Imine reductase mutant and application thereof

Abstract

The invention relates to an imine reductase mutant and application thereof, the amino acid sequence of the imine reductase mutant is obtained by mutation on the basis of the sequence shown in SEQ ID NO.1. The imine reductase VpIR with high (S) stereoselectivity is obtained by taking imine as a substrate and screening an imine reductase library; the imine reductase mutant with high catalytic efficiency and high selectivity is obtained by mutating key sites of enzyme molecules, and the application potential of the imine reductase in industrial application is expanded.

Description

[0001] This application claims the priority of patent application No. 202411715960.6 (the filing date of the prior application is November 27, 2024, and the name of the invention is an imine reductase mutant and its application). Technical Field

[0002] The invention relates to the field of biotechnology, in particular to an imine reductase mutant and application thereof. Background Art

[0003] Chiral amines and their derivatives are an important branch of chiral drugs. They are important structural units of many bioactive molecules, natural products, intermediates and decomposers. They are widely used in the synthesis of drugs, agricultural chemicals and materials, as well as in asymmetric catalysis. Chiral amines are structural units of many pharmaceutical intermediates and agricultural chemicals. Chiral amine drugs include neurological, antihypertensive and cardiovascular drugs. The preparation of chiral amine compounds by enzyme catalysis has attracted widespread attention from academia and industry due to its high efficiency, environmental friendliness and high economic efficiency, such as the application of transaminases in drugs such as sitagliptin. However, due to the limitation of the reaction mechanism, transaminases are limited 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 have excellent properties such as high catalytic efficiency, strong regioselectivity and stereoselectivity, and stand out among many methods for synthesizing chiral amines, attracting the research attention of scientific researchers. (S)-2-aryl substituted pyrrolidines are common in a variety of natural products, drug molecules and active intermediates. Functionalized chiral 2-aryl substituted pyrrolidines have been shown to have a variety of biological activities and are widely used in a variety of drugs, such as larotrectinib chiral intermediates. Currently, the methods for synthesizing (S)-2-aryl substituted pyrrolidines are mostly chemical methods, which have cumbersome synthesis steps, low economic benefits, and have the disadvantages of harsh reaction conditions and the need to use expensive precious metal catalysts. The enzymatic method has attracted widespread attention from researchers due to its relatively mild reaction conditions, but there are few studies on the synthesis of (S)-2-aryl substituted pyrrolidines using the enzymatic method. In addition, the IREDs reported so far generally have problems such as narrow substrate spectrum, poor stability, and low enzyme activity. Therefore, the development of IREDs with excellent performance has gradually attracted people's attention.

[0005] CN116218803A discloses an imine reductase and a preparation method thereof and a DNA encoding the imine reductase. The imine reductase has a high specific enzyme activity, high thermal stability and a large optimum pH range. When used to catalyze myosamine to prepare (S)-nornicotine, the conversion rate and enantiomeric excess percentage are high. CN116286700A discloses the use of an imine reductase mutant in the synthesis of nicotine key intermediate (S)-3-(pyrrolidin-2-yl)pyridine. The optional mutation point includes at least one of the following sites: the 246th position A or the 285th position D is mutated to V. The imine reductase mutant improves the yield of the catalytic reduction reaction and shortens the reaction time.

[0006] In summary, providing a new type of IREDs with excellent performance, improving substrate spectrum, stability and enzyme activity, etc., is of great significance for the production and application of chiral amines, and has become one of the problems to be solved urgently in this field. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides an imine reductase mutant and its application. In view of the problems of narrow substrate spectrum, poor stability and low enzyme activity of existing imine reductase, a new imine reductase mutant is developed to improve the substrate catalytic performance and expand the application prospects of imine reductase.

[0008] To achieve this object, the present invention adopts the following technical solutions:

[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 a combination of at least two of T18, I32, R62, Q107, F117, E119, G132, M138, V148, S167, N168, I180, R192, S228, K232, M238, S255, D273, A275 or L282;

[0011] (II) obtained by substituting, deleting or adding one or at least two amino acid residues from the amino acid sequence obtained in (I), and having the same or similar function as the amino acid sequence obtained in (I);

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

[0013] The invention improves the catalytic performance of substrates, broadens the substrate spectrum and expands the application prospect of imine reductase by directing evolution of imine reductase VpIR from Variovorax paradoxus of Burkholderiaceae.

[0014] In the present invention, a specific mutation is introduced into the wild-type imine reductase to obtain an imine reductase mutant, which can improve its catalytic activity and expand its substrate spectrum. It can be understood that based on the imine reductase mutant, those skilled in the art can use the general technical means in the art to replace, 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, and the mutation includes:

[0017] Any one of 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, or a combination of at least two of them.

[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) The combination of T18A, M138Y and K232R, (15) The combination of T18A, R62P and M138Y, (16) The combination of M138Y, K232R and S255R, (17) The combination of M138Y, S167C and K232R, (18) The combination of M138Y, E119D and K232R, (19) The combination of R62P, M138Y and K232R, (20) The combination of M138Y, E119D and S255R, (21) The combination of M138Y, E119D and S167C, (22) The combination of R62P, E119D and M138Y, (23) The combination of M138Y, S167D, S228R and K232R.

[0020] In the present invention, for ease of writing, the point mutation combination "the combination of T18A and M138Y" is written as "T18A+M138Y", and the subsequent combinations are written in the same way.

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

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

[0023] In a third aspect, the present invention provides a recombinant vector comprising the nucleic acid molecule described in the second aspect.

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

[0025] Preferably, the starting plasmid of the recombinant plasmid includes 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(+) , 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, 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] In a fourth aspect, the present invention provides a recombinant cell, wherein the recombinant cell contains the nucleic acid molecule described in the second aspect or the recombinant vector described in the third aspect.

[0027] Preferably, the starting cell of the recombinant cell comprises a eukaryotic cell or a prokaryotic cell.

[0028] Preferably, the eukaryotic cell comprises yeast.

[0029] Preferably, the prokaryotic cell comprises 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] In a fifth aspect, the present invention provides a method for preparing the imine reductase mutant as described in the first aspect, the preparation method comprising:

[0032] Inserting the nucleic acid molecule encoding the imine reductase mutant described in the first aspect into an expression vector to obtain a recombinant vector, introducing the recombinant vector into a host cell to obtain a recombinant cell, culturing and purifying the product to obtain the imine reductase mutant.

[0033] In the present invention, the method of inducing the culture of the recombinant cells and the method of isolating the imine reductase from the culture can adopt conventional methods in the art. The culture medium used when the recombinant cells express the imine reductase can be a culture medium in the art that can grow the recombinant cells and produce the imine reductase mutant of the present invention, such as LB culture medium.

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

[0035] It should be understood that the imine reductase mutant of the present invention can be used in the form of whole cells of engineered bacteria, in the form of crude enzymes without purification, or in the form of partially purified or completely purified enzymes. The imine reductase mutant of the present invention can also be prepared into a catalyst in the form of an immobilized enzyme or an immobilized cell using immobilization techniques known in the art.

[0036] In a seventh aspect, the present invention provides a method for producing a chiral amine, the method comprising using the imine reductase mutant 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, furyl, pyridyl or thienyl.

[0040] Preferably, the method comprises reducing 5-(2-tert-butylphenyl)-3,4-dihydro-2H-pyrrole to (S)-2-(2-tert-butylphenyl)pyrrolidine 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, and the coenzyme includes any one of NADP, NAD, NADPH or NADH, or a combination of at least two of them.

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

[0043] The invention uses imine as a substrate and obtains an imine reductase VpIR with high (S) stereoselectivity by screening an existing imine reductase library. The imine reductase VpIR is derived from Variovorax paradoxus. Saturation mutation and further combination mutation are performed on key sites around the active pocket of the structural model and on the protein surface and the subunit interface to obtain an imine reductase mutant with high catalytic efficiency and high selectivity, thereby broadening the substrate spectrum and expanding the application potential of the enzyme in industrial applications. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0045] Figure 2 This is the HPLC spectrum of (S)-2-(2-tert-butylphenyl)pyrrolidine standard.

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

[0047] Figure 4 This is the chiral HPLC spectrum of (S)-2-(2-tert-butylphenyl)pyrrolidine standard.

[0048] Figure 5 This is the chiral HPLC spectrum of the VpIR enzyme-catalyzed reaction solution in Example 1. DETAILED DESCRIPTION

[0049] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0050] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0051] The invention screens wild-type imine reductases from Variovorax paradoxus, Cupriavidus sp., Aeromonasveronii, Dadobacter endophyticus, and Luteolibacter luteus, and finds that the imine reductase from Variovorax paradoxus has relatively good stereoselectivity (S), but exhibits poor activity in the target reaction and low conversion rate. Therefore, the invention uses the amino acid sequence of Variovorax paradoxus shown in SEQ ID NO.1 as the starting gene of the invention, further uses directed evolution to transform, and obtains 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 an amino acid sequence in which the sequence shown in SEQ ID NO.1 is mutated, 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] Or the amino acid sequence of the imine reductase mutant has a mutated amino acid site, and has 90%, 95% or 99% or more homology with the sequence shown in SEQ ID NO.1, and has an amino acid sequence with imine reductase catalytic activity. The imine reductase mutant provided by the present invention can efficiently and stereoselectively synthesize (S)-2-arylpyrrolidine, and is suitable for industrial production.

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

[0055] The mutation is one of the following single site mutations or combined 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. The DNA molecule encodes any one of the above-mentioned imine reductase mutants. The above-mentioned imine reductase mutants encoded by the DNA molecule have higher catalytic activity and selectivity.

[0057] The above-mentioned DNA molecules of the present invention can also exist in the form of "expression cassettes". "Expression cassettes" refer to linear or circular nucleic acid molecules, covering DNA and RNA sequences that can direct the expression of specific nucleotide sequences in appropriate host cells. In general, it includes a promoter that is effectively connected to the target nucleotide, which is optionally effectively connected to a termination signal and / or other regulatory elements. The expression cassette can also include sequences required for the correct translation of the nucleotide sequence. The coding region usually encodes the target protein, but also encodes a target functional RNA in the sense or antisense direction, such as antisense RNA or non-translated RNA. The expression cassette 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. The expression cassette can also be naturally occurring, but obtained in an effective recombinant formation 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 above-mentioned DNA molecules. The DNA molecules in the recombinant plasmid are placed in the appropriate position of the recombinant plasmid so that the DNA molecules can be correctly and smoothly replicated, transcribed or expressed.

[0059] Although the qualifier used in the present invention to define the above-mentioned DNA molecules is "containing", it does not mean that other sequences unrelated to its function can be added at both ends of the DNA sequence at will. Those skilled in the art know that in order to meet the requirements of the recombination operation, it is necessary to add suitable restriction endonuclease cutting sites at both ends of the DNA sequence, or to add additional start codons, stop codons, etc. Therefore, if a closed expression is used to define it, it will not be able to truly cover these situations.

[0060] The term "plasmid" as used in the present invention includes any plasmid, cosmid, phage 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 expression plasmid or a eukaryotic expression plasmid, but preferably 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 cell is Escherichia coli DH5α, Top10, BL21 DE3 or Escherichia coli Rosetta DE3 cell; the eukaryotic cell is a yeast cell.

[0062] According to a typical embodiment of the present invention, a method for producing chiral amines is provided, which comprises the step of reducing an imine substrate using an imine reductase, wherein the imine reductase is any of the above-mentioned imine reductase mutants of the present 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, furyl, pyridyl or thienyl.

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

[0067] The beneficial effects of the present invention will be further described below in conjunction with embodiments.

[0068] Example 1

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

[0070]

[0071] Weigh 10.0 mg of imine reductase (VpIR, CsIR, AvIR, DeIR and LlIR) into the corresponding 5.0 mL centrifuge tubes, add 2.0 mg of GDH, 40.0 mg of glucose, 2.0 mg of NADP, and 10.0 mg of iodine reductase (VpIR, CsIR, AvIR, DeIR and LlIR) into the corresponding 5.0 mL centrifuge tubes, and ...+ , 10.0 g / L substrate 1a and 20.0 μL DMSO solution in 0.1 M pH 7.0 phosphate buffer, fixed to 1.0 mL. Then placed in a 30°C shaker for 24 h, and detected by ultra-high performance liquid chromatography (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 spectrum of substrate 5-(2-tert-butylphenyl)-3,4-dihydro-2H-pyrrole is as follows Figure 1 As shown, the HPLC spectrum of (S)-2-(2-tert-butylphenyl)pyrrolidine standard is as follows Figure 2 As shown, the HPLC spectrum of the VpIR enzyme catalytic reaction solution is as follows Figure 3 As shown, the chiral HPLC spectrum of (S)-2-(2-tert-butylphenyl)pyrrolidine standard is as follows Figure 4 As shown, the chiral HPLC spectrum of the VpIR enzyme catalyzed reaction solution is as follows Figure 5 The detection conversion rate (product and raw material peak area percentage) and ee value (R and S configuration percentage difference) are shown in Table 1.

[0073] Table 1

[0074] Enzymes source Conversion rate (%) ee(%) Vp Variovorax paradoxus 92.4 >99.0％(S) C Cupriavidus sp. 80.4％ >99.0％(S) AvI Aeromonas veronii 40.4％ 87.4％(S) DeIR Dyadobacter endophyticus 88.6％ 69.2％(R) LqCy Luteolibacter luteus 82.4％ 76.0％(S)

[0075] As can be seen from Table 1, VpIR, CsIR, AvIR and LlIR can obtain (S)-2-(2-tert-butylphenyl)pyrrolidine (1b), among which VpIR has the best catalytic efficiency, and DeIR has high catalytic efficiency but obtains the opposite configuration. However, when VpIR attempts to increase the substrate loading (such as 20.0 g / L or 30.0 g / L), the catalytic activity is greatly reduced, and it is judged that the catalytic activity for the substrate is insufficient. Therefore, the imine reductase derived from Variovorax paradoxus (having the amino acid sequence shown in SEQ ID NO.1) is used as the starting enzyme of the present invention, and directed evolution is carried out to improve the catalytic activity of the target enzyme.

[0076] Example 2

[0077] In this example, a mutant library of Variovorax paradoxus imine reductase was constructed. Non-conserved residue sites near the VpIR substrate binding pocket and 21 sites on the protein surface and subunit interface (T18, I32, R62, Q107, F117, E119, G132, M138, V148, S167, N168, I180, R192, S228, K232, M238, S255, E260, D273, A275 and L282) were selected for site-directed saturation mutagenesis, and the positive mutants obtained above were combined to improve the enzyme catalytic activity. A complete linear fragment was obtained by whole-plasmid PCR. The PCR product was digested with DpnⅠ to remove the maternal template of the starting gene, and then transformed into Escherichia coli BL21 (DE3), spread on an LB culture dish containing 50.0 μg / mL kanamycin, and cultured at 37°C overnight. After induction of expression in a 96-well plate, ultra-performance liquid chromatography high-throughput screening was performed to select mutants with higher activity than the maternal version, and the mutation site was determined by gene sequencing.

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

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

[0080] The specific operation process of saturated mutation library culture and feeding reaction is as follows: the obtained monoclonal colony is picked into 600.0μL LB in a 96-well deep-well plate for culture, shaken and cultured at 37°C until OD600 is 0.6, IPTG is added to a final concentration of 0.5mM, and induced expression is carried out at 25°C overnight. The next day, the 96-well plate is centrifuged to remove the supernatant culture medium, 100.0μL lysozyme solution (lysozyme 10.0mg / mL, pH=7.0) is added to each well, and the mixture is kept at 30°C for 2h. Then, the feeding amounts added to each well are 1.6mg of substrate 1a, 4.0μL DMSO, 8.0mg glucose, 0.4mg GDH, and 0.4mg NADP. + The volume was fixed to 200.0 μL with PBS buffer and reacted at 30°C for 24 h. The next day, 1.0 mL / well acetonitrile solution was added to terminate the reaction. The reaction was centrifuged at 4000 rpm for 15 min, and 1.0 mL of the supernatant was filtered and the conversion rate was detected by ultra-high performance liquid chromatography. The results are shown in Table 2.

[0081] Table 2

[0082]

[0083]

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

[0085] From the data in Table 2, it can be seen that when the imine substrate concentration is 20.0 g / L, the mutant enzymes of 11 sites, including T18A, I32V, R62P, Q107A, E119D, M138Y, S167C, S167D, S228R, K232R and S225R, have an activity of more than 2.0 times that of the VpIR wild type and maintain excellent stereoselectivity > 99.0%. However, when the concentration is increased to 30.0 g / L, the conversion rate of the wild type and its mutants drops to <80.0%, among which the conversion rate of M138Y can reach up to 77.7%. Therefore, the next round of mutation attempts to combine the above 10 sites with M138Y as a template, hoping to obtain positive mutants that tolerate higher substrate concentrations. The results of the combined mutation test are shown in Table 3.

[0086] Table 3

[0087]

[0088]

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

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

[0091] The M138Y+E119D combination with the highest conversion rate was selected and compared with the catalytic performance of the wild-type VpIR, and the results are shown in Table 4. The wild enzyme had a conversion rate of 92.4% after 24 hours at a substrate loading of 10.0 g / L, and the conversion rate was only 10.7% when the substrate loading increased to 30.0 g / L. When the loading of the mutant M138Y increased to 20.0 g / L, the conversion rate was >99.0% after 24 hours. It is worth noting that at a substrate loading of 30.0 g / L, M138Y+E119D can convert all substrates within 8 hours, which fully demonstrates that the catalytic performance of the mutant has been greatly improved compared to the parent.

[0092] Table 4

[0093] Enzyme number Substrate loading (g / L) Reaction time (h) Conversion rate (%) ee(%) Vp 10.0 24 92.4 >99.0(S) Vp 20.0 24 46.6 >99.0(S) Vp 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] In this example, (S)-2-arylpyrrolidine was prepared by enzymatic method on a 100-gram scale. 300.0 mg of substrate 1a and 2% v / v DMSO, 50.0 mg of NADP were added to two 50.0 mL four-necked bottles in sequence. + , 50.0 mg GDH, 2.0 g glucose, 0.1 M pH 7.0 phosphate buffer was diluted to 30.0 mL, and the reaction was started by stirring. The temperature was controlled at 30° C. in a constant temperature water bath, and the pH was controlled at 7.0 with a 3N sodium carbonate solution in a titrator. Finally, 150% w / w dry bacterial powder containing VpIR and M138Y+E119D mutant imine reductase was added to the three reaction systems respectively. Among them, the M138Y+E119D mutant imine reductase had Conv (%)>99.0% and ee>99.0% (S) detected after 8 hours, and Conv (%) 40.0% and ee>99.0% (R) detected by VpIR reaction after 24 hours, indicating that the enzyme activity of the imine reductase mutant constructed by the present invention was significantly improved, and the reaction time was effectively shortened.

[0096] Example 4

[0097] This example explores the substrate spectrum of mutant M138Y+E119D, and selects substrates 2a, 3a and 4a, whose structures are shown below.

[0098]

[0099] Weigh raw materials 2a, 3a and 4a and add 10.0 mg of dry bacterial powder containing mutation M138Y+E119D into 5.0 mL centrifuge tubes, add 2.0 mg GDH, 40.0 mg glucose, 2.0 mg NADP, etc. + and 20.0 μL DMSO were added to pH=7.0 PBS buffer solution and the volume was fixed to 1.0 mL. The mixture was placed in a shaker at 30°C for 24 h to synthesize 2b, 3b and 4b by enzymatic method. The samples were diluted with acetonitrile to detect the conversion rate and ee value. The test results are shown in Table 5.

[0100] Table 5

[0101]

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

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

[0104] In summary, the present invention screened wild-type imine reductases from Variovorax paradoxus, Cupriavidus sp., Aeromonas veronii, Dadobacter endophyticus, and Luteolibacter luteus, and found that the imine reductase VpIR activity of Variovorax paradoxus from the Burkholderiaceae family was relatively good. Therefore, the present invention uses Variovorax paradoxus having an amino acid sequence shown in SEQ ID NO.1 as the starting enzyme of the present invention, and further uses directed evolution to obtain a series of imine reductase mutants with high catalytic efficiency and selectivity and substrate spectrum.

[0105] The applicant declares that the above is only a specific implementation mode 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 thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope 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 any one of the following sequences: (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 a combination of at least two of T18, I32, R62, Q107, F117, E119, G132, M138, V148, S167, N168, I180, R192, S228, K232, M238, S255, D273, A275 or L282; (II) obtained by substituting, deleting or adding one or at least two amino acid residues from the amino acid sequence obtained in (I), and having the same or similar function as the amino acid sequence obtained in (I); (III) has at least 90% sequence homology with the amino acid sequence obtained from (I) or (II), and has the same or similar functions as the amino acid sequence obtained from (I).

2. The imine reductase mutant according to claim 1, characterized in that 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, and the mutation includes: Any one of 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, or a combination of at least two thereof; Preferably, the combination of mutations includes any one of the following: (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) The combination of T18A, M138Y and K232R, (15) The combination of T18A, R62P and M138Y, (16) The combination of M138Y, K232R and S255R, (17) The combination of M138Y, S167C and K232R, (18) The combination of M138Y, E119D and K232R, (19) The combination of R62P, M138Y and K232R, (20) The combination of M138Y, E119D and S255R, (21) The combination of M138Y, E119D and S167C, (22) The combination of R62P, E119D and M138Y, (23) The combination of M138Y, S167D, S228R and K232R.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the imine reductase mutant according to claim 1 or 2.

4. A recombinant vector, characterized in that: The recombinant vector contains the nucleic acid molecule according to claim 3.

5. The recombinant vector according to claim 4, characterized in that The recombinant vector comprises a recombinant plasmid; Preferably, the starting plasmid of the recombinant plasmid includes 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(+) , 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, 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 cell contains the nucleic acid molecule according to claim 3 or the recombinant vector according to claim 4 or 5.

7. The recombinant cell according to claim 6, characterized in that The starting cell of the recombinant cell includes a eukaryotic cell or a prokaryotic cell; Preferably, the eukaryotic cell comprises yeast; Preferably, the prokaryotic cell comprises Escherichia coli; Preferably, the Escherichia coli includes any one of Escherichia coli DH5α, Escherichia coli Top10, Escherichia coli BL21-DE3 or Escherichia coli Rosetta-DE3.

8. Use of the imine reductase mutant according to claim 1 or 2, the nucleic acid molecule according to claim 3, the recombinant vector according to claim 4 or 5, or the recombinant cell according to claim 6 or 7 in producing chiral amines.

9. A method for producing a chiral amine, characterized in that: The method comprises using the imine reductase mutant according to claim 1 or 2 to catalyze a reduction reaction of an imine substrate; The structural formula of the imine substrate is shown in Formula I; 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, furyl, pyridyl or thienyl.

10. The method for producing chiral amines according to claim 9, characterized in that The method comprises reducing 5-(2-tert-butylphenyl)-3,4-dihydro-2H-pyrrole to (S)-2-(2-tert-butylphenyl)pyrrolidine using the imine reductase mutant of claim 1 or 2; Preferably, the reaction system of the reduction reaction further includes a coenzyme or a coenzyme regeneration system, and the coenzyme includes any one of NADP, NAD, NADPH or NADH, or a combination of at least two of them.

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