Heme enzyme mutants and uses thereof

Abstract

This invention provides a hydantoin mutant, related products, and their application in the production of the pharmaceutical intermediate (R)-3-(carbamoylmethyl)-5-methylhexanoic acid or its analogues and downstream products. Compared with wild-type hydantoin, the hydantoin mutant of this invention exhibits significantly enhanced catalytic activity, and can stably maintain a high enantiomeric excess (ee value) during the catalytic synthesis of (R)-3-(carbamoylmethyl)-5-methylhexanoic acid. Therefore, using the hydantoin mutant of this invention, high-purity R-3-(carbamoylmethyl)-5-methylhexanoic acid or its analogues can be produced in a low-cost and high-efficiency manner, thus possessing outstanding application value and broad industrialization prospects.

Description

Technical Field

[0001] This invention relates to the field of biocatalysis technology, specifically to hydantoin mutants and their application in the catalytic production of (R)-3-(carbamoylmethyl)-5-methylhexanoic acid or its analogues and downstream products. Background Technology

[0002] Pregabalin (PGB, brand name Lyrica) is a lipophilic γ-aminobutyric acid analog with a unique mechanism of action. Clinically, it is primarily used to treat neuropathic pain, partial seizures, and fibromyalgia. Specifically, pregabalin binds tightly to the α2-δ subunit of voltage-dependent calcium channels in the central nervous system, effectively reducing calcium ion influx and consequently decreasing the release of excitatory neurotransmitters such as glutamate and norepinephrine. This mechanism of action not only helps control neuropathic pain but also has a significant therapeutic effect on specific types of epileptic seizures. Compared to traditional treatments, pregabalin, as a second-generation antiepileptic drug, stands out due to its significant efficacy and relatively low toxicity.

[0003] Currently, the main production routes for pregabalin are divided into resolution and asymmetric synthesis. Resolution, as a relatively mature technology, is widely used in industrial production; this method utilizes chiral resolving agents or the selective action of enzymes to separate racemic starting materials into two stereoconfigurations. Asymmetric synthesis, on the other hand, uses chiral reagents to desymmetrize and ring-open racemic starting materials, directly generating optically active chiral compounds. This is an important method for synthesizing pregabalin and its intermediates.

[0004] Studies have shown that enzymes such as imidases and hydantoins can asymmetricly hydrolyze 3-isobutylglutarimide to synthesize the highly optically active pregabalin intermediate (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid, exhibiting high enantioselectivity. This method avoids the steps of resolution and racemization, effectively shortening the reaction pathway, improving the utilization rate of raw materials, and is more environmentally friendly, thus possessing significant commercial value in the preparation of pregabalin and its intermediates.

[0005] However, with the increasing market demand for pregabalin, the existing enzyme catalytic efficiency and enantiomeric excess (ee) values ​​are no longer sufficient to meet the requirements of large-scale industrial production. To further improve production efficiency and reduce costs, it is urgent to use protein engineering methods to modify enzyme activity, optimize enzyme structure and function, and improve its catalytic activity and ee value, thereby better adapting to the needs of pregabalin industrial production. Summary of the Invention

[0006] To address the deficiencies and problems existing in the prior art, this invention provides a hydantoin mutant with excellent catalytic activity and capable of stably achieving a high ee value, its related products, and their application in the production of (R)-3-(carbamoylmethyl)-5-methylhexanoic acid or its analogues and downstream products, a key chiral intermediate of pregabalin, as well as a method for producing (R)-3-(carbamoylmethyl)-5-methylhexanoic acid or its analogues or downstream products using the hydantoin mutant and its related products.

[0007] Specifically, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides a hydantoin mutant having the following amino acid sequence: an amino acid sequence formed by mutation of the wild-type hydantoin amino acid sequence as shown in SEQ ID NO:2 at one or more of the following sites: position 68, position 190, position 216, position 316 and position 338.

[0009] The amino acid sequence of wild-type hydantoin, as shown in SEQ ID NO:2, can be encoded by the DNA sequence shown in SEQ ID NO:1.

[0010] Preferably, the mutation is selected from the following:

[0011] (1) The methionine at position 68 is mutated to phenylalanine;

[0012] (2) Leucine at position 190 is mutated to isoleucine;

[0013] (3) The glutamic acid at position 216 is mutated to proline;

[0014] (4) Threonine at position 316 is mutated to serine;

[0015] (5) The aspartic acid at position 338 is mutated to glycine.

[0016] More preferably, compared to the amino acid sequence shown in SEQ ID NO:2, the amino acid sequence of the hydantoin mutant contains amino acid mutations selected from the following:

[0017] i) Replace the methionine at position 68 of the amino acid sequence shown in SEQ ID NO:2 with phenylalanine;

[0018] ii) Replace the methionine at position 68 of the amino acid sequence shown in SEQ ID NO:2 with phenylalanine and the leucine at position 190 with isoleucine;

[0019] iii) The methionine at position 68 of the amino acid sequence shown in SEQ ID NO:2 is replaced with phenylalanine, and the glutamic acid at position 216 is replaced with proline;

[0020] iv) Replace the methionine at position 68 of the amino acid sequence shown in SEQ ID NO:2 with phenylalanine, and replace the threonine at position 316 with serine;

[0021] v) Replace the methionine at position 68 of the amino acid sequence shown in SEQ ID NO:2 with phenylalanine, the leucine at position 190 with isoleucine, and the glutamic acid at position 216 with proline.

[0022] vi) Replace the methionine at position 68 of the amino acid sequence shown in SEQ ID NO:2 with phenylalanine, the leucine at position 190 with isoleucine, and the threonine at position 316 with serine.

[0023] vii) As shown in SEQ ID NO:2, the methionine at position 68 is replaced with phenylalanine, the leucine at position 190 is replaced with isoleucine, the glutamic acid at position 216 is replaced with proline, and the threonine at position 316 is replaced with serine.

[0024] viii) As shown in SEQ ID NO:2, the methionine at position 68 is replaced with phenylalanine, the leucine at position 190 is replaced with isoleucine, the glutamic acid at position 216 is replaced with proline, and the aspartic acid at position 338 is replaced with glycine.

[0025] ix) As shown in SEQ ID NO:2, the methionine at position 68 is replaced with phenylalanine, the leucine at position 190 is replaced with isoleucine, the glutamic acid at position 216 is replaced with proline, the threonine at position 316 is replaced with serine, and the aspartic acid at position 338 is replaced with glycine.

[0026] In a second aspect, the present invention provides a fusion protein, which is a protein formed by fusing the hydantoin mutant described in the first aspect above with a tag protein.

[0027] In a feasible implementation, the tag protein is any one or more of Poly-Arg, Poly-His, c-myc, and HA.

[0028] Thirdly, the present invention provides an enzyme agent or enzyme composition comprising the hydantoin mutant as described in the first aspect above or the fusion protein as described in the second aspect above.

[0029] Fourthly, the present invention provides a polynucleotide encoding a hydantoin mutant as described in the first aspect above or a fusion protein as described in the second aspect above.

[0030] Fifthly, the present invention provides nucleic acid constructs, recombinant expression vectors, or transformed host cells comprising the polynucleotides described in the fourth aspect above.

[0031] In a feasible implementation, the recombinant expression vector is obtained by cloning the nucleotide sequence encoding the hydantoin mutant into various expression vectors using methods conventional in the art. More preferably, the expression vector includes various vectors conventional in the art, such as commercially available plasmids, phages, or viral vectors, with plasmid pET-28a(+) being preferred.

[0032] In a feasible implementation, the transformed host cells can be obtained by transforming the recombinant expression vector into host cells. The host cells are conventional host cells in the art, as long as the recombinant expression vector can stably replicate spontaneously and the gene of the hydantoin mutant of the present invention carried by it can be effectively expressed. The host cells are preferably *Escherichia coli*, more preferably *E. coli* BL21(DE3). Transforming the recombinant expression vector into *E. coli* BL21(DE3) yields the preferred transformed host cells of the present invention. The transformation method used can be a conventional method in the art, such as heat shock, electroporation, etc., more preferably heat shock.

[0033] The hydantoin mutant of this invention can be prepared using conventional methods in the art. Preferably, the transformed host cells described above can be cultured to express the hydantoin mutant of this invention, and then the cells can be lysed and the recombinantly expressed hydantoin mutant of this invention can be isolated from the lysate. The culture medium used to culture the transformed host cells can be any culture medium in the art that allows the transformed host cells to grow and produce the hydantoin mutant of this invention. The culture medium is preferably LB medium, with the following formulation: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0. The culture method and conditions can be appropriately selected according to different host cell types and culture methods, as long as the transformed host cells can grow and produce the hydantoin mutant. The specific operations for culturing the transformed host cells can be performed according to conventional procedures in the art. As a preferred method, for example, the transformed *E. coli* is inoculated into LB medium containing kanamycin and cultured at 37°C. When the OD600 of the culture medium reaches 0.6–0.8, β-D-isopropyl-thiogalactopyranoside (IPTG) at a final concentration of 0.1–0.5 mmol / L is added for induction. The culture is then continued at 25°C for 12–16 h to efficiently express the hydantoin mutant described in this invention. After the culture is completed, the precipitated bacterial cells are collected by centrifugation, which are the resting cells recombinantly expressing the hydantoin mutant described in this invention. The obtained cells are suspended in sodium phosphate buffer (0.1 mol / L, pH 7.5), sonicated, and the lysate is centrifuged. The supernatant is collected to obtain the crude enzyme solution containing the hydantoin mutant.

[0034] In a feasible implementation, the enzyme agent or enzyme composition described in the third aspect above is in the form of: culturing the transformed host cells as described in the fifth aspect above to express the hydantoin mutant of the present invention, and then lysing the cells and collecting the supernatant to obtain a crude enzyme solution containing the hydantoin mutant.

[0035] In a sixth aspect, the present invention provides the use of the hydantoin mutant as described in the first aspect above, the fusion protein as described in the second aspect above, the enzyme 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 expression vector or the transformed host cell as a catalyst in the production of R-3-(carbamoylmethyl)-5-methylhexanoic acid or its analogues and its downstream products.

[0036] In a feasible implementation, the substrate for the catalytic reaction is selected from 3-isobutylglutarimide, 3-(2-methylpropyl)glutarimide and their analogues.

[0037] 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.

[0038] 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 the hydantoin mutant as described in the first aspect above, the fusion protein as described in the second aspect above, and / or the enzyme agent or enzyme composition as described in the third aspect above as catalysts to carry out a catalytic reaction to generate R-3-(carbamoylmethyl)-5-methylhexanoic acid or an analogue thereof.

[0039] Preferably, the substrate for the catalytic reaction is selected from 3-isobutylglutarimide, 3-(2-methylpropyl)glutarimide and their analogues.

[0040] The conditions for the catalytic reaction can be carried out under the conventional conditions for such reactions in this field.

[0041] In a preferred embodiment, the method may include the following steps: adding whole *E. coli* cells expressing the recombinant hydantoin mutant of the present invention and the substrate to a reaction buffer, and mixing and reacting at a certain temperature, preferably 30–50°C. The reaction buffer is a standard laboratory buffer with a pH range of 6.0–8.0, preferably sodium phosphate buffer, and a preferred concentration of 0.1–0.2 mol / L.

[0042] In this invention, the chemical equation for the preparation of R-3-(carbamoylmethyl)-5-methylhexanoic acid using the hydantoin mutant catalyzed by the present invention is as follows:

[0043]

[0044] Eighthly, the present invention provides a method for producing pregabalin, the method comprising:

[0045] (1) To produce R-3-(carbamoylmethyl)-5-methylhexanoic acid or an analogue thereof according to the method described in the seventh aspect above;

[0046] (2) The R-3-(carbamoylmethyl)-5-methylhexanoic acid or its analogue obtained in step (1) is rearranged by Hoffmann to generate pregabalin;

[0047] Optionally, before performing the Hoffmann rearrangement on (R)-3-(carbamoylmethyl)-5-methylhexanoic acid or its analogues, the method further includes the steps of purification and crystallization.

[0048] Beneficial effects

[0049] Compared to wild-type hydantoin, the hydantoin mutant provided by this invention exhibits significantly enhanced catalytic activity, maintaining a consistently high enantiomeric excess (ee) value during the catalytic synthesis of (R)-3-(carbamoylmethyl)-5-methylhexanoic acid. These two core advantages significantly reduce reaction time, improve catalytic synthesis efficiency, and effectively lower production costs. Furthermore, the product catalyzed by this mutant possesses extremely high stereoselectivity. Therefore, using the hydantoin mutant of this invention, high-purity R-3-(carbamoylmethyl)-5-methylhexanoic acid or its analogues can be produced in a low-cost, high-efficiency manner, demonstrating outstanding application value and broad industrialization prospects in the chemical and pharmaceutical industries. Detailed Implementation

[0050] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are provided for illustrative purposes only 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.

[0051] Example 1: Preparation of recombinant expression plasmid of wild-type hydantoin from Pseudomonadota bacterium strain

[0052] The wild-type hydantoinase encoding gene (GenBank: MEM7282693.1) from the *Pseudomonadota bacterium* strain was obtained from the NCBI GenBank nucleic acid database. Its DNA coding sequence (as shown in SEQ ID NO: 1) was obtained through codon optimization. This DNA coding sequence was artificially synthesized and cloned into the pET28a(+) plasmid to obtain a recombinant expression plasmid for the wild-type hydantoinase. This recombinant expression plasmid was transformed into *E. coli* BL21(DE3) competent cells, and the transformed *E. coli* cells were plated on LB agar plates containing 50 μg / mL kanamycin and incubated overnight at 37°C. Single colonies were selected and cultured overnight on LB medium (containing 50 μg / mL kanamycin) at 37°C. The recombinant plasmid was then extracted using a plasmid miniprep kit and sequenced for verification, yielding the recombinant expression plasmid for the wild-type hydantoinase (hereinafter referred to as H1).

[0053] Example 2: Construction of recombinant expression plasmid of heinz mutant

[0054] Construct a structural model of H1 and dock it with a substrate molecule, focusing on the substrate binding site. We select amino acid residues within a certain range and, in conjunction with multiple sequence alignment, design NNK degenerate codons at appropriate sites for site-directed saturation mutation.

[0055] In this embodiment, the selected mutation sites are M68, L190, E216, T316, and N338. Upstream and downstream primers were designed for these mutation sites, and the sequences of the upstream and downstream primers are shown in Table 1.

[0056] Table 1. Upstream and downstream primer sequences designed for the proposed mutation site.

[0057]

[0058] In the primer sequences in Table 1 above, "N" represents any nucleotide A, T, C or G, "K" represents nucleotide T or G, and "M" represents nucleotide A or C; among them, "MNN" and "NNK" are reverse complementary.

[0059] Using the wild-type hydantoin recombinant expression plasmid constructed in Example 1 as a template, PCR was performed using the primers shown in Table 1. The PCR system consisted of: 10 μL PrimeSTAR (HS), 6 μL ddH2O, 1 μL DMSO, 1 μL each of the upstream and downstream primers, and 1 μL of the template plasmid. The PCR amplification program was as follows: after pre-denaturation at 98℃ for 3 min, 20 cycles of the following were performed: denaturation at 98℃ for 10 s, annealing at 55℃ for 15 s, extension at 72℃ for 7 min, and final incubation at 72℃ for 5 min. PCR product digestion: After PCR amplification, 1 μL of DpnI and 2 μL of Cutsmart were added, and the mixture was incubated at 37℃ for 1 h to obtain the recombinant expression plasmid of the hydantoin mutant.

[0060] Example 3: Screening for Heinz mutants

[0061] The recombinant expression plasmids of each hydantoin mutant prepared in Example 2 were transformed into Escherichia coli BL21(DE3) competent cells, and the cells were plated on LB agar plates containing 50 μg / mL kanamycin and incubated overnight at 37°C. Single colonies from LB agar were inoculated into 96-well plates. Each well was pre-filled with 600 μL of LB medium containing 50 μg / mL kanamycin. The plates were incubated at 37°C with shaking at 220 rpm for 3 h. Then, IPTG (0.1 mM) was added as an inducer, and the plates were incubated at 25°C with shaking at 220 rpm for 15 h. The cells were collected by centrifugation at 6000 × g for 10 min. The supernatant was discarded, and the cells were resuspended in sodium phosphate buffer (100 mM, pH 7.5). Lysozyme (1.5 mg / mL) was added for disruption. Then, 3-isobutylglutarimide (10 mg / mL) was added, and the reaction was carried out at 37°C for 4 h. After terminating the reaction, the plates were diluted 2-fold, centrifuged, and the supernatant was collected. The yield of the product R-3-(carbamoylmethyl)-5-methylhexanoic acid was determined by HPLC, and its conversion rate was calculated using the following formula as an indicator of its catalytic activity:

[0062]

[0063] In the above formula, the substrate refers to 3-isobutylglutarimide.

[0064] Based on the preliminary screening results, and by combining and stacking effective single-point mutations, nine mutants with significantly enhanced catalytic activity compared to the parent H1 were obtained, numbered H1-1 to H1-9. The number of amino acid mutations and their catalytic activities in these nine mutants compared to the parent H1 are shown in Table 2 below.

[0065] Table 2. Number of mutations and catalytic activity of the nine mutants.

[0066] Mutant number Number of mutations catalytic activity of mutants H1 0 - H1-1 1 + H1-2 2 + H1-3 2 + H1-4 2 + H1-5 3 + H1-6 3 + H1-7 4 ++ H1-8 4 + H1-9 5 ++

[0067] In the mutant activity column of Table 2, one plus sign "+" indicates that the mutant catalytic activity is increased by 1-2 times compared with the parent H1; two plus signs "++" indicate that the mutant catalytic activity is increased by 2-5 times compared with the parent H1.

[0068] Furthermore, the specific mutation details of mutants H1-1 to H1-9 are as follows:

[0069] H1-1: The methionine at position 68 of the amino acid sequence shown in SEQ ID NO:2 is replaced with phenylalanine;

[0070] H1-2: As shown in SEQ ID NO:2, the methionine at position 68 is replaced with phenylalanine, and the leucine at position 190 is replaced with isoleucine;

[0071] H1-3: As shown in SEQ ID NO:2, the methionine at position 68 is replaced with phenylalanine, and the glutamic acid at position 216 is replaced with proline;

[0072] H1-4: As shown in SEQ ID NO:2, the methionine at position 68 is replaced with phenylalanine, and the threonine at position 316 is replaced with serine;

[0073] H1-5: As shown in SEQ ID NO:2, the methionine at position 68 is replaced with phenylalanine, the leucine at position 190 is replaced with isoleucine, and the glutamic acid at position 216 is replaced with proline.

[0074] H1-6: As shown in SEQ ID NO:2, the methionine at position 68 is replaced with phenylalanine, the leucine at position 190 is replaced with isoleucine, and the threonine at position 316 is replaced with serine.

[0075] H1-7: As shown in SEQ ID NO:2, the methionine at position 68 is replaced with phenylalanine, the leucine at position 190 is replaced with isoleucine, the glutamic acid at position 216 is replaced with proline, and the threonine at position 316 is replaced with serine.

[0076] H1-8: As shown in SEQ ID NO:2, the methionine at position 68 is replaced with phenylalanine, the leucine at position 190 is replaced with isoleucine, the glutamic acid at position 216 is replaced with proline, and the aspartic acid at position 338 is replaced with glycine.

[0077] H1-9: As shown in SEQ ID NO:2, the methionine at position 68 is replaced with phenylalanine, the leucine at position 190 is replaced with isoleucine, the glutamic acid at position 216 is replaced with proline, the threonine at position 316 is replaced with serine, and the aspartic acid at position 338 is replaced with glycine.

[0078] Example 4: Preparation of crude enzyme solution of hydantoin mutant

[0079] E. coli BL21(DE3) cells transformed with a recombinant expression plasmid containing the hydantoin mutant were inoculated into LB tubes containing 50 μg / mL kanamycin and cultured at 37°C with shaking for 8–12 h. Then, 1% (v / v) of the bacterial culture was added to LB flasks containing 50 μg / mL kanamycin and cultured at 37°C with shaking for approximately 3 h until the OD600 reached 0.6–0.8. IPTG (0.1 mM) was then added, and the cells were cultured at 25°C with shaking for approximately 12–16 h. After culturing, the cells were collected by centrifugation at 8000 rpm for 10 min at 4°C and resuspended in 10 mL of sodium phosphate buffer (100 mM, pH 7.5) to obtain wet hydantoin mutant cells. The cell resuspension was subjected to ultrasonic disruption (ultrasonic conditions: 150W power, 3s operation, 3s interval, 10min total) to obtain the crude enzyme solution of the Heinz mutant.

[0080] Using the above procedure, crude enzyme solutions of wild-type hydantoin H1 and hydantoin mutants H1-1 to H1-9 obtained in Example 3 were prepared.

[0081] Example 5: An example of the reaction of wild-type hydantoin H1 and its mutant H1-9 catalyzing the production of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid from 3-isobutylglutarimide.

[0082] Prepare a 10 mL reaction system. Specifically, the 10 mL reaction system includes: 0.2 g of substrate 3-isobutylglutarimide, 0.1 g of crude enzyme solution of wild-type hydantoin H1 or its mutant H1-9 (as prepared in Example 4), and 10 mL of sodium phosphate buffer (100 mM, pH 7.5). The reaction system was incubated at 40 °C. During the reaction, the pH of the reaction system was controlled by adding 2 M NaOH to maintain it at approximately 7.5. The reaction was terminated when the pH of the reaction system no longer changed. The conversion rate and ee value of wild-type hydantoin H1 and its mutant H1-9 were detected by HPLC, and the results are shown in Table 3.

[0083] The conversion rate calculation formula is the same as in Example 3.

[0084] The formula for calculating the ee value is: Where [R] represents the concentration of the R configuration product and [S] represents the concentration of the S configuration product.

[0085] Table 3. Reactions of wild-type hydantoinase H1 or its mutant H1-9 catalyzing 3-isobutylglutarimide.

[0086] Hydantoin Reaction time (h) Conversion rate (%) ee value (%) H1 4 45 99 H1-9 4 96 99

[0087] As shown in Table 3, the conversion rate of the hydantoin mutant H1-9 is more than twice that of the wild-type hydantoin, indicating that its catalytic activity is significantly improved compared to the wild-type hydantoin. Furthermore, the hydantoin mutant H1-9 achieves a higher ee value while maintaining high catalytic activity. Therefore, in terms of overall catalytic performance, the hydantoin mutant of this invention significantly improves upon the wild-type hydantoin, which is of crucial significance for industrial applications.

[0088] 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.

[0089] The sequences discussed in this article:

[0090] SEQ ID NO:1

[0091] ATGAGCTCTCTGCTGGTTAAAGGTGGCACTGTGGTAACCGCCGAACACACTTTCTC

[0092] TGCTGATGTCCTGTGTGTGGACGGCAAGATTCAGGCGGTTGCAGAAAACCTGGAT

[0093] GCTCCTTCCGGCAGCACCATTGTGGATGCAGACTCCCAGTACGTGATGCCAGGCG

[0094] GCATTGATCCACACATCCCACATGCAACTGCCATTCATGGGCACTGTTGCGTCTGAG

[0095] GACTTTTATACCGGTACCGCAGCAGGTCTGGCTGGCGGCACTACCATGATTATCGAT

[0096] TTCGTTATTCCGGACCCGCAGCAGAACATTTGGGATGCGTATCAGCAGTGGGCGTTC

[0097] CTGGTCTGAGAAGTCCGTGTCCGATTACTCTTTCCACGTGGCAATTACGTGGTGGG

[0098] ATGAAACTGTAAGCCATGATATGGGTCGTATCGTTACCGAAGGCGGCGTGAACAGC

[0099] TTCAAACACTTTATGGCGTATAAGAACGCGATCATGTGCAGCGACGAAACCCTGGT

[0100] TAACTCTTTTTCCCGTGCTTCCGAACTGGGCGCGATCGTTACCGTGCATGCGGAAA

[0101] ACGGTGATCTGGTTTTCCGTCTGCAGGACGAACTGCACCGTATGGGTATCACTGGT

[0102] CCGGAGGGTCATCCTCTGTCTCGTCCGCCTGAAGTGGAGGGCGAAGCTGCAAATC

[0103] GTGCTATCCGCATCGCAGAGGTCCTGAACGTTCCACTGTACGTCGTTCATGTTAGC

[0104] GCGCGTCAGGCGCTGGAGGCTATTACGCGTGCGCGTAACGAAGGTCAGCGTGTGT

[0105] TTGGCGAAGTTCTGGCAGGCCACCTGCTGATCGATGATTCCGTTTACCGTCACCCG

[0106] GATTTCGACGTTGCGGCAGCGCATGTTATGAGCCCGCCGTTTCGCTCCCCGGAACA

[0107] CCAGGTCGCCCTGTGGCACGGTCTGCAGTCTGGTAATCTGCAGACTACCGCGACC

[0108] GATCACTGCTGCTTCTGCGCGGACCAGAAAGCCGCTGGCAAGGAAGATTTCCGTC

[0109] TGATCCCGAACGGCACCGCTGGTATTGAAAACCGTATGGAAGTTCTGTGGCACGA

[0110] GGGTGTTTCCACCGGTCGCCTGACCATGAACGAGTTCGTTCGTGTTACCTCCACCG

[0111] CGGCGGCGCAAATCTTCAATATCTACCCGCGTAAAGGTTCCATTAGCGTGGGTGCG

[0112] GACGCCGATATCGTGGTGTGGGATCCGAAAGCGTCTAAGACCATTTCCGCGAAAA

[0113] CTCATAAACAGAACGTAGACTACAACATCTTCGAGGGTCGCACTGTAATCGGTTGC

[0114] GCGTCTAACACCATCAGCCAGGGTAAACTGGTATTCAGCGACGGTGAGCTGAACG

[0115] TAGAACGTGGTGCAGGCCGCTTCGTTAAACGTCCACCGTTCGCGCCGTTCTATGAT

[0116] GCAATCGAAAAACAGAACGTACTGTCTACGCCGAAACCGGTTGAACGCCTGCGTT

[0117] GGCAGGGTCGCCCAATCCTGCGCAAGGATGCA;

[0118] SEQ ID NO:2

[0119] MSSLLVKGGTVVTAEHTFSADVLCVDGKIQAVAENLDAPSGSTIVDADSQYVMPGGI

[0120] DPHTHMQLPFMGTVASEDFYTGTAAGLAGGTTMIIDFVIPDPQQNIWDAYQQWRSWS

[0121] EKSVSDYSFHVAITWWDETVSHDMGRIVTEGGVNSFKHFMAYKNAIMCSDETLVNSF

[0122] SRASELGAIVTVHAENGDLVFRLQDELHRMGITGPEGHPLSRPPEVEGEAANRAIRIA

[0123] EVLNVPLYVVHVSARQALEAITRARNEGQRVFGEVLAGHLLIDDSVYRHPDFDVAAA

[0124] HVMSPPFRSPEHQVALWHGLQSGNLQTTATDHCCFCADQKAAGKEDFRLIPNGTAGI

[0125] ENRMEVLWHEGVSTGRLTMNEFVRVTSTAAAQIFNIYPRKGSISVGADADIVVWDPK

[0126] ASKTISAKTHKQNVDYNIFEGRTVIGCASNTISQGKLVFSDGELNVERGAGRFVKRPPF

[0127] APFYDAIEKQNVLSTPKPVERLRWQGRPILRKDA*;

[0128] SEQ ID NO:3

[0129] CAACTGCCATTCNNKGGCACTGTTGCGTCTG;

[0130] SEQ ID NO:4

[0131] CAGACGCAACAGTGCCMNNGAATGGCAGTTG;

[0132] SEQ ID NO:5

[0133] GGAAAACGGTGATNNKGTTTTCCGTCTGCAG;

[0134] SEQ ID NO:6

[0135] CTGCAGACGGAAAACMNNATCACCGTTTTCC;

[0136] SEQ ID NO:7

[0137] CTCGTCCGCCTNNKGTGGAGGGCGAAGCTG;

[0138] SEQ ID NO:8

[0139] CAGCTTCGCCCTCCACMNNAGGCGGACGAG;

[0140] SEQ ID NO:9

[0141] CAGACTACCGCGNNKGATCACTGCTGCTTCTG;

[0142] SEQ ID NO:10

[0143] CAGAAGCAGCAGTGATCMNNCGCGGTAGTCTG;

[0144] SEQ ID NO:11

[0145] CGTCTGATCCCGNNKGGCACCGCTGGTATTG;

[0146] SEQ ID NO:12

[0147] CAATACCAGCGGTGCCMNNCGGGATCAGACG。

Claims

1. A hydantoin mutant, characterized in that, Compared to the amino acid sequence shown in SEQ ID NO:2, the amino acid sequence of the hydantoin mutant is selected from the following amino acid mutations: i) Replace the methionine at position 68 of the amino acid sequence shown in SEQ ID NO:2 with phenylalanine; ii) The methionine at position 68 of the amino acid sequence shown in SEQ ID NO:2 is replaced with phenylalanine, and the leucine at position 190 is replaced with isoleucine; iii) The methionine at position 68 of the amino acid sequence shown in SEQ ID NO:2 is replaced with phenylalanine, and the glutamic acid at position 216 is replaced with proline; iv) Replace the methionine at position 68 of the amino acid sequence shown in SEQ ID NO:2 with phenylalanine and the threonine at position 316 with serine. v) Replace the methionine at position 68 of the amino acid sequence shown in SEQ ID NO:2 with phenylalanine, the leucine at position 190 with isoleucine, and the glutamic acid at position 216 with proline. vi) Replace the methionine at position 68 of the amino acid sequence shown in SEQ ID NO:2 with phenylalanine, the leucine at position 190 with isoleucine, and the threonine at position 316 with serine. vii) As shown in SEQ ID NO:2, the methionine at position 68 is replaced with phenylalanine, the leucine at position 190 is replaced with isoleucine, the glutamic acid at position 216 is replaced with proline, and the threonine at position 316 is replaced with serine. viii) As shown in SEQ ID NO:2, the methionine at position 68 is replaced with phenylalanine, the leucine at position 190 is replaced with isoleucine, the glutamic acid at position 216 is replaced with proline, and the aspartic acid at position 338 is replaced with glycine. ix) As shown in SEQ ID NO:2, the methionine at position 68 is replaced with phenylalanine, the leucine at position 190 is replaced with isoleucine, the glutamic acid at position 216 is replaced with proline, the threonine at position 316 is replaced with serine, and the aspartic acid at position 338 is replaced with glycine.

2. An enzyme agent or enzyme composition comprising the hydantoin mutant as described in claim 1.

3. A polynucleotide encoding the hydantoin mutant as described in claim 1.

4. A nucleic acid construct, recombinant expression vector, or transformed host cell comprising the polynucleotide as described in claim 3.

5. The use of the hydantoin mutant as described in claim 1, the enzyme agent or enzyme composition as described in claim 2 as a catalyst in the production of (R)-3-(carbamoylmethyl)-5-methylhexanoic acid and its downstream products, wherein the downstream product of (R)-3-(carbamoylmethyl)-5-methylhexanoic acid is pregabalin.

6. The application according to claim 5, characterized in that, The substrate for the catalytic reaction is 3-isobutylglutarimide.

7. A method for producing (R)-3-(carbamoylmethyl)-5-methylhexanoic acid, characterized in that, The method includes: using the hydantoin mutant of claim 1 and / or the enzyme agent or enzyme composition of claim 2 as a catalyst to carry out a catalytic reaction to generate (R)-3-(carbamoylmethyl)-5-methylhexanoic acid; the substrate of the catalytic reaction is 3-isobutylglutarimide.

8. A method for producing pregabalin, characterized in that, The method includes: (1) Production of (R)-3-(carbamoylmethyl)-5-methylhexanoic acid according to claim 7; (2) The (R)-3-(carbamoylmethyl)-5-methylhexanoic acid obtained in step (1) is subjected to Hofmann rearrangement to generate pregabalin.

9. The method according to claim 8, characterized in that, Before performing the Hoffmann rearrangement on (R)-3-(carbamoylmethyl)-5-methylhexanoic acid, the process also includes purification and crystallization steps.

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