstract

This invention provides a dihydropyrimidine enzyme mutant, related products, and their application in the production of the pharmaceutical intermediate (R)-3-(carbamoylmethyl)-5-methylhexanoic acid or its analogues. This invention is the first to discover that the wild-type dihydropyrimidine enzyme, as shown in SEQ ID NO: 1, and the dihydropyrimidine enzyme mutant of this invention modified from it, can effectively catalyze the production of the pharmaceutical intermediate (R)-3-(carbamoylmethyl)-5-methylhexanoic acid from substrates. In particular, compared to the wild-type dihydropyrimidine enzyme, the dihydropyrimidine enzyme mutant of this invention exhibits significantly improved dihydropyrimidine enzyme activity and stronger tolerance to organic solvents and high temperatures. Furthermore, the product obtained by its catalysis shows high stereoselectivity, thus enabling the production of (R)-3-(carbamoylmethyl)-5-methylhexanoic acid or its analogues at lower cost and higher efficiency, demonstrating high industrial value.

Description

Technical Field

[0001] This invention relates to the field of biocatalysis technology, specifically to dihydropyrimidine enzyme mutants and their application in the production of (R)-3-(carbamoylmethyl)-5-methylhexanoic acid. Background Technology

[0002] Dihydropyrimidinase (EC 3.5.2.2) is a key hydrolytic enzyme in the pyrimidine metabolic pathway. It catalyzes the ring-opening hydrolysis of dihydropyrimidine compounds to generate β-ureapropionic acid, playing an important role in the metabolic cycle of uracil in organisms.

[0003] (R)-3-(carbamoylmethyl)-5-methylhexanoic acid, as a chiral drug intermediate, has irreplaceable industrial value in the synthesis of important pharmaceutical products such as pregabalin, a drug for treating neuropathic pain. The unique R configuration of this compound endows the final product with excellent pharmacological activity, while traditional chemical synthesis routes suffer from inherent limitations in stereoselectivity. For example, the chemical catalytic systems disclosed in patent documents such as US20030225161A1 and US5616793A all generate racemic mixtures, requiring complex column chromatography or chiral resolution processes to obtain a single-configuration product. This not only leads to at least 50% waste of raw materials but also involves the use of large amounts of organic solvents, significantly increasing production costs and causing environmental burden.

[0004] In comparison, biocatalysis exhibits significant advantages due to its excellent stereoselectivity and green process characteristics. Among existing technologies, the hydantoin catalytic system disclosed in CN111944856A can yield a product with a 99% ee value, but its substrate loading is only 2 g / L. While the amide hydrolase mutant technology reported in patents CN114164198A and CN117106759A has made progress in stereoselectivity, it still suffers from issues with substrate conversion rate and enzyme utilization efficiency. Typical examples show that the enzyme-to-substrate mass ratio is as high as 1:1 to 2:1, and the substrate concentration is generally low. This high catalyst input and low production density directly leads to a high cost of enzyme preparation per unit product, becoming a key technical obstacle limiting the large-scale application of this intermediate. Summary of the Invention

[0005] To address the deficiencies and problems existing in the prior art, this invention provides a dihydropyrimidine enzyme mutant with high dihydropyrimidine enzyme activity and good tolerance to organic solvents and / or high temperatures, its related products, and their application in the production of (R)-3-(carbamoylmethyl)-5-methylhexanoic acid and its downstream products, as well as a method for producing (R)-3-(carbamoylmethyl)-5-methylhexanoic acid and its downstream products using the dihydropyrimidine enzyme mutant and its related products.

[0006] Specifically, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides a dihydropyrimidine enzyme mutant having an amino acid sequence selected from the following:

[0008] An amino acid sequence having dihydropyrimidinease activity as shown in SEQ ID NO:1, formed by substitution, deletion and / or addition of one or more amino acids as shown in SEQ ID NO:1.

[0009] In a feasible implementation, the dihydropyrimidine enzyme mutant contains an amino acid mutation selected from at least one of the following sites compared to the amino acid sequence shown in SEQ ID NO:1:

[0010] S36, A37, M63, F65, V89, V135, F149, N202, A347, Y379, Q381, V400, L404;

[0011] Preferably, compared to the amino acid sequence shown in SEQ ID NO:1, the dihydropyrimidine enzyme mutant comprises any one or a combination of amino acid mutations selected from the following:

[0012] S36E, A37E, M63A, M63I, M63E, F65H, V89I, V135E, F149L, N202K, A347S, Y379F, Q381K, V400I, L404I;

[0013] More preferably, compared to the amino acid sequence shown in SEQ ID NO:1, the dihydropyrimidine enzyme mutant contains an amino acid mutation selected from the following:

[0014] M63A+V89I;

[0015] M63E+F65H;

[0016] M63E+F65H+S36E;

[0017] M63E+F65H+S36E+A37E;

[0018] M63A+F65H+S36E+V89I;

[0019] M63A+F65H+S36E+V89I+A37E;

[0020] M63A+F149L;

[0021] F65H+N202K;

[0022] F149L+N202K+A347S;

[0023] F149L+N202K+A347S+Y379F;

[0024] F65H+F149L+N202K+A347S+Y379F;

[0025] F149L+N202K+A347S+V400I;

[0026] F65H+F149L+N202K+A347S+Y379F+V400I;

[0027] F65H+N202K+A347S+Y379F;

[0028] F149L+A347S+V400I;

[0029] F65H+A347S+V400I;

[0030] More preferably, the dihydropyrimidine enzyme mutant comprises an amino acid mutation selected from the following:

[0031] M63E+F65H+S36E+A37E;

[0032] M63A+F65H+S36E+V89I+A37E;

[0033] F65H+F149L+N202K+A347S+Y379F;

[0034] F65H+F149L+N202K+A347S+Y379F+V400I;

[0035] F65H+N202K+A347S+Y379F.

[0036] In a second aspect, the present invention provides a fusion protein, which is a protein obtained by fusing a dihydropyrimidine enzyme mutant as described in the first aspect above with a protein tag.

[0037] In a feasible implementation, the protein tag is any one or more of Poly-Arg, Poly-His, c-myc, and HA.

[0038] Thirdly, the present invention provides an enzyme agent or enzyme composition comprising the dihydropyrimidine enzyme mutant as described in the first aspect above or the fusion protein as described in the second aspect above.

[0039] Fourthly, the present invention provides a polynucleotide encoding a dihydropyrimidine enzyme mutant as described in the first aspect above or a fusion protein as described in the second aspect above.

[0040] Fifthly, the present invention provides nucleic acid constructs, recombinant vectors, or transformed host cells comprising the polynucleotides described in the fourth aspect above.

[0041] In a feasible implementation, the recombinant vector is a recombinant expression vector.

[0042] In feasible implementations, the host cell is Escherichia coli, Bacillus subtilis, yeast, mold, etc.

[0043] In a sixth aspect, the present invention provides the use of the dihydropyrimidine enzyme mutant as described in the first aspect above, the fusion protein as described in the second aspect above, the enzyme agent or enzyme composition as described in the third aspect above, the polynucleotide as described in the fourth aspect above and / or the nucleic acid construct as described in the fifth aspect above, the recombinant vector or the transformed host cell as a catalyst in the production of R-3-(carbamoylmethyl)-5-methylhexanoic acid or its analogues and their downstream products.

[0044] In a feasible implementation, the substrate for the catalytic reaction is selected from 3-isobutylglutarimide, 3-(2-methylpropyl)glutarimide and their analogues.

[0045] In a feasible implementation, the downstream products of the R-3-(carbamoylmethyl)-5-methylhexanoic acid or its analogues are selected from: pregabalin, gabapentin, anti-HIV drugs and β-lactam antibiotics, etc.

[0046] In a seventh aspect, the present invention provides a method for producing R-3-(carbamoylmethyl)-5-methylhexanoic acid or an analogue thereof, the method comprising: using a dihydropyrimidine enzyme as shown in SEQ ID NO:1, a dihydropyrimidine enzyme mutant as described in the first aspect above, a fusion protein as described in the second aspect above, and / or an enzyme agent or enzyme composition as described in the third aspect above as a catalyst to carry out a catalytic reaction to generate R-3-(carbamoylmethyl)-5-methylhexanoic acid or an analogue thereof;

[0047] Preferably, the substrate for the catalytic reaction is selected from 3-isobutylglutarimide, 3-(2-methylpropyl)glutarimide and their analogues.

[0048] Eighthly, the present invention provides a method for producing pregabalin, the method comprising:

[0049] (1) To produce R-3-(carbamoylmethyl)-5-methylhexanoic acid or an analogue thereof according to the method described in the seventh aspect above;

[0050] (2) The R-3-(carbamoylmethyl)-5-methylhexanoic acid or its analogue obtained in step (1) is purified, crystallized, and then subjected to Hofmann rearrangement to generate pregabalin.

[0051] Beneficial effects

[0052] Through scientific design and extensive experimental screening, the inventors obtained the dihydropyrimidine enzyme mutant of this invention. Compared with wild-type dihydropyrimidine enzyme, the dihydropyrimidine enzyme mutant of this invention has the following advantages:

[0053] (1) It has significantly improved dihydropyrimidine enzyme activity, thereby greatly shortening the reaction time and reducing production costs.

[0054] (2) It has strong tolerance to organic solvents and high temperature; that is, in a reaction system with high organic solvent content, it can still retain high enzyme activity at high temperature. Combined with the fact that the substrate 3-isobutylglutarimide is insoluble in water at room temperature (which can significantly improve the solubility of the substrate at high temperature and high organic solvent concentration), the high activity of this enzyme at high temperature and high organic solvent content significantly improves the overall catalytic reaction rate and significantly shortens the reaction time.

[0055] In a catalytic system, increasing the content of organic solvents (including but not limited to lower alcohols, lower ketones, dimethyl sulfoxide, and N,N-dimethylformamide) and / or increasing the reaction temperature can improve the solubility of the substrate, thereby increasing the probability of contact between the substrate and the enzyme and accelerating the reaction rate. The dihydropyrimidine enzyme mutant of the present invention can still retain high enzyme activity in a reaction system with high organic solvent content and at a high reaction temperature. This means that its tolerance to organic solvents and high temperatures will be more conducive to the efficiency of the catalytic reaction.

[0056] In summary, in addition to its high enzyme activity, the dihydropyrimidine enzyme mutant of the present invention exhibits greater tolerance to organic solvents and high temperatures, which is more conducive to the efficiency of its catalytic reaction. Furthermore, the product obtained by its catalysis has high stereoselectivity. Therefore, using the dihydropyrimidine enzyme mutant of the present invention, R-3-(carbamoylmethyl)-5-methylhexanoic acid or its analogues can be produced at a lower cost and higher efficiency, thus possessing high industrial value. Attached Figure Description

[0057] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative examples are not intended to limit the embodiments. The term "illustrative" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "illustrative" is not necessarily to be construed as superior to or better than other embodiments.

[0058] Figure 1The NMR spectrum of the product (R)-3-(carbamoylmethyl)-5-methylhexanoic acid obtained in Example 7 is shown, where M is the product label and M+H is the molecular weight of the product with one hydrogen atom added.

[0059] Figure 2 The HPLC identification chromatogram of the product obtained in Example 7 is shown, wherein the peak at 4.183 min corresponds to the product (R)-3-(2-amino-2-oxoethyl)-5-methylhexanoic acid, and the peak at 8.839 min corresponds to the substrate 3-isobutylglutarimide. Detailed Implementation

[0060] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative and not intended to limit the scope of the invention. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0061] In a feasible specific embodiment, the method for preparing the dihydropyrimidine enzyme mutant of the present invention and using it to catalyze the synthesis of R-3-(carbamoylmethyl)-5-methylhexanoic acid or its analogues includes:

[0062] First, using molecular biology gene cloning technology, nucleotides encoding wild-type dihydropyrimidine enzymes are integrated into plasmid vectors to obtain expression plasmids, or the nucleotide sequence is integrated into the host chromosome. Then, using the principle of homologous recombination, single-point or multi-point editing of the bases on the original dihydropyrimidine enzyme gene fragment is performed to obtain plasmid vectors containing single-point or multi-point gene mutations (i.e., nucleotides encoding dihydropyrimidine enzyme mutants). These plasmid vectors are then transformed into competent host cells to obtain production strains of the dihydropyrimidine enzyme mutants. These strains are then fermented to obtain cells containing the corresponding dihydropyrimidine enzyme mutants. Finally, the product solution is obtained by catalyzing the substrate with enzymes in the form of cells containing dihydropyrimidine enzyme mutants, cell lysates, purified enzymes, immobilized enzymes, or immobilized cells.

[0063] The vectors mentioned herein may include DNA constructs containing a polynucleotide sequence encoding a target protein, the polynucleotide sequence being operatively linked to a suitable expression regulatory region (or expression regulatory sequence) to enable expression of the target protein in a suitable host. The expression regulatory region may contain a promoter capable of initiating transcription, any operon sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences regulating transcription and translation termination. The vector may be transformed into a suitable host cell and then replicate and function independently of the host genome, or the vector may be integrated into the genome itself.

[0064] There are no particular limitations on the vectors used in this document, and any vector known in the art may be used. Examples of commonly used vectors include natural or recombinant plasmids, viruses, and bacteriophages. For example, pWE15 M13, MBL3, MBL4, IXII, ASHII, Charon21A, etc., can be used as phage vectors or granular vectors, and the pDZ system, pBR system, pUC system, pBluescript II system, pGEM system, pTZ system, pCL system, pET system, etc., can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors, etc., can be used.

[0065] In this invention, a nucleotide chain encoding a target protein can also be inserted into a vector, and this vector can be integrated into a chromosome. Integration of the nucleotide chain into the chromosome can be achieved using any known method (e.g., homologous recombination), but is not limited thereto. The vector may contain selection markers for screening, and selection markers can be used to confirm the insertion of the target nucleic acid molecule. Markers that confer selectable phenotypes such as drug resistance, auxotrophic phenotype, cytotoxic agent resistance, or expression of surface peptides can be used.

[0066] In this invention, the term "conversion" refers to the process of introducing a vector containing a target nucleotide chain into a host cell through cell membrane permeability or integrating it into a chromosome, thereby enabling the target nucleotide sequence to be expressed and produced using the host's translation and transcription system. Polynucleotides can be introduced into the host cell in their own form and operatively linked to the desired sequence for expression in the host cell, but are not limited thereto.

[0067] In this invention, the term "mutant" refers to a polypeptide whose amino acid sequence is significantly altered by substituting or modifying one or more amino acids in the target amino acid sequence. Such mutants can typically be evaluated by altering the amino acid sequence to assess the role of each amino acid and select a suitable shape to catalyze the target substrate. Tags can be added to one or both ends of the mutant amino acid sequence to facilitate the identification, purification, or synthesis of the produced protein. As an example, the mutant of this invention can exhibit dihydropyrimidine enzyme activity and can exhibit increased activity, improved thermostability, and enhanced solvent tolerance compared to the wild-type enzyme, resulting in increased production capacity of (R)-3-(carbamoylmethyl)-5-methylhexanoic acid.

[0068] The term "amino acid" includes both naturally occurring and non-natural amino acids found in proteins. The single-letter and three-letter names for naturally occurring amino acids in proteins follow the conventional names used in the field, as seen in Sam brook, et al. (Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

[0069]

[0070]

[0071] In this invention, the sequence of the wild-type dihydropyrimidine enzyme (as shown in SEQ ID NO:1) can be obtained from the NCBI GenBank database (WP_387311288.1); this invention is the first to apply this dihydropyrimidine enzyme to catalyze the substrate 3-isobutylglutarimide to synthesize (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid.

[0072] In the description of the amino acid sequence of the dihydropyrimidine enzyme mutant of the present invention, the description of its mutation site and type is based on the amino acid sequence shown in SEQ ID NO:1 as a reference sequence. For example, M63A means that the methionine (M) at position 63 of SEQ ID NO:1 is mutated to alanine (A), V89I means that the valine (V) at position 89 of SEQ ID NO:1 is mutated to isoleucine (I), and M63A+V89I means that the above mutations are present at the same time.

[0073] In this invention, the term "culture" refers to the growth of a strain containing a gene encoding the target protein under appropriately controlled environmental conditions. The culturing process can be carried out using known culture media and conditions. Those skilled in the art can easily adapt and use this culturing process based on the selected strain.

[0074] In this invention, "culture medium" refers to a mixture containing nutrients as the main components for culturing organisms containing dihydropyrimidine enzyme expression proteins disclosed herein, and the culture medium contains nutrients, growth factors, etc., essential for host survival and protein expression, including water. The host organisms containing the target gene disclosed herein can be cultured under aerobic conditions with controlled temperature, pH, etc., in a common culture medium containing appropriate carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids, and / or vitamins. In this disclosure, carbon sources include: carbohydrates such as glucose, glycerol, saccharose, lactose, fructose, sucrose, and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; and amino acids such as glutamic acid, methionine, and lysine. Natural organic nutrients such as starch hydrolysate, molasses, saccharin paste, and corn steep liquor can be used. As nitrogen sources, the following can be used: inorganic nitrogen sources, such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; and organic nitrogen sources, such as amino acids (such as glutamic acid, methionine, and glutamine), peptone, NZ-amines, meat extracts, yeast extracts, malt extracts, corn steep liquor, casein hydrolysate, fish or its decomposition products, and defatted soybean meal or its decomposition products. These nitrogen sources can be used individually or in combination.

[0075] During the cultivation of hosts containing genes encoding the target protein, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, hydrochloric acid, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid in an appropriate manner. Foaming can be eliminated during cultivation using antifoaming agents such as ethers. Oxygen or oxygen-containing gases can be injected into the culture medium to maintain its aerobic state, but methods for maintaining this state are not limited to these.

[0076] In the culture, the culture temperature can be maintained between 20°C and 45°C, specifically 25°C to 40°C, and the strain can be cultured for approximately 10 to 50 hours, but the culture conditions are not limited to these. The target protein produced by the culture can be secreted into the culture medium or retained in the cells.

[0077] In this invention, the enzymes used are cells or cell lysates or purified enzyme solutions or immobilized enzymes obtained by shaking flasks or fermentation of strains containing enzyme expression plasmids.

[0078] In this invention, the target protein can be recovered from the organism expressing the target protein or its culture medium by suitable methods known in the art; for example, a combination of centrifugation, filtration, treatment with a protein precipitant, extraction, ultrasonic disruption, ultrafiltration, dialysis, etc., can be used.

[0079] In this invention, the protein used in the catalytic system can be any known form of protein. The protein can be in a free, partially fixed, or completely immobilized state to participate in the catalysis of 3-isobutylglutarimide to generate (R)-3-(carbamoylmethyl)-5-methylhexanoic acid.

[0080] In this invention, the method for producing (R)-3-(carbamoylmethyl)-5-methylhexanoic acid may further include a purification step. Purification can be performed by suitable methods known in the art. In one example, when the method of this disclosure for producing (R)-3-(carbamoylmethyl)-5-methylhexanoic acid includes a recovery step and a purification step, the recovery step and the purification step may be performed sequentially or discontinuously, or simultaneously or by combining them into a single step, but the manner in which the steps are performed is not limited thereto.

[0081] The chemical equation for the preparation of R-3-(carbamoylmethyl)-5-methylhexanoic acid by catalyzing 3-isobutylglutarimide with wild-type dihydropyrimidine enzyme or the dihydropyrimidine enzyme mutant described in this invention is as follows:

[0082]

[0083] The specific catalytic system is as follows: 3-Isobutylglutarimide is used as the substrate in phosphate buffer or aqueous solution, with dihydropyrimidine enzyme or its mutant added as a biocatalyst, and an appropriate amount of organic solvent added. The catalytic reaction is carried out under shaking and stirring conditions at 30-60℃ and pH 7.0-9.0. After the reaction is complete, impurities are removed by solvent extraction and crystallization to obtain the target product. Further optimization of the catalytic system determined that the optimal temperature for this reaction is 40-50℃, and the optimal pH is 7.5-9.0.

[0084] This invention utilizes the high solvent tolerance of the modified dihydropyrimidine enzyme, allowing the addition of suitable organic solvents such as lower alcohols, tetrahydrofuran, ethyl acetate, acetone, DMSO, and DMF to the catalytic system. One or more organic solvents can be added, with isopropanol and methanol being preferred. The amount of solvent added is 0.3-15% of the reaction system, preferably 1-10%.

[0085] In the enzyme catalytic system of the present invention, the substrate concentration ranges from 10 to 200 g / L, preferably 30 to 100 g / L; the catalytic time is 5 to 20 h, preferably 8 to 15 h. In the catalytic system of the present invention, the substrate conversion rate is 80 to 99%, preferably not less than 90%; in the catalytic system of the present invention, the enzyme is added in cellular form, with the cell addition amount being 1 to 10%, preferably 1 to 5%; after catalysis, the product ee value is not less than 99%.

[0086] In order to optimize a process route suitable for industrial production, this invention also purifies the enzyme catalytic reaction solution after the catalytic reaction is completed. This is mainly done by using physical methods and solvents to remove impurities, adjusting the pH of the reaction solution, and crystallizing to obtain a qualified product.

[0087] The preferred embodiments of the present invention will be described in detail below with reference to examples. It should be understood that the following embodiments are given only for illustrative purposes and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0088] Example 1: Method for constructing a dihydropyrimidine enzyme mutant expression strain

[0089] The nucleotide sequence of wild-type dihydropyrimidine enzyme from Neobacillus sp. NPDC058068 (as shown in SEQ ID NO:2) was synthesized by Anshengda Biotechnology Co., Ltd. The synthesized fragment was ligated into the pET28a(+) vector using seamless cloning technology to obtain a recombinant plasmid. The recombinant plasmid was then introduced into Escherichia coli BL21(DE3) competent cells for transformation. After incubation on ice for 30 min, the cells were heat-shocked in a water bath at 42℃ for 90 s, followed by an ice bath for 1.5 min. LB medium was then added, and the cells were incubated in a shaker at 37℃ for 1 h. After centrifugation, a small amount of the bacterial culture was evenly spread on a solid plate containing kanamycin resistance and incubated at 37℃ for 18 h to obtain colonies. The above single colonies were picked and inoculated into 10 mL of LB liquid culture medium containing 50 mg / L kanamycin. After culturing at 37°C and 220 rpm for 12 hours, the bacterial culture was transferred to 2*LB medium at a ratio of 3% and cultured at 37°C and 220 rpm until the OD600 reached about 1-1.2. IPTG was added to a final concentration of 0.1 mM for induction. After induction at 25°C for 16 hours, the culture was terminated. Then, the wet cells containing dihydropyrimidine enzyme were collected by centrifugation. The amino acid sequence of the dihydropyrimidine enzyme is shown in SEQ ID NO.1.

[0090] A certain amount of the above-cultured wet cells were used for plasmid extraction. The resulting plasmid was an expression plasmid containing the wild-type dihydropyrimidine enzyme nucleotide sequence, which was used as a template for subsequent PCR amplification. Then, mutation primers were designed according to the nucleotide sequence SEQ ID NO.2 derived from the wild-type dihydropyrimidine enzyme. The designed primers were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0091] Finally, amplification was performed according to the molecular biology PCR system, as shown in Table 1 below:

[0092] Table 1

[0093] Components Increased volume ul <![CDATA[ddH2O]]> 8.5 Prime STAR 10 Primer F 0.5 Primer R 0.5 plasmid template 0.5

[0094] Add the reagents listed in the table above to a 120 μL PCR tube in the order from top to bottom, vortex to mix thoroughly, and perform PCR amplification on a PCR device. The amplification conditions are shown in Table 2 below:

[0095] Table 2

[0096]

[0097]

[0098] After PCR, 3 μL of the PCR product was taken for nucleic acid electrophoresis to detect the PCR bands. A single bright band with a position greater than 5000 markers was considered a successful PCR. Next, 2 μL of CutOne buffer and 1 μL of DpnI enzyme for rapid enzyme digestion to eliminate the template plasmid were added to the PCR product tube. The mixture was shaken and placed in a 37°C water bath for 1 hour. The enzyme-digested PCR product was then transformed into the host BL21(DE3) according to the method used in the construction of the wild-type expression strain. The strain was cultured to induce enzyme production, and plasmids were extracted from the culture to verify successful mutation by sequencing. The strains that were verified to have the target mutation were cultured for 2*LB, and the bacterial cells were collected for enzyme activity assay.

[0099] Example 2: Method for determining the enzyme activity of dihydropyrimidine enzyme mutants

[0100] In the following examples, the enzyme activity of the bacterial cells obtained in Example 1 was detected using the following method to reflect the enzyme activity of the corresponding dihydropyrimidine enzyme mutant.

[0101] Specifically, 1g of bacterial cells were added to 10ml of phosphate buffer with a pH of 7.5 and sonicated at 150W for 10min, with a 3s interval between sonication intervals. After disruption, the crude enzyme solution was used to determine enzyme activity.

[0102] Take 10 mL of phosphate buffer (0.2 M, pH 7.5), add 0.1 g of 3-isobutylglutarimide and 0.5 mL of the crude enzyme solution obtained above, and let the mixture react in a device with constant stirring and constant temperature for 30 min. Accurately transfer 0.5 mL of the reaction solution and add 0.5 mL of sulfuric acid solution to terminate the reaction with solvent. Dilute the terminated reaction solution by a certain factor and centrifuge to remove impurities. Detect the concentration and chiral value of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid in the supernatant by liquid chromatography.

[0103] Enzyme activity is defined as the production of 1 μmol of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid per minute by 1 gram of wet cells expressing dihydropyrimidine enzyme.

[0104] In this invention, the percentage increase or decrease in enzyme activity is calculated based on the wild-type dihydropyrimidine enzyme activity being 100%; for example, a 150% increase in enzyme activity in mutant strain 1 means that the enzyme activity of this mutant strain is 150% higher than that of the wild type.

[0105] Example 3: Construction and screening of dihydropyrimidine enzyme mutants with better solvent tolerance

[0106] The three-dimensional structure of the dihydropyrimidine enzyme shown in SEQ ID NO: 1 was simulated and resolved using the Alphafold prediction platform to identify key amino acid residues that could alter the hydrophilicity of the protein surface. These residues were then subjected to site-directed mutagenesis to design corresponding dihydropyrimidine enzyme mutants. Strains expressing these dihydropyrimidine enzyme mutants were then constructed according to the construction method described in Example 1. Finally, the enzyme activity of these dihydropyrimidine enzyme mutants under organic solvent pressure was determined according to the enzyme activity assay method described in Example 2 (except for the addition of different types and concentrations of organic solvents to the reaction system), thereby determining the organic solvent tolerance of these dihydropyrimidine enzyme mutants.

[0107] Table 5 below shows the enzyme activity test results of the dihydropyrimidine enzyme mutant with good solvent tolerance.

[0108] Table 3

[0109]

[0110] As shown in Table 3, in catalytic reaction systems containing different proportions of methanol, isopropanol, or DMSO, the enzyme activities of the dihydropyrimidine enzyme mutants M63I, M63A, and M63A+V89I were significantly improved compared to the wild-type dihydropyrimidine enzyme. In particular, the enzyme activity of the dihydropyrimidine enzyme mutant M63A+V89I was still more than 30% higher than that of the wild-type enzyme when the organic solvent content of the catalytic reaction system was three times that of the wild-type enzyme. Especially for the catalytic reaction system containing isopropanol, the enzyme activity of mutant M63A+V89I was 80% higher than that of the wild-type enzyme when the isopropanol content was three times that of the wild-type enzyme, showing extremely excellent enzyme activity and organic solvent resistance.

[0111] Example 4: Construction and screening of dihydropyrimidine enzyme mutants with good heat tolerance

[0112] In this embodiment, based on SEQ ID NO: 1, some amino acids at the active sites were mutated to glutamic acid, histidine, aspartic acid, arginine, threonine, etc., to design dihydropyrimidine enzyme mutants; then, strains expressing these dihydropyrimidine enzyme mutants were constructed according to the construction method described in Example 1; finally, the enzyme activity of these dihydropyrimidine enzyme mutants was measured in the temperature range of 30-55℃ according to the enzyme activity assay method described in Example 2, thereby determining the high temperature tolerance of these dihydropyrimidine enzyme mutants.

[0113] Table 4 below shows the enzyme activity test results of the dihydropyrimidine enzyme mutant with good high temperature tolerance.

[0114] Table 4

[0115]

[0116]

[0117] As shown in Table 4, within the temperature range of 30-55℃, the enzyme activities of the dihydropyrimidine enzyme mutants M63E, M63E+F65H, M63E+F65H+S36E, and M63E+F65H+S36E+A37E were significantly improved compared with the wild-type dihydropyrimidine enzyme. In particular, the dihydropyrimidine enzyme mutant M63E+F65H+S36E+A37E showed that at the relatively high reaction temperature of 55℃, its enzyme activity was more than three times higher than that of the wild-type enzyme, demonstrating extremely excellent enzyme activity and heat resistance.

[0118] Example 5: Construction and screening of dihydropyrimidine enzyme mutants with significantly improved solvent tolerance and high-temperature tolerance

[0119] Some amino acid mutations from Examples 3 and 4 were combined to integrate dihydropyrimidine enzyme mutants with both high solvent tolerance and high temperature tolerance. Strains expressing these dihydropyrimidine enzyme mutants were constructed according to the construction method described in Example 1, and the enzyme activity of these dihydropyrimidine enzyme mutants was measured in high solvent content and at a temperature range of 40-55°C according to the enzyme activity assay method described in Example 2, thereby determining the solvent tolerance and high temperature tolerance of these dihydropyrimidine enzyme mutants.

[0120] Table 5 below shows the enzyme activity detection results of the dihydropyrimidine enzyme mutants that have good solvent tolerance and high temperature tolerance.

[0121] Table 5

[0122]

[0123] As shown in Table 5, within the temperature range of 40-55℃, in catalytic reaction systems containing different proportions of organic solvents, the enzyme activities of the dihydropyrimidine enzyme mutants M63A, M63A+F65H, M63A+F65H+S36E, M63A+F65H+S36E+V89I, and M63A+F65H+S36E+V89I+A37E were significantly improved compared to the wild-type dihydropyrimidine enzyme. In particular, the dihydropyrimidine enzyme mutant M63A+F65H+S36E+V89I+A37E, at a relatively high reaction temperature of 55℃, showed an enzyme activity nearly four times higher than that of the wild-type enzyme in a catalytic reaction system containing 100 mL of solvent, demonstrating extremely excellent enzyme activity and thermostability.

[0124] Example 6: Construction and screening of dihydropyrimidine enzyme mutants with significantly improved solvent tolerance, high temperature tolerance, and enzyme activity.

[0125] Based on the mutation sites of the mutant enzymes with high temperature stability and solvent tolerance screened in Examples 3-5 above, PyMOL software was used to further analyze the effective mutant structures, determine the trend of quaternary structural changes caused by the mutations, and further identify new mutation sites. The aim was to obtain further combined mutations with strong high-temperature tolerance and solvent tolerance and improved enzyme activity. Strains expressing these dihydropyrimidine enzyme mutants were constructed according to the construction method described in Example 1, and the enzyme activity of these dihydropyrimidine enzyme mutants was measured at high solvent content and high temperature according to the enzyme activity assay method described in Example 2. Simultaneously, the stereoselectivity of the products was detected, thereby obtaining dihydropyrimidine enzyme mutants with strong solvent tolerance and high-temperature tolerance, high enzyme activity, and good product stereoselectivity. In this example, the assay conditions used were as follows: 50‰ isopropanol as the organic solvent, 50℃ as the reaction temperature, and 7.5-8.0 as the reaction pH.

[0126] Table 6 below shows the enzyme activity detection results of dihydropyrimidine enzyme mutants that simultaneously exhibit good solvent and high temperature tolerance, high enzyme activity, and good product stereoselectivity.

[0127] Table 6

[0128]

[0129] As shown in Table 6, in catalytic systems with high solvent content and at higher reaction temperatures, the enzyme activities of all dihydropyrimidine enzyme mutants shown in Table 6 are significantly higher than those of wild-type enzymes. In particular, mutants F65H+F149L+N202K+A347S+Y379F, F65H+F149L+N202K+A347S+Y379F+V400I and F65H+N202K+A347S+Y379F have enzyme activities that are several times higher than those of wild-type enzymes under the same conditions, demonstrating extremely excellent enzyme activity, high temperature resistance and solvent resistance.

[0130] Example 7: Catalytic Examples of Dihydropyrimidine Enzyme Mutants

[0131] The *E. coli* expression strain M-6 (i.e., F65H+F149L+N202K+A347S+Y379F), as described in Example 6, was cultured in shake flask medium to induce enzyme production. The bacterial cells were collected by centrifugation at 8000 rpm for 10 min, and the cells were broken up in the manner described in Example 2 to obtain a crude enzyme solution containing the dihydropyrimidine enzyme mutant.

[0132] Add 80 ml of 0.1 M pH 7.5 phosphate buffer to a 250 ml three-necked flask, add 10 g of the substrate 3-isobutylglutarimide, add 5 ml of isopropanol solution, and add 5 g of cell lysate containing the dihydropyrimidine enzyme mutant protein. Initiate the catalytic reaction in a 50 °C water bath, maintaining the pH of the reaction solution at 8.0-8.5 with 30% sodium hydroxide solution during the reaction. After 10-12 h, when the substrate conversion rate is greater than 99%, stop the reaction. Centrifuge the reaction solution to remove cell residue. Concentrate the supernatant by vacuum distillation to 2-3 times its volume, then add 1-2 times its volume of methanol. Let stand at room temperature for 5-6 h, centrifuge to remove impurities, collect the supernatant, concentrate the solution containing methanol, water, and product, adjust the pH of the concentrate with acid, and crystallize by stirring in a low-temperature water bath. Separate the solid and liquid to obtain the crude product. Further pulping and crystallization of the crude product are performed, followed by identification by nuclear magnetic resonance (NMR) and high-performance liquid chromatography (HPLC). The results of the former are as follows: Figure 1 As shown, the latter result is as follows Figure 2 As shown; by Figure 1 and Figure 2It can be seen that (R)-3-(carbamoylmethyl)-5-methylhexanoic acid with a purity and content greater than 99% was obtained; the product yield was greater than 80%.

[0133] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

[0134] The sequences discussed in this article:

[0135] SEQ IDNO:1

[0136] MKKIIKNGTIVTASDTFQAELLIEDGKITQIGSNLSAIGAEVIDAKGCLVFPGGIDPHTH

[0137] LDMPFGGTVTKDDFESGTMAAAFGGTTTVIDFCLTNKGEPLKNAIQTWHDKSKDKA

[0138] VIDYGFHLMISEINENVLNELPQVINEEGISSFKVFMAYKNVFQADDETLFKTLVTAK

[0139] DLGALVMVHAENGDVIDYLTKKALDEGNTEPIYHALTRPPELEGEEATGRAARFTGLA

[0140] NSQLYVVHVSCADAAEKIAEARSKGFDVWGETCPQYLVLDQSYLEKPNFEGAKYV

[0141] WSPPLREKWNQEALWNALKSGQLQTIGSDQCSFDFNGQKDLGRDDFTKIPNGGPIIE

[0142] DRLAIIFSEGVKKGRISLNQFVDLTSTRAAKLFGLYPQKGTIAVGADADLVIFDPNVER

[0143] VLSAETHHMAVDYNAFEGMEVTGEPVSVLVRGEYVVRDKQFVGKPGAGQYLKRAKYRTTTPVNQNETLSI;

[0144] SEQIDNO:2

[0145] ATGAAAAAAATTTAAACGGCACCATTGTGACCGCGAGCGATACCTTTCAAG

[0146] CGGAACTGCTGATTGAAGATGGCAAAATTACGCAGATTGGCAGCAACCTGAGCG

[0147] CGATTGGCGCGGAAGTGATTGATGCGAAAGGCTGCCTGGTGTTTCCGGGCGGCAT

[0148] TGATCCGCATACCCATCTGGATATGCCATTTGGCGGCACCGTGACGAAAGATGAT

[0149] TTTGAAAGCGGCACCATGGCGGCCGCGTTCGGTGGCACCACGACCGTGATTGATT

[0150] TTTGCCTGACCAACAAAGGCGAACCGCTGAAAAACGCGATTCAGACCTGGCATG

[0151] ATAAAAGCAAAGATAAAGCGGTGATTGATTATGGTTTTCATCTGATGATTAGCGA

[0152] AATTAACGAAAACGTGCTGAACGAACTGCCGCAAGTGATTAACGAAGAAGGCAT

[0153] TAGCAGCTTTAAAGTGTTTATGGCGTATAAAACGTGTTTCAAGCGGATGATGAA

[0154] ACCCTGTTTAAAACCCTGGTGACCGCGAAGGACCTGGGCGCGTTAGTGATGGTGC

[0155] ATGCGGAAAACGGCGATGTGATCGACTATCTGACCAAAAAGCGCTGGATGAAG

[0156] GCAACACCGAACCGATTTATCATGCGCTGACCCGCCCACCGGAACTGGAGGGCG

[0157] AAGCGACCGGTCGCGCGGCGCGCTTTACCGGCCTGGCGAACAGTCAGCTGTATGT

[0158] GGTGCATGTGAGCTGCGCGGATGCGGCGGAAAAAATTGCGGAAGCGCGCAGCAA

[0159] AGGCTTTGATGTGTGGGGCGAAACCTGCCCGCAGTATCTGGTGCTGGATCAGAGC

[0160] TATCTGGAAAAACCGAACTTTGAAGGCGCGAAATATGTGTGGAGCCCGCCGCTGC

[0161] GCGAAAAATGGAACCAAGAAGCGCTGTGGAACGCGCTGAAAAGCGGTCAGCTGC

[0162] AGACCATTGGCAGCGATCAGTGCAGCTTTGATTTTAACGGTCAGAAAGACCTGGG

[0163] CCGTGACGATTTTACCAAAATTCCGAACGGCGGCCCGATTATTGAAGATCGCCTG

[0164] GCGATTATTTTTAGCGAAGGCGTGAAAAAAGGCCGCATTAGCCTGAATCAGTTTG

[0165] TGGATCTGACGAGCACCCGCGCGGCGAAACTGTTTGGCCTGTATCCGCAGAAAGG

[0166] CACCATTGCGGTGGGCGCGGACGCGGACCTGGTGATTTTTGATCCGAACGTGGAA

[0167] CGCGTGCTGAGCGCGGAAACCCATCATATGGCGGTGGATTATAACGCGTTTGAAG

[0168] GCATGGAAGTGACCGGCGAACCGGTGAGCGTGCTGGTGCGCGGCGAATATGTGG

[0169] TGCGCGATAAACAGTTTGTGGGCAAACCGGGCGCGGGTCAGTATCTGAAACGCG

[0170] CGAAATATCGCACCACGACCCCGGTGAATCAGAACGAAACCCTGAGCATT。

Claims

1. A dihydropyrimidinease mutant, characterized in that, The dihydromimutase mutant is obtained by amino acid mutation selected from the following based on the amino acid sequence shown in SEQ ID NO: 1: M63A + V89I; M63E + F65H; M63E + F65H + S36E; M63E + F65H + S36E + A37E; M63A + F65H + S36E + V89I; M63A + F65H + S36E + V89I + A37E; M63A + F149L; F65H + N202K; F149L + N202K + A347S; F149L + N202K + A347S + Y379F; F65H + F149L + N202K + A347S + Y379F; F149L + N202K + A347S + V400I; F65H + F149L + N202K + A347S + Y379F + V400I; F65H + N202K + A347S + Y379F; F149L + A347S + V400I; F65H + A347S + V400I.

2. The dihydropyrimidinease mutant according to claim 1, characterized in that, The amino acid mutation is selected from: M63E + F65H + S36E + A37E; M63A + F65H + S36E + V89I + A37E; F65H + F149L + N202K + A347S + Y379F; F65H + F149L + N202K + A347S + Y379F + V400I; F65H + N202K + A347S + Y379F.

3. A fusion protein which is a protein obtained by fusing the dihydromimutase mutant of claim 1 or 2 with a protein tag.

4. An enzyme composition comprising the dihydromimutase mutant of claim 1 or 2 or the fusion protein of claim 3.

5. A polynucleotide encoding the dihydromimutase mutant of claim 1 or 2 or encoding the fusion protein of claim 3.

6. A nucleic acid construct, a recombinant vector or a transformed host cell comprising the polynucleotide of claim 5.

7. Use of the dihydromimutase mutant of claim 1 or 2, the fusion protein of claim 3 and / or the enzyme composition of claim 4 as a catalyst in the production of (R)-3- (carbamoylmethyl)-5-methylhexanoic acid.

8. Use according to claim 7, characterized in that, The catalytic reaction substrate is 3-isobutyl glutarimide.

9. A process for the production of (R)-3-(carbamoylmethyl)-5- methylhexanoic acid, characterized in that, The method comprises: using the dihydromimutase mutant of claim 1 or 2, the fusion protein of claim 3 and / or the enzyme composition of claim 4 as a catalyst, using 3-isobutyl glutarimide as a substrate, and performing a catalytic reaction to generate (R)-3- (carbamoylmethyl)-5-methylhexanoic acid.

10. A method of producing pregabalin, characterized by, The method comprises: (1) using the dihydropyrimidine enzyme mutant according to claim 1 or 2, the fusion protein according to claim 3 and / or the enzyme composition according to claim 4 as a catalyst, performing a catalytic reaction using 3-isobutyl glutarimide as a substrate to generate (R)-3-(carbamoylmethyl)-5-methylhexanoic acid; (2) purifying and crystallizing the (R)-3-(carbamoylmethyl)-5-methylhexanoic acid obtained in step (1), and then performing a Hofmann rearrangement to generate pregabalin.

Images