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Amine transaminase AcATA mutant and application thereof in preparation of sitagliptin intermediate

A technology of amine aminotransferase and sitagliptin, which is applied in the application field of amine aminotransferase AcATA mutant and the preparation of sitagliptin intermediates, can solve the problems of narrow substrate spectrum, limited application scope and the like, and achieves the improvement of the overall conversion rate , the effect of shortened response time

Active Publication Date: 2020-08-18
ZHEJIANG UNIV OF TECH +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] However, there are few reports on natural ω-transaminases with R-type selective transamination, and these ω-transaminases catalyze a narrow spectrum of substrates, which are often the most suitable biocatalysts for specific reactions, thus greatly limiting their use. Application range

Method used

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  • Amine transaminase AcATA mutant and application thereof in preparation of sitagliptin intermediate
  • Amine transaminase AcATA mutant and application thereof in preparation of sitagliptin intermediate
  • Amine transaminase AcATA mutant and application thereof in preparation of sitagliptin intermediate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Homology modeling and substrate docking of wild-type sequences

[0040] Using the crystal structure of AcATA (PDB-ID: 3WWJ) as a template, SWISS-MODEL was used for homology modeling, and the AcATA model structure was superimposed on the wild-type model structure using a proprietary algorithm based on the comparison of secondary structure elements, and passed through FoldX The software is optimized. The optimized homology model was docked with the substrate through Autodock software, and the docking result with the lowest energy was selected for key site analysis ( figure 1 ).

Embodiment 2

[0041] Example 2: Alanine scanning mutagenesis and recombinant E. coli cell culture

[0042] All the amino acid positions obtained after docking the substrate of Example 1 were scanned for alanine using the QuikChange site-directed mutagenesis kit and Phanta Max ultra-fidelity DNA polymerase (Vazyme, Nanjing, China).

[0043] Some primers are:

[0044] Primer 1: M122 PrGAGCTGCGTGAAGCGGCGGTAACTGTTACCATC

[0045] M122 Pf GATGGTAACAGTTACCGCCGCTTCACGCAGCTC

[0046] Primer 2: G224PrCTGGCGGAAGGTCCGGCTTTCAACGTAGTAGTG

[0047] G224 Pf CACTACTACGTTGAAAGCCGGACCTTCCGCCAG

[0048] Primer 3: W192 Pr GTGAAAAACTTCCAGGCGGGTGATCTGATTCGT

[0049] W192 Pf ACGAATCAGATCACCCGCCTGGAAGTTTTTCAC

[0050] Primer 4: T126 Pr GCGATGGTAACTGTTGCCATCACTCGTGGTTAC

[0051] T126 Pf GTAACCACGAGTGATGGCAACAGTTACCATCGC

[0052] Primer 5: S150 Pr CCTCAGGTGTACATGGCTGCATGTCCGTACCAG

[0053] S150 Pf CTGGTACGGACATGCAGCCATGTACACCTGAGG

[0054] Primer 6: F60 PrATCTTCGACCAGGGCGCTTATACTTCCGATGCG

[0055] F60 Pf CGC...

Embodiment 3

[0063] Example 3: Induced expression of amine transaminase AcATA

[0064] The ω-transaminase mutant containing the complete open reading frame was cultivated according to the method of obtaining wet cells in Example 2, and the obtained wet cells could be directly used as a biocatalyst for subsequent enzyme activity determination or protein purification.

[0065] The preparation method of wet thallus (that is: genetically engineered bacteria) is: inoculate the recombinant Escherichia coli containing the ω-transaminase mutant coding gene to the LB liquid medium containing 50 μg / ml ampicillin, cultivate 8- 10h, obtain the seed liquid; then inoculate the seed liquid with a volume concentration of 1-2% inoculum into fresh LB liquid medium containing 50 μg / ml ampicillin resistance, and cultivate it at 37°C and 150 rpm until the cell OD 600 After reaching 0.6-0.8, add IPTG with a final concentration of 0.1mM, induce culture at 28°C for 12h, centrifuge at 8000rpm at 4°C for 10min, dis...

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Abstract

The invention discloses an amine transaminase AcATA mutant and application of the amine transaminase AcATA mutant in preparation of a sitagliptin intermediate. The amine transaminase AcATA mutant is obtained by single mutation at the 122th site of an amino acid sequence shown in SEQ ID No.2. The amino acid sequence of the amine transaminase AcATA mutant is shown in SEQ ID No.2. The amino acid sequence of the amine transaminase AcATA mutant is shown in the description, wherein methionine at the 122 position is mutated into histidine, valine or phenylalanine. According to the invention, sites possibly influencing the catalytic activity are obtained through molecular docking, homologous modeling and other methods, and site-specific mutagenesis is carried out, so that the finally obtained aminotransferase AcATA mutant has high enzyme activity, and the enzyme activity is higher than 460U / g and is more than 4 times of that of a wild type; the catalyst can efficiently catalyze the sitagliptin intermediate precursor ketone 1-piperidine-4-(2, 4, 5-trifluorophenyl)-1, 3-dibutanone to synthesize the sitagliptin intermediate (R)-3-amino-1-piperidine-4-(2, 4, 5-trifluorophenyl)-1-butanone, and the 24-hour conversion rate is up to 90%.

Description

technical field [0001] The invention relates to the field of biochemical technology, in particular to an aminotransaminase AcATA mutant and its application in the preparation of a sitagliptin intermediate. Background technique [0002] Sitagliptin Phosphate (Sitagliptin Phosphate) was developed and developed by Merck and Codexis in the United States. It is the first dipeptidyl peptidase-4 (DPP-4) inhibitor approved by the FDA for the treatment of type 2 diabetes (2016 October). Sitagliptin phosphate is the active ingredient of JANUVIA (Sitagliptin Hydrochloride Tablets, JANUVIA), an oral treatment drug for type II diabetes. It mainly inhibits DPP-4 on glucagon-like peptide-1 (GLP-1 ) and glucose-dependent insulinotropic peptide (GIP) degradation to achieve therapeutic purposes, slightly increase the content of GLP-1 and weaken the antagonism of GLP-1 metabolites. While the sitagliptin hydrochloride effectively exerts the hypoglycemic effect, it will not cause side effects ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P17/12C12R1/19
CPCC12N9/1096C12N15/70C12P17/12C12Y206/01
Inventor 柳志强程峰李明友张晓健贾东旭郑裕国何人宝金逸中林娇华
Owner ZHEJIANG UNIV OF TECH
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