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Recombinant R-Omega-transaminase, mutant and application of recombinant R-Omega-transaminase and mutant in asymmetrically synthesizing sitagliptin

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

Active Publication Date: 2020-04-03
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-Omega-transaminase, mutant and application of recombinant R-Omega-transaminase and mutant in asymmetrically synthesizing sitagliptin
  • Recombinant R-Omega-transaminase, mutant and application of recombinant R-Omega-transaminase and mutant in asymmetrically synthesizing sitagliptin
  • Recombinant R-Omega-transaminase, mutant and application of recombinant R-Omega-transaminase and mutant in asymmetrically synthesizing 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] The novel R-omega-TA screened was subjected to single-point mutation, according to the amino acid sequence of GzTA of NCBI (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...

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 3 minutes; (95° C. for 15 s, 50° C. for 15 s, 63° C. for 6.5 minutes) 30 cycles; 72° C. for 5 minutes.

[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 kana...

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Abstract

The invention relates to application of novel recombinant R-Omega-transaminase and a high-activity mutant of the recombinant R-Omega-transaminase in asymmetrically synthesizing sitagliptin by catalyzing sitagliptin precursor ketone. The amino acid sequence of the recombinant R-Omega-transaminase is shown by SEQ ID:1, and the mutant of the recombinant R-Omega-transaminase is obtained by performingsingle-site mutation or multisite mutation on one or more of a 60th site, 113th site, 178th site, 233th site, 146th site, 214th site and 186th site of the amino acid sequence shown by SEQ ID:1. The mutant of the novel R-Omega-transaminase with high activity and high stereoselectivity is provided; the mutant has milestone significance in realizing autonomization and localization of a biological catalysis preparation technology for the sitagliptin; and the technology monopoly situation that an enzyme source is single in the process of asymmetrically synthesizing the sitagliptin at present can bechanged.

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 Applications(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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