Imine reductase mutant and application thereof in synthesis of chiral 1, 3-disubstituted tetrahydroisoquinoline

Abstract

The invention discloses an imine reductase mutant and application thereof in synthesis of chiral 1, 3-disubstituted tetrahydroisoquinoline, and belongs to the technical field of bioengineering. According to the invention, imine reductases from different sources are screened, the imine reductase mutant SnIR-L175A / M245A is constructed, a substrate racemic imine can be converted into (1S, 3R)-4a, the conversion rate is increased to 50% compared with that of a starting sequence, the constructed imine reductase mutant is used for synthesizing 1, 3-disubstituted THIQ by taking imine as a raw material, the de value and ee value can reach 99%, and the 1, 3-disubstituted THIQ can be used for preparing the (1S, 3R)-4a. Good application prospects are realized.

Description

Technical Field

[0001] The invention relates to an imine reductase mutant and application thereof in synthesizing chiral 1,3-disubstituted tetrahydroisoquinoline, belonging to the technical field of bioengineering. Background Art

[0002] Tetrahydroisoquinoline (THIQ) alkaloids are one of the largest natural products and play an indispensable role in drug development. THIQ natural products have been intensively studied in recent decades and have a wide range of structural diversity and biological activities.

[0003] Among these different THIQs, 1,3-disubstituted THIQs with at least two chiral carbon centers are a special class of THIQ alkaloids that are different from tetrahydroisoquinolines, which are generally believed to be formed through the Pictet-Spengler (PS) reaction between aldehydes and aromatic amino acid-derived arylethylamines, and are further generated by polyketides formed by the acetate-malonate pathway, which involves an as-yet-unidentified polyketide synthase and other enzymes. However, the unusual substitution pattern of 1,3-disubstituted THIQs, especially the presence of methyl groups at the C1 and C3 positions, poses a huge obstacle to the formation of these important compounds through the traditional PS route due to the difficulty in biocatalytically pre-installing α-methyl groups on aromatic amino acids to construct C3 methyl groups and the NCS substrate-limited acetaldehyde to construct C1 methyl groups.

[0004] Although their biosynthesis remains challenging, many pioneering chemical synthetic methods have been developed to construct these valuable chiral structures, including transition metal-catalyzed asymmetric reduction of 1,3-disubstituted isoquinolines, asymmetric nucleophilic addition of chiral 3,4-dihydroisoquinolines, alkylation of chiral tetrahydroisoquinolines, PS reactions between chiral phenylethylamine derivatives and aldehydes, Bischler-Napieralski (BN) / reduction cascade reactions of chiral acylated phenylethylamines, and palladium-catalyzed three-component Catellani / gold-catalyzed cyclization / reduction cascade reactions. Although these strategies are effective, they often rely on complex substrates with pre-constructed chiral centers, heavy metal catalysts, flammable and hazardous hydrogen gas, and harsh conditions of high temperature and pressure. Therefore, it is highly desirable to develop a novel, efficient, and green method to construct chiral 1,3-disubstituted THIQs from simple achiral substrates. Summary of the invention

[0005] The present invention provides an imine reductase mutant, which has an imine reductase with an amino acid sequence as shown in SEQ ID NO.3 as a parent and has one or more mutations among (a)-(f):

[0006] (a) mutating the threonine at position 123 of the parent to alanine;

[0007] (b) mutating the proline at position 124 of the parent to alanine;

[0008] (c) mutating the leucine at position 175 of the parent to alanine;

[0009] (d) mutating the methionine at position 216 of the parent to alanine;

[0010] (e) mutating the parent threonine 223 to alanine;

[0011] (f) Mutating the methionine at position 245 of the parent to alanine.

[0012] The present invention also provides a gene encoding the imine reductase mutant.

[0013] The invention also provides a recombinant plasmid carrying the gene.

[0014] The present invention also provides a recombinant microorganism expressing the imine reductase mutant.

[0015] In one embodiment, the microorganism includes but is not limited to Escherichia coli.

[0016] In one embodiment, Escherichia coli BL21 (DE3) is used as a host and pET-21b (+) is used as a vector to express the imine reductase mutant.

[0017] The present invention also provides a method for preparing the imine reductase mutant.

[0018] In one embodiment, the method is to inoculate the recombinant Escherichia coli expressing the transaminase into a culture medium, culture the strain to a logarithmic growth phase, induce, collect the bacteria, resuspend, break, centrifuge to collect the lysate, and purify the enzyme protein.

[0019] The present invention also provides an enzyme preparation containing the imine reductase mutant.

[0020] The present invention also provides application of the imine reductase mutant in synthesizing 1,3-disubstituted THIQ.

[0021] In one embodiment, the application comprises the following steps:

[0022] (1) mixing the transaminase protein with a substrate in the presence of a lactate dehydrogenase (LDH) circulation system to react and prepare an amine 2; the substrate is a ketone 1, D-alanine or L-alanine, and pyridoxal phosphate (PLP); the LDH circulation system includes lactate dehydrogenase (LDH), glucose dehydrogenase (GDH), NADH, and glucose;

[0023] (2) reacting the amine 2 obtained in step (1) with an acyl chloride, collecting the reaction product, and then reacting the product with phosphorus oxychloride to obtain an imine 3;

[0024] (3) using the imine 3 obtained in step (2) as a substrate, and catalyzing the reaction with an imine reductase protein and a GDH circulation system to obtain 1,3-disubstituted THIQ; the GDH circulation system includes GDH, NADP + ,glucose.

[0025] In one embodiment, the transaminase is derived from Arthrobacter sp., and its amino acid sequence is shown in SEQ ID NO.1.

[0026] In one embodiment, the glucose dehydrogenase is derived from Bacillus subtilis, and its amino acid sequence is shown in SEQ ID NO.5.

[0027] In one embodiment, the lactate dehydrogenase is derived from Lactobacillus fermentum JN248, and its amino acid sequence is shown in SEQ ID NO.7.

[0028] In one embodiment, the amount of imine 3 added in step (3) is 1 mM, the amount of imine reductase mutant added is 1.6-3 mol%, the amount of GDH added is 1 mol%, and the amount of NADP + The added amount of is 10 mol %, the added amount of glucose is 100 mM; wherein all molar percentages are calculated based on the substrate imine 3.

[0029] In one embodiment, the reaction system of step (2) contains 0.5M amine 2 dissolved in dichloromethane, 1.5 equivalents of triethylamine, at least 1 equivalent of acyl chloride, 0.5M amide dissolved in acetonitrile, and 5 equivalents of phosphorus oxychloride; the acetonitrile is preferably dry acetonitrile; the phosphorus oxychloride is preferably freshly distilled phosphorus oxychloride.

[0030] In one embodiment, the reaction temperature in step (2) is 90°C.

[0031] In one embodiment, the substrate comprises ketone 1, D-alanine or L-alanine, pyridoxal phosphate (PLP).

[0032] In one embodiment, the LDH circulation system includes LDH, glucose dehydrogenase (GDH), NADH, and glucose.

[0033] In one embodiment, the reaction temperature is 20-40°C, and the reaction pH is 7.0-8.0.

[0034] In one embodiment, the reaction time is 12-24 hours, or the reaction time is not less than 12 hours.

[0035] In one embodiment, the added amount of the ketone 1 is 5-10 mM; the added amount of the D-alanine or L-alanine is 100 mM, the added amount of the PLP is 1 mM, the added amount of the glucose is 100 mM, and the added amount of the NADH is 1 mM.

[0036] In one embodiment, the amount of the transaminase added is 0.2-0.4 mol%, the amount of the GDH added is 0.15-0.3 mol%, and the amount of the LDH added is 0.03-0.06 mol%, wherein all molar percentages are calculated based on the substrate ketone 1.

[0037] In one embodiment, the added amount of the ketone 1 is 5-10 mM; the added amount of the D-alanine or L-alanine is 100 mM, the added amount of the PLP is 1 mM, the added amount of the glucose is 100 mM, and the added amount of the NADH is 1 mM.

[0038] In one embodiment, the amount of the transaminase added is 0.2-0.4 mol%, the amount of the GDH added is 0.15-0.3 mol%, and the amount of the LDH added is 0.03-0.06 mol%.

[0039] In one embodiment, the ketone 1 includes at least one of the following 1a-1d, 1f:

[0040]

[0041] In one embodiment, the acyl chloride comprises at least one of the following AG:

[0042]

[0043] In one embodiment, the imine 3 comprises at least one of the following 3a-3l:

[0044]

[0045] In one embodiment, the 1,3-disubstituted THIQ structure is as follows:

[0046]

[0047] The present invention also provides the use of the imine reductase mutant or the gene encoding the imine reductase mutant, or the recombinant cell expressing the mutant in the synthesis of 1,3-disubstituted THIQ.

[0048] The present invention also provides application of the mutant, or the recombinant cell, or the method in preparing 1,3-disubstituted THIQ.

[0049] Beneficial effects:

[0050] (1) The present invention screened imine reductases from different sources, among which the imine reductase SnIR could convert the substrate racemic imine into (1S, 3R)-4a with a conversion rate of 11%, a de value of 44, and an ee value of 59%;

[0051] (2) The present invention screened and obtained imine reductase SnIR based on mutation, and the constructed imine reductase mutant SnIR-L175A / M245A can convert the substrate racemic imine into (1S, 3R)-4a with a conversion rate of 50%, a de value of >99%, and an ee value of >99%;

[0052] (3) The present invention also provides a method for synthesizing 1,3-disubstituted THIQ using imine as raw material. The HPLC yields of (1S,3R)-4a-(1S,3R)-4l are 85%, 83%, 92%, 36%, 98%, 68%, 92%, 95%, 84%, 95%, 99%, 99%, respectively. The de values ​​are >99%, >99%, >99%, >99%, >99%, >99%, >99%, >99%, >99%, >99%, >99%, >99%, respectively. The ee values ​​are >99%, >99%, 97%, >99%, >99%, >99%, >99%, 97%, >99%, >99%, >99%, >99%, >99%, >99%, respectively.

[0053] (4) The method of the present invention uses ketone 1a as raw material to synthesize 179.8 mg of (1S, 3R)-4a by chemical enzyme modular cascade with a yield of 81%, >99de, and >99ee. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 The present invention is a synthetic route for 1,3-disubstituted THIQ.

[0055] Figure 2 This is the liquid phase analysis diagram of the imine reductase module.

[0056] Figure 3 Chiral identification for the enzyme-catalyzed synthesis of (1S,3R)-4a.

[0057] Figure 4 NMR spectroscopy analysis of enzyme-catalyzed synthesis of (1S,3R)-4a 1 H NMR and 13 C NMR.

[0058] Figure 5 NOE spectrum of nuclear magnetic resonance spectroscopy analysis for the enzyme-catalyzed synthesis of (1S,3R)-4a.

[0059] Figure 6 To expand the substrate spectrum using imine reductase mutants. DETAILED DESCRIPTION

[0060] Table 1 Amino acid sequences of related proteins

[0061]

[0062]

[0063] Table 2 Gene sequences encoding related proteins

[0064]

[0065]

[0066] DETAILED DESCRIPTION

[0068] The present invention is further described below in conjunction with specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0069] Reagents and materials purchased from: The antibiotics used in this invention, such as ampicillin sodium, kanamycin sulfate, and streptomycin sulfate, are all from Shanghai Biotechnology; NADP + , NADH comes from Shanghai BiDe Pharmaceutical Technology Co., Ltd.; the PCR enzyme involved in the molecular experiment in the present invention is purchased from Bao Biotechnology (Dalian) Co., Ltd.; the chemical reagents used in the present invention, such as methanol, dichloromethane, ethyl acetate (EtOAc), petroleum ether (PE), acetonitrile (MeCN), sodium sulfate, sodium carbonate, sodium bicarbonate, diethylamine (DEA), ammonia, sodium hydroxide, hydrochloric acid, etc., are all purchased from China Pharmaceutical Group Co., Ltd. and Shanghai Titan Technology Co., Ltd.

[0070] Transaminase module enzyme activity detection: Substrate 1a (10mM), transaminase (0.2mol%), GDH (0.15mol%), LDH (0.03mol%), D,L-alanine (100mM), PLP (1mM), glucose (100mM), NADH (1mM) and DMSO (5% v / v) were cultured in a 200μL reaction system (pH=8) containing 100mM at 30℃ for 24h. After the reaction, 24 times the volume of methanol was added to quench the reaction, shake, centrifuge at low temperature and high speed, centrifuge at 20000g for 5min, and take 20μL sample to load the chromatographic column to analyze the conversion rate. Then, the remaining reaction solution was adjusted to pH 10 with 5M sodium hydroxide solution and extracted with ethyl acetate three times. The organic layers were combined and concentrated in vacuo to remove the organic solvent, and the ee value of the product was analyzed by normal HPLC.

[0071] Chemical module detection: Amine 2a (1 equivalent) and triethylamine (TEA) (1.5 equivalents) were dissolved in dichloromethane (0.5M), and acetyl chloride was added dropwise at 0°C. After the reaction was completed, the solution was diluted with dichloromethane, washed twice with brine, dried over sodium sulfate, filtered and concentrated to obtain the crude amide, which was used directly in the next step without purification. A two-necked round-bottom flask (50mL) was equipped with a magnetic stirring bar and thoroughly flushed with argon for 3 cycles. The crude amide (1 equivalent) was dissolved in dry MeCN (0.5M), and then phosphorus oxychloride (5 equivalents) was added. Add to the mixture and reflux at 90°C for 2 hours. Take 1μL sample and dilute 1000 times with chromatography-grade methanol for liquid phase analysis of the reaction.

[0072] Imine reductase activity detection: imine rac-3a (1mM), imine reductase (1.6mol%), GDH (1mol%), NADP + (10mol%), glucose (100mM) and DMSO (5% v / v) were added and incubated at 30°C for 24 hours in a 200μL reaction system containing 100mM Tris-HCl. After the reaction was completed, 30μL of the reaction mixture was diluted to 300μL with chromatography-grade methanol, and the conversion rate and de value were determined by HPLC and a standard curve. Then, the remaining reaction solution was adjusted to pH 10 with 5M sodium hydroxide solution and extracted 3 times with ethyl acetate. The organic layers were combined and concentrated in vacuo to remove the organic solvent, and the ee value of the product was analyzed by positive HPLC.

[0073] High performance liquid chromatography (HPLC) detection conditions: ZORBAX Eclipse XDB-C18 column, column temperature of 25°C, detection wavelength of 280nm. The detection method uses a three-phase solvent system of methanol (containing 0.1% (v / v) trifluoroacetic acid), acetonitrile (containing 0.1% (v / v) trifluoroacetic acid) and double distilled water (containing 0.1% (v / v) trifluoroacetic acid) for gradient separation, and the mobile phase flow rate is 1mL min -1 . The linear gradient is as follows:

[0074] Table 3 High performance liquid chromatography (HPLC) detection conditions

[0075]

[0076] Protein purification method: Resuspend the collected bacteria in lysis buffer (50mmol / L Tris-HCl, 300mmol / L NaCl, 20mM imidazole, pH=8), and then crush the resuspension by high-pressure homogenizer. The crushed resuspension is centrifuged at low temperature and high speed, 4°C, 10000rpm for 30min to obtain a crude enzyme solution. Desalt by nickel column affinity chromatography and desalting column (HistrapTM 5mL Desalting) to finally obtain the purified protein.

[0077] Example 1: SnIR protein expression and purification

[0078] 1. Obtaining SnIR plasmid

[0079] The codon of the target gene SnIR from Stackebrandtia nassauensis was optimized to obtain the gene sequence shown in SEQ ID NO.4, and the gene was synthesized and connected to the pET-21b(+) vector to obtain the plasmid pET-21b-SnIR.

[0080] 2. Protein expression and purification

[0081] A single colony of E. coli BL21 carrying the target gene was picked up and placed in 5 mL LB medium containing antibiotics (ampicillin), cultured on a shaker at 37°C and 220 rpm for 8 h, then transferred to 500 mL 2xYT medium at a 1% (v / v) inoculation amount, and 500 ul 100 mg / mL ampicillin was added to the medium and cultured at 37°C until the optical density of the bacteria at 600 nm (OD600) was 0.5-0.7, then the culture was cooled to 18°C, and isopropyl-β-d-thiogalactoside (IPTG) was added to a final concentration of 0.2 mM for protein induction expression.

[0082] The above culture was centrifuged (8000rpm, 5min, 4°C) to collect cells and discard the supernatant. The cell pellet was then resuspended in 35mL pre-cooled (4°C) Tris-HCl buffer (25mM, pH 8.0), which contained 0.1mM pyridoxal-5-phosphate (PLP), 0.3M sodium chloride, 20mM imidazole and 10% (v / v) glycerol. The resuspended cells were broken by a high-pressure homogenizer (800bar) and centrifuged (18300rpm, 30min, 4°C) to remove cell debris. The protein was purified by metal affinity chromatography (IMAC), and the nickel column was balanced with 10 column volumes of lysate. Non-specifically bound proteins were removed by applying ten column volumes of 20mM imidazole. The specifically bound proteins were eluted from the nickel column with two column volumes of 250mM imidazole. The purified transaminase fractions were pooled, then concentrated using a 30 KDa ultrafiltration tube and replaced with a storage solution (25 mM Tris-HCl, 150 mM NaCl, 10% (v / v) glycerol, 0.1 mM PLP, pH 8.0). The purified enzyme solution was aliquoted at a final concentration of 10 mg / mL and stored at -80°C until use.

[0083] According to the above-mentioned similar method, the imine reductase GF3546 from Streptomyces sp. (amino acid sequence as shown in SEQ ID NO.9) and the imine reductase CfIR from Cystobacter ferrugineus (amino acid sequence as shown in SEQ ID NO.10) were expressed in Escherichia coli to prepare the imine reductase protein. Example 2: Expression and purification of transaminase protein

[0084] 1. Construction of plasmid expressing transaminase

[0085] Taking ArR-TA as an example, the target gene ArR-TA from Arthrobacter sp was codon optimized to obtain the gene sequence shown in SEQ ID NO.2, and its gene was synthesized and connected to the pET-21b(+) vector to obtain the plasmid pET-21b-ArR-TA.

[0086] 2. Protein expression and purification

[0087] The recombinant plasmids constructed in step 1 were transformed into E. coli BL21 competent cells, and the correct strain was verified to be the recombinant E. coli expressing the corresponding transaminase. Protein expression and purification were performed according to the method of Example 1 to obtain ArR-TA enzyme protein, which was packaged and adjusted to a final concentration of 10 mg / mL and stored at -80°C for use.

[0088] Example 3: Preparation of LDH and GDH

[0089] 1. Construction of plasmids expressing LDH and GDH

[0090] The target gene GDH from Bacillus subtilis was codon optimized to obtain the gene sequence shown in SEQ ID NO.6, and the gene was synthesized and connected to the pET-21b(+) vector to obtain the plasmid pET-21b-GDH.

[0091] The target gene LDH from Lactobacillus fermentum JN248 was codon optimized to obtain the gene sequence shown in SEQ ID NO.8, and the gene was synthesized and connected to the pET-21b(+) vector to obtain the plasmid pET-21b-LDH.

[0092] 2. Protein expression and purification

[0093] The proteins were expressed and purified according to the method of Example 1 to obtain LDH with a specific enzyme activity of 903 U / mg and GDH with a specific enzyme activity of 885 U / mg, respectively. The proteins were packaged and adjusted to a final concentration of 10 mg / mL and stored at -80°C until use.

[0094] Example 4: Imine reductase screening

[0095] Rac-3a is used as a substrate and converted into the corresponding 4a through the action of imine reductase, glucose, GDH and cofactor NADH.

[0096] The specific steps are as follows: imine rac-3a (1 mM), imine reductase (1.6 mol%), GDH (1 mol%), NADP + (10 mol%), glucose (100 mM) and DMSO (5% v / v), wherein the molar percentage of each reactant is based on the substrate imine rac-3a. Incubate at 30°C for 24 hours in a 200 μL reaction system containing 100 mM Tris-HCl. After the reaction is completed, 30 μL of the reaction mixture is diluted to 300 μL with chromatography-grade methanol, and the conversion rate and de value are determined by HPLC and a standard curve. Then, the remaining reaction solution is adjusted to pH 10 with 5 M sodium hydroxide solution and extracted 3 times with ethyl acetate. The organic layers are combined and concentrated in vacuo to remove the organic solvent, and the ee value of the product is analyzed by normal HPLC.

[0097] Table 4 Results of screening of imine reductase using 3a as substrate

[0098]

[0099]

[0100] Example 5: Construction of imine reductase mutants

[0101] The rate-limiting step enzyme SnIR in the chemoenzyme modular cascade reaction pathway was modified. First, the substrate (R)-3a was docked into the substrate pocket of the SnIR protein using molecular docking software (Discovery Studio). Next, the substrate (R)-3a was selected. Alanine mutations were performed on 15 amino acids within the region.

[0102] The primers used for SnIR mutant design are shown in Table 2.

[0103] Table 5 Primer sequences of SnIR mutants

[0104]

[0105] The reaction was carried out according to the steps of Example 4, and the conversion rate was determined using the standard curve of the substrate rac-3a to represent the catalytic efficiency of different mutants.

[0106] As shown in Table 6, five single mutants with higher conversion rates were obtained, SnIR-T123A, SnIR-P124A, SnIR-L175A, SnIR-M216A and SnIR-T223A, among which SnIR-L175A had higher de and ee values. Secondly, the optimal mutant SnIR-L175A / M245A was obtained by semi-rational design and combined mutation strategies. Among them, SnIR-L175A / T223A, SnIR-L175A / L225A, SnIR-L175A / M246A and SnIR-L175A / W287A also had good results. Compared with the wild-type SnIR, the activity and stereoselectivity of the mutant SnIR-L175A / M245A have been greatly improved.

[0107] Table 6 Relative enzyme activities of SnIR mutants

[0108]

[0109]

[0110] like Figure 1As shown, the first step (verification of the transaminase module): (3,5-dimethoxyphenyl)ethanone (1a) is used as a model substrate, and is converted into the corresponding amine (R-(2a) or S-(2a)) by transaminase, cofactor PLP, D,L-alanine, glucose, LDH and GDH constructed in Example 3, and cofactor NADH. The first step (verification of the chemical module): racemic (3,5-dimethoxyphenyl)ethanamine 2a reacts with acyl chloride to form an amide, and then generates racemic 6,8-dimethoxy-1,3-dimethyl-3,4-dihydroisoquinoline (rac-3a) under the action of phosphorus oxychloride. The third step (verification of the imine reductase module): rac-3a is used as a substrate and converted into the corresponding 4a by imine reductase, glucose, GDH, and cofactor NADH.

[0111] Example 6: Application of Imine Reductase Mutants in the Preparation of 1,3-Disubstituted THIQ

[0112] (1) First step reaction: substrate 1a (10mM), ArR-TA (0.2mol%), GDH (0.15mol%, specific enzyme activity of 885U / mg), LDH (0.03mol%, specific enzyme activity of 903U / mg), D,L-alanine (100mM), PLP (1mM), glucose (100mM), NADH (1mM) and DMSO (5% v / v) were cultured in a 200μL reaction system (pH=8) containing 100mM at 30℃ for 24 hours. After the reaction, 24 times the volume of methanol was added to quench the reaction, shaken, centrifuged at low temperature and high speed, centrifuged at 20000g for 5min, and 200μL of sample was loaded on the chromatographic column to analyze the conversion rate. The remaining reaction mixture was then alkalized to pH 10 with 5M sodium hydroxide solution and extracted 3 times with dichloromethane. The solvent of the combined organic layer was removed by vacuum concentration, and the ee value was determined by normal HPLC. The results showed that xx

[0113] (2) Second step reaction: Amine 2a (1 equivalent) and triethylamine (TEA) (1.5 equivalents) were dissolved in dichloromethane (0.5 M), and acetyl chloride was added dropwise at 0°C. After the reaction was completed, the solution was diluted with dichloromethane, washed twice with brine, dried over sodium sulfate, filtered and concentrated to obtain the crude amide, which was used directly in the next step without purification. A two-necked round-bottom flask (50 mL) was equipped with a magnetic stirring bar and thoroughly flushed with argon for 3 cycles. The crude amide (1 equivalent) was dissolved in dry MeCN (0.5 M), and then phosphorus oxychloride (5 equivalents) was added. Add to the mixture and reflux at 90°C for 2 hours. Take 1 μL sample and dilute 1000 times with chromatography-grade methanol for liquid phase analysis of the reaction.

[0114] (3) Step 3: Imine rac-3a (1 mM), imine reductase mutant SnIR-L175A / M245A (1.6 mol%), GDH (1 mol%), NADP + (10 mol%), glucose (100 mM) and DMSO (5% v / v), wherein the molar percentage of each reactant is based on the substrate imine rac-3a. Incubate at 30°C for 24 hours in a 200 μL reaction system containing 100 mM Tris-HCl. After the reaction is completed, 30 μL of the reaction mixture is diluted to 300 μL with chromatography-grade methanol, and the conversion rate and de value are determined by HPLC and a standard curve. Then, the remaining reaction solution is adjusted to pH 10 with 5 M sodium hydroxide solution and extracted 3 times with ethyl acetate. The organic layers are combined and concentrated in vacuo to remove the organic solvent, and the ee value of the product is analyzed by normal HPLC.

[0115] Example 7: Substrate spectrum expansion

[0116] According to the experimental results, the main rate-limiting step is the imine reductase module. The yield calculation in the expansion of the substrate spectrum is mainly focused on the imine reduction module. The de value and ee value can represent the results of the chemical enzyme modular cascade reaction to prepare the final product with the substrate ketone. The structural formula of the corresponding product is as follows Figure 6 shown.

[0117] Preparation of enantiomerically enriched imine 3:

[0118] Substrate 1 (5 mM), ArR-TA (0.4-1 mol%), GDH (0.15-0.3 mol%), LDH (0.03-0.06 mol%), D, L-alanine (100 mM), PLP (1 mM), glucose (100 mM), NADH (1 mM) and DMSO (5% v / v) were cultured in a 200 μL reaction system (pH = 8) containing 100 mM at 30°C for 24 hours. After the reaction, the reaction solution was adjusted to pH 10 with 5 M sodium hydroxide solution and extracted with dichloromethane three times. The organic layers were combined, dried over sodium sulfate, and concentrated in vacuo to remove the organic solvent. The crude amine 2 was directly used in the next reaction.

[0119] Amine 2 (1 eq.) and triethylamine (TEA) (1.5 eq.) were dissolved in dichloromethane (0.5 M) and acid chloride (≥1 eq.) was added dropwise at 0 °C. After the reaction was complete, the solution was diluted with dichloromethane, washed twice with brine, dried over sodium sulfate, filtered and concentrated to give the crude amide, which was used directly in the next step without purification. A two-necked round-bottom flask (50 mL) was equipped with a magnetic stirring bar and thoroughly flushed with argon for 3 cycles. The crude amide (1 eq.) was dissolved in dry MeCN (0.5 M) and phosphorus oxychloride (5 eq.) was added. Add to the mixture and reflux at 90 °C for 2 h. The solvent was evaporated in vacuo, the residue was quenched with aqueous ammonia, extracted 3 times with ethyl acetate, washed 3 times with saturated sodium bicarbonate, dried over sodium sulfate, the solvent was evaporated in vacuo, and column chromatography was used to obtain imine 3.

[0120] Imine 3 (1 mM), imine reductase mutant SnIR-L175A / M245A (1.6 mol%), GDH (1 mol%), NADP + (10mol%), glucose (100mM) and DMSO (5% v / v) were added and incubated at 30°C for 24 hours in a 200μL reaction system containing 100mM Tris-HCl. After the reaction was completed, 30μL of the reaction mixture was diluted to 300μL with chromatography-grade methanol, and the conversion rate and de value were determined by HPLC and a standard curve. Then, the remaining reaction solution was adjusted to pH 10 with 5M sodium hydroxide solution and extracted 3 times with ethyl acetate. The organic layers were combined and concentrated in vacuo to remove the organic solvent, and the ee value of the product was analyzed by positive HPLC.

[0121] The results showed that all substrates ( Figure 6 All of the above compounds (a1) and (b1) can be converted into the corresponding products (1S,3R)-4a to (1S,3R)-4l. The specific yields, de values ​​and ee values ​​are shown in the following table.

[0122] Table 7 Yield, de and ee of 1,3-disubstituted THIQ

[0123]

[0124] Comparative Example 1: Comparison of imine reductase before and after mutation

[0125] The specific implementation method is the same as Example 5, except that a mutant Y171A was also constructed and the reaction was carried out under the same conditions as Example 4. The results showed that the activity of the single point mutation SnIR-Y171A was basically lost, which may be related to the fact that it is a key catalytic site.

[0126] Table 8 Relative enzyme activity of imine reductase mutants

[0127]

[0128] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An imine reductase mutant, characterized in that The imine reductase with the amino acid sequence as shown in SEQ ID NO.3 is used as a parent, and has one or more mutations in (a)-(f): (a) mutating the threonine at position 123 of the parent to alanine; (b) mutating the proline at position 124 of the parent to alanine; (c) mutating the leucine at position 175 of the parent to alanine; (d) mutating the methionine at position 216 of the parent to alanine; (e) mutating the parent threonine 223 to alanine; (f) Mutating the methionine at position 245 of the parent to alanine.

2. A gene encoding the imine reductase mutant according to claim 1.

3. A recombinant plasmid carrying the gene according to claim 2.

4. A recombinant microorganism expressing the imine reductase mutant according to claim 1.

5. A recombinant Escherichia coli, characterized in that The imine reductase mutant according to claim 1 is expressed by using Escherichia coli BL21 (DE3) as a host and pET-21b (+) as a vector.

6. A method for preparing the imine reductase mutant according to claim 1, characterized in that: The recombinant Escherichia coli according to claim 5 is cultured in a culture medium to induce enzyme production, and the imine reductase mutant is collected.

7. An enzyme preparation containing the imine reductase according to claim 1.

8. Use of the imine reductase mutant according to claim 1 in the synthesis of 1,3-disubstituted THIQ, characterized in that: The 1,3-disubstituted THIQ is not limited to any of the following:

9. The use according to claim 8, characterized in that: The application comprises the following steps: (1) mixing a transaminase protein with a substrate in the presence of a lactate dehydrogenase circulation system to react and prepare an amine 2; the substrate is a ketone 1, D-alanine or L-alanine, and pyridoxal phosphate; the LDH circulation system comprises lactate dehydrogenase, glucose dehydrogenase, NADH, and glucose; (2) reacting the amine 2 obtained in step (1) with an acyl chloride, collecting the reaction product, and then reacting the product with phosphorus oxychloride to obtain an imine 3; (3) using the imine 3 obtained in step (2) as a substrate, and performing a catalytic reaction with the imine reductase mutant according to claim 1 and the GDH circulation system to obtain 1,3-disubstituted THIQ; the GDH circulation system comprises GDH, NADP + ,glucose.

10. The use according to claim 9, characterized in that: The ketone 1 includes at least one of the following 1a-1f: The acyl chloride comprises at least one of the following AG: The imine comprises at least one of the following:

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