A kind of aminotransferase mutant derived from Bacillus megaterium and its application

Abstract

The present invention discloses an aminotransferase mutant derived from Bacillus megaterium and its application. Wherein, the mutation site of the aminotransferase mutant is selected from one or more of the 59th, 60th, 144th, 161st, 164th, 169th, 174th, 240th, 244th, 327th, 330th, 331st, 373rd, 387th, 410th, 436th, 437th and 442nd positions of the amino acid sequence shown in SEQ ID NO: 1. The aminotransferase mutant of the present invention can catalyze the synthesis of cyclohexanone compounds into trans-aminocyclohexyl compounds, and the yield is greater than 80%, the purity is greater than 98.0%, and the proportion of trans-aminocyclohexyl compounds in the product is greater than 90%, and it has high industrial application value.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, and particularly relates to an aminotransferase mutant derived from Bacillus megaterium and application thereof. Background Art

[0002] Trans-aminocyclohexyl compounds are an important precursor for drug synthesis and an important building block for many innovative chemical drugs in the clinical stage. The previous synthesis method was to start with cyclohexanone compounds, form cis / trans mixed aminocyclohexyl compounds through chemical catalysis, and then remove the unwanted cis configuration through one or more recrystallizations. This method has many production steps and low yields (usually less than 50%). The reaction uses non-recyclable precious metals such as palladium, platinum, rhodium, and ruthenium as catalysts, which are costly, or heavy metals such as nickel as catalysts, which will pollute the environment. In addition, high-pressure hydrogen needs to be used in a closed reaction device during the process, which poses a high safety risk.

[0003] Compared with chemical methods, the enzyme catalysis method has the characteristics of good specificity, high purity, mild reaction and low cost. It is a new green process that can replace the above chemical methods. The natural aminotransferases that are currently being studied and applied more are UniProt IDF7J696 (Biotechnol Lett, 2003, 25: 1843-1846), UniProt: F2XBU9 (Biotechnol Bioeng, 1999, 65, 206-211), UniProt ID: Q0C8G1 (Nat Chem Biol, 2010, 6: 807-813.), GenBank Accession No. KJ496414.1 (Appl Microbiol Biotechnol, 2014, 98 (17): 7399-7408), and UniProt ID: Q7NWG4 (Enzyme Microb Tech, 2007, 41: 628-637), but the stereospecificity for the above reactions is still not high enough. The stereoselectivity of the aminotransferase from Arthrobater Sp. Knk168 (UniProt ID: F7J696) to this reaction is about 66%, while the stereoselectivity of the aminotransferase from Vibro fluvialis JS17 (UniProt: F2XBU9) to this reaction is only about 1%. Summary of the invention

[0004] The technical problem to be solved by the present invention is to solve the defect of low catalytic activity of enzymes in the prior art for catalyzing cyclohexanone compounds to synthesize trans-aminocyclohexane compounds. Therefore, the present invention screens out an aminotransferase from Bacillus megaterium with good stereoselectivity for the amino activity of cyclohexyl ketone compounds from currently known natural aminotransferases, and obtains several mutants with higher stereoselectivity and better catalytic activity through directional transformation. On this basis, a synthetic process for preparing trans-aminocyclohexane compounds by enzyme-catalyzed reaction is established.

[0005] The present invention provides an aminotransferase mutant derived from Bacillus megaterium, wherein the mutation site of the aminotransferase mutant is selected from one or more of positions 59, 60, 144, 161, 164, 169, 174, 240, 244, 327, 330, 331, 373, 387, 410, 436, 437 and 442 of the amino acid sequence shown in SEQ ID NO: 1.

[0006] In the present invention, preferably, the mutation site of the aminotransferase mutant is selected from one or more of positions 60, 144, 164, 169, 387, 410, 436, 437 and 442 of the amino acid sequence as shown in SEQ ID NO:1.

[0007] In the present invention, the amino acid sequence of the aminotransferase is as shown in SEQ ID NO: 1 or the aminotransferase of Bacillus megaterium (GI No. 1048348995).

[0008] In the present invention, the mutation site of the aminotransferase mutant is the 59th, 60th, 161st, 164th, 169th, 174th, 240th, 244th, 327th, 330th, 331st, 373rd, 387th, 410th, 436th, 437th or 442nd of the amino acid sequence as shown in SEQ ID NO:1; preferably, the mutation site of the aminotransferase mutant is the 60th, 387th, 436th or 442nd of the amino acid sequence as shown in SEQ ID NO:1.

[0009] In the present invention, when the aminotransferase mutant mutates at multiple sites in the amino acid sequence as shown in SEQ ID NO: 1, the multiple sites are 2, 3, 4, 5, 6, 7, 8, 9, 10 or 12; preferably, the multiple sites are 2, 3 or 5.

[0010] In the present invention, the mutation site of the aminotransferase mutant is selected from SEQ ID 161 and 327, 161 and 330, 164 and 169, 174, 240, 244, 327, 330, 331, 373, 387, 410, 436, 437 and 442 of the amino acid sequence shown in NO:1; for example, at position 59 and 164, position 59 and 174, position 59 and 164, 60 and 436, 60 and 442, 144 and 437, 161 and 327, 161 and 330, 164 and 169, 164 and 240, 164 and 437, 169 and 240, 169 and 436, 169 and 437, 174 and 244, 174 and 331, 174 and 410, 240 and 410, 240 and 437, 244 and 436, 244 and 437, 327 and 373, 327 and 436, 327 and 437, 330 and 373, 330 and 437, 387 and 436, 387 and 437, 410 and 436 or 437 and 442 are mutated; preferably, the mutation site of the aminotransferase mutant is selected from SEQ Two of positions 60, 164, 169, 410, 437 and 442 of the amino acid sequence shown in ID NO:1.

[0011] In the present invention, the mutation sites of the aminotransferase mutant are selected from 3 of positions 59, 60, 144, 161, 164, 169, 174, 240, 244, 327, 330, 373, 387, 410, 436, 437 and 442 of the amino acid sequence shown in SEQ ID NO: 1.

[0012] In the present invention, the mutation sites of the aminotransferase mutant are selected from 4 of positions 59, 60, 144, 161, 164, 169, 174, 240, 244, 327, 330, 373, 387, 410, 436, 437 and 442 of the amino acid sequence shown in SEQ ID NO: 1.

[0013] In the present invention, the mutation site of the aminotransferase mutant is selected from 5 of positions 59, 60, 144, 161, 164, 169, 174, 240, 244, 327, 373, 387, 410, 436, 437 and 442 of the amino acid sequence as shown in SEQ ID NO: 1; preferably, the mutation site of the aminotransferase mutant is positions 60, 144, 164, 169 and 442 of the amino acid sequence as shown in SEQ ID NO: 1.

[0014] In the present invention, the mutation sites of the aminotransferase mutant are selected from 6 of positions 59, 60, 161, 164, 169, 174, 240, 244, 327, 373, 387, 410, 436, 437 and 442 of the amino acid sequence shown in SEQ ID NO: 1.

[0015] In the present invention, the mutation sites of the aminotransferase mutant are selected from 7 of positions 144, 161, 164, 169, 174, 240, 244, 327, 330, 331, 373, 387, 410, 436, 437 or 442 of the amino acid sequence shown in SEQ ID NO: 1.

[0016] In the present invention, the mutation sites of the aminotransferase mutant are selected from 8 of positions 59, 60, 144, 161, 164, 174, 240, 244, 327, 330, 331, 373, 387, 410, 437 and 442 of the amino acid sequence shown in SEQ ID NO: 1.

[0017] In the present invention, when the mutation site of the aminotransferase mutant is selected from 9 of positions 60, 161, 164, 174, 240, 244, 327, 331, 373, 387, 410, 436 and 437 of the amino acid sequence shown in SEQ ID NO: 1.

[0018] In the present invention, the mutation site of the aminotransferase mutant is selected from 10 of positions 60, 169, 174, 240, 244, 327, 331, 373, 387, 410, 436, 437 and 442 of the amino acid sequence as shown in SEQ ID NO: 1; for example, "position 60, 240, 244, 327, 331, 373, 387, 410, 437 and 442" or "position 60, 169, 174, 240, 244, 327, 373, 387, 410, 436 and 442".

[0019] In the present invention, the mutation site of the aminotransferase mutant is selected from SEQ ID 11 of the 59th, 161st, 164th, 169th, 174th, 240th, 244th, 327th, 330th, 373rd, 387th, 410th, 436th, and 442nd of the amino acid sequence shown in NO:1; for example, “the 59th, 161st, 164th, 169th, 174th, 240th, 244th, 327th, 330th, 373rd, 387th, 410th, and 442nd position” or “the 59th, 169th, 174th, 240th, 244th, 327th, 330th, 373rd, 387th, 410th, and 436th position”.

[0020] In the present invention, the mutation sites of the aminotransferase mutant are positions 59, 147, 161, 164, 174, 240, 244, 327, 331, 373, 387, 410 and 436 of the amino acid sequence shown in SEQ ID NO: 1.

[0021] In the present invention, the aminotransferase mutant is an amino acid sequence as shown in SEQ ID NO: 1, in which the amino acid residue at position 59 is mutated from L to V, P, Q, R, C, F, S, D, M, K or T; preferably, the amino acid residue at position 59 is mutated from L to V.

[0022] In the present invention, the aminotransferase mutant is an amino acid sequence as shown in SEQ ID NO: 1, in which the amino acid residue at position 60 is mutated from Y to C, T, H, D, A, F, W, L, V, I, P, S, N, M or K; preferably, the amino acid residue at position 60 is mutated from Y to F, V or N.

[0023] In the present invention, the aminotransferase mutant is an amino acid sequence as shown in SEQ ID NO: 1, in which the amino acid residue at position 144 is mutated from R to A, K, G, D, E, W, F or T; preferably, the amino acid residue at position 144 is mutated from R to G.

[0024] In the present invention, the aminotransferase mutant is a mutant in which the amino acid residue at position 161 of the amino acid sequence shown in SEQ ID NO: 1 is mutated from L to C, G, K, Q, D, A or F.

[0025] In the present invention, the aminotransferase mutant is an amino acid sequence as shown in SEQ ID NO: 1, in which the amino acid residue at position 164 is mutated from Y to A, C, G, I, R, F, S, P or D; preferably, the amino acid residue at position 164 is mutated from Y to C or G.

[0026] In the present invention, the aminotransferase mutant is an amino acid sequence as shown in SEQ ID NO: 1, in which the amino acid residue at position 169 is mutated from V to L, F, A, P, H, D, S, R or G; preferably, the amino acid residue at position 169 is mutated from V to L or H.

[0027] In the present invention, the aminotransferase mutant is an amino acid sequence as shown in SEQ ID NO: 1, in which the amino acid residue at position 174 is mutated from E to C, H, S, T, A, W, R, F, G or Y; preferably, the amino acid residue at position 174 is mutated from E to G.

[0028] In the present invention, the aminotransferase mutant is an amino acid sequence as shown in SEQ ID NO: 1, in which the amino acid residue at position 240 is mutated from Q to I, D, R, G, H, K, W, C, T, F, A or P; preferably, the amino acid residue at position 240 is mutated from Q to F.

[0029] In the present invention, the aminotransferase mutant is an amino acid sequence as shown in SEQ ID NO: 1, in which the 244th amino acid residue is mutated from S to Q, L, F, K, A, P, W, R, Y, G, I, H, T or D.

[0030] In the present invention, the aminotransferase mutant is an amino acid sequence as shown in SEQ ID NO: 1, in which the amino acid residue at position 327 is mutated from S to H, K, G, D, A, T, Y, V, R, F, P or I; preferably, the amino acid residue at position 327 is mutated from S to H.

[0031] In the present invention, the aminotransferase mutant is an amino acid sequence as shown in SEQ ID NO: 1, in which the amino acid residue at position 330 is mutated from T to K, P, I, W, S, P, A, H, F or G.

[0032] In the present invention, the aminotransferase mutant is an amino acid sequence as shown in SEQ ID NO: 1, in which the amino acid residue at position 331 is mutated from Y to D, S, W, Q, R, P, I or H; preferably, the amino acid residue at position 331 is mutated from Y to H.

[0033] In the present invention, the aminotransferase mutant is an amino acid sequence as shown in SEQ ID NO: 1, in which the amino acid residue at position 373 is mutated from E to Q, C, D, W, P, G, T, Y or I; preferably, the amino acid residue at position 373 is mutated from E to R.

[0034] In the present invention, the aminotransferase mutant is an amino acid sequence as shown in SEQ ID NO: 1, in which the amino acid residue at position 387 is mutated from L to T, R, A, S, D, F, Q, P or H; preferably, the amino acid residue at position 387 is mutated from L to A, D or Q.

[0035] In the present invention, the aminotransferase mutant is an amino acid sequence as shown in SEQ ID NO: 1, in which the amino acid residue at position 410 is mutated from H to L, G, Q, R, W, P, D, T, S, F, Y or I; preferably, the amino acid residue at position 410 is mutated from H to L, R or Y.

[0036] In the present invention, the aminotransferase mutant is an amino acid sequence as shown in SEQ ID NO: 1, in which the amino acid residue at position 436 is mutated from V to P, F, C, Q, S, W, G, Y or A; preferably, the amino acid residue at position 436 is mutated from V to P or C.

[0037] In the present invention, the aminotransferase mutant is an amino acid sequence as shown in SEQ ID NO: 1, in which the amino acid residue at position 437 is mutated from M to F, D, A, P, K, G, L, H, R, T, V, Q, F or C; preferably, the amino acid residue at position 437 is mutated from M to A.

[0038] In the present invention, the aminotransferase mutant is an amino acid sequence as shown in SEQ ID NO: 1, in which the amino acid residue at position 442 is mutated from R to G, I, D, W, K, S, A, Q, V, T, N, L, H or Y; preferably, the amino acid residue at position 436 is mutated from R to K or I.

[0039] In the present invention, the mutation positions and types of the aminotransferase mutants are shown in the following table: (as shown in Table 1);

[0040] Table 1

[0041]

[0042]

[0043]

[0044]

[0045]

[0046]

[0047]

[0048] Preferably, the aminotransferase mutant is mutant 48, mutant 28, mutant 29, mutant 46, mutant 83, mutant 183, mutant 110, mutant 64, mutant 144, mutant 171, mutant 33, mutant 29 or mutant 5.

[0049] The present invention also provides a use of the aminotransferase mutant in preparing trans-aminocyclohexyl compounds.

[0050] The present invention also provides a method for preparing a trans-aminocyclohexyl compound, comprising the following steps: in the presence of an aminotransferase, subjecting a cyclohexyl ketone compound as shown in formula I and an amino donor to an amination reaction as shown below, to obtain a trans-aminocyclohexyl compound as shown in formula II;

[0051]

[0052] Where R is -COOR 1 or -CN; R 1 is hydrogen or C1-C6 alkyl; the enzyme is an aminotransferase derived from Bacillus megaterium with an amino acid sequence as shown in SEQ ID NO: 1 and / or the aminotransferase mutant as described above.

[0053] In a certain embodiment, the definitions of certain conditions in the preparation method may be as follows, and the definitions of other conditions may be as described in any of the above embodiments (hereinafter referred to as "in a certain embodiment"): When the R 1 When it is a C1~C6 alkyl group, the C1~C6 alkyl group may be a C1~C4 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl or tert-butyl, and also such as methyl or ethyl.

[0054] In one embodiment, R is -COOH, -COOMe, -COOEt or -CN.

[0055] In a certain embodiment, the amino donor is a conventional amino donor in the art. Preferably, the amino donor is one or more of isopropylamine, L-alanine, DL-alanine, L-lysine, DL-lysine, (S)-1-phenylethylamine, (R)-1-phenylethylamine, (RS)-1-phenylethylamine, pentamethylenediamine, o-phenylenediamine, o-phenylenediamine hydrochloride, 2-(4-nitrobenzene)ethylamine, and 2-(4-nitrobenzene)ethylamine hydrochloride; more preferably, the amino donor is isopropylamine.

[0056] In one embodiment, the molar ratio of the amino donor to the cyclohexyl ketone compound as shown in Formula I is a conventional molar ratio in the art; preferably (1-4):1, such as 1:1, 2:1, 3:1 or 4:1.

[0057] In a certain embodiment, the reaction solvent of the amination reaction is a buffer solution, or a mixture of a buffer solution and a conventional organic solvent in the art; the organic solvent is methyl tert-butyl ether, isopropyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, dimethyl sulfoxide, N,N-dimethylformamide, methanol, ethanol, isopropanol or n-butanol; preferably, the reaction solvent of the amination reaction is a buffer solution.

[0058] In a certain embodiment, when the reaction solvent of the amination reaction is a buffer solution and a conventional organic solvent in the art, the ratio of the organic solvent to the total reaction volume is 0 to 250 mL / L.

[0059] In a certain embodiment, the buffer is a conventional buffer in the art, preferably one or more of phosphate buffered saline (PBS), tris(hydroxymethyl)methylamine salt buffer (TRIS) and triethanolamine salt buffer (TOEA); more preferably phosphate buffer, tris(hydroxymethyl)methylamine salt buffer or triethanolamine salt buffer.

[0060] In a certain embodiment, the volume-to-mass ratio of the buffer solution to the cyclohexyl ketone compound as shown in Formula I is a conventional volume-to-mass ratio in the art; preferably 5 to 100 mL / g.

[0061] In a certain embodiment, the pH value of the amination reaction is a conventional pH value in the art, preferably 6.0 to 9.0; more preferably 7.0 to 9.0; for example 7.0, 7.5 or 9.0.

[0062] In one embodiment, the reaction temperature of the amination reaction is a conventional reaction temperature in the art, preferably 25°C to 40°C; more preferably 30°C.

[0063] In one embodiment, the reaction time of the amination reaction is the conventional reaction time in the art, which is usually related to the reaction scale, and is preferably 16 to 20 hours; for example, 16 hours, 18 hours or 20 hours.

[0064] In a certain embodiment, the method for preparing the trans-aminocyclohexyl compound comprises the following steps: adding the cyclohexyl ketone compound as shown in formula I and the aminotransferase to the mixed solution of the buffer solution and the amino donor, mixing evenly and reacting at 25°C to 40°C.

[0065] In one embodiment, after the amination reaction is completed, the following post-treatment steps are further included: adjusting the pH of the reaction solution to 4 with an acid, adding an ether solvent for extraction and separation, adding an ether solvent to the aqueous phase and adjusting the pH to 13 with a base, and drying the organic phase to remove the ether solvent.

[0066] In a certain embodiment, the acid is a conventional acid in the art, preferably hydrochloric acid.

[0067] In a certain embodiment, the ether solvent is a conventional ether solvent in the art, preferably tert-butyl methyl ether.

[0068] In a certain embodiment, the drying is carried out by a conventional drying method in the art, preferably drying with a desiccant; the desiccant is a conventional desiccant in the art, preferably anhydrous sodium sulfate.

[0069] In a certain embodiment, the base is a conventional base in the art, preferably sodium hydroxide or potassium hydroxide, such as potassium hydroxide.

[0070] In one embodiment, the method for removing the ether solvent is conventional in the art, preferably vacuum distillation, such as vacuum distillation at a pressure of -0.09 MPa.

[0071] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0072] The reagents and raw materials used in the present invention are commercially available.

[0073] The amino acid abbreviations in the present invention are conventional in the art unless otherwise specified, and the amino acids corresponding to the specific abbreviations are shown in Table 2.

[0074] Table 2

[0075]

[0076]

[0077] The positive and progressive effects of the present invention are:

[0078] The present invention uses genetic engineering to direct evolution of an aminotransferase to obtain several mutants with higher activity and stereospecificity. Based on the aminotransferase mutants, the present invention develops a reaction process for synthesizing trans-aminocyclohexyl compounds from cyclohexanone compounds in one step, with a yield of more than 80%, a purity of more than 98.0%, and a proportion of trans-aminocyclohexyl compounds in the product of more than 90%. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 This is a gas chromatogram of the reaction liquid in Example 6;

[0080] Figure 2 is the gas chromatogram of cyclohexanonecarboxylic acid ethyl ester standard;

[0081] Figure 3 is the gas chromatogram of trans-p-aminocyclohexylcarboxylic acid ethyl ester standard;

[0082] Figure 4 This is a supercritical fluid chromatography detection diagram of the product trans-p-aminocyclohexylcarboxylic acid ethyl ester of Example 6;

[0083] Figure 5 This is the supercritical fluid chromatography detection diagram of trans- and cis-aminocyclohexylcarboxylic acid ethyl ester standards;

[0084] Figure 6 This is the NMR image of the product trans-p-aminocyclohexylcarboxylic acid ethyl ester of Example 6;

[0085] Figure 7 is a gas chromatogram of the reaction liquid of Example 14;

[0086] Figure 8 is a gas chromatogram of the reaction liquid in Example 15;

[0087] Fig. 9 is a gas chromatogram of the reaction liquid in Example 16;

[0088] Fig.10 is a gas chromatogram of cyclohexanonecarboxylic acid methyl ester standard;

[0089] Fig.11 This is a supercritical fluid chromatography detection diagram of the product trans-p-aminocyclohexylcarboxylic acid methyl ester of Example 16;

[0090] Fig.12 This is the supercritical fluid chromatography detection diagram of trans-p-aminocyclohexyl carboxylic acid methyl ester standard;

[0091] Fig.13 is a gas chromatogram of the reaction liquid in Example 17;

[0092] Fig.14 is a gas chromatogram of the reaction liquid in Example 21;

[0093] Fig.15 is a liquid chromatogram of the product trans-p-aminocyclohexylcarboxylic acid ethyl ester of Example 21;

[0094] Fig.16 is a gas chromatogram of the reaction liquid in Example 22;

[0095] Fig.17 is a liquid chromatogram of the product trans-p-aminocyclohexylcarboxylic acid ethyl ester of Example 22;

[0096] Fig.18 is a gas chromatogram of the reaction liquid of Example 23;

[0097] Fig.19 This is the liquid chromatogram of the product of Example 23, trans-p-aminocyclohexylcarboxylic acid ethyl ester. DETAILED DESCRIPTION

[0098] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0099] Example 1

[0100] Aminotransferase catalyzes the synthesis of trans-p-aminocyclohexylcarboxylic acid ethyl ester from compound 1 in PBS buffer. Add 0.015 g of pyridoxal phosphate and 0.2 mol / L, 30 mL of PBS buffer containing isopropylamine at pH = 7.5 to a 100 mL beaker (the ratio of amino donor to compound 1 is 4.0 mol / mol), and stir until all pyridoxal phosphate is dissolved to obtain a uniform solution. Take out 1.0 mL of the solution from the beaker and add it to a 4.0 mL reaction bottle, and add 0.1 g of compound 1 and 0.1 g of aminotransferase with mutant 48 to the solution. Vortex the resulting solution and react at 30 ° C for 16 hours. After completion, the reaction solution was taken out and the pH value of the reaction solution was adjusted to 4 with hydrochloric acid, and the acetone was removed by reduced pressure distillation at a vacuum degree of -0.09 MPa (gauge pressure) for 0.5 to 1 h, and then 1.0 mL of water was added, and extraction was carried out with 2.0 mL of methyl tert-butyl ether (pH was adjusted to 13 with potassium hydroxide). After extraction, the organic phase was taken out and anhydrous sodium sulfate was added to remove water, and then filtered to obtain a filtrate, and the filtrate was distilled under a vacuum degree of -0.09 MPa (gauge pressure). After purification by column chromatography, 0.0980 g of the product was finally obtained, and the yield was 98.0%. The product was detected by nuclear magnetic resonance, and the results showed that the purity of trans-p-aminocyclohexylcarboxylic acid ethyl ester was greater than 98%.

[0101] Example 2

[0102] Aminotransferase catalyzes the synthesis of trans-p-aminocyclohexylcarboxylic acid ethyl ester from compound 1 in TOEA buffer.

[0103] Add 0.015 g of pyridoxal phosphate and 0.2 mol / L, 30 mL of pH = 7.5 TOEA buffer containing isopropylamine (the ratio of amino donor to compound 1 is 4.0 mol / mol) to a 100 mL beaker, and stir until the pyridoxal phosphate is completely dissolved to obtain a uniform solution. Take out 1.0 mL of the solution from the beaker and add it to a 4.0 mL reaction bottle, and add 0.1 g of compound 1 and 0.1 g of aminotransferase with mutant 28 to the solution. Vortex the resulting solution and react at 30 ° C for 16 hours. After completion, the reaction solution was taken out and the pH value of the reaction solution was adjusted to 4 with hydrochloric acid, 4.0 mL of methyl tert-butyl ether was added for extraction, and the aqueous phase was taken out after extraction and distilled under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure) for 0.5 to 1 h to remove acetone, and then 1.0 mL of water was added, and extraction was carried out with 2.0 mL of methyl tert-butyl ether (pH was adjusted to 13 with potassium hydroxide), and the organic phase was taken out after extraction and anhydrous sodium sulfate was added to remove water, and then filtered to obtain a filtrate, and distilled under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure), purified by column chromatography, and finally 0.0819 g of the product was obtained, with a yield of 81.9%, and the product was detected by nuclear magnetic resonance, and the results showed that the purity of trans-aminocyclohexylcarboxylic acid ethyl ester was greater than 98%.

[0104] Example 3

[0105] Aminotransferase catalyzes the synthesis of trans-p-aminocyclohexylcarboxylic acid ethyl ester from compound 1 in Tris buffer.

[0106] Add 0.015 g of pyridoxal phosphate and 0.2 mol / L, 30 mL of Tris buffer containing isopropylamine (the ratio of amino donor to compound 1 is 4.0 mol / mol) at pH = 7.5 to a 100 mL beaker, and stir until the pyridoxal phosphate is completely dissolved to obtain a uniform solution. Take out 1.0 mL of the solution from the beaker and add it to a 2.0 mL reaction bottle, and add 0.1 g of compound 1 and 0.1 g of aminotransferase with mutant 183 to the solution. Vortex the resulting solution and react at 30 ° C for 18 hours. After completion, the reaction solution was taken out and the pH value of the reaction solution was adjusted to 4 with hydrochloric acid, 4.0 mL of methyl tert-butyl ether was added for extraction, and the aqueous phase was taken out after extraction and distilled under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure) for 0.5 to 1 h to remove acetone, and then 1.0 mL of water was added, and extraction was carried out with 2.0 mL of methyl tert-butyl ether (pH=13 was adjusted with potassium hydroxide), and the organic phase was taken out after extraction and anhydrous sodium sulfate was added to remove water, and then filtered to obtain a filtrate, and distilled under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure), purified by column chromatography, and finally 0.0842 g of the product was obtained, and the yield was 84.2%. The product was detected by nuclear magnetic resonance, and the results showed that the purity of trans-p-aminocyclohexylcarboxylic acid ethyl ester was greater than 98%.

[0107] Example 4

[0108] The aminotransferase catalyzes the synthesis of trans-p-aminocyclohexylcarboxylic acid ethyl ester from compound 1 under the condition that the ratio of amino donor isopropylamine to compound 1 is 2.0 mol / mol.

[0109] Add 0.015 g of pyridoxal phosphate and 200 mmol / L, 30 mL of PBS buffer containing isopropylamine (the ratio of amino donor to compound 1 is 1.0 mol / mol) at pH = 7.5 to a 100 mL beaker, and stir until the pyridoxal phosphate is completely dissolved to obtain a uniform solution. Take out 1.0 mL of the solution from the beaker and add it to a 4.0 mL reaction bottle, and add 0.1 g of compound 1 and 0.1 g of aminotransferase with mutant 110 to the solution. Vortex the resulting solution and react at 30°C for 18 h. After completion, the reaction solution was taken out and the pH value of the reaction solution was adjusted to 4 with hydrochloric acid, and the acetone was removed by reduced pressure distillation at a vacuum degree of -0.09 MPa (gauge pressure) for 0.5 to 1 h, and then 1.0 mL of water was added, and extraction was carried out with 2.0 mL of methyl tert-butyl ether (pH was adjusted to 13 with potassium hydroxide). After extraction, the organic phase was taken out and anhydrous sodium sulfate was added to remove water, and then filtered to obtain a filtrate, and the product was finally obtained. 0.0962 g of product was obtained with a yield of 96.2%. The product was detected by nuclear magnetic resonance, and the results showed that the purity of trans-p-aminocyclohexylcarboxylic acid ethyl ester was greater than 98%.

[0110] Example 5

[0111] At pH = 7.0, aminotransferase catalyzes compound 1 to synthesize trans-p-aminocyclohexylcarboxylic acid ethyl ester.

[0112] Add 0.015 g of pyridoxal phosphate and 0.2 mol / L, 30 mL of PBS buffer containing isopropylamine (the ratio of amino donor to compound 1 is 4.0 mol / mol) at pH = 7.0 to a 100 mL beaker, and stir until the pyridoxal phosphate is completely dissolved to obtain a uniform solution. Take out 1.0 mL of the solution from the beaker and add it to a 4.0 mL reaction bottle, and add 0.1 g of compound 1 and 0.1 g of aminotransferase with mutant 64 to the solution. Vortex the resulting solution and react at 30°C for 16 hours. After completion, the reaction solution was taken out and the pH value of the reaction solution was adjusted to 4 with hydrochloric acid, and the acetone was removed by reduced pressure distillation at a vacuum degree of -0.09 MPa (gauge pressure) for 0.5 to 1 h, and then 1.0 mL of water was added, and extraction was carried out with 2.0 mL of methyl tert-butyl ether (pH was adjusted to 13 with potassium hydroxide). After extraction, the organic phase was taken out and anhydrous sodium sulfate was added to remove water, and then filtered to obtain a filtrate, and the product was finally obtained. 0.0975 g, the yield was 97.5%, and the product was detected by nuclear magnetic resonance, and the results showed that the purity of trans-p-aminocyclohexylcarboxylic acid ethyl ester was greater than 98%.

[0113] Example 6

[0114] In 200 mL of reaction solution, aminotransferase catalyzes compound 1 to synthesize trans-p-aminocyclohexylcarboxylic acid ethyl ester.

[0115] 0.10 g of pyridoxal phosphate and 0.2 mol / L, 200 mL of PBS buffer containing isopropylamine (the ratio of amino donor to compound 1 is 4.0 mol / mol) at pH = 7.5 were added to a 500 mL jacketed bottle, and 20.0 g of compound 1 and 20.0 g of aminotransferase having mutant 144 were added to the solution, mechanically stirred until all solids were dissolved to form a uniform solution, and reacted at 30° C. for 18 h. 1.0 mL of the reaction solution was taken out and extracted with 1.0 mL of methyl tert-butyl ether (pH = 13 was adjusted with potassium hydroxide). The gas chromatography of the extract of methyl tert-butyl ether showed that the conversion rate of aminotransferase to compound 1 was 99.65% (gas chromatography as shown in FIG. 2 ). Figure 1 The retention time of ethyl aminocyclohexylcarboxylate is 10.230; the gas chromatograms of the standard products of ethyl cyclohexanonecarboxylate (retention time 10.70) and ethyl aminocyclohexylcarboxylate (retention time 10.32) are shown as follows: Figure 2 and Figure 3 As shown; Figure 1 The content of ethyl p-aminocyclohexylcarboxylate in the total raw material is 99.65%), all the solution in the jacketed bottle is rotary evaporated at 40°C for 4-6h to remove acetone, then 200mL of water is added, and extraction is carried out with 400mL of methyl tert-butyl ether (pH is adjusted to 13 with potassium hydroxide), after extraction, the organic phase is taken out and anhydrous sodium sulfate is added to remove moisture, and finally the filtrate is obtained by filtration and rotary evaporated at 35°C, and the final product is 19.64g, and the yield is 98.2%; the product is detected by supercritical fluid chromatography, and the stereopurity is: trans: cis = 98.72: 1.28 (supercritical fluid chromatography as Figure 4 ; Supercritical fluid chromatography of trans:cis standards such as Figure 5 ; The retention time of the trans standard is about 2.119min; the retention time of the cis standard is about 2.689min); the product was tested by nuclear magnetic resonance, and the results showed that the purity of trans-p-aminocyclohexylcarboxylic acid ethyl ester was greater than 98% (the nuclear magnetic spectrum is as shown Figure 6 shown).

[0116] Example 7

[0117] At pH = 9.0, aminotransferase catalyzes compound 1 to synthesize trans-p-aminocyclohexylcarboxylic acid ethyl ester.

[0118] Add 0.015 g of pyridoxal phosphate and 200 mmol / L, 30 mL of PBS buffer containing isopropylamine (the ratio of amino donor to compound 1 is 4.0 mol / mol) at pH = 9.0 to a 100 mL beaker, and stir until the pyridoxal phosphate is completely dissolved to obtain a uniform solution. Take out 1.0 mL of the solution from the beaker and add it to a 4.0 mL reaction bottle, and add 0.1 mg of compound 1 and 0.1 g of aminotransferase with mutant 37 to the solution. The obtained solution was mixed by vortexing and reacted at 30° C. for 20 h. After the reaction, the reaction solution was taken out and the pH of the reaction solution was adjusted to 4 with hydrochloric acid. 4.0 mL of methyl tert-butyl ether was added for extraction. After extraction, the aqueous phase was taken out and distilled under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure) for 0.5 to 1 h to remove acetone. Then 1.0 mL of water was added and extracted with 2.0 mL of methyl tert-butyl ether (pH was adjusted to 13 with potassium hydroxide). After extraction, the organic phase was taken out and anhydrous sodium sulfate was added to remove water. The filtrate was then filtered to obtain the filtrate, which was distilled under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure) to finally obtain 0.0975 g of the product with a yield of 97.5%. The product was detected by nuclear magnetic resonance, and the results showed that the purity of trans-p-aminocyclohexylcarboxylic acid ethyl ester was greater than 98%.

[0119] Example 8

[0120] Under the condition that the ratio of buffer to compound 1 is 5 mL / g, aminotransferase catalyzes compound 1 to synthesize trans-p-aminocyclohexylcarboxylic acid ethyl ester.

[0121] Add 0.015 g of pyridoxal phosphate and 200 mmol / L, 30 mL of PBS buffer containing isopropylamine (the ratio of amino donor to compound 1 is 4.0 mol / mol) at pH = 7.5 to a 100 mL beaker, and stir until the pyridoxal phosphate is completely dissolved to obtain a uniform solution. Take out 0.5 mL of the solution from the beaker and add it to a 2.0 mL reaction bottle, and add 0.1 g of compound 1 and 0.1 g of aminotransferase with mutant 156 to the solution. The obtained solution was mixed by vortexing and reacted at 30° C. for 18 h. After the reaction, the reaction solution was taken out and the pH of the reaction solution was adjusted to 4 with hydrochloric acid. 4.0 mL of methyl tert-butyl ether was added for extraction. After extraction, the aqueous phase was taken out and distilled under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure) for 0.5 to 1 h to remove acetone. Then 1.0 mL of water was added and extracted with 2.0 mL of methyl tert-butyl ether (pH was adjusted to 13 with potassium hydroxide). After extraction, the organic phase was taken out and anhydrous sodium sulfate was added to remove water. The filtrate was then filtered to obtain a filtrate, which was distilled under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure). Purification by column chromatography finally obtained 0.0886 g of the product with a yield of 88.6%. The product was detected by nuclear magnetic resonance, and the results showed that the purity of trans-p-aminocyclohexylcarboxylic acid ethyl ester was greater than 98%.

[0122] Example 9

[0123] Under the condition that the ratio of buffer to compound 1 is 20 mL / g, aminotransferase catalyzes compound 1 to synthesize trans-p-aminocyclohexylcarboxylic acid ethyl ester.

[0124] Add 0.015 g of pyridoxal phosphate and 0.2 mol / L, 30 mL of PBS buffer containing isopropylamine (the ratio of amino donor to compound 1 is 4.0 mol / mol) at pH = 7.5 to a 100 mL beaker, and stir until the pyridoxal phosphate is completely dissolved to obtain a uniform solution. Take out 1.0 mL of the solution from the beaker and add it to a 4.0 mL reaction bottle, and add 0.1 g of compound 1 and 0.1 g of aminotransferase with mutant 171 to the solution. The obtained solution was mixed by vortexing and then reacted at 30° C. for 18 h. After the reaction, the reaction solution was taken out and the pH of the reaction solution was adjusted to 4 with hydrochloric acid. The acetone was removed by reduced pressure distillation at a vacuum degree of -0.09 MPa (gauge pressure) for 0.5 to 1 h, and then 1.0 mL of water was added, and extraction was performed with 2.0 mL of methyl tert-butyl ether (pH was adjusted to 13 with potassium hydroxide). After extraction, the organic phase was taken out and anhydrous sodium sulfate was added to remove water, and then the filtrate was obtained by filtration. The product was finally obtained by reduced pressure distillation at a vacuum degree of -0.09 MPa (gauge pressure) 0.0980 g, and the yield was 98.0%. The product was detected by nuclear magnetic resonance, and the results showed that the purity of trans-p-aminocyclohexylcarboxylic acid ethyl ester was greater than 98%.

[0125] Example 10

[0126] At 20°C, aminotransferase catalyzed compound 1 to synthesize trans-p-aminocyclohexylcarboxylic acid ethyl ester.

[0127] 0.015 g of pyridoxal phosphate and 0.2 mol / L, 30 mL of PBS buffer containing isopropylamine (the ratio of amino donor to compound 1 is 4.0 mol / mol) at pH = 7.5 were added to a 100 mL beaker, and stirred until the pyridoxal phosphate was completely dissolved to obtain a uniform solution. 1.0 mL of the solution was taken out from the beaker and added to a 4.0 mL reaction bottle, and 0.1 g of compound 1 and 0.1 g of aminotransferase having mutant 33 were added to the solution. The obtained solution was mixed by vortexing and then reacted at 20°C for 18 hours. After the reaction, the reaction solution was taken out and the pH of the reaction solution was adjusted to 4 with hydrochloric acid. The acetone was removed by reduced pressure distillation at a vacuum degree of -0.09 MPa (gauge pressure) for 0.5 to 1 hour, and then 1.0 mL of water was added, and extraction was performed with 2.0 mL of methyl tert-butyl ether (pH was adjusted to 13 with potassium hydroxide). After extraction, the organic phase was taken out and anhydrous sodium sulfate was added to remove water, and then the filtrate was obtained by filtration. The product was finally obtained by reduced pressure distillation at a vacuum degree of -0.09 MPa (gauge pressure) 0.0973 g, and the yield was 97.3%. The product was detected by nuclear magnetic resonance, and the results showed that the purity of trans-p-aminocyclohexylcarboxylic acid ethyl ester was greater than 98%.

[0128] Embodiment 11

[0129] At 40°C, aminotransferase catalyzed compound 1 to synthesize trans-p-aminocyclohexylcarboxylic acid ethyl ester.

[0130] 0.015 g of pyridoxal phosphate and 0.2 mol / L, 30 mL of PBS buffer containing isopropylamine (the ratio of amino donor to compound 1 is 4.0 mol / mol) at pH = 7.5 were added to a 100 mL beaker, and stirred until the pyridoxal phosphate was completely dissolved to obtain a uniform solution. 1.0 mL of the solution was taken out from the beaker and added to a 4.0 mL reaction bottle, and 0.1 g of compound 1 and 0.1 g of aminotransferase with mutant 29 were added to the solution. The obtained solution was mixed by vortexing and reacted at 40° C. for 20 h. After the reaction, the reaction solution was taken out and the pH of the reaction solution was adjusted to 4 with hydrochloric acid. 4.0 mL of methyl tert-butyl ether was added for extraction. After extraction, the aqueous phase was taken out and distilled under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure) for 0.5 to 1 h to remove acetone. Then 1.0 mL of water was added and extracted with 2.0 mL of methyl tert-butyl ether (pH was adjusted to 13 with potassium hydroxide). After extraction, the organic phase was taken out and anhydrous sodium sulfate was added to remove water. The filtrate was then filtered to obtain a filtrate, which was distilled under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure). Purification by column chromatography finally obtained 0.0826 g of the product. The yield of the product was 82.6% after purification by column chromatography. The product was detected by nuclear magnetic resonance. The results showed that the purity of trans-p-aminocyclohexylcarboxylic acid ethyl ester was greater than 98%.

[0131] Example 12

[0132] Under the condition that the ratio of aminotransferase added to buffer was 1:50, compound 1 was catalyzed to synthesize trans-p-aminocyclohexylcarboxylic acid ethyl ester.

[0133] 0.015 g of pyridoxal phosphate and 0.2 mol / L, 30 mL of PBS buffer containing isopropylamine (the ratio of amino donor to compound 1 is 4.0 mol / mol) at pH = 7.5 were added to a 100 mL beaker, and stirred until the pyridoxal phosphate was completely dissolved to obtain a uniform solution. 1.0 mL of the solution was taken out from the beaker and added to a 4.0 mL reaction bottle, and 0.1 g of compound 1 and 0.02 g of aminotransferase having mutant 5 were added to the solution. The obtained solution was mixed by vortexing and reacted at 30° C. for 18 h. After the reaction, the reaction solution was taken out and the pH of the reaction solution was adjusted to 4 with hydrochloric acid. 4.0 mL of methyl tert-butyl ether was added for extraction. After extraction, the aqueous phase was taken out and distilled under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure) for 0.5 to 1 h to remove acetone. Then 1.0 mL of water was added and extracted with 2.0 mL of methyl tert-butyl ether (pH=13 was adjusted with potassium hydroxide). After extraction, the organic phase was taken out and anhydrous sodium sulfate was added to remove water. Then, the filtrate was obtained by filtration, and distilled under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure). Purification was performed by column chromatography. Finally, 0.0834 g of the product was obtained with a yield of 83.4%. The product was detected by nuclear magnetic resonance. The results showed that the purity of trans-p-aminocyclohexylcarboxylic acid ethyl ester was greater than 98%.

[0134] Embodiment 13

[0135] Compound 1 was used to catalyze the synthesis of trans-p-aminocyclohexylcarboxylic acid ethyl ester under the condition that the ratio of the amino donor to compound 1 was 0.8:1.

[0136] Add 0.015 g of pyridoxal phosphate and 0.2 mol / L, 30 mL of PBS buffer containing isopropylamine (the ratio of amino donor to compound 1 is 0.8 mol / mol) at pH = 7.5 to a 100 mL beaker, and stir until the pyridoxal phosphate is completely dissolved to obtain a uniform solution. Take out 1.0 mL of the solution from the beaker and add it to a 4.0 mL reaction bottle, and add 0.1 g of compound 1 and 0.1 g of aminotransferase with mutant 64 to the solution. Vortex the resulting solution and react at 30 ° C for 16 hours. After the reaction, the reaction solution was taken out and the pH value of the reaction solution was adjusted to 4 with hydrochloric acid, and the acetone was removed by reduced pressure distillation at a vacuum degree of -0.09 MPa (gauge pressure) for 0.5 to 1 h, and then 1.0 mL of water was added, and extraction was carried out with 2.0 mL of methyl tert-butyl ether (pH was adjusted to 13 with potassium hydroxide). The extract of methyl tert-butyl ether was analyzed by gas chromatography, and the conversion rate of aminotransferase to compound 1 was 48.8%; after extraction, the organic phase was taken out and anhydrous sodium sulfate was added to remove water, and then the filtrate was obtained by filtration, and the filtrate was distilled under a vacuum degree of -0.09 MPa (gauge pressure), and purified by column chromatography to finally obtain 0.0453 g of the product with a yield of 45.3%. The product was detected by nuclear magnetic resonance, and the results showed that the purity of trans-p-aminocyclohexylcarboxylic acid ethyl ester was greater than 98%.

[0137] Embodiment 14

[0138] Under the condition that the ratio of buffer to compound 1 is 3 mL / g, aminotransferase catalyzes compound 1 to synthesize trans-p-aminocyclohexylcarboxylic acid ethyl ester.

[0139] Add 0.015 g of pyridoxal phosphate and 0.2 mol / L, 30 mL of PBS buffer containing isopropylamine (the ratio of amino donor to compound 1 is 4.0 mol / mol) at pH = 7.5 to a 100 mL beaker, and stir until the pyridoxal phosphate is completely dissolved to obtain a uniform solution. Take out 0.3 mL of the solution from the beaker and add it to a 4.0 mL reaction bottle, and add 0.1 g of compound 1 and 0.1 g of aminotransferase with mutant 144 to the solution. Vortex the resulting solution and react at 30 ° C for 16 hours. After the reaction was completed, the reaction solution was taken out and the pH of the reaction solution was adjusted to 4 with hydrochloric acid. The acetone was removed by reduced pressure distillation at a vacuum degree of -0.09 MPa (gauge pressure) for 0.5 to 1 h. Then 1.0 mL of water was added and extracted with 2.0 mL of methyl tert-butyl ether (pH was adjusted to 13 with potassium hydroxide). The gas chromatography of the extract of methyl tert-butyl ether showed that the conversion rate of aminotransferase to compound 1 was 49.1% (gas chromatography as shown in FIG. Figure 7As shown, the retention time of ethyl p-aminocyclohexylcarboxylate is 10.23, the retention time of the raw material is 10.58, and the peak area ratio of the two substances is 49.1:50.9); after extraction, the organic phase is taken out and anhydrous sodium sulfate is added to remove water, and then the filtrate is obtained by filtration, and the filtrate is distilled under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure), and purified by column chromatography to finally obtain 0.0446 g of the product with a yield of 44.6%. The product is detected by nuclear magnetic resonance, and the results show that the purity of trans-ethyl p-aminocyclohexylcarboxylate is greater than 98%.

[0140] Embodiment 15

[0141] Under the condition of pH=10, aminotransferase catalyzes compound 1 to synthesize trans-p-aminocyclohexylcarboxylic acid ethyl ester.

[0142] 0.015 g of pyridoxal phosphate and 0.2 mol / L, 30 mL of PBS buffer containing isopropylamine (the ratio of amino donor to compound 1 is 4.0 mol / mol) at pH = 10 were added to a 100 mL beaker, and stirred until the pyridoxal phosphate was completely dissolved to obtain a uniform solution. 1.0 mL of the solution was taken out from the beaker and added to a 4.0 mL reaction bottle, and 0.1 g of compound 1 and 0.1 g of aminotransferase having mutant 33 were added to the solution. The obtained solution was mixed by vortexing and reacted at 20°C for 18 hours. After the reaction, the reaction solution was taken out and the pH of the reaction solution was adjusted to 4 with hydrochloric acid. The acetone was removed by reduced pressure distillation at a vacuum degree of -0.09 MPa (gauge pressure) for 0.5 to 1 hour. Then 1.0 mL of water was added and extracted with 2.0 mL of methyl tert-butyl ether (pH was adjusted to 13 with potassium hydroxide). The gas chromatography of the extract of methyl tert-butyl ether showed that the conversion rate of aminotransferase to compound 1 was 81.8% (gas chromatography as shown in Figure 1). Figure 8 As shown, the retention time of aminocyclohexylcarboxylic acid ethyl ester is 10.21, the retention time of the raw material is 10.57, and the peak area ratio of the two substances is 81.8:18.2) After extraction, the organic phase is taken out and anhydrous sodium sulfate is added to remove water, and then the filtrate is obtained by filtration. The filtrate is distilled under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure), and purified by column chromatography to finally obtain 0.0783 g of the product with a yield of 78.3%. The product is detected by nuclear magnetic resonance, and the results show that the purity of trans-p-aminocyclohexylcarboxylic acid ethyl ester is greater than 98%.

[0143] Example 16

[0144] At pH = 7.5, aminotransferase catalyzes compound 1 to synthesize trans-p-aminocyclohexylcarboxylic acid methyl ester.

[0145] Add 0.015 g of pyridoxal phosphate and 0.2 mol / L, 30 mL of PBS buffer containing isopropylamine (the ratio of amino donor to compound 1 is 4.0 mol / mol) at pH = 7.5 to a 100 mL beaker, and stir until the pyridoxal phosphate is completely dissolved to obtain a uniform solution. Take out 20.0 mL of the solution from the beaker and add it to a 40.0 mL reaction bottle, and add 2 g of compound 1 and 2 g of aminotransferase with mutant 83 to the solution. Vortex the resulting solution and react at 30 ° C for 16 hours. After the reaction was completed, the reaction solution was taken out and the pH of the reaction solution was adjusted to 4 with hydrochloric acid. The acetone was removed by reduced pressure distillation at a vacuum degree of -0.09 MPa (gauge pressure) for 0.5 to 1 h. Then, 20.0 mL of water was added and extracted with 40.0 mL of methyl tert-butyl ether (pH was adjusted to 13 with potassium hydroxide). The gas chromatography of the extract of methyl tert-butyl ether showed that the conversion rate of aminotransferase to compound 1 was 99.6% (gas chromatography as shown in FIG. Fig. 9 As shown; the gas chromatogram of cyclohexanone carboxylic acid methyl ester standard is as follows Fig.10 After extraction, the organic phase was taken out and anhydrous sodium sulfate was added to remove moisture, and then the filtrate was filtered to obtain a filtrate, which was distilled under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure) to finally obtain 0.0953 g of the product with a yield of 95.3%. The product was detected by supercritical fluid chromatography, and the stereo purity was: trans: cis = 96.96:3.04 (supercritical fluid chromatography as shown Fig.11 As shown; the supercritical fluid chromatography of the trans standard is as shown Fig.12 The product was detected by nuclear magnetic resonance, and the results showed that the purity of trans-p-aminocyclohexylcarboxylic acid methyl ester was greater than 98%.

[0146] Embodiment 17

[0147] At pH=10, aminotransferase catalyzes compound 1 to synthesize trans-p-aminocyclohexylcarboxylic acid methyl ester.

[0148] Add 0.015 g of pyridoxal phosphate and 0.2 mol / L, 30 mL of PBS buffer containing isopropylamine (the ratio of amino donor to compound 1 is 4.0 mol / mol) at pH = 10 to a 100 mL beaker, and stir until the pyridoxal phosphate is completely dissolved to obtain a uniform solution. Take out 20.0 mL of the solution from the beaker and add it to a 40.0 mL reaction bottle, and add 2 g of compound 1 and 2 g of aminotransferase with mutant 83 to the solution. Vortex the resulting solution and react at 30 ° C for 16 hours. After the reaction was completed, the reaction solution was taken out and the pH of the reaction solution was adjusted to 4 with hydrochloric acid. The acetone was removed by reduced pressure distillation at a vacuum degree of -0.09 MPa (gauge pressure) for 0.5 to 1 h, and then 20.0 mL of water was added and extracted with 40.0 mL of methyl tert-butyl ether (pH was adjusted to 13 with potassium hydroxide). The gas chromatography of the extract of methyl tert-butyl ether showed that the conversion rate of aminotransferase to compound 1 was 52.83% (gas chromatography as shown in FIG. Fig.13 As shown, the retention time of aminocyclohexyl carboxylic acid methyl ester is 10.20, the retention time of the raw material is 10.57, and the peak area ratio of the two substances is 52.83:41.17); after extraction, the organic phase is taken out and anhydrous sodium sulfate is added to remove water, and then the filtrate is obtained by filtration, and the filtrate is distilled under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure), and purified by column chromatography to finally obtain 0.0469 g of the product with a yield of 46.9%. The product is detected by nuclear magnetic resonance, and the results show that the purity of trans-p-aminocyclohexyl carboxylic acid methyl ester is greater than 98%.

[0149] Embodiment 18

[0150] At pH = 7.5, aminotransferase catalyzes compound 1 to synthesize trans-p-aminocyclohexyl cyanide.

[0151] 0.015 g of pyridoxal phosphate and 0.2 mol / L, 30 mL of PBS buffer containing isopropylamine (the ratio of amino donor to compound 1 is 4.0 mol / mol) at pH = 7.5 were added to a 100 mL beaker, and stirred until the pyridoxal phosphate was completely dissolved to obtain a uniform solution. 1.0 mL of the solution was taken out from the beaker and added to a 4.0 mL reaction bottle, and 0.1 g of compound 1 and 0.1 g of aminotransferase with mutant 10 were added to the solution. The obtained solution was mixed by vortexing and then reacted at 20°C for 18 hours. After the reaction, the reaction solution was taken out and the pH of the reaction solution was adjusted to 4 with hydrochloric acid. The acetone was removed by reduced pressure distillation at a vacuum degree of -0.09 MPa (gauge pressure) for 0.5 to 1 hour, and then 1.0 mL of water was added, and extraction was performed with 2.0 mL of methyl tert-butyl ether (pH was adjusted to 13 with potassium hydroxide). After extraction, the organic phase was taken out and anhydrous sodium sulfate was added to remove water, and then filtered to obtain a filtrate, which was distilled under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure). Purification by column chromatography finally obtained 0.0912 g of the product with a yield of 91.2%. The product was detected by nuclear magnetic resonance, and the results showed that the purity of the trans-p-aminocyclohexylcarboxylic acid cyano group was greater than 98%.

[0152] Embodiment 19

[0153] At pH = 7.5, aminotransferase catalyzes compound 1 to synthesize trans-p-aminocyclohexylcarboxylic acid.

[0154] 0.015 g of pyridoxal phosphate and 0.2 mol / L, 30 mL of PBS buffer containing isopropylamine (the ratio of amino donor to compound 1 is 4.0 mol / mol) at pH = 7.5 were added to a 100 mL beaker, and stirred until the pyridoxal phosphate was completely dissolved to obtain a uniform solution. 1.0 mL of the solution was taken out from the beaker and added to a 4.0 mL reaction bottle, and 0.1 g of compound 1 and 0.1 g of aminotransferase having mutant 46 were added to the solution. The obtained solution was mixed by vortexing and then reacted at 20° C. for 18 h. After the reaction, the reaction solution was taken out and the pH of the reaction solution was adjusted to 4 with hydrochloric acid. The acetone was removed by reduced pressure distillation at a vacuum degree of -0.09 MPa (gauge pressure) for 0.5 to 1 h, and then 1.0 mL of water was added, and extraction was performed with 2.0 mL of methyl tert-butyl ether (pH=13 was adjusted with potassium hydroxide). After extraction, the organic phase was taken out and anhydrous sodium sulfate was added to remove water, and then filtered to obtain a filtrate, which was distilled under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure). Purification by column chromatography finally obtained 0.0956 g of the product with a yield of 95.6%. The product was detected by nuclear magnetic resonance, and the results showed that the purity of trans-p-aminocyclohexyl carboxylic acid was greater than 98%.

[0155] Embodiment 20

[0156] Under the condition that the ratio of buffer to compound 1 is 3 mL / g, aminotransferase catalyzes compound 1 to synthesize trans-p-aminocyclohexylcarboxylic acid ethyl ester.

[0157] 0.015 g of pyridoxal phosphate and 0.2 mol / L, 30 mL of PBS buffer containing isopropylamine (the ratio of amino donor to compound 1 is 4.0 mol / mol) at pH = 7.5 were added to a 100 mL beaker, and stirred until the pyridoxal phosphate was completely dissolved to obtain a uniform solution. 0.3 mL of the solution was taken out from the beaker and added to a 4.0 mL reaction bottle, and 0.1 g of compound 1 and 0.1 g of aminotransferase having mutant 46 were added to the solution. The obtained solution was mixed by vortexing and then reacted at 20° C. for 18 h. After the reaction, the reaction solution was taken out and the pH of the reaction solution was adjusted to 4 with hydrochloric acid. The acetone was removed by reduced pressure distillation at a vacuum degree of -0.09 MPa (gauge pressure) for 0.5 to 1 h, and then 1.0 mL of water was added, and extraction was performed with 2.0 mL of methyl tert-butyl ether (pH was adjusted to 13 with potassium hydroxide). After extraction, the organic phase was taken out and anhydrous sodium sulfate was added to remove water, and then filtered to obtain a filtrate, which was distilled under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure). Purification by column chromatography finally obtained 0.0786 g of the product with a yield of 78.6%. The product was detected by nuclear magnetic resonance, and the results showed that the purity of trans-p-aminocyclohexyl carboxylic acid was greater than 98%.

[0158] In the present invention, the methods for detecting the stereoselectivity of the final product trans-para-aminocyclohexyl compounds in Examples 1-5 and Examples 7-20 are the same as that in Example 6, and both can achieve a trans-stereoselectivity of 98%.

[0159] Embodiment 21

[0160] Aminotransferase catalyzes the synthesis of trans-p-aminocyclohexylcarboxylic acid ethyl ester from compound 1 in PBS buffer. Add 0.015 g of pyridoxal phosphate and 0.2 mol / L, 30 mL of PBS buffer containing isopropylamine at pH = 7.5 to a 100 mL beaker (the ratio of amino donor to compound 1 is 4.0 mol / mol), and stir until all pyridoxal phosphate is dissolved to obtain a uniform solution. Take out 1.0 mL of the solution from the beaker and add it to a 4.0 mL reaction bottle, and add 0.1 g of compound 1 and 0.1 g of aminotransferase from Bacillus megaterium (SEQ ID NO: 1, GI No.: 1048348995) to the solution. Vortex the resulting solution and react at 30 ° C for 16 hours. After the reaction, the reaction solution was taken out and the pH of the reaction solution was adjusted to 4 with hydrochloric acid, and the acetone was removed by reduced pressure distillation at a vacuum degree of -0.09 MPa (gauge pressure) for 0.5 to 1 h. Then 1.0 mL of water was added and extracted with 2.0 mL of methyl tert-butyl ether (pH was adjusted to 13 with potassium hydroxide). The gas chromatography of the extract of methyl tert-butyl ether showed that the conversion rate of aminotransferase to compound 1 was 98.1% (gas chromatography as shown in FIG. Fig.14 As shown, the retention time of aminocyclohexyl carboxylic acid ethyl ester is 10.114, the retention time of the raw material is 10.456, and the peak area ratio of the two substances is 98.1:1.9); after extraction, the organic phase is taken out and anhydrous sodium sulfate is added to remove moisture, and then the filtrate is filtered to obtain a filtrate, and the product is finally obtained by reduced pressure distillation at a vacuum degree of -0.09MPa (gauge pressure) 0.0960g, the yield is 96.0%, and the product is detected by nuclear magnetic resonance. The results show that the purity of aminocyclohexyl carboxylic acid ethyl ester is greater than 98%. The product is detected by supercritical fluid chromatography, and the stereo purity is: trans: cis = 78.38:21.62 (supercritical fluid chromatography as shown Fig.15 The product was detected by nuclear magnetic resonance, and the results showed that the purity of trans-p-aminocyclohexylcarboxylic acid ethyl ester was greater than 98%.

[0161] Embodiment 22

[0162] Aminotransferase catalyzes the synthesis of trans-p-aminocyclohexylcarboxylic acid ethyl ester from compound 1 in PBS buffer. Add 0.015 g of pyridoxal phosphate and 0.2 mol / L, 30 mL of PBS buffer containing isopropylamine at pH = 7.5 to a 100 mL beaker (the ratio of amino donor to compound 1 is 4.0 mol / mol), and stir until all pyridoxal phosphate is dissolved to obtain a uniform solution. Take out 1.0 mL of the solution from the beaker and add it to a 4.0 mL reaction bottle, and add 0.1 g of compound 1 and 0.1 g of aminotransferase (Uniport ID: F7J696) from Arthrobater Sp.Knk168 to the solution. Vortex the resulting solution and react at 30 ° C for 16 hours. After the reaction was completed, the reaction solution was taken out and the pH of the reaction solution was adjusted to 4 with hydrochloric acid, and the acetone was removed by reduced pressure distillation at a vacuum degree of -0.09 MPa (gauge pressure) for 0.5 to 1 h. Then 1.0 mL of water was added and extracted with 2.0 mL of methyl tert-butyl ether (pH was adjusted to 13 with potassium hydroxide). The gas chromatography of the extract of methyl tert-butyl ether showed that the conversion rate of aminotransferase to compound 1 was 99.8% (gas chromatography as shown in FIG. Fig.16 As shown, the retention time of aminocyclohexylcarboxylic acid ethyl ester is 10.319, the retention time of the raw material is 10.679, and the peak area ratio of the two substances is 99.78:0.22); the product is detected by liquid chromatography, and the stereo purity is: trans: cis = 66.17:33.83 (liquid chromatography as Fig.17 shown);

[0163] Embodiment 23

[0164] Aminotransferase catalyzes compound 1 to synthesize ethyl p-aminocyclohexylcarboxylate in PBS buffer. Add 0.015 g of pyridoxal phosphate and 0.2 mol / L, 30 mL of PBS buffer containing isopropylamine at pH = 7.5 to a 100 mL beaker (the ratio of amino donor to compound 1 is 4.0 mol / mol), and stir until all pyridoxal phosphate is dissolved to obtain a uniform solution. Take out 1.0 mL of the solution from the beaker and add it to a 4.0 mL reaction bottle, and add 0.1 g of compound 1 and 0.1 g of aminotransferase (Uniport ID: F2XBU9) from Vibrio fluvialis JS17 to the solution. Vortex the resulting solution and react at 30 ° C for 16 hours. After the reaction was completed, the reaction solution was taken out and the pH of the reaction solution was adjusted to 4 with hydrochloric acid, and the acetone was removed by reduced pressure distillation at a vacuum degree of -0.09 MPa (gauge pressure) for 0.5 to 1 h. Then 1.0 mL of water was added and extracted with 2.0 mL of methyl tert-butyl ether (pH was adjusted to 13 with potassium hydroxide). The gas chromatography of the extract of methyl tert-butyl ether showed that the conversion rate of aminotransferase to compound 1 was 99.8% (gas chromatography as shown in FIG. Fig.18 As shown, the retention time of aminocyclohexylcarboxylic acid ethyl ester is 10.528, the retention time of the raw material is 10.945, and the peak area ratio of the two substances is 99.78:0.22); the product is detected by liquid chromatography, and the stereo purity is: trans: cis = 1.0:99.0 (liquid chromatography as shown Fig.19 shown);

[0165] Example 24 Construction of aminotransferase mutant library

[0166] One or more of positions 59, 60, 144, 161, 164, 169, 174, 240, 244, 327, 330, 331, 373, 387, 410, 436, 437 and 442 of SEQ ID NO: 1 were mutated, and mutants 2 to mutant 196 with higher catalytic activity were screened out. The specific mutation sites and types are shown in Table 3. The reaction conditions are the same as those in Example 1, except that the mutant of the catalytic enzyme of the reaction is replaced by the mutant in Table 3. The reaction effects are shown in Table 3:

[0167] Table 3

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176] SEQUENCE LISTING <110> Shanghai Hequan Pharmaceutical Research and Development Co., Ltd. Shanghai Hequan Pharmaceutical Co., Ltd. <120> A kind of aminotransferase mutant derived from Bacillus megaterium and its application <130> P20016335C <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 483 <212> PRT <213> Bacillus megaterium <400> 1 Met Ser Leu Thr Val Gln Lys Ile Asn Trp Glu Gln Val Lys Glu Trp 1 5 10 15 Asp Arg Lys Tyr Leu Met Arg Thr Phe Ser Thr Gln Asn Glu Tyr Gln 20 25 30 Pro Val Pro Ile Glu Ser Thr Glu Gly Asp Tyr Leu Ile Met Pro Asp 35 40 45 Gly Thr Arg Leu Leu Asp Phe Phe Asn Gln Leu Tyr Cys Val Asn Leu 50 55 60 Gly Gln Lys Asn Gln Lys Val Asn Ala Ala Ile Lys Glu Ala Leu Asp 65 70 75 80 Arg Tyr Gly Phe Val Trp Asp Thr Tyr Ala Thr Asp Tyr Lys Ala Lys 85 90 95 Ala Ala Lys Ile Ile Ile Glu Asp Ile Leu Gly Asp Glu Asp Trp Pro 100 105 110 Gly Lys Val Arg Phe Val Ser Thr Gly Ser Glu Ala Val Glu Thr Ala 115 120 125 Leu Asn Ile Ala Arg Leu Tyr Thr Asn Arg Pro Leu Val Val Thr Arg 130 135 140 Glu His Asp Tyr His Gly Trp Thr Gly Gly Ala Ala Thr Val Thr Arg 145 150 155 160 Leu Arg Ser Tyr Arg Ser Gly Leu Val Gly Glu Asn Ser Glu Ser Phe 165 170 175 Ser Ala Gln Ile Pro Gly Ser Ser Tyr Asn Ser Ala Val Leu Met Ala 180 185 190 Pro Ser Pro Asn Met Phe Gln Asp Ser Asp Gly Asn Leu Leu Lys Asp 195 200 205 Glu Asn Gly Glu Leu Leu Ser Val Lys Tyr Thr Arg Arg Met Ile Glu 210 215 220 Asn Tyr Gly Pro Glu Gln Val Ala Ala Val Ile Thr Glu Val Ser Gln 225 230 235 240 Gly Ala Gly Ser Ala Met Pro Pro Tyr Glu Tyr Ile Pro Gln Ile Arg 245 250 255 Lys Met Thr Lys Glu Leu Gly Val Leu Trp Ile Asn Asp Glu Val Leu 260 265 270 Thr Gly Phe Gly Arg Thr Gly Lys Trp Phe Gly Tyr Gln His Tyr Gly 275 280 285 Val Gln Pro Asp Ile Ile Thr Met Gly Lys Gly Leu Ser Ser Ser Ser 290 295 300 Leu Pro Ala Gly Ala Val Leu Val Ser Lys Glu Ile Ala Ala Phe Met 305 310 315 320 Asp Lys His Arg Trp Glu Ser Val Ser Thr Tyr Ala Gly His Pro Val 325 330 335 Ala Met Ala Ala Val Cys Ala Asn Leu Glu Val Met Met Glu Glu Asn 340 345 350 Phe Val Glu Gln Ala Lys Asp Ser Gly Glu Tyr Ile Arg Ser Lys Leu 355 360 365 Glu Leu Leu Gln Glu Lys His Lys Ser Ile Gly Asn Phe Asp Gly Tyr 370 375 380 Gly Leu Leu Trp Ile Val Asp Ile Val Asn Ala Lys Thr Lys Thr Pro 385 390 395 400 Tyr Val Lys Leu Asp Arg Asn Phe Thr His Gly Met Asn Pro Asn Gln 405 410 415 Ile Pro Thr Gln Ile Ile Met Lys Lys Ala Leu Glu Lys Gly Val Leu 420 425 430 Ile Gly Gly Val Met Pro Asn Thr Met Arg Ile Gly Ala Ser Leu Asn 435 440 445 Val Ser Arg Gly Asp Ile Asp Lys Ala Met Asp Ala Leu Asp Tyr Ala 450 455 460 Leu Asp Tyr Leu Glu Ser Gly Glu Trp Gln Ala Leu Glu His His His 465 470 475 480 His His His

Claims

1. A kind of Bacillus megaterium ), characterized in that The aminotransferase mutant is mutated based on the amino acid sequence shown in SEQ ID NO: 1; The mutation positions and types of the aminotransferase mutants are shown in the following table: 。 2. Use of the aminotransferase mutant according to claim 1 in the preparation of trans-aminocyclohexyl compounds.

3. A method for preparing a trans-aminocyclohexyl compound, characterized in that: It includes the following steps: In the presence of an aminotransferase, a cyclohexyl ketone compound as shown in Formula I and an amino donor are subjected to an amination reaction as shown below to obtain a trans-aminocyclohexyl compound as shown in Formula II; Where R is -COOR 1 or -CN; R 1 is hydrogen or C1~C6 alkyl; the aminotransferase is the aminotransferase mutant as claimed in claim 1.

4. The method for preparing a trans-aminocyclohexyl compound according to claim 3, wherein: The R 1 is methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl or tert-butyl; And / or, the amino donor is one or more of isopropylamine, L-alanine, DL-alanine, L-lysine, DL-lysine, (S)-1-phenylethylamine, (R)-1-phenylethylamine, (RS)-1-phenylethylamine, pentamethylenediamine, o-phenylenediamine, o-phenylenediamine hydrochloride, 2-(4-nitrobenzene)ethylamine, and 2-(4-nitrobenzene)ethylamine hydrochloride; And / or, the molar ratio of the amino donor to the cyclohexyl ketone compound as shown in Formula I is (1-4):1; And / or, the reaction solvent of the amination reaction is a buffer solution, or a mixture of a buffer solution and an organic solvent; the organic solvent is methyl tert-butyl ether, isopropyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, 1,4-dioxane, ethyl acetate, dimethyl sulfoxide, N,N-dimethylformamide, methanol, ethanol, isopropanol or n-butanol; And / or, the ratio of the organic solvent to the total reaction volume is 0-250 mL / L; And / or, the volume mass ratio of the buffer solution to the cyclohexyl ketone compound as shown in Formula I is 5-100 mL / g; And / or, the reaction temperature of the amination reaction is 25°C to 40°C; And / or, the reaction time of the amination reaction is 16 to 20 hours.

5. The method for preparing a trans-aminocyclohexyl compound according to claim 4, characterized in that: The amino donor is isopropylamine; And / or, the molar ratio of the amino donor to the cyclohexyl ketone compound of formula I is 1:1, 2:1, 3:1 or 4:1; And / or, the reaction solvent of the amination reaction is a buffer solution; And / or, the reaction temperature of the amination reaction is 30°C; And / or, the reaction time of the amination reaction is 16 h, 18 h or 20 h.

6. The method for preparing a trans-aminocyclohexyl compound according to claim 5, characterized in that: The C1~C6 alkyl group is a methyl group or an ethyl group.

7. The method for preparing a trans-aminocyclohexyl compound according to claim 4, wherein: R is -COOH, -COOMe, -COOEt or -CN; And / or, the pH value of the amination reaction is 6.0-9.0; And / or, the buffer is one or more of a phosphate buffer, a tris(hydroxymethyl)methylamine salt buffer and a triethanolamine salt buffer; And / or, the preparation method of the trans-aminocyclohexyl compound comprises the following steps: adding the cyclohexyl ketone compound as shown in formula I and the aminotransferase to a mixture of the buffer solution and the amino donor, mixing well and reacting at 25°C to 40°C.

8. The method for preparing a trans-aminocyclohexyl compound according to claim 7, characterized in that: The pH value of the amination reaction is 7.0-9.0; And / or, the buffer is a phosphate buffer, a tris(hydroxymethyl)methylamine salt buffer or a triethanolamine salt buffer.

9. The method for preparing a trans-aminocyclohexyl compound according to claim 8, characterized in that: The pH value of the amination reaction is 7.0, 7.5 or 9.

0.

10. The method for preparing a trans-aminocyclohexyl compound according to any one of claims 3 to 9, characterized in that: After the amination reaction is completed, the following post-treatment steps are further included: adjusting the pH of the reaction solution to 4 with an acid, adding an ether solvent for extraction and separation, adding an ether solvent to the aqueous phase and adjusting the pH to 13 with a base, and drying the organic phase to remove the ether solvent.

11. The method for preparing a trans-aminocyclohexyl compound according to claim 10, characterized in that: The acid is hydrochloric acid; And / or, the drying is carried out by using a desiccant; And / or, the base is sodium hydroxide or potassium hydroxide; And / or, the method of removing the ether solvent is reduced pressure distillation.

12. The method for preparing a trans-aminocyclohexyl compound according to claim 11, characterized in that: The desiccant is anhydrous sodium sulfate; And / or, the base is potassium hydroxide; And / or, the method of removing the ether solvent is vacuum distillation at a pressure of -0.09 Mpa.

Images