Imine reductase mutants and their application in the synthesis of sterically hindered secondary amines

Abstract

The present invention relates to an imine reductase mutant and its application in the catalytic reductive amination of a carbonyl group and a large steric hindrance amino group to synthesize a secondary amine, belonging to the field of biotechnology. The specific activity of the imine reductase mutant described in the present invention is as high as 8.1 U / mg, and it can efficiently catalyze the reductive amination of 3-(3-trifluoromethylphenyl) propionaldehyde and (R)-1-(1-naphthyl) ethylamine to synthesize cinacalcet drug. The substrate concentration in the enzymatic reaction is as high as 500 mM, the product concentration is as high as 168 g / L, and the space-time yield is 75.6 g / L / d. In addition, the imine reductase and its mutant also have reductive amination activity towards some other carbonyl compounds and large steric hindrance amino donors, and the conversion rate is as high as over 99%. The imine reductase and its mutant described in the present invention have high catalytic activity, strong stability and high selectivity, and show industrial application prospects in the synthesis of large steric hindrance secondary amines.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and particularly relates to an imine reductase PcIRED mutant derived from Penicillium camemberti strain and its encoding gene, a recombinant expression vector and a recombinant expression transformant containing the imine reductase mutant gene, a preparation method of the recombinant imine reductase mutant, and its application in the reductive amination synthesis of cinacalcet and other large steric hindrance secondary amines by using 3-(3-trifluoromethylphenyl) propionaldehyde and its analogues and (R)-1-(1-naphthyl) ethylamine and its analogues. Background Art

[0002] Amines are an important class of structural building blocks and are widely used in the synthesis of various pharmaceutical molecules. It is reported that about 40% of chiral drugs on the market contain at least one chiral amine structural unit, and these drugs cover anti-allergic drugs, antibacterial drugs, anti-tumor drugs, and cardiovascular disease treatment drugs, etc. For example, the cinacalcet drug containing an R-configuration chiral amine unit developed by NPS Pharmaceuticals is a calcium mimetic that acts on calcium ion receptors in the body and can reduce the secretion of parathyroid hormone. This drug was approved by the FDA and EMA for marketing in 2004 and is currently mainly used for the treatment of primary hyperparathyroidism, secondary hyperparathyroidism, and parathyroid carcinoma. Its structural formula is as follows:

[0003]

[0004] Structural formula of cinacalcet

[0005] Methods for chemically synthesizing chiral amines include asymmetric hydrogenation of imines or enamines, reductive amination, asymmetric hydroamination of olefins, and C-H bond amination, etc. Among them, reductive amination is a classic and practical amine synthesis method. It is reported that about 25% of carbon-nitrogen bond-forming reactions in the pharmaceutical field are achieved through reductive amination. Chemical reductive amination often has problems such as harsh reaction conditions, use of expensive transition metal catalysts, and a large amount of waste emissions. For example, Patent Application WO03066613A1 uses Pd / C as a catalyst to achieve the reductive amination reaction of formylbenzoic acid and N-methylpiperazine under the conditions of 80 °C and pressurized hydrogen (5 bar), and 10.9 g of the key precursor of the drug imatinib is obtained by crystallization method, with a yield of 70%. Patent Application WO2012007954-A1 uses sodium cyanoborohydride as a reducing agent to catalyze the reductive amination of (R)-1-(1-naphthyl) ethylamine and 3-(3-trifluoromethylphenyl) propionaldehyde (1.2 equivalents) to obtain a cinacalcet hydrochloride product, with a yield of 76–80%.

[0006] Compared with chemical methods, enzymatic reductive amination has the advantages of mild reaction conditions, simple process, environmental friendliness, etc. Amino acid dehydrogenases, amine dehydrogenases, and imine reductases (including reductive amination enzymes) have been used to catalyze intermolecular reductive amination reactions. However, the currently reported imine reductases mainly use amines with less steric hindrance as donors for reductive amination. For example, in 2017, the Turner research group reported that the reductive amination enzyme AspRedAm from Aspergillus oryzae could use amines with small steric hindrance such as methylamine, ethylamine, cyclopropylamine, and allylamine as amino donors to catalyze the reductive amination reactions of carbonyl substrates such as cyclohexanone and phenylacetaldehyde (Nat. Chem., 2017, 9, 961–969); recently, this research group reported the use of amines with small steric hindrance to catalyze the reductive amination of α-keto esters and β-keto esters to synthesize chiral amino ester products (Nat. Chem., 2021, 13, 140–148; Angew. Chem. Int. Ed. 2021, 60, 8717–8721). There are still few reports on the use of imine reductases to catalyze the reductive amination of carbonyl substrates with amino donors with large steric hindrance, especially for the synthesis of pharmaceutical intermediates or bioactive molecules. In 2019, GlaxoSmithKline (GSK) Pharmaceuticals obtained an imine reductase mutant with high reductive amination activity for the large steric hindrance amine substrate (1R,2S)-2-phenylcyclopropylamine and the precursor aldehyde substrate through screening of an imine reductase library and directed evolution of the enzyme for the lysine-specific demethylase-1 inhibitor GSK2879552, thus realizing the enzymatic synthesis of the inhibitor GSK2879552 with a product yield of 84% (Nat. Catal., 2019, 2, 909–915).

[0007] Therefore, for the efficient synthesis of structurally diverse large steric hindrance amine molecules, especially pharmaceutical intermediates or drug molecules, it is of great research significance and application value to develop enzyme catalysts with high activity for the reductive amination of carbonyl substrates and large steric hindrance amino donors. Summary of the Invention

[0008] The purpose of the present invention is to overcome the existing technical problems and deficiencies, and through screening of a natural enzyme library and protein engineering modification technology, obtain an imine reductase mutant that can efficiently catalyze the reductive amination of carbonyl substrates and large steric hindrance amino donors, and realize an enzymatic synthesis route for the reductive amination of 3-(3-trifluoromethylphenyl)propanal and its analogs with (R)-1-(1-naphthyl)ethylamine and its analogs to synthesize cinacalcet and other large steric hindrance secondary amines.

[0009] The purpose of the present invention can be achieved by the following technical solutions:

[0010] One of the technical solutions adopted by the present invention: providing an imine reductase PcIRED derived from Penicillium camemberti, which has natural reductive amination activity towards carbonyl substrates and large steric hindrance amino donors.

[0011] Using 3-(3-trifluoromethylphenyl)propanal and (R)-1-(1-naphthyl)ethylamine as substrates, the enzyme library of imine reductase stored in the laboratory was screened for activity, and it was found that the imine reductase PcIRED from Penicillium camemberti strain has relatively high natural activity towards the above reaction.

[0012] The imine reductase PcIRED has an amino acid sequence shown in SEQ ID No.2. The nucleotide sequence of the edited imine reductase PcIRED is shown in SEQ ID No.1.

[0013] Another technical solution adopted by the present invention: providing an imine reductase PcIRED mutant with significantly improved reductive amination activity towards 3-(3-trifluoromethylphenyl)propanal and (R)-1-(1-naphthyl)ethylamine.

[0014] The imine reductase PcIRED with the amino acid sequence shown in SEQ ID No.2 was mutated, and a derivative protein with a new amino acid sequence formed by replacing one or more amino acid residues among glutamine at position 244, leucine at position 180, histidine at position 250, valine at position 210, methionine at position 216, serine at position 247, serine at position 181, and cysteine at position 174 in the amino acid sequence shown in SEQ ID No.2 with other amino acid residues.

[0015] Single-site saturation mutagenesis was carried out on the imine reductase PcIRED, and the conversion rate of the mutant-catalyzed reductive amination reaction of 3-(3-trifluoromethylphenyl)propanal and (R)-1-(1-naphthyl)ethylamine was detected to screen mutants with improved conversion rates. Specifically, the imine reductase mutant is a mutant obtained by mutating the amino acid sequence of the wild-type imine reductase PcIRED in any of the following situations:

[0016] (1) Replacing glutamine at position 244 in the amino acid sequence shown in SEQ ID No.2 with alanine;

[0017] (2) Replacing glutamine at position 244 in the amino acid sequence shown in SEQ ID No.2 with alanine; replacing leucine at position 180 with valine;

[0018] (3) Replace glutamine at position 244, leucine at position 180, and histidine at position 250 in the amino acid sequence shown in SEQ ID No.2 with alanine, valine, and asparagine, respectively;

[0019] (4) Replace glutamine at position 244, leucine at position 180, histidine at position 250, and valine at position 210 in the amino acid sequence shown in SEQ ID No.2 with alanine, valine, asparagine, and isoleucine, respectively;

[0020] (5) Replace glutamine at position 244, leucine at position 180, histidine at position 250, valine at position 210, and methionine at position 216 in the amino acid sequence shown in SEQ ID No.2 with alanine, valine, asparagine, isoleucine, and threonine, respectively;

[0021] (6) Replace glutamine at position 244, leucine at position 180, histidine at position 250, valine at position 210, methionine at position 216, and serine at position 247 in the amino acid sequence shown in SEQ ID No.2 with alanine, valine, asparagine, isoleucine, threonine, and alanine, respectively;

[0022] (7) Replace glutamine at position 244, leucine at position 180, histidine at position 250, valine at position 210, methionine at position 216, serine at position 247, and serine at position 181 in the amino acid sequence shown in SEQ ID No.2 with alanine, valine, asparagine, isoleucine, threonine, alanine, and alanine, respectively;

[0023] (8) Replace glutamine at position 244, leucine at position 180, histidine at position 250, valine at position 210, methionine at position 216, serine at position 247, serine at position 181, and cysteine at position 174 in the amino acid sequence shown in SEQ ID No.2 with alanine, valine, asparagine, isoleucine, threonine, alanine, alanine, and asparagine, respectively.

[0024] The specific method for obtaining the mutant is as follows:

[0025] Using the gene sequence of wild-type imine reductase PcIRED from Penicillium camemberti as a template, a random mutation library was constructed by site-directed saturation mutagenesis strategy, and high-throughput screening was performed on each round of the constructed mutation library to obtain the above various mutants.

[0026] The full plasmid PCR amplification for constructing the site-saturated mutation library is a conventional technique in the art. The system of the PCR amplification reaction is as follows: 1.0 ng of template, 0.5 μL (10 μM) of each of a pair of mutant primers, 12.5 μL of PrimeSTAR HS (Premix), and add sterilized distilled water to 25 μL.

[0027] The procedure of the PCR amplification is as follows: (1) Denaturation at 98 °C for 10 s; (2) Denaturation at 98 °C for 10 s, (3) Annealing at 55 °C for 10 s, (4) Extension at 72 °C for 7 min. Steps (2)–(4) are carried out for 20 cycles in total, and finally, extension at 72 °C for 7 min, and preservation of the PCR amplification product at 12 °C.

[0028] Technical solution three of the present invention: Provide an isolated nucleic acid, and the nucleic acid encodes the imine reductase mutant described above.

[0029] The preparation method of the nucleic acid of the present invention is a conventional preparation method in the art. The preparation method includes: obtaining a nucleic acid molecule encoding the imine reductase mutant through gene cloning technology, or obtaining a nucleic acid molecule encoding the imine reductase mutant through the method of artificial total sequence synthesis.

[0030] The preparation method of the nucleic acid of the present invention is a conventional preparation method in the art. The preferred preparation method is: Using the mutant DNA sequence obtained in technical solution two as a template, performing PCR amplification on the desired target gene to obtain the nucleic acid molecule with point mutations.

[0031] Wherein the PCR amplification technology is a conventional technology in the art. The PCR amplification system is as follows: 1.0 ng of template, 0.5 μL (10 μM) of each of a pair of mutant primers, 12.5 μL of PrimeSTAR HS (Premix), and add sterilized distilled water to 25 μL.

[0032] The procedure of the PCR amplification is as follows: (1) Denaturation at 98 °C for 10 s; (2) Denaturation at 98 °C for 10 s, (3) Annealing at 55 °C for 10 s, (4) Extension at 72 °C for 7 min. Steps (2)–(4) are carried out for 20 cycles in total, and finally, extension at 72 °C for 7 min, and preservation of the product at 12 °C.

[0033] Technical solution four of the present invention: Provide a recombinant expression vector containing the above nucleic acid.

[0034] The recombinant expression vector can be obtained by conventional methods in the art, that is, by ligating the nucleic acid molecule of the imine reductase mutant gene of the present invention to various commercially available expression vectors. The expression vector of the present invention is preferably plasmid pET-28a(+). The recombinant expression vector of the present invention can be prepared by the following method: The nucleic acid product obtained by PCR amplification and the expression vector pET-28a are respectively double digested with restriction endonucleases Nde I and Xhol I to form complementary sticky ends, and then ligated with T4 DNA ligase to form a recombinant expression plasmid containing the imine reductase gene of the present invention.

[0035] Technical solution five of the present invention: Provide a recombinant expression transformant containing the above recombinant expression vector.

[0036] The preparation method of the recombinant expression transformant is preferably: transforming the above recombinant expression vector into a host microorganism. The host microorganism is preferably: Escherichia coli (abbreviated as E. coli), more preferably Escherichia coli BL21(DE3) or Escherichia coli DH5α. Transforming the aforementioned recombinant expression plasmid into Escherichia coli E. coli BL21(DE3) to obtain the preferred genetically engineered strain of the present invention. The plasmid transformation method can be selected from conventional methods in the art, such as electroporation, heat shock method, etc., and preferably the heat shock method is selected for transformation. Mix the plasmid solution with competent cells, heat shock at 42°C for 90 seconds, then ice bath for 3 minutes, and then resuscitate at 37°C for 1 hour. Spread on an LB agar medium plate containing kanamycin and culture to obtain the target recombinant expression transformant.

[0037] Technical solution six of the present invention: Provide a preparation method of an imine reductase mutant, including the following steps: culturing the recombinant expression transformant and obtaining the recombinant imine reductase mutant from the culture.

[0038] The preparation method of the recombinant imine reductase mutant is preferably: inoculating the recombinant Escherichia coli into an LB medium containing kanamycin (50 μg / mL), culturing overnight at 37°C with shaking, inoculating at an inoculation amount of 1% (v / v) into a 500 mL Erlenmeyer flask containing 100 mL of LB medium, and culturing with shaking in a shaker at 37°C and 180 rpm. When the absorbance OD of the culture solution 600When it reaches 0.6 - 0.8, isopropyl-β-D-thiogalactopyranoside (IPTG) with a final concentration of 0.2 mM is added for induction. The induction temperature is 25 °C. After 24 hours of induction, the culture solution is centrifuged and washed twice with physiological saline to obtain resting cells. The obtained resting cells are suspended in potassium phosphate buffer (KPB, 100 mM, pH 7.0), ultrasonically disrupted in an ice-water bath, and the supernatant is collected by centrifugation, which is the crude enzyme solution of the recombinant enzyme. The crude enzyme solution is analyzed by polyacrylamide gel electrophoresis, and the recombinant protein exists in a completely soluble form in the cells. The recombinant protein can be purified by nickel column affinity chromatography, and gradient elution with different concentrations of imidazole is used to obtain the target protein fraction with high purity.

[0039] In the present invention, the biocatalyst for catalyzing the reductive amination of 3-(3-trifluoromethylphenyl) propionaldehyde and its analogs with (R)-1-(1-naphthyl) ethylamine and its analogs to synthesize cinacalcet and other sterically hindered secondary amines can be the above-mentioned recombinant imine reductase mutant protein, or the resting cells of the recombinant expression transformant containing the recombinant imine reductase mutant or its cell lysate.

[0040] The seventh technical solution adopted in the present invention: Provide the imine reductase mutant or the whole recombinant Escherichia coli cells as a catalyst to catalyze the reductive amination of 3-(3-trifluoromethylphenyl) propionaldehyde and its analogs with (R)-1-(1-naphthyl) ethylamine and its analogs to synthesize cinacalcet and other sterically hindered secondary amines.

[0041] One of the following compounds is the 3-(3-trifluoromethylphenyl) propionaldehyde and its analogs.

[0042]

[0043] One of the following compounds is the (R)-1-(1-naphthyl) ethylamine and its analogs.

[0044]

[0045] In the application of the imine reductase mutant or the whole recombinant Escherichia coli cells of the present invention as a catalyst to catalyze the reductive amination of 3-(3-trifluoromethylphenyl) propionaldehyde and its analogs with (R)-1-(1-naphthyl) ethylamine and its analogs to synthesize cinacalcet and other sterically hindered secondary amines, using 3-(3-trifluoromethylphenyl) propionaldehyde and its analogs as carbonyl substrates and (R)-1-(1-naphthyl) ethylamine and its analogs as amino donors, in the presence of coenzyme NADPH, the imine reductase mutant or the cell lysate of the recombinant Escherichia coli cells is used to catalyze the reductive amination of 3-(3-trifluoromethylphenyl) propionaldehyde and its analogs with (R)-1-(1-naphthyl) ethylamine and its analogs to synthesize cinacalcet and other sterically hindered secondary amines, and at the same time NADPH is oxidized to NADP+ During the reaction, the glucose dehydrogenation reaction catalyzed by coupled glucose dehydrogenase reduces the coenzyme NADP + to regenerate NADPH.

[0046] The conditions of the reductive amination reaction described in the present invention can be selected according to the conventional conditions of such reactions in the art. Preferably, the application is as follows: the reaction is carried out in an aqueous phase, the concentration of the substrate is 10 - 500 mM, the addition concentration of the cosolvent DMSO is 0–20% (v / v), the dosage of the pure enzyme of the imine reductase mutant is 0.5–1 g / L, or the resting cells of the imine reductase mutant or its cell lysate form are used, and the dosage is 30–60 g wet cells / L. An additional coenzyme NADP + has a concentration of 1 mM, the concentration of the cosubstrate glucose is 30–750 mM, the addition amount of the crude enzyme powder of glucose dehydrogenase is 3 g / L, or the resting cells of glucose dehydrogenase or its cell lysate form are used, and the dosage is 30 g wet cells / L. The reaction pH is 7.0–8.0, and the reaction temperature is 20–40 °C.

[0047] The multiple imine reductase mutants described in the present invention are suitable for catalyzing the reductive amination of 3-(3-trifluoromethylphenyl) propionaldehyde and its analogs with (R)-1-(1-naphthyl) ethylamine and its analogs to synthesize cinacalcet and other sterically hindered secondary amines.

[0048] Compared with the prior art, the present invention has the following advantages and effects: The imine reductase mutant described in the present invention has high catalytic activity and thermal stability. The activity of catalyzing the reductive amination of 3-(3-trifluoromethylphenyl) propionaldehyde and (R)-1-(1-naphthyl) ethylamine is as high as 8.1 U / mg, which is much higher than the reported imine reductases. It can efficiently catalyze the reductive amination of 3-(3-trifluoromethylphenyl) propionaldehyde and (R)-1-(1-naphthyl) ethylamine to synthesize cinacalcet. The substrate concentration in the enzymatic reaction is as high as 500 mM, and the space-time yield of the product is as high as 75.6 g / L / d. It is the first reported process of synthesizing cinacalcet by imine reductase catalysis. Compared with the chemical synthesis method, the enzymatic catalytic reaction described in the present invention has the advantages of mild reaction conditions, simple and safe operation, and green process, and has good industrial application prospects. Detailed Description of the Invention

[0049] The present invention will be described in detail below with reference to specific examples.

[0050] Example 1 Screening of the Imine Reductase Enzyme Library

[0051] The recombinant E. coli BL21(DE3) strain containing imine reductase was inoculated into deep well plates for cultivation. Each well of the primary plate contained 300 μL of LB medium (containing 50 μg / mL kanamycin) and was cultured overnight at 37 °C with 800 rpm. 50 μL of the primary seed solution was pipetted and transferred to the secondary plate with each well containing 400 μL of LB medium (containing 50 μg / mL kanamycin), and cultured at 37 °C with 800 rpm for 3 - 4 h. 50 μL of 2 mM IPTG (final concentration 0.2 mM) was added to each well for induction, and the culture continued at 20 °C for 16 - 18 h. After the cultivation was completed, the deep well plates were centrifuged at 3500 rpm and 4 °C for 10 min, and the supernatant was discarded. 200 μL of lysis solution (KPB solution (100 mM, pH 7.0) containing 1.5 g / L lysozyme and 0.01 mg / L DNase I enzyme) was added to each well, and the cells were shaken thoroughly to be fully suspended and incubated at 37 °C for 1 h. 200 μL of KPB solution (100 mM, pH 7.0) was added to each well, and centrifuged at 3500 rpm and 4 °C for 15 min. Activity screening was carried out by the following method: 190 μL of the supernatant after centrifugation of the above cell lysate was taken from each well and transferred to a new deep well plate, and 1 mM NADP + , 30 mM glucose, 5% (v / v) DMSO, 3 mg / mL crude glucose dehydrogenase powder, 10 mM 3-(3-trifluoromethylphenyl) propionaldehyde and 10 mM (R)-1-(1-naphthyl) ethylamine were added. The volume of each well was made up to 400 μL with KPB solution (100 mM, pH 7.0), and the reaction was carried out at 37 °C with 800 rpm for 24 h. 240 μL of acetic acid and 320 μL of acetonitrile were added to each well to terminate the reaction, and shaken at 800 rpm for 5 min. Then the deep well plates were placed at 3500 rpm and 4 °C and centrifuged for 15 min. 200 μL was taken from each well and transferred to an enzyme-linked immunosorbent assay (ELISA) plate, and the conversion rate was analyzed by ultra performance liquid chromatography (UPLC).

[0052] The UPLC analysis method is as follows: An analysis is performed using a Shimadzu LC-20AD ultra-high pressure liquid chromatograph equipped with a Shimadzu shim-pack GIST-HP column (2.1×50 mm, 3 μm). Mobile phase A is a 0.05% trifluoroacetic acid / water solution, and mobile phase B is a 0.05% trifluoroacetic acid / acetonitrile solution. The flow rate is set at 1.5 mL / min, the column temperature is 50 °C, the detection wavelength is 220 nm, and the injection volume is 2 μL. The initial mobile phase is 90% solution A / 10% solution B, which is maintained for 0.27 min; the concentration of solution B is increased to 25% within 0.8 min; the concentration of solution B is increased to 30% within 0.53 min; the concentration of solution B is increased to 34% within 1.6 min; the concentration of solution B is increased to 95% within 0.67 min and maintained for 0.66 min; the concentration of solution B is decreased to 10% within 0.4 min and maintained for 0.67 min. Peak elution times: 3-(3-trifluoromethylphenyl)propanal 3.14 min, (R)-1-(1-naphthyl)ethylamine 1.18 min, cinacalcet 3.75 min.

[0053] Example 2 Site-directed saturation mutagenesis of imine reductase PcIRED

[0054] The site-directed saturation mutagenesis of the imine reductase PcIRED gene sequence is carried out using the whole plasmid PCR amplification technique, and the template used is the recombinant plasmid pET28a-PcIRED of imine reductase PcIRED.

[0055] The system of the PCR reaction is as follows: 1.0 ng of template, 0.5 μL of each of a pair of mutant primers (10 μM), 12.5 μL of PrimeSTAR HS (Premix), and sterile distilled water is added to make up to 25 μL.

[0056] The procedure of the PCR amplification is as follows: (1) Denaturation at 98 °C for 10 s; (2) Denaturation at 98 °C for 10 s, (3) Annealing at 55 °C for 10 s, (4) Extension at 72 °C for 7 min. Steps (2)–(4) are carried out for 20 cycles in total, and finally, extension is carried out at 72 °C for 7 min, and the product is stored at 12 °C.

[0057] After purification of the PCR amplification products, the PCR amplification products were digested with the restriction endonuclease Dpn I at 37 °C for 2 h, then transformed into E. coli BL21(DE3) competent cells, and evenly spread on an LB agar medium plate containing 50 μg / mL kanamycin, and cultured overnight at 37 °C to construct a random mutant library. Single colonies were picked into deep well plates for culture. Each well of the primary plate contained 300 μL of LB medium (containing 50 μg / mL kanamycin), and was cultured overnight at 37 °C and 800 rpm. 50 μL of the primary seed solution was aspirated and transferred to a secondary plate with 400 μL of LB medium (containing 50 μg / mL kanamycin) per well, and cultured at 37 °C and 800 rpm for 3 - 4 h. 50 μL of 2 mM IPTG (final concentration 0.2 mM) was added to each well for induction, and the culture was continued at 20 °C for 16 - 18 h. The deep well plates were centrifuged at 3500 rpm and 4 °C for 10 min, the supernatant was discarded, and 200 μL of lysis solution (KPB solution (100 mM, pH 7.0) containing 1.5 g / L lysozyme and 0.01 mg / L DNase I) was added to each well, and the cells were shaken to be fully suspended and incubated at 37 °C for 1 h. 200 μL of KPB solution (100 mM, pH 7.0) was added to each well, and centrifuged at 3500 rpm and 4 °C for 15 min. Activity screening was carried out using one of the following four methods:

[0058] (1) Take 190 μL of the supernatant after centrifugation of the above cell lysate from each well and transfer it to a new deep well plate, add 1 mM NADP + , 30 mM glucose, 5% (v / v) DMSO, 3 mg / mL crude glucose dehydrogenase powder, 10 mM 3-(3-trifluoromethylphenyl) propionaldehyde and 10 mM (R)-1-(1-naphthyl) ethylamine, and the volume of each well was made up to 400 μL with KPB solution (100 mM, pH 7.0), and reacted at 37 °C and 800 rpm for 24 h. 30 μL of 10 M sodium hydroxide solution was added to each well to terminate the reaction, and extracted with 500 μL of methyl tert-butyl ether. The organic phase was dried with anhydrous magnesium sulfate and analyzed for the conversion rate by gas chromatography.

[0059] (2) Take 190 μL of the supernatant after centrifugation of the above cell lysate from each well and transfer it to a new deep well plate, add 1 mM NADP +, 30 mM glucose, 5% (v / v) DMSO, 3 mg / mL crude glucose dehydrogenase powder, 10 mM 3-(3-trifluoromethylphenyl) propionaldehyde, and 10 mM (R)-1-(1-naphthyl) ethylamine. The volume of each well was made up to 400 μL with KPB solution (100 mM, pH 7.0), and the reaction was carried out at 37 °C and 800 rpm for 24 h. 240 μL of acetic acid and 320 μL of acetonitrile were added to each well to terminate the reaction, and the mixture was shaken at 800 rpm for 5 min. Then the deep well plate was centrifuged at 3,500 rpm and 4 °C for 15 min. 200 μL was taken from each well and transferred to an ELISA plate, and the conversion rate was analyzed by UPLC.

[0060] (3) 190 μL of the supernatant after centrifugation of the above cell lysate was taken from each well and transferred to a new deep well plate, and 1 mM NADP was added. + , 75 mM glucose, 20% (v / v) DMSO, 3 mg / mL crude glucose dehydrogenase powder, 50 mM 3-(3-trifluoromethylphenyl) propionaldehyde, and 50 mM (R)-1-(1-naphthyl) ethylamine. The volume of each well was made up to 400 μL with KPB solution (100 mM, pH 7.0), and the reaction was carried out at 37 °C and 800 rpm for 24 h. 240 μL of acetic acid and 320 μL of acetonitrile were added to each well to terminate the reaction, and the mixture was shaken at 800 rpm for 5 min. Then the deep well plate was centrifuged at 3,500 rpm and 4 °C for 15 min. 200 μL was taken from each well and transferred to an ELISA plate, and the conversion rate was analyzed by UPLC.

[0061] (4) 190 μL of the supernatant after centrifugation of the above cell lysate was taken from each well and transferred to a new deep well plate, and 1 mM NADP was added. + , 150 mM glucose, 30% (v / v) DMSO, 3 mg / mL crude glucose dehydrogenase powder, 100 mM 3-(3-trifluoromethylphenyl) propionaldehyde, and 100 mM (R)-1-(1-naphthyl) ethylamine. The volume of each well was made up to 400 μL with KPB solution (100 mM, pH 7.0), and the reaction was carried out at 37 °C and 800 rpm for 24 h. 240 μL of acetic acid and 560 μL of acetonitrile were added to each well to terminate the reaction, and the mixture was shaken at 800 rpm for 5 min. Then the deep well plate was centrifuged at 3,500 rpm and 4 °C for 15 min. 200 μL was taken from each well and transferred to an ELISA plate, and the conversion rate was analyzed by UPLC.

[0062] The UPLC analysis method was the same as described before.

[0063] Example 3 Expression, purification and enzyme activity determination of imine reductase PcIRED and its mutants

[0064] Pick a single colony of the recombinant expression of the imine reductase mutant growing on the plate and inoculate it into a 4 mL LB test tube containing kanamycin (50 μg / mL). Place it in a shaker at 37°C and culture it at 200 rpm for 12 - 16 h. Then transfer the bacterial solution to 100 mL of LB medium containing 50 ng / μL kanamycin at an inoculation ratio of 1:100. Place it in a shaker at 37°C and culture it at 200 rpm for 3 h. Wait until its OD 600 reaches 0.6 - 0.8, add 20 μL of 1 M IPTG (final concentration 0.2 mM) to induce the expression of the target protein. Culture it at 25°C and 200 rpm for 12 - 13 h, then centrifuge to collect the bacterial cells. Suspend the obtained resting cells in KPB buffer (100 mM, pH 7.0), ultrasonically disrupt them in an ice-water bath, centrifuge to collect the supernatant, and obtain the cell lysate of the recombinant imine reductase mutant. Freeze-dry the cell lysate to obtain the crude enzyme powder.

[0065] Purify the imine reductase mutant protein using Ni column affinity chromatography. The specific method is as follows:

[0066] (1) Equilibrate the affinity Ni column (packing volume 5 ml) with Tris-HCl buffer (50 mM, pH 7.5, containing 300 mM NaCl, 20 mM imidazole);

[0067] (2) Resuspend the obtained resting cells with Tris-HCl buffer (50 mM, pH 7.5, containing 300 mM NaCl, 20 mM imidazole), ultrasonically disrupt them, centrifuge at 8000 rpm and 4°C for 45 min, collect the supernatant, and pass it through the Ni column at a flow rate of 1 mL / min to bind the target protein to the Ni column;

[0068] (3) Elute the heteroproteins that have no binding ability to the Ni column with Tris-HCl buffer (50 mM, pH 7.5, containing 300 mM NaCl) containing 20 - 70 mM imidazole;

[0069] (4) Then elute the target protein with Tris-HCl buffer (50 mM, pH 7.5, containing 300 mM NaCl) containing 200 - 300 mM imidazole;

[0070] (5) Detect the protein sample collected by SDS-PAGE electrophoresis. Take 20 μL of the sample and add 5 μL of 5× SDS loading buffer, perform heat treatment, and the electrophoresis loading volume is 10 μL. The standard molecular weight protein (Protein Molecular Weight Marker) is purchased from Thermo Company, USA. The concentration of the SDS-PAGE gel is 12%, select a voltage of 90 V for concentration, and then change to a voltage of 120 V for separation.

[0071] The activity assay system is a 400 μL KPB solution (100 mM, pH 7.0) containing 10 mM 3-(3-trifluoromethylphenyl) propionaldehyde, 10 mM (R)-1-(1-naphthyl) ethylamine, 1 mM NADP + , 30 mM glucose, 25% (v / v) DMSO, 0.2 mg / mL pure imine reductase, and 3 mg / mL crude glucose dehydrogenase powder. The reaction was carried out at 37 °C and 800 rpm, and the conversion rate at the initial stage of the reaction was measured. 30 μL of 10 M sodium hydroxide solution was added to each well to terminate the reaction, and extraction was performed with 500 μL of methyl tert-butyl ether. The organic phase was dried with anhydrous magnesium sulfate and the conversion rate was analyzed by gas chromatography. The specific enzyme activity was calculated based on the conversion rate. The results are shown in Table 1.

[0072] Table 1 Enzyme activity of PcIRED and its mutants

[0073]

[0074] Example 4 Reductive amination reaction of ketones with bulky amino donors catalyzed by PcIRED and its mutants

[0075] The conversion effects of PcIRED and its mutants catalyzing the reductive amination of different ketone substrates 2–10 with bulky amino substrates b–p were determined. The reaction was carried out according to one of the following five reaction systems. System 1: A reaction system of KPB buffer (100 mM, pH 7.0) with a total volume of 500 μL containing 10 mM ketone substrate, 2 equivalents of amino donor, 1 mM NADP + , 30 mM glucose, 20% (v / v) DMSO, 0.5 mg / mL pure imine reductase (PcIRED or its mutant), and 3 mg / mL crude glucose dehydrogenase. System 2: A reaction system of KPB buffer (100 mM, pH 7.0) with a total volume of 500 μL containing 10 mM ketone substrate, 1 equivalent of amino donor, 1 mM NADP + , 30 mM glucose, 20% (v / v) DMSO, 1.0 mg / mL pure imine reductase (PcIRED or its mutant), and 3 mg / mL crude glucose dehydrogenase. System 3: A reaction system of KPB buffer (100 mM, pH 7.0) with a total volume of 500 μL containing 10 mM ketone substrate, 2 equivalents of amino donor, 1 mM NADP + , 30 mM glucose, 20% (v / v) DMSO, 1.0 mg / mL pure imine reductase (PcIRED or its mutant), and 3 mg / mL crude glucose dehydrogenase. System 4: A reaction system of KPB buffer (100 mM, pH 7.0) with a total volume of 500 μL containing 10 mM ketone substrate, 5 equivalents of amino donor, 1 mM NADP +, a reaction system of KPB buffer (100 mM, pH 7.0) with a total volume of 500 μL containing 30 mM glucose, 20% (v / v) DMSO, 0.5 mg / mL pure imine reductase (PcIRED or its mutant), and 3 mg / mL crude glucose dehydrogenase. System Five: containing 10 mM keto substrate, 5-fold equivalent amino donor, 1 mM NADP + , a reaction system of KPB buffer (100 mM, pH 7.0) with a total volume of 500 μL containing 30 mM glucose, 20% (v / v) DMSO, 1.0 mg / mL pure imine reductase (PcIRED or its mutant), and 3 mg / mL crude glucose dehydrogenase. The reaction system was placed in an oscillator and reacted at 30 °C and 800 rpm for 24 h. Then, 40 μL of 10 M sodium hydroxide solution was added to terminate the reaction, and it was extracted with 600 μL of methyl tert-butyl ether. The organic phase was dried with anhydrous magnesium sulfate and analyzed by gas chromatography. The gas chromatography analysis method is shown in Table 2, and the reaction results are shown in Tables 3 and 4.

[0076] Table 2 Gas Chromatography Analysis Method for the Reaction of Keto Substrate and Amino Donor

[0077]

[0078]

[0079]

[0080]

[0081] Table 3 Reductive Amination Reactions of Cyclohexanone or Six-Membered Heterocyclic Ketone with Large-Steric-Hindrance Amino Substrates Catalyzed by PcIRED and Its Mutants (Reaction System One)

[0082]

[0083]

[0084] Table 4 Reductive Amination Reactions of Several Large-Steric-Hindrance Keto Substrates with Large-Steric-Hindrance Amino Substrates Catalyzed by PcIRED and Its Mutants (Reaction Systems Two to Five)

[0085]

[0086]

[0087] Example 5 Reductive Amination Reactions of Various Aldehyde Substrates with Large-Steric-Hindrance Amino Donors Catalyzed by PcIRED and Its Mutants

[0088] The conversion effects of PcIRED and its mutants in catalyzing the reductive amination of different aldehyde substrates with large steric hindrance amino substrates were determined. The reaction was carried out according to the following reaction system: a reaction system of KPB buffer (100 mM, pH 7.0) with a total volume of 500 μL containing 10 mM ketone substrate, 1 equivalent of amino donor, 1 mM NADP + 、30 mM glucose, 20% (v / v) DMSO, 0.5 mg / mL imine reductase pure enzyme (PcIRED or its mutant), and 3 mg / mL glucose dehydrogenase crude enzyme. The reaction system was placed in an oscillator and reacted at 30 °C and 800 rpm for 24 h. Then, 40 μL of 10 M sodium hydroxide solution was added to terminate the reaction, and it was extracted with 600 μL of methyl tert-butyl ether. The organic phase was dried with anhydrous magnesium sulfate and analyzed by gas chromatography. The gas chromatography analysis method is shown in Table 5, and the reaction results are shown in Table 6.

[0089] Table 5 Gas Chromatography Analysis Method for the Reaction of Aldehyde Substrate and Amino Donor

[0090]

[0091]

[0092]

[0093] Table 6 Reductive Amination Reactions of Several Aldehyde Substrates with Large Steric Hindrance Amino Substrates Catalyzed by PcIRED and Its Mutants

[0094]

[0095]

[0096] Example 6 Enzymatic Preparation and Synthesis of Cinacalcet

[0097] Prepare a solution containing 500 mM (R)-1-(1-naphthyl)ethylamine, 600 mM 3-(3-trifluoromethylphenyl)propanal, 1 mM NADP +, A reaction system with a total volume of 100 mL of KPB buffer (100 mM, pH 7.0) consisting of 750 mM glucose, 20% (v / v) DMSO, the cell disruption supernatant of 6 g of the imine reductase mutant PcIRED-M3 (Q244A / L180V / H250N / V210I / M216T / S247A / S181A / C174N) wet cells and the cell disruption supernatant of 3 g of glucose dehydrogenase wet cells was reacted at 30 °C in a water bath under mechanical stirring at 200 rpm for 48 h. Then 10 mL of 10 M sodium hydroxide solution was added to terminate the reaction, and it was extracted four times with methyl tert-butyl ether (200 mL each time). The extractants were combined, dried with anhydrous magnesium sulfate, and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography (eluent: DCM / MeOH 200:1–80:1) to obtain 15.1 g of cinacalcet product as a yellow oil, and the separation yield was 85%.

[0098] The NMR spectrum results of the enzymatically synthesized product cinacalcet are as follows: 1 H NMR(CDCl3,400MHz):δppm=8.18(d,J=8.2Hz,1H),7.88(dd,J=7.6,1.8Hz,1H),7.75(d,J=8.2Hz,1H),7.65(d,J=7.1Hz,1H),7.56-7.38(m,5H),7.38-7.26(m,2H),4.63(q,J=6.6Hz,1H),2.79-2.54(m,4H),1.84(quint,J=7.5Hz,2H),1.50(t,J=6.6Hz,3H). 13 C NMR(CDCl3,101MHz):δppm=143.2,141.3,134.1,131.9,131.4,130.61(q,J=31.9Hz),129.1,128.7,127.3,125.84,125.78,125.4,125.1(q,J=3.6Hz),124.4(q,J=272.3Hz),123.0,122.71,122.70(q,J=3.9Hz),53.8,47.3,33.5,32.0,23.7.

[0099] The above description of the embodiments is for the convenience of those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative labor. Therefore, the present invention is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art without departing from the scope of the present invention according to the disclosure of the present invention should be within the protection scope of the present invention.

Claims

1. An imine reductase mutant, characterized in that, The imine reductase mutant is selected from Pc IRED-M1, Pc IRED-M2 or Pc IRED-M3. Pc The amino acid sequence of IRED-M1 is: glutamine at position 244 of the amino acid sequence shown in SEQ ID No. 2 is replaced with alanine, and leucine at position 180 is replaced with valine; Pc The amino acid sequence of IRED-M2 is as follows: Glutamine at position 244 of the amino acid sequence shown in SEQ ID No. 2 is replaced with alanine, leucine at position 180 is replaced with valine, histidine at position 250 is replaced with asparagine, and valine at position 210 is replaced with isoleucine; Pc The amino acid sequence of IRED-M3 is as follows: glutamine at position 244 of the amino acid sequence shown in SEQ ID No. 2 is replaced with alanine; leucine at position 180 is replaced with valine; histidine at position 250 is replaced with asparagine; valine at position 210 is replaced with isoleucine; methionine at position 216 is replaced with threonine; serine at position 247 is replaced with alanine; serine at position 181 is replaced with alanine; cysteine at position 174 is replaced with asparagine.

2. An isolated nucleic acid, characterized in that: The nucleic acid encodes the imine reductase mutant as described in claim 1.

3. A recombinant expression vector comprising the nucleic acid as described in claim 2.

4. A recombinant expression transformant bacterium comprising the recombinant expression vector as described in claim 3.

5. Use of the imine reductase mutant according to claim 1 in the reductive amination of a carbonyl substrate and an amino donor to synthesize a secondary amine, characterized in that: In the presence of coenzyme NADPH, an imine reductase mutant is used to catalyze the reductive amination of a carbonyl substrate and an amino donor to prepare a secondary amine, while NADPH is oxidized to generate NADP + ; The carbonyl substrate is selected as 3-(3-trifluoromethylphenyl)propanal, and the amino donor is selected as ( R )-1-(1-naphthyl)ethylamine. At this time, the secondary amine is cinacalcet.

6. Use of the imine reductase mutant according to claim 1 in the reductive amination of a carbonyl substrate and an amino donor to synthesize a secondary amine, characterized in that: In the presence of coenzyme NADPH, an imine reductase mutant is used to catalyze the reductive amination of a carbonyl substrate and an amino donor to prepare a secondary amine, while NADPH is oxidized to generate NADP + ; The imine reductase mutant is selected from Pc IRED-M1 or Pc IRED-M2. At this time, The carbonyl substrate is selected as compound 11, and the amino donor is selected as compound q or compound a, or, The carbonyl substrate is selected as compound 1, and the amino donor is selected as compound q, compound a, compound n, compound o, or, The carbonyl substrate is selected as compound 13, and the amino donor is selected as compound s, or, The carbonyl substrate is selected as compound 14, and the amino donor is selected as compound l; The structures of compounds 1, 11, 13, and 14 are shown as follows: The structures of compounds a, n, o, q, s, and l are shown as follows: 。 7. Use of the imine reductase mutant according to claim 1 in the reductive amination of a carbonyl substrate and an amino donor to synthesize a secondary amine, characterized in that: In the presence of coenzyme NADPH, an imine reductase mutant is used to catalyze the reductive amination of a carbonyl substrate and an amino donor to prepare a secondary amine, while NADPH is oxidized to generate NADP + ; The imine reductase mutant is selected from Pc IRED-M3, at this time, The carbonyl substrate is selected as compound 2, and the amino donor is selected as compound l or compound p, or, The carbonyl substrate is selected as compound 5, 6, or 7, and the amino donor is selected as compound g, or, The carbonyl substrate is selected as compound 8, and the amino donor is selected as compound g, or, The carbonyl substrate is selected as compound 9, and the amino donor is selected as compound c, f, g, j, k, n, o, or, The carbonyl substrate is selected as compound 10, and the amino donor is selected as compound g, n, or o, or, The carbonyl substrate is selected as compound 11, and the amino donor is selected as compound q or compound a, or, The carbonyl substrate is selected as compound 1, and the amino donor is selected as compound q, compound a, compound n, compound o, or, The carbonyl substrate is selected as compound 13, and the amino donor is selected as compound s, or, The carbonyl substrate is selected as compound 14, and the amino donor is selected as compound l; The structures of compounds 1, 2, 5, 6, 7, 8, 9, 10, 11, 13, and 14 are shown as follows: The structures of compounds a, c, f, g, j, k, l, n, o, p, q, and s are shown as follows: 。

Images