Recombinant r-ω-transaminase, mutant and its application in asymmetric synthesis of sitagliptin

A technology for sitagliptin and mutants, applied in the field of recombinant R-ω-transaminase, mutants and asymmetric synthesis of sitagliptin, to achieve high stereoselective effect

Active Publication Date: 2021-08-27
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, there is no report on the successful preparation of sitagliptin by other R-ω-TA

Method used

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  • Recombinant r-ω-transaminase, mutant and its application in asymmetric synthesis of sitagliptin
  • Recombinant r-ω-transaminase, mutant and its application in asymmetric synthesis of sitagliptin
  • Recombinant r-ω-transaminase, mutant and its application in asymmetric synthesis of sitagliptin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Screening of novel ω-TA, determination of stereoselectivity and precise determination of enzyme activity

[0027] 1. Enzyme source and gene synthesis

[0028] Using the amino acid sequence of the commercial enzyme R-ω-TA 117 as a template, three ω-TA strains were obtained by gene mining from the NCBI database, namely Gibberella zeae TA (GzTA, GenBank No. XP_381942.1), Mycobacterium vanbaalenii TA (MvTA, GenBank No. WP_011781668.1) and Neosartorya fischeri TA (NfTA, GenBank Accession XP_001261640.1). The homology between the above three enzymes and R-ω-TA 117 is 44%, 52% and 38%, respectively. Codon optimization was carried out according to the codon preference of E.coli, and the nucleotide sequences of the three enzymes were synthesized by the method of whole gene synthesis, which are respectively represented by SEQ ID NO.2, SEQ ID NO.4 and SEQ ID NO.6 shown; the amino acid sequences encoding the enzymes are shown in SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.5...

Embodiment 2

[0043] Example 2: Construction and screening of GzTA single point mutants

[0044] 1. Mutant construction

[0045] Carry out single point mutation to the novel R-omega-TA screened, according to the amino acid sequence of NCBI's GzTA (GenBank number is XP_381942.1, the amino acid sequence is shown in SEQ ID NO: 1, and the nucleotide sequence is SEQ ID NO : 2 shows) design the mutation primer of site-directed mutation, utilize fast PCR technique, take recombinant vector pET28b / GzTA as template, introduce single mutation to the 60th position of GzTA amino acid sequence, primer is:

[0046] Forward primer GACCTGACCTACGAC NNK CCAGCTGTGTGGGAC (the underline is the mutated base)

[0047] Reverse primer GTCCCACACAGCTGG MNN GTCGTAGGTCAGGTC (the underline is the mutated base)

[0048] PCR reaction system: 2×Phanta Max Buffer (containing Mg 2+ ) 25μL, dNTPs 10mM, forward primer 2μL, reverse primer 2μL, template DNA 1μL, Phanta Max Super-Fidelity DNA Polymerase 50U, add ddH 2 0 to...

Embodiment 3

[0062] Example 3: Construction and screening of GzTA two-site mutants

[0063] The single mutant GzTA constructed according to Example 2 1 Sequence design of mutation primers for site-directed mutagenesis, using rapid PCR technology to recombinant vector pET28b / GzTA 1 as template, for GzTA 1 A single mutation is introduced at position 113 of the amino acid sequence, and the primers are:

[0064] Forward primer ATCAAAGACGCT NNK GTTGAACTGATCGTT (the underline is the mutant base)

[0065] reverse primer GATCAGTTCAAC MNN AGCGTCTTTGATACC (the underline is the mutated base)

[0066] The PCR reaction system is the same as the "construction of mutants" in Example 2.

[0067] The PCR amplification conditions were 95°C for 3min; (95°C for 15s, 50°C for 15s, 63°C for 6.5min) for 30 cycles; 72°C for 5min.

[0068] The PCR product was transformed into E.coli BL21(DE3) competent cells, and a single clone was picked in LB liquid medium containing 50 μg / mL kanamycin, and cultured ove...

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Abstract

The invention relates to the application of a novel recombinant R-ω-transaminase and its high-activity mutant in catalyzing the asymmetric synthesis of sitagliptin precursor ketone. The amino acid sequence of the recombinant R-ω-transaminase is shown in SEQ ID: 1, and its mutant is the 60th, 113, 178, 233, 146, 214 or 186 of the amino acid sequence shown in SEQ ID: 1 One or more single-site mutations or multi-site mutations are obtained. The present invention provides a novel R-omega-TA mutant with high activity and high stereoselectivity, which is a milestone in realizing the self-management and localization of sitagliptin biocatalytic preparation technology, and can change the current asymmetric synthesis of sitagliptin The technical monopoly situation of a single enzyme source in the process of Gliptin.

Description

[0001] (1) Technical field [0002] The invention relates to a new recombinant R-ω-transaminase and its mutant, and the application of the R-ω-transaminase and the mutant in asymmetric synthesis of sitagliptin. [0003] (2) Background technology [0004] Sitagliptin, English name sitagliptin, full name (3R)-3-amino-1-[3-(trifluoromethyl)-5,6,7,8-tetrahydro-1,2,4-triazolo [4,3-a]pyrazin-7-yl]-4-(2,4,5-trifluorophenyl)butan-1-one, a dipeptidyl group researched and developed by Merck and Codexis Peptidase-4 (DPP-4) inhibitors can control blood sugar levels by protecting endogenous incretins and enhancing their effects. Compared with sulfonylurea drugs and metformin drugs, it has more stable hypoglycemic function, fewer side effects, and better clinical effect, and can be effectively used in the treatment of type II diabetes, and is a therapeutic drug with great potential for type II diabetes. [0005] The preparation of sitagliptin mainly includes chemical synthesis and asymmetr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12P17/18
CPCC12N9/1096C12P17/182C12Y206/01
Inventor 柳志强李军良贾东旭郑裕国张晓健徐海鹏彭晨
Owner ZHEJIANG UNIV OF TECH
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