An imine reductase mutant, its preparation method and uses

Abstract

The application discloses an imine reductase mutant, a preparation method and application thereof, and belongs to the field of synthesis and application of chiral amine catalyzed by biological enzymes. A series of advantageous mutants are obtained by a method of directed evolution on the basis of imine reductase ThIRED from Torrubiella hemipterigena. The imine reductase advantageous mutant obtained by the application has high catalytic activity, high selectivity and wide substrate applicability, and has wide application prospects in industrialized production of high-activity imine reductase.

Description

Technical Field

[0001] This invention belongs to the field of bioenzymatic synthesis of chiral amines and their applications, specifically relating to an imine reductase mutant, its preparation method, and its uses. Background Technology

[0002] Reductive amination is an important reaction in amine synthesis. In the presence of a reducing agent, alkylated chiral amines are directly synthesized from prochiral ketones or aldehydes with amines. Chiral amines are widely found in the functional groups of various natural products and active pharmaceutical ingredients (APIs). Therefore, the asymmetric synthesis of amines is extremely important in industrial production within synthetic chemistry. Currently, the synthesis methods for chiral amines include chemical methods and enzyme-catalyzed synthesis. Chemical methods include chiral ligand-assisted methods, CN-bond formation reactions (hydrogenation amination, carbene insertion into NH bonds, nitrile insertion into CH bonds), hydrogenation of imines / enamines and nucleophilic addition of imines, as well as transition metal-catalyzed synthesis methods.

[0003] Compared to chemical synthesis methods, enzymatic catalysis offers advantages such as simplicity, speed, and low cost, enabling green and pollution-free processes. Imine reductases (IREDs) catalyze the reductive amination of ketones or aldehydes with amines, forming nitrogen-containing chiral compounds. Reported imine reductases possess a defined substrate spectrum, catalyzing the formation of various chiral compounds and exhibiting high enantioselectivity in the synthesis of unprotected chiral amines. In recent years, groundbreaking research on imine reductases has been achieved both domestically and internationally, highlighting their crucial role in chiral amine synthesis. For instance, the development and modification of imine reductases (IREDs) and reductive aminases (RedAMs) have garnered significant attention and application for enzymatic synthesis of chiral amines. Currently, several drug synthesis routes involve the use of imine reductase as a catalyst to form chiral amines, such as hypnotics (suvorexant), antidiabetic drugs (sitagliptin), antidepressants (setratline, escitalopram), anesthetics (levobupivacaine), dementia treatments (rivastigmine), LSD1 inhibitor GSK2879552, JAK1 inhibitor abrocitinib, and key intermediates for leniolisib and JAK1 inhibitors.

[0004] Natural imine reductases are hampered by low catalytic efficiency, low chiral selectivity, and poor stability, hindering their widespread industrial application. Therefore, modifying imine reductases to obtain mutants with high catalytic activity, high stability, and high specificity is of great value in making them suitable for industrial applications. Summary of the Invention

[0005] In order to solve the above-mentioned problems in the prior art, the purpose of this invention is to provide an imine reductase mutant, its preparation method and uses.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This invention provides an imine reductase mutant, which is obtained by mutation based on wild-type imine reductase. The mutation site is at least one of the following sites: position 127, position 128, position 134, position 135, position 140, position 187, position 191, position 223, position 224, position 226, position 227, position 230, position 249, position 253, position 254, position 256, and position 260.

[0008] Furthermore, the amino acid sequence of the wild-type imine reductase is shown in SEQ ID NO:2, and the DNA sequence encoding the wild-type imine reductase is shown in SEQ ID NO:1.

[0009] Further, the mutation includes at least one of the following mutation methods: replacing the 127th amino acid residue with A or S; replacing the 128th amino acid residue with G or K; replacing the 134th amino acid residue with D, L, N, P, S, or Y; replacing the 135th amino acid residue with A or V; replacing the 140th amino acid residue with N, S, or T; replacing the 187th amino acid residue with A or L; replacing the 191st amino acid residue with I, V, T, S, H, M, or L; replacing the 223rd amino acid residue with A; replacing the 224th amino acid residue with A or V; Replace the 226th amino acid residue with D or E; Replace the 227th amino acid residue with E, K, P, V, M or F; Replace the 230th amino acid residue with C, E, F, H, K, L, M, N, Q, T or V; Replace the 249th amino acid residue with S; Replace the 253rd amino acid residue with E, N, P, Q or V; Replace the 254th amino acid residue with E, K, N or S; Replace the 256th amino acid residue with H, C, F, I, L, N, Q, K, T, V or Y; Replace the 260th amino acid residue with K, L, M or V.

[0010] Further, the mutation includes at least one of the following single point mutations or combinations of mutations: G127A, Q134D, Q134L, Q134N, Q134S, Q134Y, M135V, M135A, M187L, M187A, F191I, F191L, F191H, F191T, F191M, F191V, V223A, M224A, M224V, L227M, L227F, L227E, L227K, L227P, L227V, Y230C, Y230E, Y230F, Y230H, Y230K, Y230L, Y230M, Y 230N, Y230Q, Y230T, Y230V, N249S, M253E, M253N, M253P, M253Q, M253V, Q254E, Q254K, Q254N, Q254S, A256C, A256F, A256H, A256I, A256K , A256L, A256N, A256Q, A256T, A256V, A256Y, N260K, N260L, N260M, N260V, M187L+F191V, N249S+M187L, N249S+M187L+F191I, N249S+M18 7L+F191H, N249S+M187L+F191L, N249S+M187L+F191V, N249S+Y230H, N249S+A226E+Q140N, N249S+A226D+Q140N, N249S+A226E+Q140S, N249S+A226D+Q140S, N249S+A226E+Q140T, N249S+A226D+Q140T, N249S+M187L+F191S, N249S+F191L, N249S+M187L+F191M, N249S+F191 V、N249S+F191T、N249S+Y230E、N249S+L227M+Y230Q、N249S+M187L+F191V+Y230E、N249S+M187L+F191L+Y230E、N249S+M187L+F191L+Y2 30H, N249S+M187L+F191L+L227M+Y230Q, N249S+M187L+F191I+Y230H, N249S+M187L+F191I+Y230E, N249S+M187L+F191I+L227M+Y230Q,

[0011] N249S+M187L+F191V+L227M+Y230Q, N249S+M187L+F191I+A256H, N249S+M187L+F191I+N260K, N249S+M187L+F191I+Q134P, N249S+M187L+F191I+Q134 D, N249S+M187L+F191I+N260K+Q134P, N249S+M187L+F191I+N260K+Q134D, N249S+M187L+F191I+A256H+Q134P, N249S+M187L+F191I+A256H+Q134D, N24 9S+M187L+F191I+N260K+G127A、N249S+M187L+F191I+N260K+G127S、N249S +M187L+F191I+N260K+A128G、N249S+M187L+F191I+N260K+A128K、N249S+M 187L+F191I+N260K+M253V+Q254S, N249S+M187L+F191I+A256K, N249S+M187L+F191I+A256T, N249S+M187L+F191I+A256V, N249S+M187L+F191I+L227M N249S+M187L+F191L+A256I, N249S+M187L+F191L+A256V, N249S+M187L+F191L+A256Y, N249S+M187L+F191L+L227M, N249S+M187L+F191V+A256I, N249 S+M187L+F191V+A256K、N249S+M187L+F191V+A256T、N249S+M187L+F191V+ A256V、N249S+M187L+F191V+A256Y、N249S+M187L+F191V+L227M、N249S+M18 7L+F191I+A256I, N249S+M187L+F191I+A256Y, N249S+M187L+F191L+A256K, N249S+M187L+F191L+A256T, N249S+M187L+F191L+L227F, N249S+M187L+F 191V+L227F, N249S+M187L+F191I+L227F+A256T, N249S+M187L+F191V+L227F+N260K, N249S+M187L+F191V+L227M+N260K, N249S+M187L+F191I+L227FN249S+M187L+F191I+L227M+A256T. ,

[0012] In the combined mutation code of this invention, "+" indicates that mutations were performed before and after "+".

[0013] Furthermore, the mutant amino acid sequence of the imine reductase has 90%, 95%, 96%, 97%, 98% or more homology with SEQ ID NO:2.

[0014] Furthermore, the amino acid sequence of the imine reductase mutant is shown in SEQ ID NO:4, and the DNA sequence encoding the imine reductase mutant is shown in SEQ ID NO:3.

[0015] The present invention also provides a gene encoding the above-mentioned imine reductase mutant.

[0016] The present invention also provides a vector containing the above-mentioned genes.

[0017] Furthermore, the vector is selected from plasmids, artificial chromosomes, bacteriophages, or viral vectors.

[0018] Furthermore, the plasmid is pET-28a plasmid.

[0019] The present invention also provides a recombinant expression system comprising the above-mentioned genes or vectors.

[0020] Furthermore, the recombinant expression system is recombinant Escherichia coli.

[0021] The present invention also provides a method for preparing the above-mentioned imine reductase mutant, the method comprising the following steps:

[0022] (1) Construct a vector containing the gene encoding the above-mentioned imine reductase mutant;

[0023] (2) The vector was transferred into the host bacteria, cultured, and an inducer was added to induce the expression of the imine reductase mutant.

[0024] This invention also provides the application of the above-mentioned imine reductase mutant in the bio-enzymatic production of chiral amines.

[0025] The present invention also provides a method for preparing chiral amines, wherein the method comprises the above-mentioned imine reductase mutant, substrate ketone, amine donor and coenzyme regeneration system, thereby obtaining chiral amines.

[0026] Furthermore, the substrate ketone is a and b are each independently selected from integers from 1 to 10; the amine donor is R1-NH2, where R1 is... c, d, and e are each independently selected from integers from 1 to 10.

[0027] Furthermore, the substrate ketone is The amine donor is The coenzyme is NADPH, and the coenzyme regeneration system is D-glucose and NADP. + And glucose dehydrogenase.

[0028] Further, the molar ratio of the imine reductase mutant, substrate ketone, amine donor, and D-glucose is (1–10) mg:(0.01–0.2 mmol):(0.01–0.3 mmol):(0.02–0.4 mmol), and the NADP... + The concentration of glucose dehydrogenase is 0.1–5 mmol / mL, the concentration of glucose dehydrogenase is 0.1–5 mg / mL, the pH of the reaction is 7.1–10.0, the temperature of the reaction is 20–50 °C, and the reaction time is 10–36 h.

[0029] Further, the molar ratio of the imine reductase mutant, substrate ketone, amine donor, and D-glucose is (2.5–5) mg:(0.02–0.1 mmol):(0.03–0.15 mmol):(0.04–0.2 mmol), and the NADP... + The concentration of glucose dehydrogenase is 1 mmol / mL, the concentration of glucose dehydrogenase is 1 mg / mL, the pH of the reaction is 8.0, the temperature of the reaction is 30°C, and the reaction time is 18–24 h.

[0030] Furthermore, the reaction, after completion, includes the following steps: the completeness of the overnight reaction solution is detected by HPLC. The completely reacted reaction solution is further processed. The processing steps are as follows: the reaction solution is quenched with acetic acid at room temperature, and the pH is adjusted to 3.0; ethyl acetate is added and stirred for 20-30 minutes; diatomaceous earth is added and stirred for 20-30 minutes; the solution is filtered and the filtrate is collected; the aqueous phase is separated and collected; the aqueous phase is alkalized with NaOH at room temperature to pH 10.0; the aqueous phase is extracted with ethyl acetate (X2); the organic phases are combined, washed with 10% NaCl solution and separated; the solvent of the combined organic extracts is removed under reduced pressure.

[0031] The present invention has achieved the following beneficial effects:

[0032] This invention, based on the imine reductase ThIRED from Torrubiella hemipterigena, obtained a series of advantageous mutants through directed evolution. The advantageous imine reductase mutants obtained in this invention possess high catalytic activity, high selectivity, and broad substrate applicability, showing great promise for industrial production of highly active imine reductases.

[0033] The substrate ketone used in the embodiments of this invention:

[0034] Substrate S1 is named N-tert-butoxycarbonyl-3-pyrrolidinone in Chinese and English, and its structural formula is as follows:

[0035]

[0036] Substrate S2 has the Chinese name N-BOC-3-aza-heptan-1-one and the English name N-BOC-3-aza-heptan-1-one, with the following structural formula:

[0037]

[0038] Amine donors used in this invention:

[0039] Amine donor A1, also known as n-butylamine, has the following structural formula:

[0040] The amine donor A2 is 1-aminopentane, and its English name is Amylamine. Its structural formula is:

[0041] Amine donor A3, whose Chinese name is cyclobutylamine and English name is Cyclobutylamine, has the following structural formula:

[0042] Amine donor A4, also known as benzylamine, has the following structural formula:

[0043] Amine donor A5, also known as cyclopropylamine, has the following structural formula:

[0044] In the mutation code of this invention, the numbers are the amino acid site numbers of the mutation, the letters before the numbers represent the amino acid before the mutation, and the letters after the numbers represent the amino acid after the mutation. For example, N249S means that the 249th amino acid is mutated from N to S, and so on.

[0045] In the mutation code of this invention, the letters before and after the numbers are amino acid abbreviations known in the art. For example, V represents Valine, A represents Alanine, M represents Methionine, L represents Leucine, Q represents Glutamine, C represents Cysteine, K represents Lysine, T represents Threonine, I represents Isoleucine, S represents Serine, H represents Histidine, E represents Glutamic acid, P represents Proline, F represents Phenylalanine, D represents Aspartic acid, Y represents Tyrosine, G represents Glycine, and N represents Asparagine.

[0046] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0047] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0048] Figure 1 This is the chiral HPLC chromatogram of the chiral S1-A1 reduced amination product catalyzed by IRED-M1 in this invention.

[0049] Figure 2 This is the chiral HPLC chromatogram of the chiral S1-A2, the reduced amination product catalyzed by IRED-M1 in this invention.

[0050] Figure 3 This is the chiral HPLC chromatogram of the chiral S1-A3, the reduced amination product catalyzed by IRED-M1 in this invention.

[0051] Figure 4 This is the chiral HPLC chromatogram of the chiral S1-A4, the reduced amination product catalyzed by IRED-M1 in this invention.

[0052] Figure 5 This is the chiral HPLC chromatogram of the chiral S1-A5, the reduced amination product catalyzed by IRED-M1 in this invention.

[0053] Figure 6 This is the chiral HPLC chromatogram of the chiral S2-A3, the reduced amination product catalyzed by IRED-M1 in this invention.

[0054] Figure 7 This is the chiral HPLC chromatogram of the chiral S2-A4, the reduced amination product catalyzed by IRED-M1 in this invention.

[0055] Figure 8 This is the chiral HPLC chromatogram of the chiral S2-A5, the reduced amination product catalyzed by IRED-M1 in this invention. Detailed Implementation

[0056] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0057] The ThIRED involved in this invention was obtained from the NCBI database using genome mining technology. The gene is from Torrubiella hemipterigena (ThIRED, GenBank: CEJ93364.1).

[0058] Example 1: Site-directed mutagenesis of imine reductase ThIRED based on sequence conservation and its catalytic activity

[0059] Through sequence conservation analysis, the conserved amino acid position 249 of the ThIRED amino acid sequence (as shown in SEQ ID NO:2) was first targeted for modification. Furthermore, N249 was mutated to three conserved amino acids S, F, and A at homologous sites, respectively, constructing three ThIRED mutants: N249S, N249F, and N249A.

[0060] The specific method for recombinant protein expression is as follows: The ThIRED wild-type or mutant gene was constructed into the *E. coli* expression vector pET-28a plasmid. The pET-8a plasmid carrying the ThIRED wild-type or mutant gene was transformed into *E. coli* BL21(DE3) competent cells for recombinant protein expression. Single colonies were picked from agar plates and placed in 5 mL of liquid LB medium containing 50 μg / mL kanamycin, and cultured overnight at 37°C and 220 rpm with shaking. The overnight culture seed culture was inoculated at a 1:100 ratio into 400 mL of TB medium containing 50 μg / mL kanamycin. The culture was carried out at 37°C and 220 rpm for 2-3 hours until the OD600 value reached 0.6-0.8. Then, 0.2 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) was added to induce recombinant protein expression. After culturing with shaking at 24°C and 220 rpm for 16 hours, the bacterial cells were collected by centrifugation at 8,500 rpm for 20 minutes and resuspended in sodium phosphate buffer (100 mM, pH 7.0). The cells were then collected again by centrifugation at 4,000 rpm for 30 minutes. The resuspended and washed cells were resuspended in sodium phosphate buffer (100 mM, pH 7.0) and sonicated. The cell lysate was centrifuged at 12,000 rpm for 30 minutes. The supernatant was then placed in a freeze dryer and freeze-dried until a lyophilized powder of the ThIRED wild-type or mutant recombinant protein was obtained.

[0061] The catalytic activity of three ThIRED mutants, N249S, N249F, and N249A, was screened in a combination of ketone substrate S1 and amine donor A5. The 1 mL reaction mixture contained 2.5 mg / mL of the imine reductase mutant lyophilized powder, 100 mM ketone substrate S1, 150 mM amine donor A5, and 1 mM NADP. + The reaction mixture consisted of D-glucose (2.0 equivalents, the amount of D-glucose being twice that of the ketone), NADPH regeneration system enzyme powder (1 mg / mL), sodium phosphate buffer (200 mM, pH 8.0), and 20% DMSO (v / v, DMSO volume to total reaction volume). The conversion rate of substrate S1 under ThIRED wild-type catalysis was 10%, and the conversion rate under the N249S mutant catalysis was 75%. In the combination of ketone substrate S1 and amine donor A3, 1 mL of the combined reaction system contained 2.5 mg / mL imine reductase mutant lyophilized powder, 50 mM ketone substrate S1, 75 mM amine donor A3, and 1 mM NADP. +The reaction mixture contained D-glucose (2.0 equivalents, the amount of D-glucose being twice that of the ketone), NADPH regeneration system enzyme powder (1 mg / mL), sodium phosphate buffer (200 mM, pH 8.0), and 20% DMSO (v / v, DMSO volume to total reaction volume). Under ThIRED wild-type catalysis, the conversion rate of substrate S1 and amine donor A3 was 22.8%. Under mutant N249S catalysis, the conversion rate of substrate S1 and amine donor A3 was 41.1%. Under mutant N249A catalysis, the conversion rate of substrate S1 and amine donor A5 was 68%, while the conversion rate of substrate S1 and amine donor A3 was only 19%. N249F lost its catalytic activity. The conversion rate was calculated according to Example 3.

[0062] This demonstrates that the catalytic activity and enantioselectivity of the N249S ThIRED mutant of this invention are significantly superior to those of the ThIRED wild type.

[0063] Example 2: Site-directed saturation mutagenesis of imine reductase ThIRED and its catalytic activity

[0064] To further enhance the catalytic activity of ThIRED, saturation mutations were performed on amino acids G127, A128, Q134, M135, M187, F191, L227, Y230, M253, Q254, A256, and N260 located in the substrate binding pocket. The specific method for expressing the ThIRED mutant recombinant protein was as described in Example 1, and the catalytic activity of the ThIRED mutant was screened using the ketone substrate S1 and the amine donor A5.

[0065] Based on the wild type as a template, saturated mutant libraries were constructed at single sites, and catalytic activity was screened using 96-well deep-well plates. From 3000 mutants, several superior mutants compared to the wild type were obtained after preliminary screening, as follows: G127A, Q134D, Q134G, Q134L, Q134N, Q134S, Q134Y, M135V, M135A, M187L, M187A, F191I, F191L, F191H, F191T, F191M, F191V, V223A, M224A, M224V, L227M, L227F, L227E, L227K, L... 227P, L227V, Y230C, Y230E, Y230F, Y230H, Y230K, Y230L, Y230M, Y230N, Y230Q, Y230T, Y230V, N249S, M253E, M253N, M253P, M253Q, M253V, Q254E, Q254K, Q254N, Q254S, A256C, A256F, A256H, A256I, A256K, A256L, A256N, A256Q, A256T, A256V, A256Y, N260K, N260L, N260M, N260V.

[0066] The thIRED unit point mutants of imine reductase screened in this embodiment all showed significantly better catalytic activity against the substrate than the wild type.

[0067] In this embodiment, the catalytic reaction of substrate S1 with different amine donors A3 and A5 was set up to evaluate the activity of the mutant. Specifically, a 1 mL reaction system of substrate S1 and amine donor A3 contained 50 mM S1, 75 mM A3, and 1 mM NADP. + The reaction mixture consisted of ThIRED mutant lyophilized powder (2.5 mg / mL), D-glucose (2.0 equivalents, the amount of D-glucose being twice that of ketone), NADPH regeneration system enzyme powder (1 mg / mL), sodium phosphate buffer (200 mM, pH 8.0), and 20% DMSO (v / v, the volume of DMSO relative to the total reaction volume); a 1 mL reaction system of substrate S1 and amine donor A5 contained 100 mM S1, 150 mM A5, and 1 mM NADP. + The following were prepared: ThIRED mutant lyophilized powder (2.5 mg / mL), D-glucose (2.0 equivalents, the amount of D-glucose being twice the amount of ketone), NADPH regeneration system enzyme powder (1 mg / mL), sodium phosphate buffer (200 mM, pH 8.0), and 20% DMSO (v / v, the volume of DMSO being the total reaction volume). The mixture was placed in a magnetic stirrer (750 rpm, 30 °C) and reacted for 18 h.

[0068] The conversion rate described in this embodiment was calculated as follows: an equal volume of acetonitrile was added to the reaction system to terminate the reaction. After centrifugation, the supernatant was collected, diluted to a certain concentration, and the product conversion rate was detected by HPLC. The conversion rate was calculated based on the product standard curve.

[0069] Table 1 Catalytic activity of dominant ThIRED imine reductase mutants

[0070]

[0071]

[0072] The results are shown in Table 1. Under the catalysis of the aforementioned point mutants, the substrate conversion rate (S1-A5 combination) can be increased by up to 75% compared to 10% for the wild type. The conversion rate was calculated according to Example 3.

[0073] The imine reductase ThIRED unit point mutants screened in this embodiment showed significantly better catalytic activity against the substrate than the wild type.

[0074] Example 3: ThIRED multi-site combined dominant mutants of imine reductase and their catalytic activity

[0075] The specific method for expressing ThIRED mutant recombinant proteins is as described in Example 1. The catalytic activity of ThIRED mutants at multiple sites was screened to obtain several superior mutants that are better than the wild type.

[0076] The specific method for determining the activity of the ThIRED imine reductase mutant in this embodiment is as follows:

[0077] In this embodiment, the catalytic reaction of substrate S1 with different amine donors A3 and A5 was set up. Specifically, a 1 mL reaction system of substrate S1 and amine donor A3 contained 50 mM S1, 75 mM A3, and 1 mM NADP. + The reaction mixture consisted of ThIRED mutant lyophilized powder (2.5 mg / mL), D-glucose (2.0 equivalents, the amount of D-glucose being twice that of ketone), NADPH regeneration system enzyme powder (1 mg / mL), sodium phosphate buffer (200 mM, pH 8.0), and 20% DMSO (v / v, the volume of DMSO relative to the total reaction volume); a 1 mL reaction system of substrate S1 and amine donor A5 contained 100 mM S1, 150 mM A5, and 1 mM NADP. +The following were prepared: ThIRED mutant lyophilized powder (2.5 mg / mL), D-glucose (2.0 equivalents, the amount of D-glucose being twice the amount of ketone), NADPH regeneration system enzyme powder (1 mg / mL), sodium phosphate buffer (200 mM, pH 8.0), and 20% DMSO (v / v, the volume of DMSO being the total reaction volume). The mixture was placed in a magnetic stirrer (750 rpm, 30 °C) and reacted for 18 h.

[0078] The conversion rate described in this embodiment was calculated as follows: an equal volume of acetonitrile was added to the reaction system to terminate the reaction. After centrifugation, the supernatant was collected, diluted to a certain concentration, and the product conversion rate was detected by HPLC. The conversion rate was calculated based on the product standard curve.

[0079] Table 2. Catalytic activity of ThIRED multisite combination mutants with superior imine reductase activity

[0080]

[0081]

[0082] The results are shown in Table 2. In the combined reaction of ketone substrate S1 and amine donor A5, the substrate conversion rate under mutant catalysis increased from 10% in the wild type to over 90%; in the combined reaction of ketone substrate S1 and amine donor A3, the substrate conversion rate under mutant catalysis increased from 22.8% in the wild type to over 80%. These superior mutants all contain the key N249, G127, A128, Q134, M187, F191, L227, Y230, A256, and N260 sites.

[0083] The ThIRED dominant mutants of imine reductase, which included key sites N249, G127, A128, Q134, M187, F191, L227, Y230, A256, and N260 in this embodiment, showed significantly better catalytic activity against the substrate than the wild type.

[0084] Example 4: Enantioselectivity of the dominant ThIRED mutant of imine reductase

[0085] For several dominant mutants in Example 3, the enantioselectivity of the mutants in the generated products was determined by reacting with substrate S1 and amine donor A3 and reacting with substrate S1 and amine donor A5, respectively.

[0086] Catalytic reaction setup:

[0087] 1 mL of the reaction system contains 100 mM S1, 150 mM A5 (or 50 mM S1, 75 mM A3), and 1 mM NADP. +ThIRED mutant lyophilized powder (2.5 mg / mL), D-glucose (2.0 equivalents, the amount of D-glucose is twice that of ketone), NADPH regeneration system enzyme powder (1 mg / mL), sodium phosphate buffer (200 mM, pH 8.0) and 20% DMSO (v / v, the volume of DMSO is the same as the total reaction volume). The mixture was placed in a magnetic stirrer (750 rpm, 30 °C) and reacted for 18 h.

[0088] Product extraction:

[0089] The reaction solution after overnight reaction (18 h) was analyzed for reaction completeness by HPLC. The completely reacted solution underwent the following treatment steps: the reaction was quenched with acetic acid at room temperature, and the pH was adjusted to 5-6; an equal volume of ethyl acetate was added for extraction, and the organic phase was collected by centrifugation. The aqueous phase was adjusted to pH 9-10 with an appropriate amount of NaOH, and then an equal volume of ethyl acetate was added for extraction. The organic phases were combined, concentrated, and redissolved in methanol to determine chirality. The analytical column was a Daicel chiralpak IG3. The ee value was calculated as: ee = ([R] / [S]) / ([R]+[S])*100%. [R] is the concentration of the R enantiomer, and [S] is the concentration of the S enantiomer.

[0090] Table 3 Enantioselectivity of dominant ThIRED imine reductase mutants

[0091]

[0092]

[0093] The results are shown in Table 3: the ee value of the catalytic product obtained by the dominant ThIRED imine reductase mutant with substrate S1 and amine donor A5 can reach 99.9%, which is better than the wild type's ee value of 99%; the ee value of the catalytic product obtained by the reaction combination of substrate S1 and amine donor A3 can reach up to 99.3%, which is better than the wild type's ee value of 25.3%. This demonstrates that the enantioselectivity of the catalytic product obtained by the dominant ThIRED imine reductase mutant is better than that of the wild type.

[0094] Example 5: Application of the imine reductase mutant IRED / M1 in the synthesis of chiral amines

[0095] Catalytic reactions were carried out using the dominant mutant M1 with different combinations of substrates (S1 and S2) and amine donors (A1, A2, A3, A4, A5). The mutant used in this example was M1, which contained mutations at N249, M187, F191, and N260 sites. Specifically, N249 was mutated to S, M187 to L, F191 to I, and N260 to K (mutants capable of catalyzing such reactions are not limited to M1; multiple mutants listed in Tables 2 and 3 can catalyze the reactions in this example).

[0096] The dominant mutant M1 has the amino acid sequence shown in SEQ ID NO:4. The DNA sequence encoding the dominant mutant M1 is shown in SEQ ID NO:3.

[0097] Catalytic reaction steps: Add the substrate ketone and the corresponding amine donor as shown in Table 4 to the reaction system, 1 mM NaDP + The mixture consisted of M1 mutant lyophilized powder (5 mg / mL), D-glucose (2.0 equivalents, the amount of D-glucose being twice that of ketone), NADPH regeneration system enzyme powder (1 mg / mL), sodium phosphate buffer (200 mM, pH 8.0), and 20% DMSO (v / v, the volume of DMSO being the total reaction volume). The mixture was reacted in a 30°C water bath at 270 rpm.

[0098] Product extraction steps: The reaction solution after overnight reaction (18 h) was analyzed for reaction completeness by HPLC. The reaction solution after complete reaction was processed as follows: the reaction was quenched with acetic acid at room temperature and the pH was adjusted to 3.0; ethyl acetate was added and stirred for 20-30 minutes; diatomaceous earth was added and stirred for 20-30 minutes; the mixture was filtered and the filtrate was collected; the aqueous phase was separated and collected; the aqueous phase was alkalized with NaOH to pH 10.0 at room temperature; the aqueous phase was extracted with ethyl acetate (X2); the organic phases were combined, washed with 10% NaCl solution and separated; the solvent of the combined organic extracts was removed under reduced pressure.

[0099] The conversion rate described in this embodiment is calculated using the following method:

[0100] Take 100 μL of the sample from the reaction system, add an equal proportion of 100 μL of acetonitrile to terminate the reaction, centrifuge, collect the supernatant, dilute to a certain concentration, and use HPLC to determine the configuration, ee value, and conversion rate of the product. Analytical methods: Analytical columns: Daicelchiralpak AD-H, Daicel chiralpak IG, Lux Cellulose-2. ee value calculation: ee = ([R] / [S]) / ([R]+[S])*100%. Conversion rate is calculated based on product formation. Separation yield: actual compound mass / theoretical compound mass*100%.

[0101] The specific substrates, their concentrations, and experimental results of this embodiment are shown in Table 4.

[0102] Table 4. Catalytic effects of the imine reductase mutant IRED / M1 on different substrates ketones and amines.

[0103] reaction Substrate ketone concentration (mM) Amine concentration (mM) Volume (mL) Amount of product (g) ee(R) Conversion rate yield 1(S1-Al) 100 150 100 1.6 ＞99.9％ ＞98％ 93％ 2(S1-A2) 100 150 100 2.3 ＞99.9％ ＞98％ 83％ 3(S1-A3) 100 100 100 2 ＞99.9％ ＞98％ 84％ 4(S1-A4) 100 150 50 1.3 ＞99.9％ ＞98％ 97％ 5(S1-A5) 100 150 100 1.86 ＞99.9％ ＞98％ 83％ 6(S2-A3) 20 30 100 1.2 >99.9％ >98％ 86％ 7(S2-A4) 20 30 150 0.82 ＞99.9％ ＞98％ 89％ 8(S2-A5) 100 150 100 2.039 99％ ＞98％ 81％

[0104] As shown in Table 4, the conversion rates of R-configured nitrogen-containing heterocyclic alkylamines produced by the imine reductase mutant IRED / M1 from different substrate ketones and amines were all above 98%, with ee values ​​greater than 99%, and separation yields all above 80%, with the highest separation yield reaching 97%. This demonstrates that the imine reductase mutant IRED / M1 of this invention has excellent application prospects in the synthesis of chiral amines.

[0105] Products 1-8 are characterized as follows:

[0106] Product 1 (S1-A1)

[0107]

[0108] A dark brown oily liquid was obtained from 1-tert-butoxycarbonyl-3 / pyrrolidone and n-butylamine, which is product 1.

[0109] 1H NMR(400MHz,Chloroform / d)δ3.63–3.40(m,2H),3.39–3.25(m,2H),3.19–2.97(m,1H),2.70–2.53(m,2H),2. 11–1.99(m,1H),1.78–1.59(m,2H),1.53–1.48(m,2H),1.46(s,9H),1.40–1.28(m,2H),0.92(t,J=7.3Hz,3H).

[0110] 13C NMR (100MHz, Chloroform / d) δ154.7,79.2,57.9,57.1,52.0,51.5,48.0,44.5,44.1,32.4,32.1,31.4,28.5,20.5,14.0.

[0111] The HPLC chromatogram of product 1 is shown in [reference needed]. Figure 1 .

[0112] Product 2 (S1-A2)

[0113]

[0114] A brown oily liquid, product 2, was obtained from 1-tert-butoxycarbonyl-3 / pyrrolidone and 1-aminopentane.

[0115] 1H NMR(400MHz,Chloroform / d)δ3.64–3.39(m,2H),3.39–3.24(m,2H),3.15–2.99(m,1H),2.66–2.54(m,2H),2. 12–1.99(m,1H),1.76–1.63(m,1H),1.53–1.47(m,2H),1.46(s,9H),1.37–1.27(m,4H),0.90(t,J=6.7Hz,3H).

[0116] 13C NMR (100MHz, Chloroform / d) δ154.6,79.1,57.9,57.1,52.0,51.5,48.4,44.5,44.1,32.2,31.4,30.0,29.5,28.5,22.6,14.0.

[0117] The HPLC chromatogram of product 2 is shown in [reference needed]. Figure 2 .

[0118] Product 3 (S1-A3)

[0119]

[0120] Using 1-tert-butoxycarbonyl-3-pyrrolidone and cyclobutylamine as raw materials, a brown oily liquid was obtained, which is product 3.

[0121] 1H NMR(400MHz,Chloroform / d)δ3.63–3.38(m,2H),3.37–3.22(m,3H),3.10–2.94 (m,1H),2.31–2.16(m,2H),2.08–1.96(m,1H),1.76–1.52(m,6H),1.45(s,9H).

[0122] 13C NMR (100MHz, Chloroform / d) δ154.6,79.1,55.9,55.1,53.0,52.1,51.7,44.4,44.0,32.5,32.0,31.9,31.8,28.5,14.8.

[0123] The HPLC chromatogram of product 3 is shown in [reference needed]. Figure 3 .

[0124] Product 4 (S1-A4)

[0125]

[0126] A reddish-brown oily liquid, product 4, was obtained from 1-tert-butoxycarbonyl-3-pyrrolidone and benzylamine.

[0127] 1H NMR(400MHz,Chloroform / d)δ7.35–7.27(m,4H),7.28–7.21(m,1H),3.83–3.72(m,2H),3.61–3.39(m,2H),3.39–3 .25(m,2H),3.12(ddd,J=32.1,10.9,5.3Hz,1H),2.08–1.96(m,1H),1.78–1.65(m,1H),1.45(s,9H),1.38(s,1H).

[0128] 13C NMR (100MHz, Chloroform / d) δ154.6,140.1,128.5,128.1,127.1,79.1,57.1,56.2,52.2,52.0,51.5,44.4,44.1,32.1,31.4,28.5.

[0129] The HPLC chromatogram of product 4 is shown in [reference needed]. Figure 4 .

[0130] Product 5 (S1-A5)

[0131]

[0132] A brown oily liquid, product 5, was obtained from 1-tert-butoxycarbonyl-3-pyrrolidone and cyclopropylamine.

[0133] 1H NMR (300MHz, CDCl3) δ3.69–3.50(m,1H),3.52–3.36(m,2H),3.37–3.25(m,1H),3.12(dd,J=16.9,7. 8Hz,1H),2.20–1.98(m,2H),1.87–1.68(m,2H),1.46(s,9H),0.52–0.42(m,2H),0.40–0.31(m,2H).

[0134] 13C NMR (75MHz, Chloroform / d) δ154.6,79.0,57.9,57.2,51.9,51.5,44.3,44.0,32.0,31.3,29.0,28.9,28.5,28.4,6.3.

[0135] The HPLC chromatogram of product 5 is shown in [reference needed]. Figure 5 .

[0136] Product 6 (S2-A3)

[0137]

[0138] Using N-BOC-3-azacycloheptanone and cyclobutylamine as raw materials, a yellow oily liquid was obtained, which is product 6.

[0139] 1H NMR(400MHz,Chloroform / d)δ3.76–3.65(m,1H),3.40–3.29(m,1H),3.22–2.99(m,1H),2.88– 2.60(m,2H),2.36–2.11(m,2H),1.95–1.52(m,8H),1.47(d,J=12.1Hz,9H),1.42–1.16(m,3H).

[0140] 13C NMR(100MHz,Chloroform / d)δ155.7,155.5,79.2,79.1,56.3,56.0,52.2,52.2,51.8,51.2 ,47.8,46.8,35.7,34.5,32.3,32.3,32.2,32.2,28.5,28.5,27.8,27.4,22.8,22.6,14.6.

[0141] The HPLC chromatogram of product 6 is shown in [reference needed]. Figure 6 .

[0142] Product 7 (S2-A4)

[0143]

[0144] A yellow oily liquid, product 7, was obtained by using N-BOC-3-azacycloheptanone and benzylamine as raw materials.

[0145] 1H NMR(400MHz,Chloroform / d)δ7.37–7.27(m,4H),7.26–7.19(m,1H),3.93–3.71(m,3H),3.71–3.58(m,0.5H),3.51–3.21(m,1H),3.21– 3.02(m,1H),2.94–2.82(m,1H),2.82–2.72(m,0.5H),1.94–1.68(m,3H),1.65–1.56(m,1H),1.46(d,J=7.5Hz,9H),1.42–1.34(m,2H).

[0146] 13C NMR(100MHz,Chloroform / d)δ155.9,155.6,140.6,140.6,128.5,128.4,128.2,128.0,127.0,126.8, 79.4,79.2,57.8,57.6,51.6,50.8,50.8,48.0,47.0,34.9,34.0,28.5,28.5,27.9,27.6,22.8,22.6.

[0147] The HPLC chromatogram of product 7 is shown in [reference needed]. Figure 7 .

[0148] Product 8 (S2-A5)

[0149]

[0150] A brown oily liquid, product 8, was obtained from N-BOC-3-azacycloheptanone and cyclopropylamine.

[0151] 1H NMR(300MHz,Chloroform / d)δ3.93–3.77(m,1H),3.70–3.36(m,2H),3.31–2.97(m,2H),2.93–2.78(m,1 H),2.31–2.11(m,1H),2.01–1.53(m,4H),1.45(d,J=4.9Hz,9H),1.40–1.20(m,2H),0.55–0.32(m,4H).

[0152] 13C NMR(75MHz,Chloroform / d)δ155.7,79.1,79.1,58.6,58.3,50.9,48.0,46.8,34.7,33.1,28.6,28.3,28.3,27.8,27.4,22.4,22.3,6.8,6.2,5.8,5.7.

[0153] The HPLC chromatogram of product 8 is shown in [reference needed]. Figure 8 .

[0154] Example 6: Substrate tolerance of imine reductase mutant IRED / M1

[0155] Wild-type imine reductases exhibit low activity and low substrate tolerance. The substrate tolerance of substrate S1 was tested using the dominant mutant M1. Specifically, a 1 mL reaction system of substrate S1 and amine donor A5 contained 100 mM, 200 mM, 300 mM, 400 mM, and 500 mM of S1, with corresponding concentrations of amine donor A5 of 150 mM, 300 mM, 450 mM, 600 mM, and 750 mM, and 1 mM NADP. + The reaction mixture consisted of lyophilized lysate of ThIRED-M1 (1.5 mg / mL), D-glucose (2.0 equivalents, the molar amount of D-glucose being twice that of ketone), NADPH regeneration system enzyme powder (1 mg / mL), sodium phosphate buffer (200 mM, pH 8.0), and 20% DMSO (v / v, the volume of DMSO relative to the total reaction volume). The catalytic reaction conditions, product extraction, and conversion rate calculation methods were as described in Example 5.

[0156] Table 5. Product conversion rates of imine reductase wild-type and mutant under different substrate concentrations.

[0157]

[0158] The reaction results are shown in Table 5. When the concentration of substrate S1 was increased from 100 mM to 500 mM, the product conversion rate of the dominant mutant M1 decreased from 97.9% to 45.9%, while the product conversion rate of the wild type decreased from 18.9% to 12.2%.

[0159] This demonstrates that the superior mutant M1 of this invention exhibits significantly better tolerance to high concentrations of substrate S1 than the wild type.

[0160] In summary, this invention has obtained a series of advantageous mutants based on the imine reductase ThIRED from Torrubiella hemipterigena through directed evolution. The advantageous imine reductase mutants obtained by this invention possess high catalytic activity, high selectivity, and broad substrate applicability, and have broad application prospects in the industrial production of highly active imine reductases.

[0161] The sequence involved in this invention is as follows:

[0162] The wild-type ThIRED has the DNA sequence shown in SEQ ID NO:1 and the protein sequence shown in SEQ ID NO:2:

[0163] SEQ ID NO:1 Artificial DNA Sequence <909bp>

[0164]

[0165] SEQ ID NO:2 Artificial amino acid sequence <302aa>

[0166]

[0167]

[0168] The ThIRED mutant M1 has the DNA sequence shown in SEQ ID NO:3 and the protein sequence shown in SEQ ID NO:4:

[0169] SEQ ID NO:3 Artificial DNA Sequence <909bp>

[0170]

[0171]

[0172] SEQ ID NO:4 Artificial amino acid sequence <302aa>

[0173]

[0174]

Claims

1. An imine reductase mutant, characterized in that, The imine reductase mutant was obtained by mutation of the wild-type imine reductase, and the amino acid sequence of the wild-type imine reductase is shown in SEQ ID. As shown in NO:2, the mutation is one of the following single point mutations or combination mutations: N249S, N249S+M187L, N249S+M187L+F191I, N249S+M187L+F191H, N249S+M187L+F191L, N249S+M187L+F191V, N249S+Y230H, N249S+A226E+Q140N, N249S+A226D+Q140N, N249S+A226E+Q140S, N249S+A226D+Q140S, N249S+A226E+Q140T, N249S+A22 6D+Q140T, N249S+M187L+F191S, N249S+F191L, N249S+M187L+F191M, N249S+F191V, N249S+F191T, N249S+Y230E, N249S+L227M+Y230Q, N249 S+M187L+F191V+Y230E, N249S+M187L+F191L+Y230E, N249S+M187L+F191L+Y230H, N249S+M187L+F191L+L227M+Y230Q, N249S+M187L+F191I+ Y230H, N249S+M187L+F191I+Y230E, N249S+M187L+F191I+L227M+Y230Q, N249S+M187L+F191V+L227M+Y230Q, N249S+M187L+F191I+A256H, N 249S+M187L+F191I+N260K, N249S+M187L+F191I+Q134P, N249S+M187L +F191I+Q134D, N249S+M187L+F191I+N260K+Q134P, N249S+M187L+F19 1I+N260K+Q134D, N249S+M187L+F191I+A256H+Q134P, N249S+M187L+F191I+A256H+Q134D, N249S+M187L+F191I+N260K+G127A, N249S+M187 L+F191I+N260K+G127S, N249S+M187L+F191I+N260K+A128G, N249S+M187L+F191I+N260K+A128K, N249S+M187L+F191I+N260K+M253V+Q254S,N249S+M187L+F191I+A256K, N249S+M187L+F191I+A256T, N249S+M187L+F191I+A256V, N249S+M187L+F191I+L227M, N249S+M187L+F191L+A256I, N249S+M187L+F191L+A256V, N249S+M187L+ F191L+A256Y, N249S+M187L+F191L+L227M, N249S+M187L+F191V+A256I, N249S+M187L+F191V+A256K, N249S+M187L+F191V+A256T, N249S+M187L+F191V+A256V, N249S+M187L+F191V+A256Y N249S+M187L+F191V+L227M, N249S+M187L+F191I+A256I, N249S+M187L+F191I+A256Y, N249S+M187L+F191L+A256K, N249S+M187L+F191L+A256T, N249S+M187L+F191L+L227F, N249S+M187L+ F191V+L227F, N249S+M187L+F191I+L227F+A256T, N249S+M187L+F191V+L227F+N260K, N249S+M187L+F191V+L227M+N260K, N249S+M187L+F191I+L227F, N249S+M187L+F191I+L227M+A256T.

2. The imine reductase mutant according to claim 1, characterized in that, The amino acid sequence of the imine reductase mutant is shown in SEQ ID NO:

4.

3. A gene encoding an imine reductase mutant according to any one of claims 1-2.

4. A recombinant expression vector, plasmid, and recombinant bacteria comprising the gene of claim 3.

5. A method for preparing the imine reductase mutant according to any one of claims 1-2, characterized in that, The method includes the following steps: (1) Construct a vector containing a gene encoding the imine reductase mutant according to any one of claims 1-2; (2) The vector was transferred into the host bacteria, cultured, and an inducer was added to induce the expression of the imine reductase mutant.

6. The application of the imine reductase mutant according to any one of claims 1-2 in the enzymatic production of chiral amines, wherein the substrate ketone for the enzymatic production of chiral amines is... or amine donor is , , , or .

7. A method for preparing chiral amines, characterized in that, The method comprises the reaction of the imine reductase mutant, substrate ketone, amine donor, and coenzyme regeneration system as described in any one of claims 1-2, thereby obtaining a chiral amine; the substrate ketone is... or The amine donor is , , , or .

8. The method according to claim 7, characterized in that, The coenzyme is NADPH, and the coenzyme regeneration system is D-glucose and NADP. + And glucose dehydrogenase.

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