A recombinant transaminase and its enzyme preparation and application

Abstract

The present invention relates to the technical field of genetic engineering and enzyme engineering, in particular to the technical field of biocatalytic asymmetric synthesis of chiral intermediates, and more specifically to a recombinant transaminase and its enzyme preparation and application. The present invention utilizes genetic engineering technology to successfully construct a recombinant bacterium capable of expressing a recombinant transaminase, and utilizes the obtained recombinant transaminase to prepare a chiral intermediate compound I of ticagrelor, wherein the chiral selectivity ee value can reach 98%, and the chemical purity is greater than 99%, and the compound is a synthetic catalyst with high catalytic efficiency and high chiral selectivity.

Description

Technical Field

[0001] The present invention relates to the technical field of gene engineering and enzyme engineering, in particular to the technical field of biocatalytic asymmetric synthesis of chiral intermediates, and more specifically to a recombinant transaminase and its enzyme preparation and application. Background Art

[0002] Transaminase, also known as aminotransferase, is a type of pyridoxal phosphate-dependent transferase that can specifically transfer the amino group to the substrate ketone in the presence of an amino donor to obtain the corresponding chiral amine. As a green catalyst, the transamination reaction process involving transaminase has the advantages of simple reaction and green environmental protection.

[0003] The catalytic activity of transaminases is closely related to the substrate. Improving the catalytic performance of natural enzymes in certain types of synthesis by molecular modification of biocatalysts is of great significance for the application of industrial enzymatic synthesis. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a new recombinant transaminase that can be used in the synthesis of a chiral intermediate of ticagrelor shown in formula I from a compound shown in formula II, thereby significantly improving the catalytic activity and efficiently preparing the target compound.

[0005] In order to solve the above technical problems, the present invention first discloses a recombinant transaminase, the amino acid sequence of the recombinant transaminase is shown in SEQ ID NO: 1.

[0006] Furthermore, the present invention also discloses a nucleotide sequence encoding the recombinant transaminase, and the nucleotide sequence encoding is shown in SEQ ID NO:2.

[0007] Furthermore, the present invention discloses a recombinant expression vector comprising the nucleotide sequence SEQ ID NO: 2 encoding the recombinant transaminase.

[0008] Preferably, the recombinant expression vector is pET-28a.

[0009] Furthermore, the present invention also discloses a recombinant host cell, wherein the recombinant host cell comprises a recombinant expression vector comprising the nucleotide sequence SEQ ID NO: 1 encoding the transaminase.

[0010] Preferably, the host cell is Escherichia coli BL21 (DE3).

[0011] Meanwhile, the present invention also discloses an enzyme preparation, which contains the recombinant transaminase with the aforementioned amino acid sequence as shown in SEQ ID NO:1.

[0012] Furthermore, the present invention also discloses a method for preparing a recombinant transaminase, wherein a recombinant bacterium carrying a coding nucleotide sequence as shown in SEQ ID NO: 2 is inoculated into an LB medium, cultured at 37° C. overnight, and then transferred to an LB medium at a 1% inoculation rate, cultured at 37° C. until OD600=0.6-0.8, IPTG is added, and induced at 18° C. for 16-20 hours. After fermentation, the wet bacteria are collected by centrifugation, washed, and ultrasonically broken to obtain a recombinant transaminase enzyme solution.

[0013] Finally, the present invention also discloses the use of the above transaminase in catalyzing the transamination reaction of ketone compounds to prepare chiral amines. In particular, the ketone compound refers to the compound of formula II or a similar compound thereof.

[0014] More preferably, the transaminase is used in the preparation of a chiral intermediate compound of ticagrelor, and the synthetic process route is as follows:

[0015]

[0016] Compound I is obtained by reacting with compound II as substrate, transaminase as catalyst, buffer solution of pH 7.0-8.5 as reaction medium, auxiliary solvent, amino donor and PLP in the reaction system, and is separated and purified.

[0017] The present invention uses genetic engineering technology to successfully construct a recombinant expression vector capable of expressing a recombinant transaminase, which can stably exist and replicate autonomously in a host cell, and the transaminase catalyst obtained by the recombinant bacteria has high catalytic efficiency and high selectivity. When the recombinant transaminase is used as a catalyst to prepare the chiral intermediate compound I of ticagrelor, the chiral selectivity ee value is greater than 98%, and the chemical purity is greater than 99%, which is a synthesis method with high catalytic efficiency and high chiral selectivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the transaminase expression vector. DETAILED DESCRIPTION

[0019] In order to better understand the present invention, the present invention is further described below in conjunction with specific embodiments.

[0020] Unless otherwise specified, the reagents used in the examples of the present invention are common commercially available products.

[0021] Example 1

[0022] After the recombinant transaminase gene was sent to Shanghai Bioengineering Gene Synthesis, the expression vector pET-28a(+) was selected, and the foreign gene was inserted through the EcoR I and Hind III double restriction sites, such as Figure 1As shown, the constructed vector was loaded into E. coli BL21 (DE3), Kna resistance plates were spread to screen positive clones, and plasmid extraction and sequencing were performed to finally confirm that the recombinant engineered bacteria containing the esterase gene were obtained. Among them, the nucleotide sequence of T-002 is shown in SEQ ID NO: 1.

[0023] Example 2 Preparation of recombinant transaminase

[0024] The recombinant bacteria constructed in Example 1 were inoculated into 10 mL of LB medium and cultured at 37°C with shaking overnight. The inoculation amount was transferred to LB medium at 1%, and cultured at 37°C until OD600 = 0.6-0.8. IPTG was added at a final concentration of 0.5 mM and induced at 20°C for 16 h. The wet cells were collected by centrifugation, washed twice, and then ultrasonically disrupted to obtain a diluted crude enzyme solution.

[0025] Example 3 Application of recombinant transaminase in the preparation of ticagrelor intermediate compound I

[0026] In a 250mL conical flask, 9mL 0.2M pH7.0 PB buffer, 0.1g isopropylamine, 10mL diluted crude enzyme solution (containing about 1g wet bacteria), 0.001g PLP, 1g substrate, and 4mL DMSO were added respectively. The reaction temperature was 40°C, 220rpm, and the shaker was used to catalyze the reaction overnight. After the substrate was completely converted, an equal volume of ethyl acetate was added for extraction twice, and the upper ethyl acetate phase was taken. According to liquid chromatography detection, the ee value was 98.9%, and the chemical purity after further purification was about 99.2%.

[0027] The above is a specific implementation of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications are also considered to be within the protection scope of the present invention.

Claims

1. Use of a recombinant transaminase in catalyzing the transamination reaction of a ketone compound to prepare a chiral amine, wherein the ketone compound is a compound of formula II, ； The amino acid sequence of the recombinant transaminase is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that: The application of the transaminase in the preparation of a chiral intermediate compound of ticagrelor, and the synthetic process route are as follows: ； Compound I is obtained by reacting with compound II as substrate, transaminase as catalyst, buffer solution of pH 7.0-8.5 as reaction medium, auxiliary solvent, amino donor and PLP in the reaction system, and is separated and purified.

3. The use according to claim 1, characterized in that: The nucleotide sequence of the recombinant transaminase gene is shown in SEQ ID NO:

2.

4. The use according to claim 1, characterized in that: The recombinant transaminase is obtained by expressing a recombinant bacterial strain.

5. The use according to claim 4, characterized in that: The recombinant strain used pET-28a plasmid as expression vector to express recombinant transaminase.

6. The use according to claim 4, characterized in that: The recombinant strain uses Escherichia coli BL21 (DE3) as a host cell.

7. The use according to claim 6, characterized in that: The recombinant strain is formed by connecting the transaminase gene shown in SEQ ID NO: 2 to the EcoR I and Hind III sites of the pET-28a plasmid and then transforming it into Escherichia coli BL21 (DE3).

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