Transaminase mutant and application thereof

Abstract

The invention discloses a mutant of transaminase and application of the mutant. The invention provides a preparation method of a compound II, which comprises the following step: in a solvent, in the presence of a transaminase mutant, carrying out transaminase reaction on a compound I and an amino donor as shown in the following formula to obtain the compound II. The chiral lactam compound prepared from the transaminase mutant provided by the invention has the yield of 85% or more and the e.e. Value of 98% or more, has the advantages of high yield, high purity, simple and convenient post-treatment and the like, is environment-friendly, and can be used as a green and sustainable synthesis route. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme engineering, and particularly relates to an aminotransferase mutant derived from Ruegeria sp. TM1040, a method for preparing chiral lactam compounds therefrom, and applications thereof. Background Art

[0002] Chiral arylamines and their analogs are important precursors for drug synthesis, important precursors for many APIs, and important building blocks for a variety of innovative chemical drugs in the clinical stage, such as atogepant (CX-8998), cinacalcet, sitagliptin, etc. Previous synthesis methods usually use non-recyclable precious metals such as palladium, platinum, rhodium, and ruthenium as catalysts, which are costly, or use heavy metals such as nickel as catalysts, which can cause environmental pollution, and high-pressure hydrogen needs to be used in a closed reaction device during the process, with relatively high safety risks. Compared with chemical methods, enzyme-catalyzed methods have the characteristics of good specificity, high purity, mild reaction, low cost, etc. Aminotransferases are an important method for synthesizing chiral arylamines and their analogs, which are green and environmentally friendly and are widely used. For example, the synthesis of sitagliptin (Science, 2010, 329, 305-309 - Biocatalytic Asymmetric Synthesis of Chiral Amines from Ketones applied to sitagliptin manufacture).

[0003] Chiral lactam compounds are important precursors for drug synthesis and important building blocks for a variety of innovative chemical drugs in the clinical stage.

[0004] Previous synthesis methods require multiple chemical reactions, with many steps, low yields, high costs, poor atom economy, and being unfriendly to the environment, and are not a green and sustainable synthesis route; synthesizing chiral lactams through an enzyme-catalyzed method can shorten the route to 1 step, with high atom economy, and the reaction mainly occurs in an aqueous solution, which is safe, environmentally friendly, and conforms to the principles of green chemistry. Summary of the Invention

[0005] The technical problem to be solved by the present invention is that the chemical methods for synthesizing chiral lactam compounds in the prior art are costly and have poor atom economy. To this end, the present invention provides a mutant of transaminase and its applications. Using this transaminase mutant to prepare chiral lactam compounds, the yield can reach more than 85%, the e.e. value can reach more than 98%, and it has the advantages of high yield, high purity, and simple post-treatment, is environmentally friendly, and can be used as a green and sustainable synthesis route.

[0006] The present invention provides a method for preparing Compound II, which comprises the following steps: in a solvent, in the presence of a mutant of transaminase, subject Compound I and an amino donor to a transamination reaction as shown in the following formula to obtain Compound II;

[0007]

[0008] Wherein,

[0009] R 1 、R 2 、R 3 、R 4 and R 5 are independently H, halogen, amino, C 1-4 alkyl, carboxyl, hydroxyl, C 1-4 alkoxy or nitro;

[0010] R 6 is C 2-6 alkyl;

[0011] The mutant of the transaminase has differences in amino acid residues at one or more of the following sites selected from the amino acid sequence shown in SEQ ID NO: 1:

[0012] Position 1, Position 2, Position 3, Position 19, Position 47, Position 49, Position 50, Position 55, Position 56, Position 59, Position 68, Position 76, Position 86, Position 87, Position 88, Position 108, Position 118, Position 120, Position 141, Position 152, Position 155, Position 157, Position 159, Position 162, Position 168, Position 170, Position 178, Position 191, Position 214, Position 231, Position 234, Position 243, Position 251, Position 303, Position 320, Position 324, Position 341, Position 376, Position 382, Position 402, Position 405, Position 416 and Position 420.

[0013] In one embodiment, the difference is deletion, addition or substitution, preferably substitution.

[0014] In one embodiment, the mutant of the transaminase may have differences in amino acid residues at one or more of the following sites selected from the amino acid sequence shown in SEQ ID NO: 1:

[0015] The 1st, 2nd, 3rd, 19th, 47th, 49th, 56th, 59th, 76th, 86th, 87th, 88th, 108th, 118th, 120th, 141st, 152nd, 155th, 157th, 159th, 162nd, 168th, 178th, 214th, 231st, 234th, 243rd, 303rd, 320th, 324th, 341st, 376th, 382nd, 405th and 420th positions.

[0016] In one embodiment, the mutant of the transaminase has an amino acid residue difference at one or more of the following sites selected from the amino acid sequence shown in SEQ ID NO: 1:

[0017] The 2nd, 3rd, 19th, 47th, 49th, 59th, 76th, 86th, 87th, 88th, 108th, 118th, 120th, 141st, 152nd, 155th, 157th, 162nd, 168th, 178th, 214th, 231st, 234th, 243rd, 303rd, 320th, 324th, 341st, 382nd and 420th positions.

[0018] In one embodiment, the mutant of the transaminase has the 1st amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 replaced by A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V or W;

[0019] and / or, the 2nd amino acid residue replaced by A, C, D, E, F, G, H, I, K, M, Q, R, S, T or Y;

[0020] and / or, the 3rd amino acid residue replaced by C, E, F, H, I, L, M, N, Q, R, S, T, V, W or Y;

[0021] and / or, the 19th amino acid residue replaced by A, D, E, G, H, I, K, L, M, N, P, Q, R, T, V, W or Y;

[0022] and / or, the 47th amino acid residue replaced by A, C, F, G, H, I, K, L, M, P, Q, R, S, T, V, W or Y;

[0023] and / or, the 49th amino acid residue replaced by A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, V, W or Y;

[0024] and / or, the 50th amino acid residue is replaced with A, C, D, E, F, H, I, L, M, N, P, Q, R, S, T, V or W;

[0025] and / or, the 55th amino acid residue is replaced with A, C, D, E, G, H, I, K, M, N, P, Q, R, S, T, V, W or Y;

[0026] and / or, the 56th amino acid residue is replaced with C, D, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y;

[0027] and / or, the 59th amino acid residue is replaced with A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V or W;

[0028] and / or, the 68th amino acid residue is replaced with A, C, D, F, G, H, I, K, L, M, N, P, R, S, T, V, W or Y;

[0029] and / or, the 76th amino acid residue is replaced with A, C, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W or Y;

[0030] and / or, the 86th amino acid residue is replaced with A, C, D, E, F, G, H, I, K, L, N, Q, R, T, V, W or Y;

[0031] and / or, the 87th amino acid residue is replaced with A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T or V;

[0032] and / or, the 88th amino acid residue is replaced with A, C, D, E, G, H, I, K, L, M, N, P, Q, S, T or Y;

[0033] and / or, the 108th amino acid residue is replaced with A, C, E, F, G, L, N, P, Q, R, S, T, V, W or Y;

[0034] and / or, the 118th amino acid residue is replaced with A, C, D, E, F, H, I, L, M, N, P, Q, R, S, T, V, W or Y;

[0035] and / or, the 120th amino acid residue is replaced with A, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W or Y;

[0036] and / or, the 141st amino acid residue is replaced with A, C, D, F, G, H, I, K, M, N, R, S, T, V, W or Y;

[0037] and / or, the 152nd amino acid residue is replaced with A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V or W;

[0038] and / or, the 155th amino acid residue is replaced with A, C, D, E, F, G, H, I, K, M, N, P, Q, R, T, V or W;

[0039] and / or, the 157th amino acid residue is replaced with A, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V or Y;

[0040] and / or, the 159th amino acid residue is replaced with A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, V, W or Y;

[0041] and / or, the 162nd amino acid residue is replaced with A, D, E, F, G, H, I, K, M, N, P, Q, S, T, V, W or Y;

[0042] and / or, the 168th amino acid residue is replaced with A, C, E, G, H, I, K, L, M, N, P, Q, R, S, T or Y;

[0043] and / or, the 170th amino acid residue is replaced with A, C, D, E, F, G, H, I, L, M, N, Q, R, S, T, V, W or Y;

[0044] and / or, the 178th amino acid residue is replaced with A, C, D, E, F, G, H, I, K, L, M, P, R, S, T, V, W or Y;

[0045] and / or, the 191st amino acid residue is replaced with A, C, D, F, H, I, K, L, M, N, P, Q, R, T, V or Y;

[0046] and / or, the 214th amino acid residue is replaced with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, W or Y;

[0047] and / or, the 231st amino acid residue is replaced with A, C, E, F, I, K, L, M, N, P, Q, R, S, V, W or Y;

[0048] and / or, the 234th amino acid residue is replaced with A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W or Y;

[0049] and / or, the 243rd amino acid residue is replaced with A, C, D, F, G, H, I, K, N, P, R, S, T, V, W or Y;

[0050] and / or, the 251st amino acid residue is replaced with A, C, D, E, F, G, H, I, L, M, N, P, R, S, T, V or W;

[0051] and / or, the 303rd amino acid residue is replaced with A, D, E, G, H, I, K, L, M, N, P, Q, R, T, V, W or Y;

[0052] and / or, the 320th amino acid residue is replaced with A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W or Y;

[0053] and / or, the 324th amino acid residue is replaced with C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T or Y;

[0054] and / or, the 341st amino acid residue is replaced with A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W or Y;

[0055] and / or, the 376th amino acid residue is replaced with A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T or W;

[0056] and / or, the 382nd amino acid residue is replaced with A, C, D, E, F, G, I, M, N, P, Q, R, S, T, V or Y;

[0057] and / or, the 402nd amino acid residue is replaced with A, C, E, F, G, H, I, M, P, Q, R, S, T, V, W or Y;

[0058] and / or, the 405th amino acid residue is replaced with A, C, D, E, F, G, H, I, K, L, M, Q, R, S, T, V, W or Y;

[0059] and / or, the 416th amino acid residue is replaced with A, D, E, F, G, H, N, P, Q, R, V, W or Y;

[0060] and / or, the 420th amino acid residue is replaced with A, D, E, F, G, H, I, K, L, M, N, P, Q, S, V, W or Y.

[0061] In one embodiment, the mutant of the transaminase may be that the 1st amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is replaced with L;

[0062] and / or, the 2nd amino acid residue is replaced with D or G;

[0063] and / or, the 3rd amino acid residue is replaced with H or N;

[0064] and / or, the 19th amino acid residue is replaced with A or W;

[0065] and / or, the 47th amino acid residue is replaced with Y;

[0066] and / or, the 49th amino acid residue is replaced with N;

[0067] and / or, the 56th amino acid residue is replaced with S;

[0068] and / or, the 59th amino acid residue is replaced with L;

[0069] and / or, the 76th amino acid residue is replaced with I;

[0070] and / or, the 86th amino acid residue is replaced with A;

[0071] and / or, the 87th amino acid residue is replaced with F;

[0072] and / or, the 88th amino acid residue is replaced with A;

[0073] and / or, the 108th amino acid residue is replaced with N;

[0074] and / or, the 118th amino acid residue is replaced with S;

[0075] and / or, the 120th amino acid residue is replaced with T;

[0076] and / or, the 141st amino acid residue is replaced with R;

[0077] and / or, the 152nd amino acid residue is replaced with F;

[0078] and / or, the 155th amino acid residue is replaced with A;

[0079] and / or, the 157th amino acid residue is replaced with V;

[0080] and / or, the 159th amino acid residue is replaced with S;

[0081] and / or, the 162nd amino acid residue is replaced with M;

[0082] and / or, the 168th amino acid residue is replaced with A;

[0083] and / or, the 178th amino acid residue is replaced with R;

[0084] and / or, the 214th amino acid residue is replaced with Y;

[0085] and / or, the 231st amino acid residue is replaced with A;

[0086] and / or, the 234th amino acid residue is replaced with M;

[0087] and / or, the 243rd amino acid residue is replaced with H;

[0088] and / or, the 303rd amino acid residue is replaced with N;

[0089] and / or, the 320th amino acid residue is replaced with F;

[0090] and / or, the 324th amino acid residue is replaced with M;

[0091] and / or, the 341st amino acid residue is replaced with M;

[0092] and / or, the 376th amino acid residue is replaced with I;

[0093] and / or, the 382nd amino acid residue is replaced with M;

[0094] and / or, the 405th amino acid residue is replaced with R;

[0095] and / or, the 420th amino acid residue is replaced with Q or Y.

[0096] In one embodiment, the mutant of the transaminase is that the 2nd amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is replaced with G;

[0097] and / or, the 3rd amino acid residue is replaced with H;

[0098] and / or, the 19th amino acid residue is replaced with W;

[0099] and / or, the 47th amino acid residue is replaced with Y;

[0100] and / or, the 49th amino acid residue is replaced with N;

[0101] and / or, the 59th amino acid residue is replaced with L;

[0102] and / or, the 76th amino acid residue is replaced with I;

[0103] and / or, the 86th amino acid residue is replaced with A;

[0104] and / or, the 87th amino acid residue is replaced with F;

[0105] and / or, the 88th amino acid residue is replaced with A;

[0106] and / or, the 108th amino acid residue is replaced with N;

[0107] and / or, the 118th amino acid residue is replaced with S;

[0108] and / or, the 120th amino acid residue is replaced with T;

[0109] and / or, the 141st amino acid residue is replaced with R;

[0110] and / or, the 152nd amino acid residue is replaced with F;

[0111] and / or, the 155th amino acid residue is replaced with A;

[0112] and / or, the 157th amino acid residue is replaced with V;

[0113] and / or, the 162nd amino acid residue is replaced with M;

[0114] and / or, the 168th amino acid residue is replaced with A;

[0115] and / or, the 178th amino acid residue is replaced with R;

[0116] and / or, the 214th amino acid residue is replaced with Y;

[0117] and / or, the 231st amino acid residue is replaced with A;

[0118] and / or, the 234th amino acid residue is replaced with M;

[0119] and / or, the 243rd amino acid residue is replaced with H;

[0120] and / or, the 303rd amino acid residue is replaced with N;

[0121] and / or, the 320th amino acid residue is replaced with F;

[0122] and / or, the 324th amino acid residue is replaced with M;

[0123] and / or, the 341st amino acid residue is replaced with M;

[0124] and / or, the 382nd amino acid residue is replaced with M;

[0125] and / or, the 420th amino acid residue is replaced with Y.

[0126] In one embodiment, the mutant of the transaminase has differences in amino acid residues at 4 to 31 positions on the amino acid sequence shown in SEQ ID NO: 1; preferably, the mutant of the transaminase has differences in amino acid residues at 11 to 27 positions on the amino acid sequence shown in SEQ ID NO: 1; for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27.

[0127] In one embodiment, the mutant of the transaminase has differences in amino acid residues selected from the following 11 to 27 positions on the amino acid sequence shown in SEQ ID NO: 1:

[0128] Position 1, Position 2, Position 3, Position 19, Position 47, Position 49, Position 56, Position 59, Position 76, Position 86, Position 87, Position 88, Position 108, Position 118, Position 120, Position 141, Position 152, Position 155, Position 157, Position 159, Position 162, Position 168, Position 178, Position 214, Position 231, Position 234, Position 243, Position 303, Position 320, Position 324, Position 341, Position 376, Position 382, Position 405, and Position 420;

[0129] Preferably, the mutant of the transaminase has differences in amino acid residues selected from the following 11 to 27 positions on the amino acid sequence shown in SEQ ID NO: 1:

[0130] Position 1, Position 2, Position 3, Position 19, Position 47, Position 49, Position 56, Position 59, Position 76, Position 86, Position 87, Position 88, Position 108, Position 118, Position 120, Position 141, Position 152, Position 155, Position 157, Position 159, Position 162, Position 168, Position 178, Position 214, Position 231, Position 234, Position 243, Position 303, Position 320, Position 324, Position 341, Position 376, Position 382, Position 405, and Position 420;

[0131] And the mutant of the transaminase has differences in amino acid residues at least at positions 19, 59, 86, 87, 88, 152, 155, 168, 231, 234, and 382.

[0132] In one embodiment, the mutant of the transaminase has differences in amino acid residues at positions selected from the following 18th to 27th positions in the amino acid sequence shown in SEQ ID NO: 1: the 2nd, 3rd, 19th, 47th, 49th, 59th, 76th, 86th, 87th, 88th, 108th, 118th, 120th, 141st, 152nd, 155th, 157th, 162nd, 168th, 178th, 214th, 231st, 234th, 243rd, 303rd, 320th, 324th, 341st, 382nd, and 420th positions;

[0133] Preferably, the mutant of the transaminase has differences in amino acid residues at positions selected from the following 18th to 27th positions in the amino acid sequence shown in SEQ ID NO: 1: the 2nd, 3rd, 19th, 47th, 49th, 59th, 76th, 86th, 87th, 88th, 108th, 118th, 120th, 141st, 152nd, 155th, 157th, 162nd, 168th, 178th, 214th, 231st, 234th, 243rd, 303rd, 320th, 324th, 341st, 382nd, and 420th positions;

[0134] And the mutant of the transaminase has differences at least at the 19th, 59th, 86th, 87th, 88th, 108th, 118th, 120th, 152nd, 155th, 168th, 231st, 234th, 303rd, 324th, 341st, 382nd, and 420th positions.

[0135] In one embodiment, the differences of the mutant of the transaminase in the amino acid sequence shown in SEQ ID NO: 1 are shown in Table 1:

[0136] Table 1

[0137]

[0138]

[0139]

[0140]

[0141]

[0142] In one embodiment, the halogen may independently be fluorine, chlorine, bromine, or iodine.

[0143] In one embodiment, the C 1-4 alkyl groups can independently be methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl or tert-butyl.

[0144] In one embodiment, the C 1-4 alkoxy groups can independently be methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, isobutoxy or tert-butoxy.

[0145] In one embodiment, the C 2-6 alkyl groups can independently be ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl or tert-butyl.

[0146] In one embodiment, R 1 , R 2 , R 3 , R 4 and R 5 are H.

[0147] In one preferred embodiment, the compound I and the compound II are selected from any one of the following embodiments:

[0148] Embodiment 1: The compound I is The compound II is

[0149] Embodiment 2: The compound I is The compound II is

[0150] Embodiment 3: The compound I is The compound II is

[0151] Embodiment 4: The compound I is The compound II is

[0152] Embodiment 5: The compound I is The compound II is

[0153] Embodiment 6: The compound I is The compound II is

[0154] The solvent is a solvent conventional for such reactions in the art. Preferably, the solvent is a buffer solution; more preferably, the solvent is selected from one or more of sodium borate buffer solution, potassium phosphate buffer solution, tris(hydroxymethyl)aminomethane hydrochloride buffer solution and triethanolamine salt buffer solution; most preferably, the solvent is sodium borate buffer solution, tris(hydroxymethyl)aminomethane hydrochloride buffer solution or triethanolamine salt buffer solution.

[0155] In one embodiment, the volume-to-mass ratio of the solvent to Compound I is (0.01 - 1) L / g; for example, (0.05 - 0.5) L / g; and more specifically, 0.05 L / g or 0.1 L / g.

[0156] In the transamination reaction, the amino donor is a conventional amino donor in this type of reaction in the art. Preferably, the amino donor is selected from one or more of isopropylamine, L-alanine, DL-alanine, L-lysine, DL-lysine, (S)-1-phenylethylamine, (R)-1-phenylethylamine, (RS)-1-phenylethylamine, pentanediamine, o-phthalylamine, o-phthalylamine hydrochloride, 2-(4-nitrophenyl)ethylamine, and 2-(4-nitrophenyl)ethylamine hydrochloride. More preferably, it is isopropylamine.

[0157] In a preferred embodiment, the molar ratio of the amino donor to Compound I is (3 - 30):1; for example, (10 - 25):1, and more specifically, 11:1, 12:1, 21:1, 22:1, or 23:1.

[0158] In a preferred embodiment, the amino donor and the solvent participate in the reaction in the form of a solution of the amino donor. Preferably, in the solution of the amino donor, the molar concentration of the amino donor is 0.1 - 1.5 mol / L, for example, 0.2 - 1 mol / L, and more specifically, 1 mol / L.

[0159] In a preferred embodiment, the pH value of the solution of the amino donor is 6.0 - 11.0, for example, 7 - 10.0, and preferably 8.5, 9, or 9.5.

[0160] In a preferred embodiment, the reaction system of the transamination reaction further includes a coenzyme, and the coenzyme is a conventional coenzyme of aminotransferase in the art, such as pyridoxal phosphate.

[0161] In a preferred embodiment, the molar ratio of the coenzyme to Compound I is (0.001 - 1):1, for example, (0.002 - 0.25):1, and more specifically, 0.009:1, 0.017:1, 0.018:1, or 0.019:1.

[0162] In a preferred embodiment, the mass ratio of the mutant of the transaminase to the mass of Compound I is (0.01 - 1):1, for example, (0.02 - 0.5):1, and more specifically, 0.02:1, 0.2:1, or 0.5:1.

[0163] In a preferred embodiment, the temperature of the transamination reaction is 20 - 55 °C, for example, 25 - 50 °C, and more specifically, 27 °C, 37 °C, or 47 °C.

[0164] The progress of the transamination reaction is detected by using conventional monitoring methods for such reactions in the art (such as TLC or UPCC). The time of the transamination reaction is determined by the disappearance or cessation of reaction of Compound I as the end point of the reaction, which can be 4 - 50 h, for example, 24 h.

[0165] In a preferred embodiment, the method for preparing Compound II comprises the following steps: in the solvent, in the presence of the mutant of the transaminase and the coenzyme, reacting Compound I with the amino donor to obtain Compound II.

[0166] In a preferred embodiment, the reaction system of the transamination reaction consists of the solvent, the mutant of the transaminase, the coenzyme, Compound I and the amino donor.

[0167] In a preferred embodiment, the method for preparing Compound II comprises the following steps:

[0168] (A) Mixing the coenzyme, buffer and amino donor to obtain a mixed solution;

[0169] (B) Mixing Compound I, the mutant of the transaminase and the mixed solution obtained in step (A), and carrying out the transamination reaction to obtain Compound II.

[0170] The transamination reaction further comprises a post-treatment step, and the post-treatment is a conventional post-treatment for such reactions in the art; preferably, the post-treatment comprises one or more of the following steps: adjusting the pH value (for example, adjusting the pH = 13 with potassium hydroxide), extraction (for example, methyl tert-butyl ether) and concentration (for example, distillation under reduced pressure).

[0171] The present invention provides a mutant of a transaminase, and the mutant of the transaminase satisfies the following conditions (1) and (2):

[0172] Condition (1): The mutant of the transaminase has amino acid residue differences at one or more sites selected from the following on the amino acid sequence shown in SEQ ID NO:1:

[0173] The 1st, 2nd, 3rd, 19th, 47th, 49th, 50th, 55th, 56th, 59th, 68th, 76th, 86th, 87th, 88th, 108th, 118th, 120th, 141st, 152nd, 155th, 157th, 159th, 162nd, 168th, 170th, 178th, 191st, 214th, 231st, 234th, 243rd, 251st, 303rd, 320th, 324th, 341st, 376th, 382nd, 402nd, 405th, 416th and 420th positions;

[0174] Condition (2): The mutant of the transaminase has at least 1 amino acid residue difference selected from the following sites in the amino acid sequence shown in SEQ ID NO: 1:

[0175] The 1 site is selected from the 1st, 2nd, 3rd, 19th, 47th, 49th, 50th, 56th, 68th, 76th, 86th, 88th, 108th, 118th, 120th, 141st, 155th, 157th, 162nd, 168th, 170th, 178th, 191st, 214th, 243rd, 251st, 303rd, 320th, 324th, 341st, 376th, 382nd, 402nd, 405th, 416th and 420th positions.

[0176] The definition of the mutant of the transaminase can also be as described in any aspect of the present invention.

[0177] The present invention also provides an isolated nucleic acid, wherein the polynucleotide sequence of the nucleic acid encodes the mutant of the transaminase as described above.

[0178] The present invention also provides a recombinant expression vector, wherein the recombinant expression vector contains the nucleic acid as described above.

[0179] The present invention also provides a transformant, wherein the transformant contains the recombinant expression vector as described above.

[0180] The present invention also provides a method for preparing the mutant of the transaminase as described above, which cultures the transformant as described above and obtains a culture containing the mutant of the transaminase.

[0181] The present invention also provides the use of the mutant of the transaminase as described above in the preparation of chiral lactam compounds. Preferably, the chiral lactam compound is compound II as described above.

[0182] The amino acid abbreviations in the present invention are all conventional in the art unless otherwise specified, and the amino acids corresponding to the specific abbreviations are shown in Table 2.

[0183] Table 2

[0184]

[0185] On the basis of not violating the common knowledge in the art, the above preferred conditions can be arbitrarily combined to obtain various preferred examples of the present invention.

[0186] The reagents and raw materials used in the present invention are all commercially available.

[0187] The positive and progressive effects of the present invention are as follows: The S-chiral lactam obtained by catalyzing from the wild-type aminotransferase in the present invention has a conversion rate of less than 1%. Through directed evolution, several mutants with better catalytic activity ((S)-chiral lactam with a conversion rate of ≥90% and e.e. ≥98%) and better thermal tolerance are obtained. The yield of the target product can reach more than 85%, which can be used as a green and sustainable synthesis route and has high industrial application value. Detailed implementation manners

[0188] The present invention will be further illustrated by the following examples, but the present invention is not limited to the scope of the examples described herein. The experimental methods without specific conditions in the following examples are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0189] The mutants of the transaminase in the following examples can be natural aminotransferases (obtained by directed evolution from Ruegeria sp. TM1040) or prepared by conventional gene synthesis methods in the art.

[0190] The buffer solution formula used in the examples of the present invention is as follows:

[0191] The formula of 50 mM / L sodium tetraborate buffer solution with pH 10.0 (sodium borate buffer solution; containing 1 M / L isopropylamine): Sodium tetraborate decahydrate (MW 381.37): 19.06 g, isopropylamine: 86 mL, adjust the pH to 10.0 with hydrochloric acid (at room temperature), and make up to 1 L with water. (If you want to prepare a buffer solution with a different pH, just adjust it to the desired pH.)

[0192] The formula of 50 mM / L potassium phosphate buffer solution with pH 10.0 (containing 1 M / L isopropylamine): Take KH2PO4 (MW: 136.09): 2.654 g; take K2HPO4·3H2O (MW: 228.22): 6.96 g, isopropylamine: 86 mL, adjust the pH to 10.0 with hydrochloric acid (at room temperature), and make up to 1 L with water. (If you want to prepare a buffer solution with a different pH, just adjust it to the desired pH.)

[0193] The formulation of 50 mM / L triethanolamine buffer at pH 10.0 (containing 1 M / L isopropylamine): Take triethanolamine (MW: 149.19): 7.46 g, isopropylamine: 86 mL, adjust the pH to 10.0 with hydrochloric acid (at room temperature), and make up to 1 L with water. (If you want to prepare buffers with different pH values, just adjust to the desired pH.)

[0194] The formulation of 50 mM / L tris(hydroxymethyl)aminomethane hydrochloride buffer at pH 10.0 (containing 1 M / L isopropylamine): Take tris(hydroxymethyl)aminomethane hydrochloride (MW: 157.6) 7.88 g, isopropylamine: 86 mL, adjust the pH to 10.0 with hydrochloric acid (at room temperature), and make up to 1 L with water. (If you want to prepare buffers with different pH values, just adjust to the desired pH.)

[0195] Example 1: In sodium borate buffer, aminotransferase catalyzes the synthesis of the (S)-configured chiral compound II-2 from compound I-2 (R1-R5 = H, R6 = i-propyl).

[0196]

[0197] Add 0.01 g of pyridoxal phosphate and 50 mmol / L, 50 mL of sodium borate buffer containing isopropylamine at pH = 9.0 to a 100 mL beaker, and stir until the pyridoxal phosphate is completely dissolved to obtain a homogeneous solution.

[0198] Take out 20 mL of the solution from the beaker and add it to a 50 mL reaction flask. Then add 0.2 g of compound I-2 and 0.1 g of aminotransferase with mutant 261 to the solution. After vortex mixing the resulting solution, react it at 47 °C for 24 h. After completion, take out the reaction solution and extract it with three times the volume of methyl tert-butyl ether (MTBE) (adjust the pH = 13 with potassium hydroxide). After extraction, take out the organic phase and perform vacuum distillation at a vacuum degree of -0.09 MPa (gauge pressure). Finally, 0.1368 g of the product is obtained, and the yield is 93.5%. After detection by UPCC, the enantiomeric excess value (e.e.) of the (S)-configured product is ≥98%.

[0199] Example 2: In sodium borate buffer, aminotransferase catalyzes the synthesis of the (S)-configured chiral compound II-2 from compound I-2 (R1-R5 = H, R6 = i-propyl).

[0200] Add 0.01 g of pyridoxal phosphate and 50 mmol / L, 50 mL of sodium borate buffer containing isopropylamine at pH = 9.5 to a 100 mL beaker, and stir until the pyridoxal phosphate is completely dissolved to obtain a homogeneous solution.

[0201] 20 mL of the solution was taken out from the beaker and added to a 50 mL reaction flask. 0.2 g of Compound I-2 and 0.1 g of aminotransferase with mutant 261 were added to the solution. After vortex mixing the resulting solution, the reaction was carried out at 37 °C for 24 h. After completion, the reaction solution was taken out and extracted with three volumes of methyl tert-butyl ether (MTBE) (adjusting the pH = 13 with potassium hydroxide). After extraction, the organic phase was taken out and distilled under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure). Finally, 0.1374 g of the product was obtained, and the yield was 93.9%. After detection by UPCC, the enantiomeric excess value (e.e.) of the (S)-configured product was ≥98%.

[0202] Example 3: Aminotransferase catalyzed the synthesis of (S)-configured chiral Compound II-1 from Compound I-1 (R1~R5 = H, R6 = ethyl) in sodium borate buffer.

[0203]

[0204] 0.01 g of pyridoxal phosphate and 50 mmol / L, 50 mL of sodium borate buffer containing isopropylamine with pH = 8.5 were added to a 100 mL beaker, and stirred until pyridoxal phosphate was completely dissolved to obtain a homogeneous solution.

[0205] 20 mL of the solution was taken out from the beaker and added to a 50 mL reaction flask. 0.2 g of Compound I-2 and 0.1 g of aminotransferase with mutant 184 were added to the solution. After vortex mixing the resulting solution, the reaction was carried out at 27 °C for 24 h. After completion, the reaction solution was taken out and extracted with three volumes of methyl tert-butyl ether (MTBE) (adjusting the pH = 13 with potassium hydroxide). After extraction, the organic phase was taken out and distilled under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure). Finally, 0.1452 g of the product was obtained, and the yield was 92.9%. After detection by UPCC, the enantiomeric excess value (e.e.) of the (S)-configured product was ≥98%.

[0206] Example 4: Aminotransferase catalyzed the synthesis of (S)-configured chiral Compound II-1 from Compound I-1 (R1~R5 = H, R6 = ethyl) in sodium borate buffer.

[0207] 0.01 g of pyridoxal phosphate and 50 mmol / L, 50 mL of sodium borate buffer containing isopropylamine with pH = 9.5 were added to a 100 mL beaker, and stirred until pyridoxal phosphate was completely dissolved to obtain a homogeneous solution.

[0208] Take out 20 mL of the solution from the beaker and add it to a 50 mL reaction flask. Then add 0.2 g of Compound I-1 and 0.1 g of aminotransferase with mutant 184 to the solution. After vortex mixing the resulting solution, react it at 47 °C for 24 h. After completion, take out the reaction solution and extract it with three volumes of methyl tert-butyl ether (MTBE) (adjust the pH to 13 with potassium hydroxide). After extraction, take out the organic phase and perform vacuum distillation under a vacuum of -0.09 MPa (gauge pressure). Finally, 0.1483 g of the product is obtained, with a yield of 94.9%. After detection by UPCC, the enantiomeric excess value (e.e.) of the (S)-configured product is ≥98%.

[0209] Example 5: Aminotransferase catalyzes the synthesis of (S)-configured chiral Compound II-3 from Compound I-3 (R1~R5 = H, R6 = n-propyl) in sodium borate buffer.

[0210]

[0211] Add 0.01 g of pyridoxal phosphate and 50 mL of 50 mmol / L sodium borate buffer containing isopropylamine with pH = 9.0 to a 100 mL beaker, and stir until the pyridoxal phosphate is completely dissolved to obtain a homogeneous solution.

[0212] Take out 20 mL of the solution from the beaker and add it to a 50 mL reaction flask. Then add 0.2 g of Compound I-3 and 0.1 g of aminotransferase with mutant 54 to the solution. After vortex mixing the resulting solution, react it at 47 °C for 24 h. After completion, take out the reaction solution and extract it with three volumes of methyl tert-butyl ether (MTBE) (adjust the pH to 13 with potassium hydroxide). After extraction, take out the organic phase and perform vacuum distillation under a vacuum of -0.09 MPa (gauge pressure). Finally, 0.1389 g of the product is obtained, with a yield of 94.9%. After detection by UPCC, the enantiomeric excess value (e.e.) of the (S)-configured product is ≥98%.

[0213] Example 6: Aminotransferase catalyzes the synthesis of (S)-configured chiral Compound II-3 from Compound I-3 (R1~R5 = H, R6 = n-propyl) in sodium borate buffer.

[0214] Add 0.01 g of pyridoxal phosphate and 50 mL of 50 mmol / L sodium borate buffer containing isopropylamine with pH = 9.5 to a 100 mL beaker, and stir until the pyridoxal phosphate is completely dissolved to obtain a homogeneous solution.

[0215] 20 mL of the solution was taken out from the beaker and added into a 50 mL reaction flask, and 0.2 g of Compound I-3 and 0.1 g of aminotransferase with mutant 54 were added to the solution. The resulting solution was vortexed and mixed evenly and then reacted at 47 °C for 24 h. After completion, the reaction solution was taken out and extracted with three volumes of methyl tert-butyl ether (MTBE) (the pH was adjusted to 13 with potassium hydroxide). After extraction, the organic phase was taken out and distilled under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure). Finally, 0.1393 g of the product was obtained, and the yield was 95.2%. After detection by UPCC, the enantiomeric excess value (e.e.) of the (S)-configuration product was ≥98%.

[0216] Example 7: Aminotransferase catalyzed the synthesis of (S)-configuration chiral compound II-4 from Compound I-4 (R1~R5 = H, R6 = n-butyl) in sodium borate buffer solution.

[0217]

[0218] 0.01 g of pyridoxal phosphate and 50 mL of 50 mmol / L sodium borate buffer solution containing isopropylamine with pH = 9.0 were added into a 100 mL beaker, and stirred until pyridoxal phosphate was completely dissolved to obtain a homogeneous solution.

[0219] 20 mL of the solution was taken out from the beaker and added into a 50 mL reaction flask, and 0.2 g of Compound I-4 and 0.1 g of aminotransferase with mutant 323 were added to the solution. The resulting solution was vortexed and mixed evenly and then reacted at 47 °C for 24 h. After completion, the reaction solution was taken out and extracted with three volumes of methyl tert-butyl ether (MTBE) (the pH was adjusted to 13 with potassium hydroxide). After extraction, the organic phase was taken out and distilled under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure). Finally, 0.1316 g of the product was obtained, and the yield was 95.7%. After detection by UPCC, the enantiomeric excess value (e.e.) of the (S)-configuration product was ≥98%.

[0220] Example 8: Aminotransferase catalyzed the synthesis of (S)-configuration chiral compound II-5 from Compound I-5 (R1~R5 = H, R6 = i-butyl) in sodium borate buffer solution.

[0221]

[0222] 0.02 g of pyridoxal phosphate and 50 mL of 50 mmol / L sodium borate buffer solution containing isopropylamine with pH = 9.5 were added into a 100 mL beaker, and stirred until pyridoxal phosphate was completely dissolved to obtain a homogeneous solution.

[0223] 10 mL of the solution was taken out from the beaker and added to a 25 mL reaction flask. 0.2 g of Compound I-5 and 0.1 g of aminotransferase with mutant 323 were added to the solution. After vortex mixing the resulting solution, the reaction was carried out at 47 °C for 24 h. After completion, the reaction solution was taken out and extracted with three volumes of methyl tert-butyl ether (MTBE) (adjusting the pH = 13 with potassium hydroxide). After extraction, the organic phase was taken out and distilled under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure). Finally, 0.1261 g of the product was obtained, and the yield was 91.7%. After detection by UPCC, the enantiomeric excess value (e.e.) of the (S)-configured product was ≥98%.

[0224] Example 9: Aminotransferase catalyzed the synthesis of (S)-configured chiral Compound II-5 from Compound I-5 (R1~R5 = H, R6 = i-butyl) in potassium phosphate buffer.

[0225] 0.01 g of pyridoxal phosphate and 50 mL of 50 mmol / L sodium borate buffer containing isopropylamine with pH = 9.0 were added to a 100 mL beaker, and stirred until pyridoxal phosphate was completely dissolved to obtain a homogeneous solution.

[0226] 20 mL of the solution was taken out from the beaker and added to a 50 mL reaction flask. 0.2 g of Compound I-5 and 0.1 g of aminotransferase with mutant 201 were added to the solution. After vortex mixing the resulting solution, the reaction was carried out at 47 °C for 24 h. After completion, the reaction solution was taken out and extracted with three volumes of methyl tert-butyl ether (MTBE) (adjusting the pH = 13 with potassium hydroxide). After extraction, the organic phase was taken out and distilled under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure). Finally, 0.1333 g of the product was obtained, and the yield was 96.8%. After detection by UPCC, the enantiomeric excess value (e.e.) of the (S)-configured product was ≥98%.

[0227] Example 10: Aminotransferase catalyzed the synthesis of (S)-configured chiral Compound II-6 from Compound I-6 (R1~R5 = H, R6 = t-butyl) in potassium phosphate buffer.

[0228]

[0229] 0.02 g of pyridoxal phosphate and 50 mL of 50 mmol / L sodium borate buffer containing isopropylamine with pH = 9.5 were added to a 100 mL beaker, and stirred until pyridoxal phosphate was completely dissolved to obtain a homogeneous solution.

[0230] 10 mL of the solution was taken out from the beaker and added into a 25 mL reaction flask. 0.2 g of Compound I-6 and 0.1 g of aminotransferase with mutant 201 were added to the solution. After vortex mixing the resulting solution, the reaction was carried out at 47 °C for 24 h. After completion, the reaction solution was taken out and extracted with three volumes of methyl tert-butyl ether (MTBE) (adjusting the pH = 13 with potassium hydroxide). After extraction, the organic phase was taken out and distilled under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure). Finally, 0.1243 g of the product was obtained, and the yield was 90.3%. After detection by UPCC, the enantiomeric excess value (e.e.) of the (S)-configured product was ≥98%.

[0231] Example 11: In a tris(hydroxymethyl)aminomethane hydrochloride buffer solution, aminotransferase catalyzed the synthesis of (S)-configured chiral Compound II-2 from Compound I-2 (R1~R5 = H, R6 = i-propyl).

[0232] 0.01 g of pyridoxal phosphate and 50 mmol / L, 50 mL of sodium borate buffer solution containing isopropylamine with pH = 9.0 were added into a 100 mL beaker, and stirred until pyridoxal phosphate was completely dissolved to obtain a homogeneous solution.

[0233] 20 mL of the solution was taken out from the beaker and added into a 50 mL reaction flask. 0.2 g of Compound I-2 and 0.1 g of aminotransferase with mutant 24 were added to the solution. After vortex mixing the resulting solution, the reaction was carried out at 47 °C for 24 h. After completion, the reaction solution was taken out and extracted with three volumes of methyl tert-butyl ether (MTBE) (adjusting the pH = 13 with potassium hydroxide). After extraction, the organic phase was taken out and distilled under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure). Finally, 0.1431 g of the product was obtained, and the yield was 97.7%. After detection by UPCC, the enantiomeric excess value (e.e.) of the (S)-configured product was ≥98%.

[0234] Example 12: In a tris(hydroxymethyl)aminomethane hydrochloride buffer solution, aminotransferase catalyzed the synthesis of (S)-configured chiral Compound II-2 from Compound I-2 (R1~R5 = H, R6 = i-propyl).

[0235] 0.02 g of pyridoxal phosphate and 50 mmol / L, 50 mL of sodium borate buffer solution containing isopropylamine with pH = 9.5 were added into a 100 mL beaker, and stirred until pyridoxal phosphate was completely dissolved to obtain a homogeneous solution.

[0236] 10 mL of the solution was taken out from the beaker and added into a 25 mL reaction flask, and 0.2 g of Compound I-2 and 0.1 g of aminotransferase with Mutant 24 were added to the solution. After vortex mixing the obtained solution, the reaction was carried out at 47 °C for 24 h. After completion, the reaction solution was taken out and extracted with three volumes of methyl tert-butyl ether (MTBE) (adjusting the pH = 13 with potassium hydroxide). After extraction, the organic phase was taken out and distilled under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure). Finally, 0.1341 g of the product was obtained, and the yield was 91.6%. After detection by UPCC, the enantiomeric excess value (e.e.) of the (S)-configured product was ≥98%.

[0237] Example 13: Aminotransferase catalyzed the synthesis of (S)-configured chiral Compound II-2 from Compound I-2 (R1-R5 = H, R6 = i-propyl) in triethanolamine buffer solution.

[0238] 0.01 g of pyridoxal phosphate and 50 mL of sodium borate buffer solution containing isopropylamine with a concentration of 50 mmol / L and pH = 9.0 were added into a 100 mL beaker, and stirred until pyridoxal phosphate was completely dissolved to obtain a homogeneous solution.

[0239] 20 mL of the solution was taken out from the beaker and added into a 50 mL reaction flask, and 0.2 g of Compound I-2 and 0.1 g of aminotransferase with Mutant 97 were added to the solution. After vortex mixing the obtained solution, the reaction was carried out at 47 °C for 24 h. After completion, the reaction solution was taken out and extracted with three volumes of methyl tert-butyl ether (MTBE) (adjusting the pH = 13 with potassium hydroxide). After extraction, the organic phase was taken out and distilled under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure). Finally, 0.1432 g of the product was obtained, and the yield was 97.8%. After detection by UPCC, the enantiomeric excess value (e.e.) of the (S)-configured product was ≥98%.

[0240] Example 14: Aminotransferase catalyzed the synthesis of (S)-configured chiral Compound II-2 from Compound I-2 (R1-R5 = H, R6 = i-propyl) in triethanolamine buffer solution.

[0241] 0.02 g of pyridoxal phosphate and 50 mL of sodium borate buffer solution containing isopropylamine with a concentration of 50 mmol / L and pH = 9.5 were added into a 100 mL beaker, and stirred until pyridoxal phosphate was completely dissolved to obtain a homogeneous solution.

[0242] 10 mL of the solution was taken out from the beaker and added into a 25 mL reaction flask. 0.2 g of Compound I-2 and 0.1 g of aminotransferase with mutant 97 were added to the solution. The resulting solution was vortexed and mixed evenly, and then reacted at 47 °C for 24 h. After completion, the reaction solution was taken out and extracted with three volumes of methyl tert-butyl ether (MTBE) (the pH was adjusted to 13 with potassium hydroxide). After extraction, the organic phase was taken out and distilled under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure). Finally, 0.1348 g of the product was obtained, and the yield was 92.1%. After detection by UPCC, the enantiomeric excess value (e.e.) of the (S)-configuration product was ≥98%.

[0243] Example 15: Aminotransferase catalyzed the synthesis of (S)-configured chiral compound II-2 from Compound I-2 (R1-R5 = H, R6 = i-propyl) in sodium borate buffer.

[0244] 0.01 g of pyridoxal phosphate and 50 mL of 50 mmol / L sodium borate buffer containing isopropylamine with pH = 9.0 were added into a 100 mL beaker, and stirred until pyridoxal phosphate was completely dissolved to obtain a homogeneous solution.

[0245] 20 mL of the solution was taken out from the beaker and added into a 50 mL reaction flask. 0.2 g of Compound I-2 and 0.04 g of aminotransferase with mutant 115 were added to the solution. The resulting solution was vortexed and mixed evenly, and then reacted at 47 °C for 24 h. After completion, the reaction solution was taken out and extracted with three volumes of methyl tert-butyl ether (MTBE) (the pH was adjusted to 13 with potassium hydroxide). After extraction, the organic phase was taken out and distilled under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure). Finally, 0.1422 g of the product was obtained, and the yield was 97.2%. After detection by UPCC, the enantiomeric excess value (e.e.) of the (S)-configuration product was ≥98%.

[0246] Example 16: Aminotransferase catalyzed the synthesis of (S)-configured chiral compound II-2 from Compound I-2 (R1-R5 = H, R6 = i-propyl) in sodium borate buffer.

[0247] 0.01 g of pyridoxal phosphate and 50 mL of 50 mmol / L sodium borate buffer containing isopropylamine with pH = 9.5 were added into a 100 mL beaker, and stirred until pyridoxal phosphate was completely dissolved to obtain a homogeneous solution.

[0248] Take out 20 mL of the solution from the beaker and add it to a 50 mL reaction flask. Then add 0.2 g of Compound I-2 and 0.04 g of aminotransferase with mutant 115 to the solution. After vortex mixing the resulting solution, react it at 47 °C for 24 h. After completion, take out the reaction solution and extract it with three volumes of methyl tert-butyl ether (MTBE) (adjust the pH to 13 with potassium hydroxide). After extraction, take out the organic phase and distill it under reduced pressure at a vacuum of -0.09 MPa (gauge pressure). Finally, 0.1422 g of the product is obtained with a yield of 97.7%. After detection by UPCC, the enantiomeric excess value (e.e.) of the (S)-configured product ≥ 98%.

[0249] Example 17: Aminotransferase catalyzes the synthesis of (S)-configured chiral Compound II-2 from Compound I-2 (R1~R5 = H, R6 = i-propyl) in sodium borate buffer.

[0250] Add 0.03 g of pyridoxal phosphate and 150 mL of 50 mmol / L sodium borate buffer containing isopropylamine with pH = 9.5 to a 200 mL beaker, and stir until the pyridoxal phosphate is completely dissolved to obtain a homogeneous solution.

[0251] Take out 100 mL of the solution from the beaker and add it to a 200 mL reaction flask. Then add 1 g of Compound I-2 and 0.2 g of aminotransferase with mutant 4 to the solution. After vortex mixing the resulting solution, react it at 47 °C for 24 h. After completion, take out the reaction solution and extract it with three volumes of methyl tert-butyl ether (MTBE) (adjust the pH to 13 with potassium hydroxide). After extraction, take out the organic phase and distill it under reduced pressure at a vacuum of -0.09 MPa (gauge pressure). Finally, 0.7208 g of the product is obtained with a yield of 98.5%. After detection by UPCC, the enantiomeric excess value (e.e.) of the (S)-configured product ≥ 98%.

[0252] Example 18: Aminotransferase catalyzes the synthesis of (S)-configured chiral Compound II-2 from Compound I-2 (R1~R5 = H, R6 = i-propyl) in sodium borate buffer.

[0253] Add 0.04 g of pyridoxal phosphate and 100 mL of 50 mmol / L sodium borate buffer containing isopropylamine with pH = 9.5 to a 200 mL beaker, and stir until the pyridoxal phosphate is completely dissolved to obtain a homogeneous solution.

[0254] 50 mL of the solution was taken out from the beaker and added into a 200 mL reaction flask. Then, 1 g of Compound I-2 and 0.2 g of aminotransferase with Mutant 4 were added to the solution. The resulting solution was vortexed and mixed evenly, and then reacted at 47 °C for 24 h. After completion, the reaction solution was taken out and extracted with three volumes of methyl tert-butyl ether (MTBE) (the pH was adjusted to 13 with potassium hydroxide). After extraction, the organic phase was taken out and distilled under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure). Finally, 0.6988 g of the product was obtained with a yield of 95.5%. After detection by UPCC, the enantiomeric excess value (e.e.) of the (S)-configuration product was ≥98%.

[0255] Example 19: Aminotransferase catalyzed the synthesis of (S)-configured chiral Compound II-2 from Compound I-2 (R1-R5 = H, R6 = i-propyl) in sodium borate buffer.

[0256] 0.22 g of pyridoxal phosphate and 2.2 L of sodium borate buffer containing isopropylamine with a concentration of 50 mmol / L and pH = 9.5 were added into a 2 L beaker, and stirred until pyridoxal phosphate was completely dissolved to obtain a homogeneous solution.

[0257] 1 L of the solution was taken out from the beaker and added into a 2 L reaction flask. Then, 10 g of Compound I-2 and 0.2 g of aminotransferase with Mutant 4 were added to the solution. The resulting solution was vortexed and mixed evenly, and then reacted at 47 °C for 24 h. After completion, the reaction solution was taken out and extracted with three volumes of methyl tert-butyl ether (MTBE) (the pH was adjusted to 13 with potassium hydroxide). After extraction, the organic phase was taken out and distilled under reduced pressure at a vacuum degree of -0.09 MPa (gauge pressure). Finally, 7.0909 g of the product was obtained with a yield of 96.9%. After detection by UPCC, the enantiomeric excess value (e.e.) of the (S)-configuration product was ≥98%.

[0258] Example 20: Aminotransferase catalyzed the synthesis of (S)-configured chiral Compound II-2 from Compound I-2 (R1-R5 = H, R6 = i-propyl) in sodium borate buffer.

[0259] 0.01 g of pyridoxal phosphate and 50 mL of sodium borate buffer containing isopropylamine with a concentration of 50 mmol / L and pH = 9.0 were added into a 100 mL beaker, and stirred until pyridoxal phosphate was completely dissolved to obtain a homogeneous solution.

[0260] Take out 20 mL of the solution from the beaker and add it to a 50 mL reaction flask. Then add 0.2 g of Compound I-2 and 0.04 g of aminotransferase with mutant 206 to the solution. After vortex mixing the resulting solution, react it at 47 °C for 24 h. After completion, take out the reaction solution and extract it with three volumes of methyl tert-butyl ether (MTBE) (adjust the pH to 13 with potassium hydroxide). After extraction, take out the organic phase and perform vacuum distillation under a vacuum of -0.09 MPa (gauge pressure). Finally, 0.1411 g of the product is obtained, with a yield of 96.4%. After detection by UPCC, the enantiomeric excess value (e.e.) of the (S)-configured product is ≥98%.

[0261] Example 21: Aminotransferase-catalyzed synthesis of (S)-configured chiral Compound II-2 from Compound I-2 (R1~R5 = H, R6 = i-propyl) in sodium borate buffer

[0262] Add 0.02 g of pyridoxal phosphate and 50 mL of 50 mmol / L sodium borate buffer containing isopropylamine with pH = 9.5 to a 100 mL beaker, and stir until the pyridoxal phosphate is completely dissolved to obtain a homogeneous solution.

[0263] Take out 10 mL of the solution from the beaker and add it to a 50 mL reaction flask. Then add 0.2 g of Compound I-2 and 0.04 g of aminotransferase with mutant 206 to the solution. After vortex mixing the resulting solution, react it at 47 °C for 24 h. After completion, take out the reaction solution and extract it with three volumes of methyl tert-butyl ether (MTBE) (adjust the pH to 13 with potassium hydroxide). After extraction, take out the organic phase and perform vacuum distillation under a vacuum of -0.09 MPa (gauge pressure). Finally, 0.1353 g of the product is obtained, with a yield of 92.4%. After detection by UPCC, the enantiomeric excess value (e.e.) of the (S)-configured product is ≥98%.

[0264] Example 22 Construction of an aminotransferase mutant library

[0265] For the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 11th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 36th, 39th, 41st, 43rd, 44th, 45th, 46th, 47th, 48th, 49th, 50th, 51st, 52nd, 53rd, 54th, 55th, 56th, 57th, 58th, 59th, 60th, 61st, 62nd, 65th, 68th, 69th, 76th, 79th, 80th, 83rd, 84th, 85th, 86th, 87th, 88th, 93rd, 94th, 99th, 101st, 104th, 106th, 108th, 109th, 114th, 115th, 116th, 117th, 118th, 119th, 120th, 121st, 122nd, 123rd, 124th, 125th, 126th, 127th, 128th, 129th, 130th, 132nd, 136th, 137th, 138th, 139th, 141st, 144th, 145th, 146th, 147th, 148th, 149th, 150th, 151st, 152nd, 153rd, 154th, 155th, 156th, 157th, 158th, 159th, 160th, 161st, 162nd, 163rd, 164th, 165th, 166th, 167th, 168th, 170th, 171st, 172nd, 175th, 177th, 178th, 179th, 181st, 188th, 191st, 193rd, 194th, 196th, 198th, 199th, 207th, 209th, 210th, 211th, 212th, 213th, 214th, 215th, 216th, 217th, 218th, 219th, 224th, 225th, 226th, 227th, 228th, 229th, 230th, 231st, 232nd, 233rd, 234th, 235th, 236th, 240th, 242nd, 243rd, 244th, 247th, 250th, 251st, 252nd, 253rd, 258th, 259th, 260th, 261st, 262nd, 263rd, 264th, 265th, 267th, 268th, 269th, 271stOne or more of the 275th, 276th, 278th, 280th, 282nd, 285th, 286th, 287th, 288th, 289th, 290th, 292nd, 293rd, 297th, 298th, 299th, 300th, 303rd, 304th, 305th, 308th, 310th, 311th, 312th, 314th, 315th, 316th, 317th, 318th, 319th, 320th, 321st, 322nd, 323rd, 324th, 325th, 326th, 327th, 328th, 329th, 341st, 344th, 345th, 346th, 347th, 350th, 354th, 357th, 365th, 366th, 368th, 369th, 371st, 376th, 377th, 380th, 382nd, 383rd, 384th, 389th, 391st, 392nd, 393rd, 397th, 398th, 400th, 401st, 402nd, 404th, 405th, 406th, 408th, 409th, 410th, 411th, 412th, 413th, 414th, 415th, 416th, 418th, 419th, 420th, 421st, 422nd, 423rd, 424th, 425th, 426th, 427th, 428th, 429th, 430th, 431st, 434th, 435th, 437th, 438th, 439th, 443rd, 446th, 450th, 453rd, 455th and 456th.

[0266] And mutants 1 - 370 with higher catalytic activity were screened out. The specific mutation sites and types are shown in Table 3. The reaction conditions were the same as in Example 1, except that the mutant of the catalytic enzyme in the reaction was replaced with the mutants in Table 3, and the reaction effects are shown in Table 3:

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280] Example 23: Confirmation of the target compound.

[0281] The configurations of the target products in Examples 1 - 21 were confirmed according to the standard product of the S configuration. Under the same conditions, the retention times of the peaks of the target products in Examples 1 - 21 were the same as those of the peaks of the S - configuration standard product.

[0282] The analysis methods and results of the substrate (Compound I - 2) and the target product (II - 2) in Example 1 are shown in Table 4.

[0283] Table 4

[0284]

[0285]

[0286] In Examples 2 - 21, the analysis method was the same as that in Example 1, and the retention times of each compound are shown in Table 5.

[0287] Table 5

[0288]

Claims

1. A method for preparing compound II, characterized in that, It includes the following steps: In a solvent, in the presence of a mutant of transaminase, compound I and an amino donor are subjected to a transamination reaction as shown in the following formula to obtain compound II; Wherein, R 1 、R 2 、R 3 、R 4 and R 5 are independently H, halogen, amino, C 1-4 alkyl, carboxyl, hydroxyl, C 1-4 alkoxy or nitro; R 6 is C 2-6 alkyl; The mutant of the transaminase has amino acid residue differences at one or more sites selected from the following on the amino acid sequence shown in SEQ ID NO: 1: Position 1, position 2, position 3, position 19, position 47, position 49, position 50, position 55, position 56, position 59, position 68, position 76, position 86, position 87, position 88, position 108, position 118, position 120, position 141, position 152, position 155, position 157, position 159, position 162, position 168, position 170, position 178, position 191, position 214, position 231, position 234, position 243, position 251, position 303, position 320, position 324, position 341, position 376, position 382, position 402, position 405, position 416, and position 420.

2. The preparation method of compound II as described in claim 1, characterized in that, It satisfies one or both of the following conditions: (1) The said difference is deletion, addition or substitution, preferably substitution; and (2) The mutant of the transaminase has amino acid residue differences at one or more sites selected from the following on the amino acid sequence shown in SEQ ID NO: 1: Position 1, position 2, position 3, position 19, position 47, position 49, position 56, position 59, position 76, position 86, position 87, position 88, position 108, position 118, position 120, position 141, position 152, position 155, position 157, position 159, position 162, position 168, position 178, position 214, position 231, position 234, position 243, position 303, position 320, position 324, position 341, position 376, position 382, position 405, and position 420; Preferably, the mutant of the transaminase has amino acid residue differences at one or more sites selected from the following on the amino acid sequence shown in SEQ ID NO: 1: Position 2, position 3, position 19, position 47, position 49, position 59, position 76, position 86, position 87, position 88, position 108, position 118, position 120, position 141, position 152, position 155, position 157, position 162, position 168, position 178, position 214, position 231, position 234, position 243, position 303, position 320, position 324, position 341, position 382, and position 420.

3. The preparation method of compound II according to claim 1, characterized in that, The mutant of the transaminase is such that the amino acid residue at the 1st position in the amino acid sequence shown in SEQ ID NO: 1 is replaced with A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, or W; and / or, the amino acid residue at the 2nd position is replaced with A, C, D, E, F, G, H, I, K, M, Q, R, S, T, or Y; and / or, the amino acid residue at the 3rd position is replaced with C, E, F, H, I, L, M, N, Q, R, S, T, V, W, or Y; and / or, the amino acid residue at the 19th position is replaced with A, D, E, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; and / or, the amino acid residue at the 47th position is replaced with A, C, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y; and / or, the amino acid residue at the 49th position is replaced with A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, V, W, or Y; and / or, the amino acid residue at the 50th position is replaced with A, C, D, E, F, H, I, L, M, N, P, Q, R, S, T, V, or W; and / or, the amino acid residue at the 55th position is replaced with A, C, D, E, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y; and / or, the amino acid residue at the 56th position is replaced with C, D, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; and / or, the amino acid residue at the 59th position is replaced with A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; and / or, the amino acid residue at the 68th position is replaced with A, C, D, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y; and / or, the amino acid residue at the 76th position is replaced with A, C, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; and / or, the amino acid residue at the 86th position is replaced with A, C, D, E, F, G, H, I, K, L, N, Q, R, T, V, W, or Y; and / or, the amino acid residue at the 87th position is replaced with A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, or V; and / or, the amino acid residue at the 88th position is replaced with A, C, D, E, G, H, I, K, L, M, N, P, Q, S, T, or Y; and / or, the amino acid residue at the 108th position is replaced with A, C, E, F, G, L, N, P, Q, R, S, T, V, W, or Y; and / or, the amino acid residue at the 118th position is replaced with A, C, D, E, F, H, I, L, M, N, P, Q, R, S, T, V, W, or Y; and / or, the amino acid residue at the 120th position is replaced with A, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W, or Y; and / or, the amino acid residue at the 141st position is replaced with A, C, D, F, G, H, I, K, M, N, R, S, T, V, W, or Y;and / or, the 152nd amino acid residue is replaced with A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V or W; and / or, the 155th amino acid residue is replaced with A, C, D, E, F, G, H, I, K, M, N, P, Q, R, T, V or W; and / or, the 157th amino acid residue is replaced with A, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V or Y; and / or, the 159th amino acid residue is replaced with A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, V, W or Y; and / or, the 162nd amino acid residue is replaced with A, D, E, F, G, H, I, K, M, N, P, Q, S, T, V, W or Y; and / or, the 168th amino acid residue is replaced with A, C, E, G, H, I, K, L, M, N, P, Q, R, S, T or Y; and / or, the 170th amino acid residue is replaced with A, C, D, E, F, G, H, I, L, M, N, Q, R, S, T, V, W or Y; and / or, the 178th amino acid residue is replaced with A, C, D, E, F, G, H, I, K, L, M, P, R, S, T, V, W or Y; and / or, the 191st amino acid residue is replaced with A, C, D, F, H, I, K, L, M, N, P, Q, R, T, V or Y; and / or, the 214th amino acid residue is replaced with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, W or Y; and / or, the 231st amino acid residue is replaced with A, C, E, F, I, K, L, M, N, P, Q, R, S, V, W or Y; and / or, the 234th amino acid residue is replaced with A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W or Y; and / or, the 243rd amino acid residue is replaced with A, C, D, F, G, H, I, K, N, P, R, S, T, V, W or Y; and / or, the 251st amino acid residue is replaced with A, C, D, E, F, G, H, I, L, M, N, P, R, S, T, V or W; and / or, the 303rd amino acid residue is replaced with A, D, E, G, H, I, K, L, M, N, P, Q, R, T, V, W or Y; and / or, the 320th amino acid residue is replaced with A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W or Y; and / or, the 324th amino acid residue is replaced with C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T or Y; and / or, the 341st amino acid residue is replaced with A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W or Y; and / or, the 376th amino acid residue is replaced with A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T or W;and / or, the 382nd amino acid residue is replaced with A, C, D, E, F, G, I, M, N, P, Q, R, S, T, V or Y; and / or, the 402nd amino acid residue is replaced with A, C, E, F, G, H, I, M, P, Q, R, S, T, V, W or Y; and / or, the 405th amino acid residue is replaced with A, C, D, E, F, G, H, I, K, L, M, Q, R, S, T, V, W or Y; and / or, the 416th amino acid residue is replaced with A, D, E, F, G, H, N, P, Q, R, V, W or Y; and / or, the 420th amino acid residue is replaced with A, D, E, F, G, H, I, K, L, M, N, P, Q, S, V, W or Y; Preferably, the mutant of the transaminase is that the amino acid residue at the 1st position in the amino acid sequence shown in SEQ ID NO: 1 is replaced with L; and / or, the amino acid residue at the 2nd position is replaced with D or G; and / or, the amino acid residue at the 3rd position is replaced with H or N; and / or, the amino acid residue at the 19th position is replaced with A or W; and / or, the amino acid residue at the 47th position is replaced with Y; and / or, the amino acid residue at the 49th position is replaced with N; and / or, the amino acid residue at the 56th position is replaced with S; and / or, the amino acid residue at the 59th position is replaced with L; and / or, the amino acid residue at the 76th position is replaced with I; and / or, the amino acid residue at the 86th position is replaced with A; and / or, the amino acid residue at the 87th position is replaced with F; and / or, the amino acid residue at the 88th position is replaced with A; and / or, the amino acid residue at the 108th position is replaced with N; and / or, the amino acid residue at the 118th position is replaced with S; and / or, the amino acid residue at the 120th position is replaced with T; and / or, the amino acid residue at the 141st position is replaced with R; and / or, the amino acid residue at the 152nd position is replaced with F; and / or, the amino acid residue at the 155th position is replaced with A; and / or, the amino acid residue at the 157th position is replaced with V; and / or, the amino acid residue at the 159th position is replaced with S; and / or, the amino acid residue at the 162nd position is replaced with M; and / or, the amino acid residue at the 168th position is replaced with A; and / or, the amino acid residue at the 178th position is replaced with R; and / or, the amino acid residue at the 214th position is replaced with Y; and / or, the amino acid residue at the 231st position is replaced with A; and / or, the amino acid residue at the 234th position is replaced with M; and / or, the amino acid residue at the 243rd position is replaced with H; and / or, the amino acid residue at the 303rd position is replaced with N; and / or, the amino acid residue at the 320th position is replaced with F; and / or, the amino acid residue at the 324th position is replaced with M; and / or, the amino acid residue at the 341st position is replaced with M; and / or, the amino acid residue at the 376th position is replaced with I; and / or, the amino acid residue at the 382nd position is replaced with M; and / or, the amino acid residue at the 405th position is replaced with R; and / or, the amino acid residue at the 420th position is replaced with Q or Y; Preferably, the mutant of the transaminase has the 2nd amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 replaced by G; and / or, the 3rd amino acid residue replaced by H; and / or, the 19th amino acid residue replaced by W; and / or, the 47th amino acid residue replaced by Y; and / or, the 49th amino acid residue replaced by N; and / or, the 59th amino acid residue replaced by L; and / or, the 76th amino acid residue replaced by I; and / or, the 86th amino acid residue replaced by A; and / or, the 87th amino acid residue replaced by F; and / or, the 88th amino acid residue replaced by A; and / or, the 108th amino acid residue replaced by N; and / or, the 118th amino acid residue replaced by S; and / or, the 120th amino acid residue replaced by T; and / or, the 141st amino acid residue replaced by R; and / or, the 152nd amino acid residue replaced by F; and / or, the 155th amino acid residue replaced by A; and / or, the 157th amino acid residue replaced by V; and / or, the 162nd amino acid residue replaced by M; and / or, the 168th amino acid residue replaced by A; and / or, the 178th amino acid residue replaced by R; and / or, the 214th amino acid residue replaced by Y; and / or, the 231st amino acid residue replaced by A; and / or, the 234th amino acid residue replaced by M; and / or, the 243rd amino acid residue replaced by H; and / or, the 303rd amino acid residue replaced by N; and / or, the 320th amino acid residue replaced by F; and / or, the 324th amino acid residue replaced by M; and / or, the 341st amino acid residue replaced by M; and / or, the 382nd amino acid residue replaced by M; and / or, the 420th amino acid residue replaced by Y.

4. The preparation method of compound II according to claim 1, characterized in that, The mutant of the transaminase has differences in amino acid residues at 4 to 31 positions in the amino acid sequence shown in SEQ ID NO: 1; Preferably, the mutant of the transaminase has differences in amino acid residues at 11 to 27 positions in the amino acid sequence shown in SEQ ID NO: 1; such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27; Preferably, the mutant of the transaminase has differences in amino acid residues at 11 to 27 positions selected from the following in the amino acid sequence shown in SEQ ID NO: 1: Position 1, Position 2, Position 3, Position 19, Position 47, Position 49, Position 56, Position 59, Position 76, Position 86, Position 87, Position 88, Position 108, Position 118, Position 120, Position 141, Position 152, Position 155, Position 157, Position 159, Position 162, Position 168, Position 178, Position 214, Position 231, Position 234, Position 243, Position 303, Position 320, Position 324, Position 341, Position 376, Position 382, Position 405 and Position 420; Preferably, the mutant of the transaminase has differences in amino acid residues selected from the following 11 to 27 positions on the amino acid sequence shown in SEQ ID NO: 1: Position 1, Position 2, Position 3, Position 19, Position 47, Position 49, Position 56, Position 59, Position 76, Position 86, Position 87, Position 88, Position 108, Position 118, Position 120, Position 141, Position 152, Position 155, Position 157, Position 159, Position 162, Position 168, Position 178, Position 214, Position 231, Position 234, Position 243, Position 303, Position 320, Position 324, Position 341, Position 376, Position 382, Position 405 and Position 420; And the mutant of the transaminase has differences in amino acid residues at least at Position 19, Position 59, Position 86, Position 87, Position 88, Position 152, Position 155, Position 168, Position 231, Position 234 and Position 382; Preferably, the mutant of the transaminase has differences in amino acid residues selected from the following 18 to 27 positions on the amino acid sequence shown in SEQ ID NO: 1: Position 2, Position 3, Position 19, Position 47, Position 49, Position 59, Position 76, Position 86, Position 87, Position 88, Position 108, Position 118, Position 120, Position 141, Position 152, Position 155, Position 157, Position 162, Position 168, Position 178, Position 214, Position 231, Position 234, Position 243, Position 303, Position 320, Position 324, Position 341, Position 382 and Position 420; Preferably, the mutant of the transaminase has differences in amino acid residues selected from the following 18 to 27 positions on the amino acid sequence shown in SEQ ID NO: 1: Position 2, Position 3, Position 19, Position 47, Position 49, Position 59, Position 76, Position 86, Position 87, Position 88, Position 108, Position 118, Position 120, Position 141, Position 152, Position 155, Position 157, Position 162, Position 168, Position 178, Position 214, Position 231, Position 234, Position 243, Position 303, Position 320, Position 324, Position 341, Position 382 and Position 420; And the mutant of the transaminase has at least differences in amino acid residues at positions 19, 59, 86, 87, 88, 108, 118, 120, 152, 155, 168, 231, 234, 303, 324, 341, 382 and 420.

5. The preparation method of compound II according to claim 1, characterized in that, The differences of the mutant of the transaminase in the amino acid sequence shown in SEQ ID NO: 1 are as shown in any of the following schemes:

6. The preparation method of Compound II according to claim 1, characterized in that, It satisfies one or more of the following conditions: (1) The halogen is independently fluorine, chlorine, bromine or iodine; (2) The said C 1-4 The alkyl groups are independently methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl or tert-butyl; (3) The C 1-4 alkoxy group is independently methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, isobutoxy or tert-butoxy; (4) The said C 2-6 The alkyl groups are independently ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl or tert-butyl; (5)R 1 , R 2 , R 3 , R 4 and R 5 is H; (6) The solvent is a buffer solution; preferably, the solvent is selected from one or more of sodium borate buffer solution, potassium phosphate buffer solution, tris(hydroxymethyl)aminomethane hydrochloride buffer solution and triethanolamine salt buffer solution; more preferably, the solvent is sodium borate buffer solution, tris(hydroxymethyl)aminomethane hydrochloride buffer solution or triethanolamine salt buffer solution; (7) The volume-mass ratio of the solvent to Compound I is (0.01-1) L / g; for example, (0.05-0.5) L / g; for another example, 0.05 L / g or 0.1 L / g; (8) The amino donor is selected from one or more of isopropylamine, L-alanine, DL-alanine, L-lysine, DL-lysine, (S)-1-phenylethylamine, (R)-1-phenylethylamine, (RS)-1-phenylethylamine, pentanediamine, o-phthalylamine, o-phthalylamine hydrochloride, 2-(4-nitrophenyl)ethylamine and 2-(4-nitrophenyl)ethylamine hydrochloride; preferably, it is isopropylamine; (9) The molar ratio of the amino donor to Compound I can be (3-30):1; for example, (10-25):1, for another example, 11:1, 12:1, 21:1, 22:1 or 23:1; (10) The amino donor and the solvent participate in the reaction in the form of a solution of the amino donor; preferably, in the solution of the amino donor, the molar concentration of the amino donor is 0.1-1.5 mol / L, for example, 0.2-1 mol / L, for another example, 1 mol / L; more preferably, the pH value of the solution of the amino donor is 6.0-11.0, for example, 7-10.0, and preferably 8.5, 9 or 9.5; (11) The reaction system of the transamination reaction further includes a coenzyme, such as pyridoxal phosphate; preferably, the molar ratio of the coenzyme to Compound I is (0.001-1):1, for example, (0.002-0.25):1, for another example, 0.009:1, 0.017:1, 0.018:1 or 0.019:1; (12) The mass ratio of the mutant of the transaminase to the mass of Compound I is (0.01-1):1, for example, (0.02-0.5):1, for another example, 0.02:1, 0.2:1 or 0.5:1; (13) The temperature of the transamination reaction is 20-55 °C, for example, 25-50 °C, for another example, 27 °C, 37 °C or 47 °C; and (14) The transamination reaction further includes a post-treatment step, and the post-treatment includes one or more of the following steps: adjusting the pH value (for example, adjusting the pH = 13 with potassium hydroxide), extraction (for example, methyl tert-butyl ether), and concentration (for example, vacuum distillation). Preferably, the method for preparing the compound II satisfies one or two of the following conditions: (1) The method for preparing the compound II includes the following steps: in the solvent, in the presence of the mutant of the transaminase and the coenzyme, performing a transamination reaction on the compound I and the amino donor to obtain the compound II; Preferably, the method for preparing the compound II includes the following steps: (A) Mixing the coenzyme, buffer solution, and amino donor to obtain a mixed solution; (B) Mixing the compound I, the mutant of the transaminase, and the mixed solution obtained in the step (A), and performing the transamination reaction to obtain the compound II; (2) The reaction system of the transamination reaction is composed of the solvent, the mutant of the transaminase, the coenzyme, the compound I, and the amino donor.

7. A mutant of a transaminase, characterized in that, The mutant of the transaminase satisfies the following conditions (1) and (2): Condition (1): The mutant of the transaminase has differences in amino acid residues at one or more sites selected from the following on the amino acid sequence shown in SEQ ID NO: 1: Position 1, Position 2, Position 3, Position 19, Position 47, Position 49, Position 50, Position 55, Position 56, Position 59, Position 68, Position 76, Position 86, Position 87, Position 88, Position 108, Position 118, Position 120, Position 141, Position 152, Position 155, Position 157, Position 159, Position 162, Position 168, Position 170, Position 178, Position 191, Position 214, Position 231, Position 234, Position 243, Position 251, Position 303, Position 320, Position 324, Position 341, Position 376, Position 382, Position 402, Position 405, Position 416, and Position 420; Condition (2): The mutant of the transaminase has differences in amino acid residues at least at one site selected from the following on the amino acid sequence shown in SEQ ID NO: 1: The one site is selected from Position 1, Position 2, Position 3, Position 19, Position 47, Position 49, Position 50, Position 56, Position 68, Position 76, Position 86, Position 88, Position 108, Position 118, Position 120, Position 141, Position 155, Position 157, Position 162, Position 168, Position 170, Position 178, Position 191, Position 214, Position 243, Position 251, Position 303, Position 320, Position 324, Position 341, Position 376, Position 382, Position 402, Position 405, Position 416, and Position 420; The definition of the mutant of the transaminase may also be as described in any one of claims 2-5.

8. An isolated nucleic acid, wherein the nucleotide sequence of the nucleic acid encodes a mutant of the transaminase as described in any one of claims 2-5.

9. A recombinant expression vector, wherein the recombinant expression vector comprises the nucleic acid as described in claim 8.

10. A transformant, wherein the transformant comprises the recombinant expression vector as described in claim 9.

11. A method for preparing a mutant of the transaminase as described in any one of claims 2-5, which comprises culturing the transformant as described in claim 10 and obtaining a culture containing the mutant of the transaminase.

12. Use of a mutant of the transaminase as described in any one of claims 1-5 or 7 in the preparation of a chiral lactam compound; preferably, the chiral lactam compound is compound II as described in claim 1 or 6.

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