Recombinant transaminase and its enzyme preparation and application
The recombinant transaminase strain was constructed through genetic engineering technology and expressed in E. coli BL21 (DE3), which solved the ticagrelor intermediate (1R,2S)-2-(3,4 difluorophenyl)cyclopropylamine in the prior art The problem of insufficient catalytic activity in the preparation of chiral amines has achieved an efficient and clean preparation method and meets the requirements of green production.
Patent Information
- Application Number
- CN202211395614.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-11-09
- Publication Date
- 2025-01-17
- Estimated Expiration
- 2042-11-09
AI Technical Summary
The prior art has failed to effectively prepare the key intermediate (1R,2S)-2-(3,4 difluorophenyl)cyclopropylamine chiral amine of ticagrelor, and the catalytic activity is insufficient, making it difficult to achieve efficient preparation.
Through genetic engineering technology, the amino acid sequence and encoding gene of recombinant aminotransferase are constructed, expressed in E. coli BL21 (DE3), the pET-30a plasmid is used as the expression vector to form a recombinant strain, and the aminotransferase preparation is produced through fermentation to catalyze the transfer of ketone compounds Ammonia reaction to prepare chiral amines.
It significantly improves catalytic activity and realizes the efficient and clean preparation of chiral amines of ticagrelor intermediate (1R,2S)-2-(3,4 difluorophenyl)cyclopropylamine, which meets the requirements of modern green production.
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Figure CN115927239B_ABST
Abstract
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 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.
[0004] Ticagrelor is a new type of cyclopentyltriazolopyrimidine oral antiplatelet drug developed by AstraZeneca in the UK. It is a selective adenosine diphosphate receptor antagonist that acts on the P2Y12ADP receptor to inhibit ADP-mediated platelet activation and aggregation. The drug was approved for marketing in China in November 2012.
[0005] (1R,2S)-2-(3,4-difluorophenyl)cyclopropylamine is a key intermediate for preparing ticagrelor. Currently, various preparation schemes for this intermediate compound have been disclosed in the prior art, but the preparation of the key intermediate (1R,2S)-2-(3,4-difluorophenyl)cyclopropylamine of ticagrelor by enzymatic method has not been reported. Summary of the invention
[0006] The technical problem to be solved by the present invention is to provide a new recombinant transaminase engineering bacterium, and obtain a chiral amine that can be used in the preparation of the key intermediate (1R, 2S)-2-(3, 4-difluorophenyl)cyclopropylamine of ticagrelor, thereby significantly improving the catalytic activity and efficiently preparing the target compound.
[0007] In order to solve the above technical problems, the present invention first discloses the amino acid sequence of the recombinant transaminase as shown in SEQ ID NO: 1.
[0008] Furthermore, the present invention also discloses the nucleotide sequence of the gene encoding the recombinant transaminase as shown in SEQ ID NO: 2.
[0009] Meanwhile, the present invention also discloses a recombinant strain, wherein the recombinant strain expresses the recombinant transaminase shown in SEQ ID NO:1.
[0010] Furthermore, the recombinant strain uses pET-30a plasmid as an expression vector to express the recombinant transaminase.
[0011] Furthermore, the recombinant strain uses Escherichia coli BL21 (DE3) as a host cell.
[0012] Furthermore, the recombinant strain is formed by connecting the transaminase gene shown in SEQ ID NO: 2 to the BamH I and Xho I sites of the pET-30a plasmid and then transforming it into Escherichia coli BL21 (DE3).
[0013] At the same time, the present invention also discloses an enzyme preparation containing the transaminase produced by fermentation of the above recombinant strain.
[0014] Furthermore, the present invention also discloses a method for producing transaminase by fermentation of recombinant bacterial strains, the specific steps of which are: transferring the seed liquid of the transaminase recombinant strain to LB culture medium at an inoculation rate of 2%, culturing at 37°C until OD600 = 0.6, adding 50 μl of 0.5 mol / L IPTG, and inducing at 18°C for 14 hours to obtain the transaminase fermentation liquid.
[0015] Finally, the present invention also discloses the use of the transaminase obtained by the above-mentioned method of producing transaminase by fermentation of the recombinant strain in catalyzing the transamination reaction of ketone compounds to prepare chiral amines, wherein the ketone compound refers to the compound of formula II or a similar compound thereof,
[0016]
[0017] Specifically, the present invention also discloses the use of the transaminase in the preparation of a chiral intermediate compound of ticagrelor, and the synthesis process route is as follows:
[0018]
[0019] The present invention utilizes genetic engineering technology, and through the recombination and construction of transaminase engineering bacteria, successfully obtains an engineering bacterium that can be applied to the preparation of chiral amines in (1R, 2S)-2-(3,4-difluorophenyl)cyclopropylamine. The preparation of the intermediate compound (1R, 2S)-2-(3,4-difluorophenyl)cyclopropylamine of ticagrelor by this method has the advantages of efficient and clean production, and is an industrial production method that meets the requirements of modern green production. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 This is the structure diagram of the Recombinant-01 plasmid. DETAILED DESCRIPTION
[0021] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.
[0022] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without making any creative work shall fall within the scope of protection of the present invention.
[0023] Example 1 Construction of transaminase recombinant strain
[0024] The upstream and downstream primers P1 and P2 of the transaminase gene were designed (as shown in Table 1), and the T-003 gene containing BamH I and XhoI restriction sites was amplified by PCR using pUC57-T-003 constructed by full gene synthesis as a template.
[0025] PCR conditions were: 98°C for 3 min, 98°C for 30 s, 55°C for 90 s, 72°C for 90 s, 30 cycles. PCR amplification system: 1.5 μL template, 1.5 μL upstream and downstream primers, 20.5 μL sterilized double distilled water, 25 μL PrimerSTAR Mix. The PCR product was purified and recovered using a gel recovery kit, and the concentration of the recovered product was tested by electrophoresis.
[0026] The gel recovery product and pET-30a plasmid (expression vector) were digested with BamH I and XhoI enzymes, and the gel recovery kit was used to purify and recover the gel recovery product after digestion. The gel recovery kit was used to purify and recover the plasmid after digestion, and the concentration of the recovered product was tested by electrophoresis. The target gene T-003 was connected to the vector pET-30a, and the connection system was: 4 μL of the target gene, 2 μL of the vector pET-30a, 2 μL of buffer, 1 μL of ligase, and connected at 16°C overnight. The constructed vector was introduced into E. coli BL21 (DE3) by chemical transformation technology, coated on an LB plate containing kanamycin, placed in a 37°C incubator overnight, and the single colony grown was subjected to plasmid extraction and sequencing, and finally a recombinant engineered bacterium containing the transaminase gene was obtained.
[0027] Among them, the amino acid sequence of the transaminase gene is SEQ ID NO.1, and the nucleotide sequence of the transaminase gene is SEQ ID NO.2.
[0028] Table 1 Primers
[0029]
[0030] Example 2 Preparation of (1R, 2S)-2-(3,4-difluorophenyl)cyclopropylamine by fermentation of recombinant bacteria
[0031] (1) Preparation of transaminase fermentation broth
[0032] The recombinant bacteria constructed in Example 1 were inoculated into 10 mL of LB medium and cultured at 37°C with shaking overnight. The cells were transferred to LB medium at a 2% inoculum size and cultured at 37°C until OD600 = 0.6. IPTG with a final concentration of 0.5 mmol / L was added and induced at 18°C for 16 h. The cells were centrifuged, washed 3 times with saline, resuspended, and ultrasonically disrupted to obtain a crude enzyme solution.
[0033] (2) Enzymatic Preparation of (1R, 2S)-2-(3,4-difluorophenyl)cyclopropylamine
[0034] Weigh 50 mL of 0.1 M pH 8.0 Tris-HCl. Then add 10 mL of the crude enzyme solution prepared in (1), 0.2 M isopropylamine, and 10 mg of PLP, and mix well. Weigh 1 g of the compound in a 5 mL centrifuge tube, add 5 mL of DMSO to dissolve, and then slowly add it to the reaction bottle. Shake at 35°C, 220 rpm to catalyze the reaction. After overnight reaction, extract with ethyl acetate, take the upper ethyl acetate phase, and send it for analysis and detection. After liquid chromatography detection, the chiral purity is 99%, and the chemical purity after recrystallization is 90.07%.
[0035] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.
Claims
1. A method for preparing a chiral amine (1R, 2S)-2-(3,4-difluorophenyl)cyclopropylamine, a key intermediate of Ticagrelor, characterized in that: The synthetic process route is as follows: ； Among them, Transaminase is a transaminase fermentation broth fermented from the seed liquid of transaminase recombinant bacteria. The specific fermentation method is: transfer the seed liquid of transaminase recombinant strain to LB medium at an inoculation rate of 2%, culture at 37°C until OD600=0.6, add 50 μl 0.5 mol / L IPTG, and induce at 18°C for 14 h, that is, transaminase fermentation broth; The transaminase recombinant bacterial seed solution is prepared by connecting the transaminase gene shown in SEQ ID NO: 2 to the BamH I and Xho I sites of the pET-30a plasmid, and then transforming it into Escherichia coli BL21 (DE3) and fermenting it.
2. A method for preparing a chiral amine (1R, 2S)-2-(3, 4-difluorophenyl)cyclopropylamine key intermediate of ticagrelor according to claim 1, characterized in that: Prepare according to the following proportions and conditions: Weigh 50 mL of 0.1 M pH 8.0 Tris-HCl, add 10 mL of crude enzyme solution and 0.2 M isopropylamine, and 10 mg of PLP, and mix well; weigh 1 g of compound II in a 5 mL centrifuge tube, add 5 mL of DMSO to dissolve, and then slowly add to the reaction bottle, shake at 35 ° C, 220 rpm, and catalyze the reaction; after the reaction, extract with ethyl acetate, and take the upper ethyl acetate phase, which is the target compound I.