Dihydropyrimidinase mutant and application thereof in production of (R)-3-(carbamylmethyl)-5-methylhexanoic acid

Abstract

The invention provides a dihydropyrimidinase mutant, a related product of the dihydropyrimidinase mutant and application of the dihydropyrimidinase mutant in production of a medical intermediate (R)-3-(carbamylmethyl)-5-methylhexanoic acid or an analogue of the (R)-3-(carbamylmethyl)-5-methylhexanoic acid. It is found for the first time that wild type dihydropyrimidinase shown in SEQ ID NO: 1 and a dihydropyrimidinase mutant modified by the wild type dihydropyrimidinase can effectively catalyze a substrate to generate a medical intermediate (R)-3-(carbamylmethyl)-5-methylhexanoic acid; particularly, compared with wild type dihydropyrimidinase, the dihydropyrimidinase mutant has the advantages that the dihydropyrimidinase activity is remarkably improved, the organic solvent tolerance and the high-temperature tolerance are relatively high, and the stereoselectivity of a product obtained by catalysis of the dihydropyrimidinase mutant is relatively high; therefore, the (R)-3-(carbamylmethyl)-5-methylhexanoic acid or the analogue thereof can be produced at lower cost and higher efficiency, and the method has higher industrial value.

Description

Technical Field

[0001] The invention relates to the technical field of biocatalysis, in particular to a dihydropyrimidinase mutant and an application thereof in producing (R)-3-(carbamoylmethyl)-5-methylhexanoic acid. Background Art

[0002] Dihydropyrimidinase (EC 3.5.2.2) is a key hydrolase in the pyrimidine metabolic pathway. It catalyzes the ring-opening hydrolysis reaction of dihydropyrimidine compounds to produce β-ureidopropionic acid, and plays an important role in the metabolic cycle of uracil in organisms.

[0003] As a chiral drug intermediate, (R)-3-(carbamoylmethyl)-5-methylhexanoic acid 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 the compound can give the final product excellent pharmacological activity, while the traditional chemical synthesis route has the inherent defect of insufficient stereoselectivity. For example, the chemical catalytic systems disclosed in patent documents such as US20030225161A1 and US5616793A all generate racemic mixtures, and a single configuration product must be obtained through complex column chromatography or chiral resolution processes, which not only leads to at least 50% of the raw materials being wasted, but also involves the use of a large amount of organic solvents, which significantly increases the production cost and causes an environmental burden.

[0004] In comparison, the biocatalytic method has shown significant advantages with its excellent stereoselectivity and green process characteristics. In the prior art, although the hydantoinase catalytic system disclosed in CN111944856A can obtain products with an ee value of 99%, its substrate loading is only 2g / L; although the amidohydrolase mutant technology reported in patents CN114164198A and CN117106759A has made progress in stereoselectivity, there are still problems with substrate conversion rate and enzyme utilization efficiency. Its typical examples show that the enzyme-substrate mass ratio is as high as 1:1 to 2:1, and the substrate concentration is generally low. This process feature of high catalyst input and low production density directly leads to a high cost of enzyme preparation per unit product, which becomes a key technical obstacle to restricting the large-scale application of this intermediate. Summary of the invention

[0005] In view of the defects and problems existing in the prior art, the present invention provides a dihydropyrimidinase mutant having high dihydropyrimidinase activity and good tolerance to organic solvents and / or high temperatures, its related products and its use 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 dihydropyrimidinase mutant and its related products.

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

[0007] In a first aspect, the present invention provides a dihydropyrimidinase mutant having an amino acid sequence selected from the group consisting of:

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

[0009] In a feasible embodiment, compared with the amino acid sequence shown in SEQ ID NO: 1, the dihydropyrimidinase mutant comprises an amino acid mutation at at least one site selected from the following:

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

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

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

[0013] Further preferably, compared with the amino acid sequence shown in SEQ ID NO: 1, the dihydropyrimidinase mutant comprises 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] Still more preferably, the dihydropyrimidinase 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 the dihydropyrimidinase mutant described in the first aspect with a protein tag.

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

[0038] In a third aspect, the present invention provides an enzyme agent or an enzyme composition, which comprises the dihydropyrimidinase mutant as described in the first aspect or the fusion protein as described in the second aspect.

[0039] In a fourth aspect, the present invention provides a polynucleotide encoding the dihydropyrimidinase mutant as described in the first aspect above or the fusion protein as described in the second aspect above.

[0040] In a fifth aspect, the present invention provides a nucleic acid construct, a recombinant vector or a transformed host cell comprising the polynucleotide as described in the fourth aspect above.

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

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

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

[0044] In a feasible embodiment, the substrate of the catalytic reaction is selected from: 3-isobutylglutarimide, 3-(2-methylpropyl)glutarimide and the like.

[0045] In a feasible embodiment, the downstream product of R-3-(carbamoylmethyl)-5-methylhexanoic acid or its analogs is 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 analog thereof, the method comprising: using the dihydropyrimidinase shown in SEQ ID NO: 1, the dihydropyrimidinase mutant described in the first aspect, the fusion protein described in the second aspect, and / or the enzyme agent or enzyme composition described in the third aspect as a catalyst to carry out a catalytic reaction to generate R-3-(carbamoylmethyl)-5-methylhexanoic acid or an analog thereof;

[0047] Preferably, the substrate of the catalytic reaction is selected from: 3-isobutylglutarimide, 3-(2-methylpropyl)glutarimide and the like.

[0048] In an eighth aspect, the present invention provides a method for producing pregabalin, the method comprising:

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

[0050] (2) purifying and crystallizing the R-3-(carbamoylmethyl)-5-methylhexanoic acid or its analogue obtained in step (1), and then performing Hofmann rearrangement to produce pregabalin.

[0051] Beneficial Effects

[0052] The inventors obtained the dihydropyrimidinase mutant of the present invention through scientific design and a large number of experimental screenings; compared with the wild-type dihydropyrimidinase, the dihydropyrimidinase mutant of the present invention has the following advantages:

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

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

[0055] In the catalytic system, increasing the content of organic solvent (organic solvent types include but are not limited to lower alcohols, lower ketones, dimethyl sulfoxide and N,N-dimethylformamide, etc.) and / or increasing the reaction temperature can increase the solubility of the substrate, thereby increasing the contact probability between the substrate and the enzyme and accelerating the reaction rate; and the dihydropyrimidinase mutant of the present invention can still retain a high enzyme activity in a reaction system with a high organic solvent content and at a higher reaction temperature, which 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 having high enzyme activity, the dihydropyrimidinase mutant of the present invention has tolerance to organic solvents and high temperatures, which will be more conducive to the efficiency of the catalytic reaction using it, and the stereoselectivity of the product obtained by catalysis is high. Therefore, using the dihydropyrimidinase mutant of the present invention, R-3-(carbamoylmethyl)-5-methylhexanoic acid or its analogues can be produced at a lower cost and higher efficiency, thereby having a higher industrial value. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] One or more embodiments are exemplarily described by the pictures in the accompanying drawings, and these exemplary descriptions do not constitute limitations on the embodiments. The special word "exemplary" here means "used as an example, embodiment or illustrative". Any embodiment described as "exemplary" here is not necessarily interpreted as being superior or better than other embodiments.

[0058] Figure 1The nuclear magnetic resonance identification spectrum of the product (R)-3-(carbamoylmethyl)-5-methylhexanoic acid obtained in Example 7 is shown, wherein 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 spectrum 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 DESCRIPTION

[0060] The present invention is further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention, rather than for limiting the scope of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the art without making creative work are within the scope of protection of the present invention.

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

[0062] First, through molecular biological gene cloning technology, the nucleotide encoding the wild-type dihydropyrimidinase is integrated into a plasmid vector to obtain an expression plasmid or the nucleotide sequence is integrated into the host chromosome; then, using the principle of gene homologous recombination, the bases on the original dihydropyrimidinase gene fragment are edited at a single point or multiple points to obtain a plasmid vector containing a single point or multiple point gene mutation (i.e., nucleotides encoding a dihydropyrimidinase mutant); the plasmid vector is transformed into a competent host cell to obtain a production strain of the dihydropyrimidinase mutant; then, such a strain is fermented and cultured to obtain cells containing the corresponding dihydropyrimidinase mutant; finally, the product solution is obtained using cells containing the dihydropyrimidinase mutant or cell fragments or purified enzymes or enzyme-catalyzed substrates in the form of immobilized enzymes and immobilized cells.

[0063] The carrier mentioned herein may include a DNA construct, which comprises a base sequence of a polynucleotide encoding a target protein, the base sequence of the polynucleotide being operably connected to a suitable expression control region (or expression control sequence) so that the target protein can be expressed in a suitable host. The expression control 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 a sequence regulating transcription and translation termination. The carrier may be transformed into a suitable host cell, then replicated and acted independently of the host genome, or the carrier may be integrated into the genome itself.

[0064] The vector used herein is not particularly limited, and any vector known in the art can 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 cosmid vectors, and 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 the present invention, the nucleotide chain encoding the target protein can also be inserted into a vector, and the vector can be integrated into the chromosome. The nucleotide chain can be integrated into the chromosome using any known method (e.g., homologous recombination), but is not limited thereto. The vector can contain a screening marker for screening, and the selection marker is used to confirm whether the target nucleic acid molecule is inserted or not, and a marker that confers a selectable phenotype such as drug resistance, auxotrophy, cytotoxic resistance, or expression of a surface polypeptide can be used.

[0066] In the present invention, the term "transformation" means that the vector containing the target nucleotide chain is introduced into the host cell through the permeability of the cell membrane, or integrated into the chromosome, so that the target nucleotide sequence utilizes the host's translation and transcription system to express and produce enzyme proteins. The polynucleotide can be introduced into the host cell in its own form and operably linked to the sequence required for expression in the host cell, but is not limited thereto.

[0067] In the present invention, the term "mutant" refers to a polypeptide whose amino acid sequence is greatly changed by replacing or modifying one or more amino acids in the target amino acid sequence. Such mutants can usually evaluate the role of each amino acid by changing the amino acid sequence, and select a suitable shape to catalyze the target substrate. A tag can be added to one or both ends of the mutant amino acid sequence so that the produced protein can be identified, purified or synthesized. As an example, the mutant of the present invention can exhibit the activity of dihydropyrimidinase, and can show increased activity, improved thermal stability and improved solvent tolerance in the production capacity of (R)-3-(carbamoylmethyl)-5-methylhexanoic acid compared with the wild-type enzyme.

[0068] The term "amino acid" includes naturally occurring amino acids and non-natural amino acids in proteins. The single-letter and three-letter nomenclature of naturally occurring amino acids in proteins adopts the names commonly used in the art, which can be found in Sambrook, 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 the present invention, the sequence of the wild-type dihydropyrimidinase (as shown in SEQ ID NO: 1) can be obtained from the GenBank database of NCBI (WP_387311288.1); the present invention is the first to apply this dihydropyrimidinase 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 dihydropyrimidinase mutant of the present invention, the description of the mutation site and type is based on the amino acid sequence shown in SEQ ID NO:1 as the 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 the present invention, the term "culture" means that the strain containing the target protein encoding gene is grown under appropriately controlled environmental conditions. The culture process can be carried out according to known culture media and culture conditions. Those skilled in the art can easily adjust and use this culture process according to the selected strain.

[0074] In the present invention, "culture medium" means a mixed substance containing nutrients required for culturing the dihydropyrimidinase-expressing protein organism disclosed herein as the main component, and the culture medium contains nutrients, growth factors, etc., which are indispensable for host survival and protein expression, including water. The host organism containing the target gene disclosed herein can be cultured in a common culture medium containing appropriate carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids and / or vitamins, etc., while controlling temperature, pH, etc. under aerobic conditions. In the present 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; amino acids, such as glutamic acid, methionine, and lysine, etc. Natural organic nutrients, such as starch hydrolyzates, molasses, blackstrap molasses, and corn syrup, etc., can be used. As nitrogen sources, 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-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition products, and defatted soybean cake or its decomposition products can be used. The above nitrogen sources can be used alone or in combination.

[0075] During the cultivation of the host containing the target protein encoding gene, 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 to the culture medium in an appropriate manner. During the cultivation period, foaming can be eliminated by using defoamers such as ethers. Oxygen or oxygen-containing gas can be injected into the culture medium to maintain the aerobic state of the culture medium, but the method for maintaining the state is not limited thereto.

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

[0077] In the present invention, the enzyme used is the cells or cell disrupted liquid or enzyme purified liquid and immobilized enzyme obtained by fermentation of a strain containing an enzyme expression plasmid in a shake flask or fermentation.

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

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

[0080] In the present invention, the method for producing (R)-3-(carbamoylmethyl)-5-methylhexanoic acid may further include a purification step. Purification may be performed by a suitable method known in the art. In an example, when the method for producing (R)-3-(carbamoylmethyl)-5-methylhexanoic acid disclosed herein includes a recovery step and a purification step, the recovery step and the purification step may be performed continuously or discontinuously in any order, or may be performed simultaneously or by merging into one step, but the manner of performing the steps is not limited thereto.

[0081] In the present invention, the chemical equation for preparing R-3-(carbamoylmethyl)-5-methylhexanoic acid by catalyzing 3-isobutylglutarimide using wild-type dihydropyrimidinase or the dihydropyrimidinase mutant of the present invention is as follows:

[0082]

[0083] The specific catalytic system is: in phosphate buffer or aqueous solution, 3-isobutylglutarimide is used as substrate, dihydropyrimidinase or its mutant is added as biocatalyst, and an appropriate amount of organic solvent is added, and the catalytic reaction is carried out at 30-60°C, pH7.0-9.0, under shaking and stirring conditions; after the reaction is completed, the target product is obtained through solvent extraction, impurity removal and crystallization purification; after further optimizing the catalytic system, it is determined that the optimum temperature of the reaction is 40-50°C, and the optimum reaction pH is 7.5-9.0.

[0084] The present invention utilizes the high solvent tolerance of the modified dihydropyrimidinase, and can add organic solvents suitable for substrate solubilization such as lower alcohols, tetrahydrofuran, ethyl acetate, acetone, DMSO and DMF to the catalytic system. One or more organic solvents can be added, preferably isopropanol and methanol; 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 range is 10-200 g / L, preferably 30-100 g / L; the catalytic time is 5-20 h, preferably 8-15 h. In the catalytic system of the present invention, the substrate conversion rate is 80-99%, preferably not less than 90%; in the catalytic system of the present invention, the enzyme is added in the form of cells, and the amount of cells added is 1-10%, preferably 1-5%; after the catalysis of the present invention is completed, the product ee value is not less than 99%.

[0086] In order to optimize and complete a set of process routes suitable for industrial production, the present invention also purifies the enzyme catalytic reaction liquid after the catalytic reaction is completed, mainly using physical methods and solvents to remove impurities, adjust the pH of the reaction liquid and crystallize to obtain a product of qualified quality.

[0087] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0088] Example 1: Construction of a dihydropyrimidinase mutant expression strain

[0089] The nucleotide sequence of the wild-type dihydropyrimidinase from Neobacillus sp. NPDC058068 (as shown in SEQ ID NO: 2) was commissioned to Anshengda Biotechnology Co., Ltd. for synthesis, and the synthetic fragment was connected to the plasmid pET28a (+) vector by seamless cloning technology to obtain a recombinant plasmid, and then the recombinant plasmid was introduced into Escherichia coli BL21 (DE3) competent cells for transformation. After an ice bath for 30 minutes, heat shock was performed in a 42°C water bath for 90 seconds, and then an ice bath was performed for 1.5 minutes. LB culture medium was added, and after recovery culture at 37°C on a shaker for 1 hour, the bacterial liquid was centrifuged and a small amount of the bacterial liquid was evenly spread on a solid plate culture medium containing kanamycin resistance, and colonies were obtained after culture at 37°C for 18 hours. The above monoclonal colony was picked and inoculated into 10 mL of LB liquid test tube culture medium containing 50 mg / L kanamycin. After culturing at 37° C. and 220 rpm for 12 hours, the bacterial liquid was transferred to 2*LB culture medium at a ratio of 3%, and cultured at 37° C. and 220 rpm until OD600 reached about 1-1.2. IPTG with a final concentration of 0.1 mM was added for induction. After induction at 25° C. for 16 hours, the culture was terminated, and the wet cells containing dihydropyrimidinase were collected by centrifugation, wherein the amino acid sequence of the dihydropyrimidinase is shown in SEQ ID NO.1.

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

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

[0092] Table 1

[0093] Components Add ul <![CDATA[ddH2O]]> 8.5 Prime STAR 10 Primer F 0.5 Primer R 0.5 Plasmid template 0.5

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

[0095] Table 2

[0096]

[0097]

[0098] After the PCR is completed, 3ul of PCR product is taken for nucleic acid electrophoresis to detect PCR bands. If there is a single brighter band and the band position is greater than 5000 markers, the PCR is considered successful; then, 2ul of CutOne buffer and 1ul of DpnI enzyme for rapid enzyme digestion to eliminate the template plasmid are added to the PCR product tube, and the mixture is shaken and mixed evenly. The mixture is placed in a 37°C water bath for 1h, and the enzyme-digested PCR product is transformed into the host BL21 (DE3) in the same way as the method used in the construction of the above-mentioned wild bacterial expression strain; the strain is cultured to induce enzyme production, and the plasmid is extracted from the culture to verify whether the mutation is successful by sequencing. The strain that has been verified to have the target mutation is cultured in 2*LB, and the bacteria are collected for enzyme activity determination.

[0099] Example 2: Method for determining the enzymatic activity of dihydropyrimidinase mutants

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

[0101] Specifically, 1 g of bacterial cells were added to 10 ml of phosphate buffer with a pH value of 7.5, and ultrasonically disrupted at 150 W for 10 min, with each ultrasonic wave lasting 3 seconds and resting for 3 seconds; after the disruption was completed, the obtained crude enzyme solution was used to determine the 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 react the resulting mixture in a device with constant stirring and constant temperature for 30 minutes, accurately pipette 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 a certain multiple and then centrifuge to remove impurities, and use liquid chromatography to detect the concentration and chirality of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid in the supernatant.

[0103] Definition of enzyme activity: 1 gram of wet cells of the dihydropyrimidinase expressing strain catalyzes the production of 1 μmol of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid per minute, which is defined as 1 enzyme activity unit.

[0104] In the present invention, the ratio of enzyme activity increase or decrease is calculated based on the enzyme activity of the wild-type dihydropyrimidinase being 100%; for example, the enzyme activity of mutant 1 is increased by 150%, which means that the enzyme activity of this mutant is 150% higher than that of the wild-type.

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

[0106] The three-dimensional structure of the dihydropyrimidinase shown in SEQ ID NO: 1 was simulated and analyzed using the Alphafold prediction platform to determine the key amino acid residues that can change the hydrophilicity of the protein surface, and site-directed mutagenesis was performed to design the corresponding dihydropyrimidinase mutants; then, strains expressing these dihydropyrimidinase mutants were constructed according to the construction method described in Example 1; finally, the enzyme activity of these dihydropyrimidinase mutants under organic solvent pressure was determined according to the enzyme activity determination method described in Example 2 (except that different types and concentrations of organic solvents were added to the reaction system), thereby determining the organic solvent tolerance of these dihydropyrimidinase mutants.

[0107] Table 5 below shows the results of enzyme activity detection of dihydropyrimidinase mutants with better solvent tolerance.

[0108] Table 3

[0109]

[0110] As can be seen from Table 3, in the catalytic reaction system containing methanol, isopropanol or DMSO with different addition ratios, the enzyme activities of the dihydropyrimidinase mutants M63I, M63A and M63A+V89I were significantly improved compared with the wild-type dihydropyrimidinase; in particular, the dihydropyrimidinase mutant M63A+V89I, when the organic solvent content of the catalytic reaction system was three times that of the wild-type enzyme, its enzyme activity was still more than 30% higher than that of the wild-type enzyme; in particular, for the catalytic reaction system containing isopropanol, the mutant M63A+V89I had an enzyme activity that was 80% higher than that of the wild-type enzyme in the catalytic reaction system with an isopropanol content that 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 dihydropyrimidinase mutants with better high temperature tolerance

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

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

[0114] Table 4

[0115]

[0116]

[0117] As can be seen from Table 4, within the temperature range of 30-55°C, compared with the wild-type dihydropyrimidinase, the enzyme activities of the dihydropyrimidinase mutants M63E, M63E+F65H, M63E+F65H+S36E, and M63E+F65H+S36E+A37E were significantly improved; in particular, the dihydropyrimidinase mutant M63E+F65H+S36E+A37E, at a higher reaction temperature of 55°C, its enzyme activity was more than three times higher than that of the wild-type enzyme, showing extremely excellent enzyme activity and high temperature resistance.

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

[0119] Some of the amino acid mutations in Example 3 and Example 4 were combined in order to integrate dihydropyrimidinase mutants having both high solvent tolerance and high temperature tolerance; strains expressing these dihydropyrimidinase mutants were constructed according to the construction method described in Example 1, and the enzyme activity of these dihydropyrimidinase mutants at high solvent content and in the temperature range of 40-55°C was determined according to the enzyme activity determination method described in Example 2, thereby determining the solvent tolerance and high temperature tolerance of these dihydropyrimidinase mutants.

[0120] Table 5 below shows the enzyme activity test results of the dihydropyrimidinase mutants having both good solvent tolerance and high temperature tolerance.

[0121] Table 5

[0122]

[0123] As can be seen from Table 5, within the temperature range of 40-55°C, in the catalytic reaction system containing different proportions of organic solvents, compared with the wild-type dihydropyrimidinase, the enzyme activities of the dihydropyrimidinase mutants M63A, M63A+F65H, M63A+F65H+S36E, M63A+F65H+S36E+V89I, and M63A+F65H+S36E+V89I+A37E were significantly improved; in particular, the dihydropyrimidinase mutant M63A+F65H+S36E+V89I+A37E, at a higher reaction temperature of 55°C, in a catalytic reaction system containing 100 mL, its enzyme activity was nearly four times higher than that of the wild-type enzyme, showing extremely excellent enzyme activity and high temperature resistance.

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

[0125] According to the mutation sites of the mutant enzymes with high temperature stability and solvent tolerance screened in the above-mentioned Examples 3-5, the effective mutation structure is further analyzed using pymol software to determine the trend of quaternary structure changes caused by mutations, and further determine new mutation sites, with the purpose of obtaining further combined mutations with strong high temperature tolerance and solvent tolerance and improved enzyme activity; strains expressing these dihydropyrimidinase mutants are constructed according to the construction method recorded in Example 1, and the enzyme activity of these dihydropyrimidinase mutants at high solvent content and high temperature is determined according to the enzyme activity determination method recorded in Example 2, and the stereoselectivity of the product is detected at the same time, so as to obtain dihydropyrimidinase mutants with strong solvent tolerance and high temperature tolerance, high enzyme activity and good product stereoselectivity. In this embodiment, the determination conditions used are as follows: the organic solvent is 50‰ isopropanol, the reaction temperature is 50°C, and the reaction pH is 7.5-8.0.

[0126] Table 6 below shows the enzyme activity test results of dihydropyrimidinase mutants having good solvent tolerance and high temperature tolerance, high enzyme activity and good product stereoselectivity.

[0127] Table 6

[0128]

[0129] As can be seen from Table 6, in a catalytic system with a high solvent content, at a higher reaction temperature, the enzyme activity of each dihydropyrimidinase mutant shown in Table 6 is significantly improved compared with the wild-type enzyme, especially the mutants F65H+F149L+N202K+A347S+Y379F, F65H+F149L+N202K+A347S+Y379F+V400I and F65H+N202K+A347S+Y379F, whose enzyme activities are several times higher than those of the wild-type enzyme under the same conditions, showing extremely excellent enzyme activity, high temperature resistance and solvent resistance.

[0130] Example 7: Catalytic example of dihydropyrimidinase mutants

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

[0132] In a 250 ml three-necked flask, 80 ml of 0.1 M pH 7.5 phosphate buffer, 10 g of substrate 3-isobutylglutarimide, 5 ml of isopropanol solution, and 5 g of cell disrupted liquid containing the dihydropyrimidinase mutant protein were added, and a catalytic reaction was carried out in a 50° C. water bath. During the reaction, 30% sodium hydroxide was used to control the pH of the reaction liquid to 8.0-8.5. The reaction was carried out for 10-12 hours. At this time, the substrate conversion rate was greater than 99%, and the reaction was stopped; the reaction liquid was centrifuged to remove cell residues; the centrifuged supernatant was concentrated by vacuum distillation to 2-3 times the volume, and then 1-2 times the volume of methanol was added, and the mixture was allowed to stand at room temperature for 5-6 hours, and impurities were removed by centrifugation. The clear liquid was collected, and the solution containing methanol, water and the product was concentrated. The pH value of the concentrated liquid was adjusted with acid, and the product was stirred and crystallized in a low-temperature water bath. The crude product was separated by solid-liquid separation to obtain a crude product. The crude product was then pulped and crystallized, and then identified by nuclear magnetic resonance and high performance liquid chromatography (HPLC). The results of the former are as follows: Figure 1 As shown, the latter result is Figure 2 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% is obtained; the product yield is 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, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

[0134] Sequences covered in this article:

[0135] SEQ ID NO: 1

[0136] MKKIIKNGTIVTASDTFQAELLIEDGKITQIGSNLSAIGAEVIDAKGCLVFPGGIDPHTH

[0137] LDMPFGGTVTKDDFESGTMAAAFGGTTTVIDFCLTNKGEPLKNAIQTWHDKSKDKA

[0138] VIDYGFHLMISEINENVLNELPQVINEEGISSFKVFMAYKNVFQADDETLFKTLVTAK

[0139] DLGALVMVHAENGDVIDYLTKKALDEGNTEPIYHALTRPPELEGEEATGRAARFTGLA

[0140] NSQLYVVHVSCADAAEKIAEARSKGFDVWGETCPQYLVLDQSYLEKPNFEGAKYV

[0141] WSPPLREKWNQEALWNALKSGQLQTIGSDQCSFDFNGQKDLGRDDFTKIPNGGPIIE

[0142] DRLAIIFSEGVKKGRISLNQFVDLTSTRAAKLFGLYPQKGTIAVGADADLVIFDPNVER

[0143] VLSAETHHMAVDYNAFEGMEVTGEPVSVLVRGEYVVRDKQFVGKPGAGQYLKRAKYRTTTPVNQNETLSI;

[0144] SEQ ID NO: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 dihydropyrimidinase mutant, characterized in that The dihydropyrimidinase mutant has an amino acid sequence selected from the group consisting of: An amino acid sequence having the dihydropyrimidinase activity as shown in SEQ ID NO: 1, formed by substitution, deletion and / or addition of one or more amino acids in the amino acid sequence shown in SEQ ID NO:

1.

2. The dihydropyrimidinase mutant according to claim 1, characterized in that Compared with the amino acid sequence shown in SEQ ID NO: 1, the dihydropyrimidinase mutant comprises an amino acid mutation at at least one site selected from the following: S36, A37, M63, F65, V89, V135, F149, N202, A347, Y379, Q381, V400, L404; Preferably, compared with the amino acid sequence shown in SEQ ID NO: 1, the dihydropyrimidinase mutant comprises any one amino acid mutation or a combination of multiple amino acid mutations selected from the following: S36E, A37E, M63A, M63I, M63E, F65H, V89I, V135E, F149L, N202K, A347S, Y379F, Q381K, V400I, L404I; Further preferably, compared with the amino acid sequence shown in SEQ ID NO: 1, the dihydropyrimidinase mutant comprises an amino acid mutation selected from the following: 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; Still more preferably, the dihydropyrimidinase mutant comprises an amino acid mutation selected from the following: 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 dihydropyrimidinase mutant according to claim 1 or 2 with a protein tag. 4 . An enzyme agent or enzyme composition, comprising the dihydropyrimidinase mutant according to claim 1 or 2 or the fusion protein according to claim 3 .

5. A polynucleotide encoding the dihydropyrimidinase mutant according to claim 1 or 2, or encoding the fusion protein according to claim 3.

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

7. Use of the dihydropyrimidinase as shown in SEQ ID NO: 1, the dihydropyrimidinase mutant as described in claim 1 or 2, the fusion protein as described in claim 3 and / or the enzyme agent or enzyme composition as described in claim 4 as a catalyst in the production of (R)-3-(carbamoylmethyl)-5-methylhexanoic acid or its analogs and its downstream products.

8. The use according to claim 7, characterized in that: The catalytic reaction substrate is selected from: 3-isobutylglutarimide, 3-(2-methylpropyl)glutarimide; And / or, the downstream product of the (R)-3-(carbamoylmethyl)-5-methylhexanoic acid or its analogs is selected from: pregabalin, gabapentin, anti-HIV drugs and β-lactam antibiotics.

9. A method for producing (R)-3-(carbamoylmethyl)-5-methylhexanoic acid or an analog thereof, characterized in that: The method comprises: using the dihydropyrimidinase as shown in SEQ ID NO: 1, the dihydropyrimidinase mutant as described in claim 1 or 2, the fusion protein as described in claim 3 and / or the enzyme agent or enzyme composition as described in claim 4 as a catalyst to carry out a catalytic reaction to generate (R)-3-(carbamoylmethyl)-5-methylhexanoic acid or an analog thereof; Preferably, the substrate of the catalytic reaction is selected from: 3-isobutylglutarimide, 3-(2-methylpropyl)glutarimide.

10. A method for producing pregabalin, characterized in that: The method comprises: (1) Producing (R)-3-(carbamoylmethyl)-5-methylhexanoic acid or its analogue by the method according to claim 9; (2) purifying and crystallizing the (R)-3-(carbamoylmethyl)-5-methylhexanoic acid or its analogue obtained in step (1), and then performing Hofmann rearrangement to produce pregabalin.

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