Imine reductase mutants and uses thereof

Abstract

The application relates to an imine reductase mutant and application thereof, the amino acid sequence of the imine reductase mutant is an amino acid sequence obtained by mutation on the basis of the sequence shown in SEQ ID NO. 1, imine is used as a substrate, a high (S) stereoselective imine reductase VpIR is obtained by screening an imine reductase library, a high catalytic efficiency and high selectivity imine reductase mutant is obtained by mutation on a key site of the enzyme molecule, and the application potential of the enzyme in industrial application is expanded.

Description

[0001] This application claims priority to patent application number 202411715960.6 (the earlier application was filed on November 27, 2024, and is entitled "An Imine Reductase Mutant and Its Application"). Technical Field

[0002] This invention relates to the field of biotechnology, and in particular to an imine reductase mutant and its applications. Background Technology

[0003] Chiral amines and their derivatives constitute an important branch of chiral drugs, serving as crucial structural units in numerous bioactive molecules, natural products, intermediates, and decomposition agents. They have wide applications in the synthesis of pharmaceuticals, agrochemicals, and materials, as well as in asymmetric catalysis. Chiral amines are structural units in many pharmaceutical intermediates and agrochemicals; chiral amine drugs include neurological, antihypertensive, and cardiovascular drugs. The preparation of chiral amine compounds via enzymatic catalysis has gained widespread attention from academia and industry due to its advantages of high efficiency, environmental friendliness, and economic efficiency, as exemplified by the application of transaminases in drugs such as sitagliptin. However, due to limitations in the reaction mechanism, transaminases are currently confined to the synthesis of chiral primary amines.

[0004] Imine reductases (IREDs) are a class of NAD(P)H-dependent oxidoreductases that catalyze the asymmetric reduction of imines to synthesize chiral amines. IREDs possess excellent characteristics such as high catalytic efficiency, strong regioselectivity, and stereoselectivity, making them stand out among numerous methods for synthesizing chiral amines and attracting the attention of researchers. (S)-2-aryl-substituted pyrrolidines are commonly found in various natural products, drug molecules, and active intermediates. Functionalized chiral 2-aryl-substituted pyrrolidines have been shown to possess diverse biological activities and are widely used in various drugs, such as the chiral intermediate of larotrectinib. Currently, most methods for synthesizing (S)-2-aryl-substituted pyrrolidines are chemical methods, which involve cumbersome synthetic steps, low economic efficiency, and have drawbacks such as harsh reaction conditions and the need for expensive noble metal catalysts. Enzymatic methods have attracted widespread attention due to their relatively mild reaction conditions, but research on the enzymatic synthesis of (S)-2-aryl-substituted pyrrolidines is limited. In addition, currently reported IREDs generally suffer from problems such as narrow substrate spectrum, poor stability, and low enzyme activity. Therefore, the development of IREDs with superior performance has gradually attracted people's attention.

[0005] CN116218803A discloses an imine reductase, its preparation method, and the DNA encoding the imine reductase. This imine reductase exhibits high specific activity, high thermal stability, and a wide optimal pH range. When used to catalyze the preparation of (S)-nornicotine from mesmin, it achieves high conversion and enantiomeric excess percentage. CN116286700A discloses the application of an imine reductase mutant in the synthesis of the key intermediate (S)-3-(pyrrolidine-2-yl)pyridine for nicotine. The optional mutation sites include at least one of the following: A at position 246 or D at position 285 mutated to V. This imine reductase mutant improves the yield of the catalytic reduction reaction and shortens the reaction time.

[0006] In summary, providing a novel type of IRED with superior performance, improving substrate spectra, stability, and enzyme activity, is of great significance for the production and application of chiral amines and has become one of the urgent problems to be solved in this field. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides an imine reductase mutant and its applications. Addressing the issues of narrow substrate spectrum, poor stability, and low enzyme activity inherent in existing imine reductases, this invention develops a novel imine reductase mutant to improve substrate catalytic performance and expand the application prospects of imine reductase.

[0008] To achieve this objective, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides an imine reductase mutant, wherein the amino acid sequence of the imine reductase mutant comprises any one of the following sequences:

[0010] (I) An amino acid sequence obtained by mutation based on the sequence shown in SEQ ID NO.1, wherein the mutation sites include any one or at least two combinations of T18, I32, R62, Q107, F117, E119, G132, M138, V148, S167, N168, I180, R192, S228, K232, M238, S255, D273, A275 or L282;

[0011] (II) The amino acid sequence obtained from (I) is obtained by substitution, deletion or addition of one or at least two amino acid residues, and has the same or similar function as the amino acid sequence obtained from (I);

[0012] (III) has at least 90% sequence homology with the amino acid sequence obtained in (I) or (II), and has the same or similar function as the amino acid sequence obtained in (I).

[0013] This invention improves the substrate catalytic performance and broadens the substrate spectrum by directed evolution of the imine reductase VpIR from Variovorax paradoxus of Burkholderiaceae, thus expanding the application prospects of imine reductase.

[0014] In this invention, a specific mutation is introduced into wild-type imine reductase to obtain an imine reductase mutant, which can improve its catalytic activity and expand its substrate spectrum. It is understood that, based on the imine reductase mutant, those skilled in the art can use common techniques in the art to substitute, delete or add one or at least two amino acid residues to obtain other sequences with the same or similar functions.

[0015] SEQ ID NO.1:MSSKQKITVILGAMGATIARLYLEQGHEVTIWNRSADKAAPLVAQGA VLADSAAAAVRASRVVLMCVYDYRAADAILGAEGVAAAMDGRRLLVQLTTGSPRDARDAQAWAQRHGATFLEGAIQAAPEQMGKGDTPILMSGDEQVFRAVEPLLAVLGGGIVYLGEKISNAAA MDLATLSTIYGTMLGFLHGARVAESEGFDVAEFGRIVAGIMPTFASFLQHEGAVIQSGDFKISQSPMRISVEATQRILQTARESGINSEFPAFAAGLFQRADAAGLGGEELAALIKLLRAPA.

[0016] Preferably, the amino acid sequence of the imine reductase mutant is an amino acid sequence obtained by mutation based on the sequence shown in SEQ ID NO.1, wherein the mutation includes:

[0017] Any one or a combination of at least two of the following: T18A, I32V, R62A, R62P, Q107A, F117Y, E119D, G132P, M138Y, V148H, S167C, S167D, N168A, N168P, I180M, I180F, R192A, S228H, S228R, K232R, M238V, M238C, M238T, M238L, S225R, S255N, S255M, D273I, D273M, A275K, A275N, A275C, or L282M.

[0018] Preferably, the combination of mutations includes any one of the following:

[0019] (1) Combination of T18A and M138Y, (2) Combination of I32V and M138Y, (3) Combination of R62P and M138Y, (4) Combination of Q107A and M138Y, (5) Combination of E119D and M138Y, (6) Combination of M138Y and S167C, (7) Combination of M138Y and S167D, (8) Combination of M138Y and S228R, (9) Combination of M138Y and K232R, (10) Combination of M138Y and S255R, (11) Combination of T18A, M138Y and S255R, (12) Combination of T18A, M138Y and E119D, (13) Combination of T18A, M138Y and S167C (14) Combination of T18A, M138Y and K232R, (15) Combination of T18A, R62P and M138Y, (16) Combination of M138Y, K232R and S255R, (17) Combination of M138Y, S167C and K232R, (18) Combination of M138Y, E119D and K232R, (19) Combination of R62P, M138Y and K232R, (20) Combination of M138Y, E119D and S255R, (21) Combination of M138Y, E119D and S167C, (22) Combination of R62P, E119D and M138Y, (23) Combination of M138Y, S167D, S228R and K232R.

[0020] In this invention, for ease of writing, the point mutation combination "combination of T18A and M138Y" is written as "T18A+M138Y", and subsequent combinations follow the same pattern.

[0021] In a second aspect, the present invention provides a nucleic acid molecule that encodes the imine reductase mutant described in the first aspect.

[0022] In this invention, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0023] Thirdly, the present invention provides a recombinant vector containing the nucleic acid molecules described in the second aspect.

[0024] Preferably, the recombinant vector comprises a recombinant plasmid.

[0025] Preferably, the starting plasmids of the recombinant plasmids include pET-21b(+), pET-22b(+), pET-3a(+), pET-3d(+), pET-11a(+), pET-12a(+), pET-14b, pET-15b(+), pET-16b(+), pET-17b(+), pET-19b(+), pET-20b(+), pET-21a(+), pET-23a(+), pET-23b(+), pET-24a(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28a(+), pET-29a(+), pET-30a(+), pET-31b(+), pET-32a(+), and pET-35b(+). , pET-38b(+), pET-39b(+), pET-40b(+), pET-41a(+), pET-41b(+), pET-42a(+), pET- 43a(+), pET-43b(+), pET-44a(+), pET-49b(+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE4 0, any one of pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-1, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-8, pPIC9k, pGAPZαA, pUC-18, or pUC-19.

[0026] Fourthly, the present invention provides a recombinant cell containing the nucleic acid molecule described in the second aspect or the recombinant vector described in the third aspect.

[0027] Preferably, the starting cells of the recombinant cells include eukaryotic cells or prokaryotic cells.

[0028] Preferably, the eukaryotic cells include yeast.

[0029] Preferably, the prokaryotic cells include Escherichia coli.

[0030] Preferably, the Escherichia coli includes any one of Escherichia coli DH5α, Escherichia coli Top10, Escherichia coli BL21-DE3, or Escherichia coli Rosetta-DE3.

[0031] Fifthly, the present invention provides a method for preparing an imine reductase mutant as described in the first aspect, the method comprising:

[0032] The nucleic acid molecule encoding the imine reductase mutant described in the first aspect is inserted into the expression vector to obtain a recombinant vector. The recombinant vector is introduced into a host cell to obtain a recombinant cell, which is then cultured and the product is purified to obtain the imine reductase mutant.

[0033] In this invention, the methods for inducing and culturing recombinant cells and for isolating imine reductase from the culture can both employ conventional methods in the art. The culture medium used when expressing imine reductase in recombinant cells can be any culture medium in the art capable of growing the recombinant cells and producing the imine reductase mutant of this invention, such as LB medium.

[0034] In a sixth aspect, the present invention provides the use of the imine reductase mutant as described in the first aspect, the nucleic acid molecule as described in the second aspect, the recombinant vector as described in the third aspect, or the recombinant cell as described in the fourth aspect in the production of chiral amines.

[0035] It should be understood that the imine reductase mutant described in this invention can be used in whole-cell engineered bacteria, in unpurified crude enzyme form, or in partially or completely purified enzyme form. Furthermore, the imine reductase mutant of this invention can be prepared into an immobilized enzyme or a catalyst in immobilized cell form using immobilization techniques known in the art.

[0036] In a seventh aspect, the present invention provides a method for producing chiral amines, the method comprising using an imine reductase mutant as described in the first aspect to catalyze a reduction reaction of an imine substrate;

[0037] The structural formula of the imine substrate is shown in Formula I;

[0038]

[0039] Wherein, n = 1, 2 or 3; m = 1, 2 or 3; R is selected from hydrogen, C1-C6 alkyl, halogen or C1-C6 alkoxy; Ar is selected from phenyl, furanyl, pyridyl or thiophene.

[0040] Preferably, the method includes reducing 5-(2-tert-butylphenyl)-3,4-dihydro-2H-pyrrole to (S)-2-(2-tert-butylphenyl)pyrrole using the imine reductase mutant described in the first aspect.

[0041] Preferably, the reaction system of the reduction reaction further includes a coenzyme or a coenzyme regeneration system, wherein the coenzyme includes any one or a combination of at least two of NADP, NAD, NADPH or NADH.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] This invention uses imine as a substrate and screens existing imine reductase libraries to obtain a highly (S) stereoselective imine reductase, VpIR, derived from Variovorax paradoxus. By performing saturation mutations and further combined mutations on key sites around the active pocket, on the protein surface, and at the subunit interface of the structural model, a highly efficient and selective imine reductase mutant was obtained, broadening the substrate spectrum and expanding the application potential of this enzyme in industrial applications. Attached Figure Description

[0044] Figure 1 The HPLC chromatogram of the substrate 5-(2-tert-butylphenyl)-3,4-dihydro-2H-pyrrole is shown.

[0045] Figure 2 The HPLC chromatogram of (S)-2-(2-tert-butylphenyl)pyrrolidine standard.

[0046] Figure 3 This is the HPLC chromatogram of the VpIR enzyme-catalyzed reaction solution in Example 1.

[0047] Figure 4 The chiral HPLC chromatogram of (S)-2-(2-tert-butylphenyl)pyrrolidine standard.

[0048] Figure 5 The image shows the chiral HPLC chromatogram of the VpIR enzyme-catalyzed reaction solution in Example 1. Detailed Implementation

[0049] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0050] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0051] This invention screened wild-type imine reductases derived from *Variovorax paradoxus*, *Cupriavidus* sp., *Aeromonasveronii*, *Dadobacter endophyticus*, and *Luteolibacter luteus*. It was found that the imine reductase from *Variovorax paradoxus* exhibited relatively good stereoselectivity (S), but showed poor activity and low conversion rate in the target reaction. Therefore, this invention used the amino acid sequence shown in SEQ ID NO.1 of *Variovorax paradoxus* as the starting gene and further employed directed evolutionary modification to obtain a series of imine reductase mutants with high catalytic efficiency and selectivity.

[0052] According to a typical embodiment of the present invention, an imine reductase mutant is provided. The amino acid sequence of the imine reductase mutant is the amino acid sequence of the sequence shown in SEQ ID NO.1 with mutations, and the mutated amino acid sites include: T18, I32, R62, Q107, F117, E119, G132, M138, V148, S167, N168, I180, R192, S228, K232, M238, S255, E260, D273, A275, L282;

[0053] Alternatively, the amino acid sequence of the imine reductase mutant may have mutated amino acid sites and possess 90%, 95%, or 99% or more homology with the sequence shown in SEQ ID NO.1, exhibiting imine reductase catalytic activity. The imine reductase mutant provided by this invention can efficiently and stereoselectively synthesize (S)-2-arylpyrrolidine, making it suitable for industrial production.

[0054] The term "homology" as used herein has the meaning commonly known in the art, and those skilled in the art are familiar with the rules and standards for determining homology between different sequences. The sequences defined by different degrees of homology in this invention must also simultaneously possess improved imine reductase catalytic activity towards the substrate. In the above embodiments, those skilled in the art can obtain such variant sequences under the guidance of the disclosure of this invention.

[0055] The mutation is one of the following single point mutations or combinations of mutations: T18A, I32V, R62A, R62P, Q107A, F117Y, E119D, G132P, M138Y, V148H, S167C, S167D, N168A, N168P, I180M, I180F, R192A, S228H, S228R, K232R, M238V. M238C, M238T, M238L, S225R, S255N, S255M, D273I, D273M, A275K, A275N, A275C, L282 M, T18A+M138Y, I32V+M138Y, R62P+M138Y, Q107A+M138Y, E119D+M138Y, M138Y+S167C , M138Y+S167D, M138Y+S228R, M138Y+K232R, M138Y+S255R, T18A+M138Y+S255R, T18A +M138Y+E119D, T18A+M138Y+S167C, T18A+M138Y+K232R, T18A+R62P+M138Y, M138Y+K 232R+S255R, M138Y+S167C+K232R, M138Y+E119D+K232R, R62P+M138Y+K232R, M138Y+ E119D+S255R, M138Y+E119D+S167C, R62P+E119D+M138Y, M138Y+S167D+S228R+K232R.

[0056] According to a typical embodiment of the present invention, a DNA molecule is provided. This DNA molecule encodes any of the aforementioned imine reductase mutants. The imine reductase mutant encoded by this DNA molecule exhibits high catalytic activity and selectivity.

[0057] The DNA molecules described above in this invention can also exist in the form of an "expression cassette." An "expression cassette" refers to a linear or circular nucleic acid molecule encompassing DNA and RNA sequences capable of directing the expression of a specific nucleotide sequence in an appropriate host cell. Generally, it includes a promoter effectively linked to the target nucleotide, optionally linked to a termination signal and / or other regulatory elements. The expression cassette may also include sequences required for the correct translation of the nucleotide sequence. The coding region typically encodes the target protein, but may also encode the target functional RNA, such as antisense RNA or untranslated RNA, in either the sense or antisense direction. Expression cassettes containing the target polynucleotide sequence can be chimeric, meaning that at least one of its components is heterologous to at least one of its other components. Expression cassettes can also be naturally occurring, but are obtained through efficient recombination for heterologous expression.

[0058] According to a typical embodiment of the present invention, a recombinant plasmid is provided. The recombinant plasmid contains any of the aforementioned DNA molecules. The DNA molecules in the recombinant plasmid are positioned at appropriate locations within the plasmid, enabling the DNA molecules to be correctly and smoothly replicated, transcribed, or expressed.

[0059] Although the present invention uses the qualifier "contains" when defining the aforementioned DNA molecule, this does not mean that other sequences unrelated to its function can be arbitrarily added to both ends of the DNA sequence. Those skilled in the art will know that, in order to meet the requirements of recombination operations, it is necessary to add suitable restriction endonuclease cleavage sites to both ends of the DNA sequence, or to add additional start codons, stop codons, etc. Therefore, using a closed-form description to define it will not truly cover these situations.

[0060] As used in this invention, the term "plasmid" includes any plasmid, granule, bacteriophage, or Agrobacterium binary nucleic acid molecule in double-stranded or single-stranded linear or circular form, preferably a recombinant expression plasmid, which can be a prokaryotic or eukaryotic expression plasmid, but is preferred to be a prokaryotic expression plasmid. In some embodiments, the recombinant plasmid is pET-21b(+), pET-22b(+), pET-3a(+), pET-3d(+), pET-11a(+), pET-12 a(+), pET-14b, pET-15b(+), pET-16b(+), pET-17b(+), pET-19b(+), pET-20b(+), pET-21a(+), pET -23a(+), pET-23b(+), pET-24a(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28a(+), pET-29a( +), pET-30a(+), pET-31b(+), pET-32a(+), pET-35b(+), pET-38b(+), pET-39b(+), pET-40b(+), pE T-41a(+), pET-41b(+), pET-42a(+), pET-43a(+), pET-43b(+), pET-44a(+), pET-49b(+), pQE2, pQ E9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-1 , pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-8, pPIC9k, pGAPZαA, pUC-18 or pUC-19.

[0061] According to a typical embodiment of the present invention, a host cell is provided, the host cell containing any of the above-mentioned recombinant plasmids. The host cells suitable for the present invention include, but are not limited to, prokaryotic cells or eukaryotic cells. Preferably, the prokaryotic cells are *Escherichia coli* DH5α, Top10, BL21 DE3, or *Escherichia coli* Rosetta DE3 cells; the eukaryotic cells are yeast cells.

[0062] According to a typical embodiment of the present invention, a method for producing chiral amines is provided. The method includes the step of reducing an imine substrate using an imine reductase, wherein the imine reductase is any of the imine reductase mutants described above in this invention.

[0063] The structural formula of the imine substrate is shown in Formula I;

[0064]

[0065] Wherein, n = 1, 2 or 3; m = 1, 2 or 3; R is selected from hydrogen, C1-C6 alkyl, halogen or C1-C6 alkoxy; Ar is selected from phenyl, furanyl, pyridyl or thiophene.

[0066] According to a typical embodiment of the present invention, the reaction system of imine reductase with imine substrates further includes a coenzyme, wherein the coenzyme includes any one or a combination of at least two of NADP, NAD, NADPH or NADH.

[0067] The beneficial effects of the present invention will be further illustrated below with reference to embodiments.

[0068] Example 1

[0069] In this embodiment, imine reductases were screened from wild-type imine reductases derived from Variovorax paradoxus, Cupriavidus sp., Aeromonas veronii, Dadobacter endophyticus, and Luteolibacterluteus (VpIR, CsIR, AvIR, DeIR, and LlIR, respectively). The screening steps are as follows: using 5-(2-tert-butylphenyl)-3,4-dihydro-2H-pyrrole (1a) as a substrate, imine reductase catalysis yielded (S)-2-(2-tert-butylphenyl)pyrrolidine (1b) as shown in the reaction formula below.

[0070]

[0071] Weigh 10.0 mg of imine reductase (VpIR, CsIR, AvIR, DeIR, and LlIR) into their respective 5.0 mL centrifuge tubes, and add 2.0 mg GDH, 40.0 mg glucose, and 2.0 mg NADP, respectively.+ 10.0 g / L substrate 1a and 20.0 μL of DMSO solution were added to 0.1 M pH 7.0 phosphate buffer and brought to a final volume of 1.0 mL. The mixture was then incubated at 30 °C in a shaker for 24 h. Detection was performed by ultra-high performance liquid chromatography (UHPLC) (C18 column, mobile phase: acetonitrile and water, flow rate 0.4 mL / min) and chiral high performance liquid chromatography (HPLC) (AY column, n-hexane:ethanol (0.1% diethylamine) = 98:2, flow rate 1 mL / min).

[0072] The HPLC chromatogram of the substrate 5-(2-tert-butylphenyl)-3,4-dihydro-2H-pyrrole is shown below. Figure 1 As shown, the HPLC chromatogram of (S)-2-(2-tert-butylphenyl)pyrrolidine standard is as follows: Figure 2 As shown, the HPLC chromatogram of the VpIR enzyme catalytic reaction solution is as follows. Figure 3 As shown, the chiral HPLC chromatogram of (S)-2-(2-tert-butylphenyl)pyrrolidine standard is as follows: Figure 4 As shown, the chiral HPLC chromatogram of the VpIR enzyme-catalyzed reaction solution is as follows: Figure 5 As shown in Table 1, the conversion rate (percentage of product and feed peak area) and ee value (difference between R and S configuration percentages) were detected.

[0073] Table 1

[0074] enzymes source Conversion rate (%) ee(%) VpIR Variovorax paradoxus 92.4 >99.0％(S) CsIR Cupriavidus sp. 80.4％ >99.0％(S) AvIR Aeromonas veronii 40.4％ 87.4％(S) DeIR Dyadobacter endophyticus 88.6％ 69.2％(R) LlIR Luteolibacter luteus 82.4％ 76.0％(S)

[0075] As shown in Table 1, VpIR, CsIR, AvIR, and LlIR can yield (S)-2-(2-tert-butylphenyl)pyrrolidine (1b), with VpIR exhibiting the best catalytic efficiency. While DeIR provides efficient catalysis, it yields the opposite configuration. However, when VpIR attempts to increase the substrate loading (e.g., 20.0 g / L or 30.0 g / L), the catalytic activity significantly decreases, indicating insufficient catalytic activity for the substrate. Therefore, using an imine reductase derived from Variovorax paradoxus (with the amino acid sequence shown in SEQ ID NO. 1) as the starting enzyme for this invention, directed evolution was performed to improve the catalytic activity of the target enzyme.

[0076] Example 2

[0077] This embodiment constructs a mutant library of Variovorax paradoxus imine reductase. Twenty-one sites (T18, I32, R62, Q107, F117, E119, G132, M138, V148, S167, N168, I180, R192, S228, K232, M238, S255, E260, D273, A275, and L282) near the VpIR substrate binding pocket and on the protein surface and subunit interface were selected for site-directed saturation mutagenesis. The resulting positive mutants were then combined to enhance enzyme catalytic activity. Complete linear fragments were obtained by whole plasmid PCR. The PCR products were digested with DpnI to remove the parental template of the starting gene and then transformed into Escherichia coli BL21(DE3). The fragments were plated in LB culture dishes containing 50.0 μg / mL kanamycin and incubated overnight at 37°C. After expression was induced in 96-well plates, mutants with increased activity compared to the parent were selected by high-throughput screening using ultra-high performance liquid chromatography. The mutation sites were then determined by gene sequencing.

[0078] The specific operating procedure is as follows: pET28a-VpIR (pET28a plasmid containing the VpIR encoding gene, the nucleic acid sequence of the VpIR encoding gene is shown in SEQ ID NO.2) is used as a template, and PCR is performed using the high-fidelity polymerase PrimeSTAR. The PCR reaction conditions are as follows: In a PCR reaction system with a total volume of 50.0 μL, add 0.5-20.0 ng of template, 25 μL of 2×PrimeSTAR (Premix), 1.0 μL (10 μM) of each of a pair of mutant primers, and add sterile distilled water to a final volume of 50.0 μL. PCR reaction program: (1) denaturation at 98℃ for 10 sec, (2) annealing at 55℃ for 30 sec, (3) extension at 72℃ for 6 min, and perform steps (1) to (3) for a total of 30 cycles. Store the product at 12℃. After the PCR product is verified by agarose gel electrophoresis, add Dpn I and digest at 37℃ for 1 h. The digestion products were transferred into E. coli BL21(DE3) competent cells and plated on a plate containing kanamycin. The cells were then incubated at 37°C for about 18 hours.

[0079] SEQ ID NO.2:atgagcagtaaacagaaaatcaccgtgattggcctgggcgcaatgggtgcaaccattgcccgtctgtatctgg aacagggccatgaagttaccatttggaatcgcagcgccgataaagccgccccgctggttgcccagggcgcagtgttagcagatagtgcagccgcagccgtgcgtgcaagccgtgtggttctgatgtgcgtttatgattatcgtgcagccgatgccattctgggcgccgaaggtgtggccgcagcaatggatggtcgtctgctggttcagctgaccaccggcagtccgcgtgatgcccgtgatgcacaggcatgggcacagcgccacggtgccacctttctggaaggcgccattcaggccgccccggaacagatgggtaaaggcgataccccgattctgatgagcggcgatgaacaggtttttcgtgcagtggaaccgctgctggcagtgctgggcggtggtattgtgtatctgggtgaaaaaattagcaatgccgccgcaatggatctggcaaccctgagcaccatctatggcaccatgctgggctttctgcatggtgcccgcgttgcagaaagtgaaggttttgatgtggccgaatttggccgtattgtggccggtattatgccgacctttgccagctttctgcagcatgaaggtgcagtgattcagagcggtgactttaaaattagtcagagtccgatgcgtattagtgttgaagcaacccagcgcattctgcagaccgcccgcgaaagtggtattaatagtgaatttccggcctttgccgcaggcctgtttcagcgtgccgatgccgccggtctgggcggtgaagaactggccgccctgattaagctgctgcgcgcaccggcctaa。

[0080] The specific procedures for saturated mutant library culture and feed reaction are as follows: The obtained single-clone colonies were picked and cultured in 600.0 μL of LB medium in a 96-well deep-well plate at 37°C with shaking until the OD600 reached 0.6. IPTG was then added to a final concentration of 0.5 mM, and expression was induced overnight at 25°C. The next day, the 96-well plate was centrifuged to remove the supernatant. 100.0 μL of lysozyme solution (lysozyme 10.0 mg / mL, pH = 7.0) was added to each well, and the plate was incubated at 30°C for 2 hours for disruption. Subsequently, the feed amounts added to each well were: substrate 1a 1.6 mg, DMSO 4.0 μL, glucose 8.0 mg, GDH 0.4 mg, and NADP 0.4 mg. + The solution was brought to a final volume of 200.0 μL with PBS buffer and reacted at 30 °C for 24 h. The reaction was terminated the next day by adding 1.0 mL / well acetonitrile solution. After centrifugation at 4000 rpm for 15 min, 1.0 mL of the supernatant was collected, filtered, and the conversion rate was determined by ultra-high performance liquid chromatography. The results are shown in Table 2.

[0081] Table 2

[0082]

[0083]

[0084] Note: a: 1a substrate loading is 20 g / L, b: 1a substrate loading is 30 g / L.

[0085] As shown in Table 2, when the imine substrate concentration was 20.0 g / L, the mutant enzymes at 11 sites (T18A, I32V, R62P, Q107A, E119D, M138Y, S167C, S167D, S228R, K232R, and S225R) exhibited more than 2.0-fold increased activity compared to the VpIR wild-type while maintaining excellent stereoselectivity (>99.0%). However, when the concentration was increased to 30.0 g / L, the transformation efficiency of the wild-type and its mutants decreased to <80.0%, with M138Y showing the highest transformation efficiency at 77.7%. Therefore, the next round of mutation attempts will use M138Y as a template to combine the above 10 sites, hoping to obtain positive mutants tolerant to higher substrate concentrations. The results of the combined mutation test are shown in Table 3.

[0086] Table 3

[0087]

[0088]

[0089] Note: a:1a substrate loading is 30.0 g / L.

[0090] As shown in Table 3, under a substrate loading of 30.0 g / L, the transformation efficiency of the mutant M138Y+E119D was 1.2 times higher than that of the parent M138Y and nearly 9.0 times higher than that of the wild type. However, when other beneficial mutants, such as K232R and S255R, were further fused into the M138Y+E119D plasmid, the transformation efficiency decreased by 10.0%. Another suboptimal double mutant, M138Y+K232R, had a transformation efficiency of 84.6%, but adding the S167D+S228R site also reduced the transformation efficiency by 20%.

[0091] The M138Y+E119D combination, which achieved the highest conversion rate, was selected and compared with the catalytic performance of the wild-type VpIR. The results are shown in Table 4. With a substrate loading of 10.0 g / L, the wild-type enzyme achieved a conversion rate of 92.4% after 24 hours. However, as the substrate loading increased to 30.0 g / L, the conversion rate dropped to only 10.7%. In contrast, for the mutant M138Y, with a loading of 20.0 g / L, the conversion rate was >99.0% after 24 hours. Notably, at a substrate loading of 30.0 g / L, M138Y+E119D could completely convert the substrate within 8 hours, demonstrating a significant improvement in the catalytic performance of the mutant compared to the parent enzyme.

[0092] Table 4

[0093] Enzyme number Substrate loading (g / L) Reaction time (h) Conversion rate (%) ee(%) VpIR 10.0 24 92.4 >99.0(S) VpIR 20.0 24 46.6 >99.0(S) VpIR 30.0 24 10.7 >99.0(S) M138Y 20.0 12 >99.0 >99.0(S) M138Y+E119D 30.0 8 >99.0 >99.0(S)

[0094] Example 3

[0095] This embodiment describes the preparation of (S)-2-arylpyrrolidase on a 100-gram scale. 300.0 mg of substrate 1a and 2% v / v DMSO, 50.0 mg of NADP were added sequentially to two 50.0 mL four-necked flasks. + 50.0 mg GDH, 2.0 g glucose, and 0.1 M pH 7.0 phosphate buffer were added to a final volume of 30.0 mL. The reaction was started by stirring. The temperature was controlled at 30 °C in a constant temperature water bath. The pH was controlled at 7.0 using a titrator with 3 N sodium carbonate solution. Finally, 150% w / w dried bacterial powder containing VpIR and M138Y+E119D mutant imine reductase was added to each of the three reaction systems. Among them, the M138Y+E119D mutant imine reductase showed Conv (%) > 99.0% and ee > 99.0% (S) after 8 h, and the VpIR reaction showed Conv (%) 40.0% and ee > 99.0% (R) after 24 h. This indicates that the enzyme activity of the imine reductase mutant constructed in this invention is significantly improved and the reaction time is effectively shortened.

[0096] Example 4

[0097] This embodiment investigated the substrate spectrum of the mutant M138Y+E119D, selecting substrates 2a, 3a, and 4a, the structures of which are shown below.

[0098]

[0099] Raw materials 2a, 3a, and 4a were weighed and added to 5.0 mL centrifuge tubes along with 10.0 mg of dried bacterial powder containing the mutant M138Y+E119D. Then, 2.0 mg of GDH, 40.0 mg of glucose, and 2.0 mg of NADP were added, respectively. + Add 20.0 μL of DMSO to a pH 7.0 PBS buffer solution and bring the volume to 1.0 mL. Incubate at 30 °C in a shaker for 24 h to synthesize 2b, 3b, and 4b enzymatically. Samples were diluted with acetonitrile to determine the conversion rate and ee value. The results are shown in Table 5.

[0100] Table 5

[0101]

[0102] Note: a: substrate loading of 20 g / L, M138Y+E119D conversion rate; b: substrate loading of 30 g / L, M138Y+E119D conversion rate.

[0103] In this embodiment, the mutant M138Y+E119D showed good catalytic activity and selectivity for 2a, 3a and 4a, indicating that the present invention, through directed evolution of VpIR, not only improved the catalytic performance but also broadened the substrate spectrum, making the application of this enzyme in industrial production more mature.

[0104] In summary, this invention, through screening wild-type imine reductases derived from *Variovorax paradoxus*, *Cupriavidus* sp., *Aeromonas veronii*, *Dadobacter endophyticus*, and *Luteolibacter luteus*, found that the imine reductase VpIR activity from *Variovorax paradoxus* (of the Burkholderiaceae family) was relatively better. Therefore, this invention uses the amino acid sequence shown in SEQ ID NO.1 of *Variovorax paradoxus* as the starting enzyme, and further employs directed evolutionary modification to obtain a series of imine reductase mutants with high catalytic efficiency, selectivity, and substrate profiles.

[0105] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. An imine reductase mutant, characterized in that, The amino acid sequence of the imine reductase mutant includes the amino acid sequence obtained by mutation based on the sequence shown in SEQ ID NO.1, which is: Combinations of M138Y, T18A, and M138Y; combinations of I32V and M138Y; combinations of R62P and M138Y; combinations of Q107A and M138Y; combinations of E119D and M138Y; combinations of M138Y and S167C; combinations of M138Y and S167D; combinations of M138Y and S228R; combinations of M138Y and K232R; combinations of M138Y and S255R; combinations of T18A, M138Y, and S167C; combinations of T18A, M138Y, and... Any combination of K232R, T18A, R62P and M138Y, M138Y, K232R and S255R, M138Y, S167C and K232R, M138Y, E119D and K232R, R62P, M138Y and K232R, M138Y, E119D and S255R, R62P, E119D and M138Y, or M138Y, S167D, S228R and K232R.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the imine reductase mutant of claim 1.

3. A recombinant vector, characterized in that, The recombinant vector contains the nucleic acid molecule as described in claim 2.

4. The recombinant vector according to claim 3, characterized in that, The recombinant vector includes a recombinant plasmid.

5. The recombinant vector according to claim 4, characterized in that, The starting plasmids of the recombinant plasmids include pET-21b(+), pET-22b(+), pET-3a(+), pET-3d(+), pET-11a(+), pET-12a(+), pET-14b, pET-15b(+), pET-16b(+), pET-17b(+), pET-19b(+), pET-20b(+), pET-21a(+), pET-23a(+), pET-23b(+), pET-24a(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28a(+), pET-29a(+), pET-30a(+), pET-31b(+), pET-32a(+), pET-35b(+), p ET-38b(+), pET-39b(+), pET-40b(+), pET-41a(+), pET-41b(+), pET-42a(+), pET-43 a(+), pET-43b(+), pET-44a(+), pET-49b(+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE40 Any one of pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-1, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-8, pPIC9k, pGAPZαA, pUC-18, or pUC-19.

6. A recombinant cell, characterized in that, The recombinant cells contain the nucleic acid molecule of claim 2 or the recombinant vector of claim 3.

7. The recombinant cell according to claim 6, characterized in that, The starting cells for the recombinant cells include eukaryotic cells or prokaryotic cells.

8. The recombinant cell according to claim 7, characterized in that, The eukaryotic cells include yeast.

9. The recombinant cell according to claim 7, characterized in that, The prokaryotic cells include Escherichia coli.

10. The recombinant cell according to claim 9, characterized in that, The *Escherichia coli* species include any one of *Escherichia coli* DH5α, *Escherichia coli* Top10, *Escherichia coli* BL21-DE3, or *Escherichia coli* Rosetta-DE3.

11. A method for producing chiral amines, characterized in that, The method includes using the imine reductase mutant of claim 1 to catalyze the reduction reaction of an imine substrate to reduce 5-(2-tert-butylphenyl)-3,4-dihydro-2H-pyrrole to (S)-2-(2-tert-butylphenyl)pyrrole.

12. The method for producing chiral amines according to claim 11, characterized in that, The reaction system of the reduction reaction also includes a coenzyme or a coenzyme regeneration system, wherein the coenzyme includes any one or a combination of at least two of NADP, NAD, NADPH or NADH.

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