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(R)-omega-transaminase mutant and application thereof

A technology of mutants and transaminases, applied in applications, transferases, enzymes, etc., can solve problems such as lack of competitive advantages, and achieve the effects of high stereoselectivity, high substrate tolerance, and high enzyme activity

Active Publication Date: 2020-07-14
ZHEJIANG UNIV OF TECH +2
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In the case that the current asymmetric synthesis of sitagliptin technology is monopolized by Merck and Codexis, the substrate concentration of other technical routes for the preparation of sitagliptin by (R)-ω-TA still does not have a competitive advantage

Method used

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  • (R)-omega-transaminase mutant and application thereof
  • (R)-omega-transaminase mutant and application thereof
  • (R)-omega-transaminase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Screening, determination of stereoselectivity and precise determination of enzyme activity of novel ω-TA

[0033] 1. Enzyme source and gene synthesis

[0034] 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 Aspergillus terreus TA (AtTA, GenBank No. XP_001209325.1), Mycolicibacterium wolinskyi TA (MwTA , GenBank accession WP_067853383) and Aspergillus parasiticus TA (ApTA, GenBank accession KJK66446).

[0035] The sequence identity rates of the above three enzymes with (R)-ω-TA 117 were 38.14%, 48.97% and 37.9%, respectively. Codon optimization was carried out according to the codon preference of E.coli, and three strains of enzymes were synthesized by the method of whole gene synthesis, 6×His-tag tags were added at the end of the nucleic acid sequence, and restriction sites Xho I and Nco I were added at both ends, The gene was cloned into the cor...

Embodiment 2

[0058] Example 2 Construction and screening of AtTA single point mutants

[0059] 1. Mutant construction

[0060] The novel (R)-ω-TA screened was subjected to single-point mutation, and mutation primers were designed according to the nucleotide sequence of AtTA (shown as SEQ ID NO.2), and the recombinant vector pET28b / AtTA was used as the recombinant vector by using rapid PCR technology. Template, introduce a single mutation at position 182 of the amino acid sequence of AtTA, and the primers are:

[0061] Forward primer: TGAAAAAT NNK CAGTGGGGTGATCTGGTGC (the underline is the mutated base, as shown in SEQ ID NO.4)

[0062] Reverse primer: CCCCACTG MNN ATTTTTCACAGTCGGGTCA (the underline is the mutant base, as shown in SEQ ID NO.5)

[0063] 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 50 μL.

[0064] The PCR amplificatio...

Embodiment 3

[0078] Example 3 Construction and screening of AtTA two-site mutants

[0079] The single mutant AtTA constructed according to Example 2 1 Sequence design of mutation primers for site-directed mutagenesis, using rapid PCR technology to recombinant vector pET28b / AtTA 1 as the template, the AtTA 1 A single mutation is introduced at position 79 of the amino acid sequence, and the primers are:

[0080] Forward primer: ACATTACG NNK CTGGAGGCTAGCTGCACCA (the underline is the mutant base, as shown in SEQ ID NO.6)

[0081] Reverse primer: GCCTCCAG MNN CGTAATGTGGTCATCCAGA (the underline is the mutant base, as shown in SEQ ID NO.7)

[0082] The PCR reaction system is the same as the "construction of mutants" in Part 1 of Example 2.

[0083] The PCR amplification conditions were: 95°C for 3min; (95°C for 15s, 50°C for 15s, 63°C for 6.5min) 30 cycles; 72°C for 5min.

[0084] The PCR product was transformed into E.coli BL21(DE3) competent cells, and a single clone was picked in LB l...

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Abstract

The invention discloses a (R)-omega-transaminase mutant and application thereof. The mutant is obtained by multi-point mutation of leucine at 182, arginine at 79, glutamine at 51, valine at 149, leucine at 235 and glycine at 216 in an amino acid sequence shown by SEQ ID NO.1. New (R)-omega-TA enzyme is screened through a gene mining technology in the invention; furthermore, molecular modificationis carried out through a protein engineering technology; a (R)-omega-TA mutant catalysis having high enzyme activity, high substrate tolerance and high stereoselectivity is obtained; the mutant can biologically catalyze precursor ketone analogue 1-(3-oxygen pyrrolidine-1-yl)-4-(2,4,5-trifluorophenyl)-1,3-butanedione synthesis sitagliptin intermediate (R)-1-[3-amino-4-(2,4,5-trifluorophenyl) butyryl] pyrrole-3-ketone; furthermore, the conversion rate is relatively high; the conversion rate can be up to 95.4%; and thus, the (R)-omega-transaminase mutant and application thereof in the invention have milestone meaning for breaking through a sitagliptin biocatalysis preparation technology.

Description

technical field [0001] The present invention relates to the field of biochemical technology, in particular to a (R)-ω-transaminase mutant and its application, especially the (R)-ω-transaminase mutant in the preparation of sitagliptin intermediate (R)-1- [3-Amino-4-(2,4,5-trifluorophenyl)butyryl]pyrrol-3-one. Background technique [0002] 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, which can control blood sugar levels by protecting endogenous incretins and enhancing their effects, are a therapeutic drug with great potential for type Ⅱ diabetes. [0003] The preparation of sitagliptin is mainly an asymmetric synthesis method, usually using transaminase as a biocatalyst for biotransformation. Transaminase (TA for short, EC 2.6.1.X) is a coenzyme-ty...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P41/00C12P17/10C12R1/19
CPCC07K2319/21C12N9/1096C12N15/70C12N2800/22C12P17/10C12P41/006C12Y206/01
Inventor 柳志强贾东旭彭晨李军良程峰张晓健郑裕国何人宝金逸中林娇华
Owner ZHEJIANG UNIV OF TECH
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